Performance of ‘Coscia’ pear (Pyrus communis) on nine rootstocks in the north of Israel

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不同品种石榴枝条的抗寒性评价

河南农业科学，2023，52（6）：120‐130Journal of Henan Agricultural Sciencesdoi ：10.15933/ki.1004‐3268.2023.06.013不同品种石榴枝条的抗寒性评价雷梦瑶1，高小峰2，白清敏3，邓珂4，左卫芳1，李玉英1（1.南阳师范学院河南省软籽石榴工程研究中心，河南省南水北调中线水源区流域生态安全国际联合实验室，河南南阳473061；2.南阳市农业科学院，河南南阳473000；3.南阳市农村能源环境保护管理站，河南南阳473000；4.南阳农业职业学院农业工程学院，河南南阳473000）摘要：为筛选抗寒石榴品种，以河南省南阳市淅川县生长的石榴品种突尼斯、中农红、红双喜和千层花1年生休眠期枝条为试材，通过模拟低温胁迫环境，分别在4℃、0℃、-3℃、-6℃、-9℃、-12℃和-15℃的低温下处理24h ，测定相对电导率（REC ），脯氨酸、可溶性糖、可溶性蛋白、丙二醛（MDA ）含量，过氧化物酶（POD ）、超氧化物歧化酶（SOD ）、过氧化氢酶（CAT ）活性等指标，并利用隶属函数法综合评价4个品种石榴枝条的抗寒性。

结果表明，千层花的半致死温度（LT 50）最低，达到-6.94℃，红双喜最高，为−2.27℃；随着温度降低，4个品种枝条POD 活性、可溶性糖含量和相对电导率整体呈上升趋势，SOD 、CAT 活性及MDA 、可溶性蛋白含量整体呈先升后降趋势，脯氨酸含量整体呈先降后升趋势。

利用隶属函数法综合评价的石榴品种抗寒性强弱顺序为千层花>红双喜>突尼斯>中农红。

关键词：石榴；低温胁迫；隶属函数法；抗寒性；评价中图分类号：S665.4文献标志码：A文章编号：1004-3268（2023）06-0120-11收稿日期：2023-01-05基金项目：河南省重点研发专项（2211111520600）；河南省科技厅高等学校学科创新引智基地项目（CXJD2019001）；河南省科技创新体系建设项目（192207310029）作者简介：雷梦瑶（1997-），女，河南周口人，在读硕士研究生，研究方向：特色植物栽培。

采前甲壳宝处理对“红丰”梨贮藏品质的影响

姜永峰，陆玉卓，邢英丽，等．采前甲壳宝处理对“红丰”梨贮藏品质的影响［Ｊ］．江苏农业科学，２０２３，５１（２４）：２１２－２１７．ｄｏｉ：１０．１５８８９／ｊ．ｉｓｓｎ．１００２－１３０２．２０２３．２４．０２９采前甲壳宝处理对“红丰”梨贮藏品质的影响姜永峰，陆玉卓，邢英丽，李宏军，郝　义（辽宁省果树科学研究所，辽宁营口１１５００９）摘要：以红丰梨为试验材料，使用３种不同浓度［４／１０００（Ｖ／Ｖ）、３／１０００（Ｖ／Ｖ）、２／１０００（Ｖ／Ｖ）］的水溶肥料“甲壳宝”稀释液对红丰梨采前１个月进行３次（７ｄ／次）喷布，研究其对红丰梨采后贮藏品质的影响。

结果表明，采前甲壳宝处理有效地提高果实采收品质和贮藏品质，浓度为４／１０００（Ｖ／Ｖ）效果最好。

采收时处理组的色差值优于对照组；处理组可溶性固形物（ＴＳＳ）和可滴定酸（ＴＡ）含量高于对照组；贮藏５０ｄ时，经过４／１０００（Ｖ／Ｖ）的甲壳宝处理的红丰梨果实ＴＳＳ含量是对照组的１．１３倍，ＴＡ含量是对照组的１．１５倍；硬度是对照组的１．３５倍；失质量率对照组５．２２％，处理组３．７２％；腐烂率对照组是处理组的１．９４倍。

通过感官评价发现，采前４／１０００（Ｖ／Ｖ）的甲壳宝处理能够有效提高红丰梨的贮藏品质；相关性分析可知红丰梨感官评价越高硬度和ＴＳＳ含量越高，腐烂率、失质量率和色差越小，处理组感官评价和各指标的相关系数均大于对照组。

关键词：红丰梨；甲壳宝；采前处理；贮藏品质；水溶肥；保鲜中图分类号：Ｓ６６１．２０９＋．３；ＴＳ２５５．３文献标志码：Ａ文章编号：１００２－１３０２（２０２３）２４－０２１２－０６收稿日期：２０２３－０２－１５基金项目：辽宁省盖州市果品保鲜科技特派团项目（编号：２０２２ＪＨ５／１０４０００６７）；乡村振兴科技支持专项；“揭榜挂帅”科技攻关专项（编号：１６５５８１５１１４４１５）。

作者简介：姜永峰（１９７２—），男，辽宁营口人，助理研究员，研究方向为果品保鲜技术示范推广。

15898468_4个肉牛品种微卫星多态性分析

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张相伦等 E 个肉牛品种微卫星多态性分析
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羧甲基纤维素钠复合天然抗氧化剂对冷鲜鸡肉的抗氧化及保水效果

櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄櫄Ｍａｃｒｏｍｏｌｅｃｕｌｅｓ，２０２０，１５５：１２５２－１２６１．［２３］ＣｈｏｑｕｅＥ，ＲｅｚｚａｎｉＧ，ＳａｌｖａｙＡＧ，ｅｔａｌ．Ｉｍｐａｃｔｏｆｆｕｎｇａｌｅｘｔｒａｃｔｓｏｎｔｈｅｐｈｙｓｉｃａｌａｎｄａｎｔｉｏｘｉｄａｎｔｐｒｏｐｅｒｔｉｅｓｏｆｂｉｏａｃｔｉｖｅｆｉｌｍｓｂａｓｅｄｏｎｅｎｚｙｍａｔｉｃａｌｌｙｈｙｄｒｏｌｙｚｅｄｙｅａｓｔｃｅｌｌｗａｌｌ［Ｊ］．ＪｏｕｒｎａｌｏｆＰｏｌｙｍｅｒｓａｎｄｔｈｅＥｎｖｉｒｏｎｍｅｎｔ，２０２１，２９（６）：１９５４－１９６２．［２４］ＨａｒｋｅｒＦＲ，ＧｕｎｓｏｎＦＡ，ＢｒｏｏｋｆｉｅｌｄＰＬ，ｅｔａｌ．Ａｎａｐｐｌｅａｄａｙ：ｔｈｅｉｎｆｌｕｅｎｃｅｏｆｍｅｍｏｒｙｏｎｃｏｎｓｕｍｅｒｊｕｄｇｍｅｎｔｏｆｑｕａｌｉｔｙ［Ｊ］．ＦｏｏｄＱｕａｌｉｔｙａｎｄＰｒｅｆｅｒｅｎｃｅ，２００２，１３（３）：１７３－１７９．［２５］Ｎａｊａｆｉ－ＭａｒｇｈｍａｌｅｋｉＳ，ＭｏｒｔａｚａｖｉＳＭＨ，ＳａｅｉＨ，ｅｔａｌ．Ｔｈｅｅｆｆｅｃｔｏｆａｌｇｉｎａｔｅ－ｂａｓｅｄｅｄｉｂｌｅｃｏａｔｉｎｇｅｎｒｉｃｈｅｄｗｉｔｈｃｉｔｒｉｃａｃｉｄａｎｄａｓｃｏｒｂｉｃａｃｉｄｏｎｔｅｘｔｕｒｅ，ａｐｐｅａｒａｎｃｅａｎｄｅａｔｉｎｇｑｕａｌｉｔｙｏｆａｐｐｌｅｆｒｅｓｈ－ｃｕｔ［Ｊ］．ＩｎｔｅｒｎａｔｉｏｎａｌＪｏｕｒｎａｌｏｆＦｒｕｉｔＳｃｉｅｎｃｅ，２０２１，２１（１）：４０－５１．［２６］ＹｌｍａｚＦＭ，ＥｒｓｕｓＢＳ．Ｕｌｔｒａｓｏｕｎｄ－ａｓｓｉｓｔｅｄｖａｃｕｕｍｉｍｐｒｅｇｎａｔｉｏｎｏｎｔｈｅｆｏｒｔｉｆｉｃａｔｉｏｎｏｆｆｒｅｓｈ－ｃｕｔａｐｐｌｅｗｉｔｈｃａｌｃｉｕｍａｎｄｂｌａｃｋｃａｒｒｏｔｐｈｅｎｏｌｉｃｓ［Ｊ］．ＵｌｔｒａｓｏｎｉｃｓＳｏｎｏｃｈｅｍｉｓｔｒｙ，２０１８，４８：５０９－５１６．［２７］ＬｉｕＸ，ＹａｎｇＱ，ＬｕＹＺ，ｅｔａｌ．Ｅｆｆｅｃｔｏｆｐｕｒｓｌａｎｅ（ＰｏｒｔｕｌａｃａｏｌｅｒａｃｅａＬ．）ｅｘｔｒａｃｔｏｎａｎｔｉ－ｂｒｏｗｎｉｎｇｏｆｆｒｅｓｈ－ｃｕｔｐｏｔａｔｏｓｌｉｃｅｓｄｕｒｉｎｇｓｔｏｒａｇｅ［Ｊ］．ＦｏｏｄＣｈｅｍｉｓｔｒｙ，２０１９，２８３：４４５－４５３．［２８］ＱｉａｏＬＰ，ＧａｏＭ，ＺｈｅｎｇＪＸ，ｅｔａｌ．Ｎｏｖｅｌｂｒｏｗｎｉｎｇａｌｌｅｖｉａｔｉｏｎｔｅｃｈｎｏｌｏｇｙｆｏｒｆｒｅｓｈ－ｃｕｔｐｒｏｄｕｃｔｓ：ｐｒｅｓｅｒｖａｔｉｏｎｅｆｆｅｃｔｏｆｔｈｅｃｏｍｂｉｎａｔｉｏｎｏｆＳｏｎｃｈｕｓｏｌｅｒａｃｅｕｓＬ．ｅｘｔｒａｃｔａｎｄｕｌｔｒａｓｏｕｎｄｉｎｆｒｅｓｈ－ｃｕｔｐｏｔａｔｏｅｓ［Ｊ］．ＦｏｏｄＣｈｅｍｉｓｔｒｙ，２０２１，３４８：１２９１３２．［２９］ＬｉＳ，ＣｈｅｎＧ，ＺｈａｎｇＣ，ｅｔａｌ．Ｒｅｓｅａｒｃｈｐｒｏｇｒｅｓｓｏｆｎａｔｕｒａｌａｎｔｉｏｘｉｄａｎｔｓｉｎｆｏｏｄｓｆｏｒｔｈｅｔｒｅａｔｍｅｎｔｏｆｄｉｓｅａｓｅｓ［Ｊ］．ＦｏｏｄＳｃｉｅｎｃｅａｎｄＨｕｍａｎＷｅｌｌｎｅｓｓ，２０１４，３（３／４）：１１０－１１６．［３０］ＹａｎｇＨＭ，ＣｈｅｎｇＳＳ，ＬｉｎＲ，ｅｔａｌ．Ｉｎｖｅｓｔｉｇａｔｉｏｎｏｎｍｏｉｓｔｕｒｅｍｉｇｒａｔｉｏｎ，ｍｉｃｒｏｓｔｒｕｃｔｕｒｅａｎｄｑｕａｌｉｔｙｃｈａｎｇｅｓｏｆｆｒｅｓｈ－ｃｕｔａｐｐｌｅｄｕｒｉｎｇｓｔｏｒａｇｅ［Ｊ］．ＩｎｔｅｒｎａｔｉｏｎａｌＪｏｕｒｎａｌｏｆＦｏｏｄＳｃｉｅｎｃｅ＆Ｔｅｃｈｎｏｌｏｇｙ，２０２１，５６（１）：２９３－３０１．王　钊，李　欣，张　乾，等．羧甲基纤维素钠复合天然抗氧化剂对冷鲜鸡肉的抗氧化及保水效果［Ｊ］．江苏农业科学，２０２２，５０（１１）：１８９－１９５．ｄｏｉ：１０．１５８８９／ｊ．ｉｓｓｎ．１００２－１３０２．２０２２．１１．０２７羧甲基纤维素钠复合天然抗氧化剂对冷鲜鸡肉的抗氧化及保水效果王　钊１，李　欣１，张　乾１，郝修振１，张晓静１，李玉江２，张本尚３，邹　建１（１．河南牧业经济学院食品与生物工程学院，河南郑州４５００４６；２．河南省科学院高新技术研究中心，河南郑州４５０００２；３．河南省科学院同位素研究所有限责任公司，河南郑州４５００１５）摘要：为提升冷鲜鸡肉品质和降低经济损失，以冷鲜鸡肉的硫代巴比妥酸值（ＴＢＡＲＳ）、失质量率和亮度值（Ｌ）为评价指标，考察天然抗氧化剂复合涂膜对冷鲜鸡肉的抗氧化和保水能力。

4个早中熟苹果品种(系)在河南商丘的表现初报

第５期
１．２生长结果状况
党伟等：４个早中熟苹果品种（系）在河南商丘的表现初报
２９
２０１３年（栽植第５年），试栽的４个品种（系）与嘎拉的平均树高都达３ｍ以上。平均干周以郑果４２、郑果４３、郑果４４三品系较大，华硕品种略小，嘎拉最小。平均单株郑果４２枝量、郑果４３、郑果４４３个品系较多，华硕，嘎拉较少。郑果４２短枝比例最高，为５８．１％，高于其余４品种（系）；长枝比例低，仅１０．７％，明显低于另４个品种（系）。新梢年生长量华硕最大，达５０．３ｃｍ，高于其余品种（系）。单叶面积郑果４３较大，华硕、郑果４Ｊ４次之，郑果４２最小。平均单株产量，以华硕最大，为２２ｋｇ，依次为郑果４４（２０ｋｇ）、郑果４３
土壤、气候条件适宜。矮化砧Ｍ２６作中间砧，株行距２ｍ× ４ｍ，东西行向，自由纺锤形整形。随机取样适期观
测各品种（系）的性状表现。
１各苹果品种（系）的性状表现
１．１果实性状与成熟期
试栽的４个品种（系）与对照品种嘎拉果形相仿，均为近圆形，表皮均着色；果个以华硕最大，平均单果
（１８ｋｇ）、郑果４２（１２ｋｇ）、嘎拉最少（１Ｏｋｇ）（表２）。

5个梨品种在熊岳地区的生长表现及果实性状评价

2021年5月北方果树NORTHERN FRUITS2021(3)17D01：10.16376/ki.bfgs.2021.03.005中图分类号：S661.2文献标识码：B文章编号：1001-5692(2021)03-0017-035个梨品种在熊岳地区的生长表现及果实性状评价姜晓艳，王家珍，李俊才，蔡忠民，李宏军，沙守峰9(辽宁省果树科学研究所，辽宁熊岳115009)摘要：为优化熊岳地区梨品种结构，同时为梨育种工作提供前期准备,2009年，辽宁省果树科学研究所梨课题从吉林省农科院果树所引进5个抗寒晚熟梨新品种'蔗梨…寒红…寒香…寒露'和'寒酥'，通过多年的生物学调查和室内测试分析，比较其物候期、生长结果习性和果实品质，认为'寒红…寒香…蔗梨'在熊岳地区表现较好，可以适当发展，同时可作为育种材料在今后的杂交育种中应用'关键词：梨；生长特性；果实品质Grow Performance and Evaluation of Fruit Characters of Five Pear Varieties inXiongyue AreaJIANG Xiao-yan,WANG Jia-zhen,LI Jun-cai,CAI Zhong-min,LI Hong-jun,SHA Shou-feng*(Liaoning Institute of Pomology,Xiongyue Liaoning115009,China)Abstract:In order to optimize the pear variety structure in Xiongyue area and provide early preparation for pear breeding.In2009,five cold resistance and late-maturing pear varieties were introduced from pomology institude academy of agriculture science of Jilin Province.They were'Zhe Pear')Hanhong Pear''Hanxiang Pear''Hanlu Pear''Hansu Pear'.Through years of biological investigation and laboratory test and analysis, the phenological period,growth and fruiting habit and fruit quality of five pear varieties were compared.The results showed that'Hanhong Pear''Hanxiang Pear'and'Zhe Pear'had better performance in Xiongyue area,and could be properly developed.At the same time,they could be used as breeding materials in future cross breeding.Key words:Pears；Growth Characters；Fruit Quality收稿日期：2021—01—21项目基金：国家现代梨产业技术体系营口综合试验站资助项目(CARS-28-30)；抗寒优质特色果树新品种创制及轻简安全高效栽培技术研究(2020JH2/ 10200019)作者简介：姜晓艳(19?2-),女，硕士，助理研究员，研究方向为梨新品种选育及栽培技术研究&E-mail：*****************通讯作者：沙守峰(1969-),男，博士，研究员，研究方向梨新品种选育及栽培技术研究&E-mail：*****************和果实发育，产量提高46.28%或31.64%,好果率提高11%或19%,糖度提高2.90%或3.47%,且延长货架期,增产提质效果显著。

基于_HS-GC-IMS_的无核黄皮果实挥发性风味物质分析

热带作物学报2022, 43(8): 1686 1695Chinese Journal of Tropical Crops基于HS-GC-IMS的无核黄皮果实挥发性风味物质分析孟祥春1，耿星1,2，黄泽鹏1，贺立红21. 广东省农业科学院果树研究所/农业农村部南亚热带果树生物学与遗传资源利用重点实验室/广东省热带亚热带果树研究重点实验室，广东广州 510640；2. 仲恺农业工程学院农业与生物学院，广东广州 510225摘要：为明确无核黄皮挥发性风味化合物组分，采用顶空气相-离子迁移色谱（headspace-gas chromatography-ion mobility spectrometry, HS-GC-IMS）技术，并结合挥发性化合物的指纹图谱、峰强度及主成分分析（principal component analysis, PCA），测定分析并比较4个不同成熟度无核黄皮果实的挥发性风味化合物组分差异。

从无核黄皮果实中共鉴定出60个已知的和3个未知的挥发性风味化合物，已知的挥发性风味化合物包含13种醛类、12种醇类、9种烯烃类、6种酮类、2种酯类、2种醚类、1种苯和1种乙酸。

挥发性风味物质指纹图谱显示，在特有的风味化合物方面，STAGE2的种类最多，STAGE4次之，STAGE1仅有2种，而STAGE3无明显的特有风味化合物。

此外，STAGE1与STAGE2共有的风味化合物种类最多，STAGE3与STAGE4仅共有乙酸和乙醇。

各化合物的信号峰强度值显示，各成熟度果实中含量均较为丰富的有己醛、戊烯醛、乙烯醇、石竹烯、α-水芹烯、柠檬烯、1-戊烯-3-酮和丁香酚甲醚。

丁香酚甲醚和乙烯醇的峰强度最高，且表现为随果实成熟度的增强，其相对含量逐渐降低。

主成分分析（PCA）显示不同成熟度果实的挥发性风味组分各自聚类，但STAGE1与STAGE2、STAGE3与STAGE4的距离分别较近。

无核黄皮果实中主要的挥发性风味化合物是醛类和醇类，其次是烯氢类和酮类，不同成熟度果实分别具有共同的及特有的挥发性风味化合物特征峰区域，成熟度越接近，其风味化合物的组成也较为相近。

肠道微生物英文文献pig

Animal(2012),6:10,pp1620–1626&The Animal Consortium2012doi:10.1017/S1751731112000481The effect of chitooligosaccharide supplementation on intestinal morphology,selected microbial populations,volatile fatty acid concentrations and immune gene expression in the weaned pig A.M.Walsh,T.Sweeney,B.Bahar,B.Flynn and J.V.O’Doherty-School of Agriculture,Food Science and Veterinary Medicine,University College Dublin,Lyons Research Farm,Newcastle,Co.Dublin,Ireland(Received24March2011;Accepted29January2012;First published online2March2012)An experiment(complete randomised design)was conducted to investigate the effects of supplementing different molecular weights (MW)of chitooligosaccharide(COS)on intestinal morphology,selected microbial populations,volatile fatty acid(VFA)concentrations and the immune status of the weaned pig.A total of28piglets(24days of age,9.1kg(6s.d.0.80)live weight)were assignedto one of four dietary treatments for8days and then sacriﬁced.The treatments were(1)control diet(0ppm COS),(2)control diet plus5to10kDa COS,(3)control diet plus10to50kDa COS and(4)control diet plus50to100kDa COS.The COS was included in dietary treatments at a rate of250mg/kg.Tissue samples were taken from the duodenum,jejunum and ileum for morphological measurements.Digesta samples were taken from the proximal colon to measure lactobacilli and Escherichia coli populations and digesta samples were taken from the caecum and proximal colon for VFA analysis.Gene expression levels for speciﬁc cytokines were investigated in colonic tissue of the pig.Supplementation of different MW of COS had no signiﬁcant effect on pig performance during the post-weaning period(days0to8;P.0.05).The inclusion of COS at all MW in the diet signiﬁcantly reduced faecal scores compared with the control treatment(P,0.01).Pigs fed the10to50kDa COS had a higher villous height(P,0.05)and villous height:crypt depth ratio(P,0.05)in the duodenum and the jejunum compared with the control treatment.Pigs fed the5to10kDa COS had a lower lactobacilli population(P,0.05)and E.coli population(P,0.05)in the colon compared with the control group.Pigs offered the5to10kDa COS had signiﬁcantly lower levels of acetic acid and valeric acid compared with the control group(P,0.05). The inclusion of different MW of COS had no signiﬁcant effect on the expression of the cytokines tumour necrosis factor-a,Interleukin (IL)-6,IL-8and IL-10in the gastro-intestinal tract of the weaned pig.The current results indicate that a lower MW of5to10kDa COS possessed an antibacterial activity,while the higher MW of10to50kDa was optimum for enhancing the intestinal structure. Keywords:chitooligosaccharide,pig,microbiology,intestinal morphologyImplicationOur results indicate that the inclusion of chitooligosaccharides (COSs)in piglet diets may moderate several gut health para-meters that contribute to some of the common problems that occur after weaning in the absence of in-feed antibiotics.It was observed that COSs with a molecular weight(MW)of5to 10kDa were more effective in reducing Escherichia coli populations while a MW of10to50kDa enhanced the intestinal structure.IntroductionThe weaning period imposes profound social and environ-mental stresses on the piglet such as removal from the sow,change in diet and mixing of piglets from different litters. Numerous studies have reported that there is a reduction in villous height(villous atrophy)and an increase in crypt depth (crypt hyperplasia)after weaning,which leads to increased susceptibility to intestinal gut dysfunction(Spreeuwenberg et al.,2001;Pierce et al.,2006).The post-weaning period is characterised by a reduction in feed intake,poor growth rates,diarrhoea and an increased risk of disease(Lalles et al., 2007).These negative effects on piglet growth during the weaning period were managed by growth-promoting anti-biotics.However,the European Union placed a total ban on the use of in-feed antibiotic growth promoters on the1st January2006due to public concerns regarding bacterial resistant and human health issues. Chitooligosaccharides(COS)may be a potential viable alternative to traditional antimicrobials in animal production.-E-mail:john.vodoherty@ucd.ie 1620Chitosan is a natural biopolymer derived by alkaline deacety-lation of chitin,which is the principal component of protective cuticles of crustaceans such as crabs,shrimps,prawns,lobsters and cell walls of some fungi such as aspergillus(Qin et al., 2006).Both chitin and chitosan are biopolymers composed of glucosamine and N-acetylated glucosamine(2-acetylamino-2-deoxy-D-glucopyranose)units linked by b(1to4)glycosidic bonds(Koide,1998).Low molecular weight(MW)COS is a water-soluble derivative of chitosan due to shorter chain lengths(Kim and Rajapakse,2005).Recently,both chitosan and its derivatives have generated considerable interest due to their biological activities,including antimicrobial,antitumour, immunoenhancing effects and the acceleration of wound healing(No et al.,2002;Liu et al.,2006)There is considerable variation in the literature on the biological properties of COS (Jeon et al.,2001;Liu et al.,2006).Most of this variation is partly due to the widely different MW used across studies.It is hypothesised that the biological properties of COS may be inﬂuenced by its MW and COS will enhance selected indices of health in weaned piglets.Material and methodsAll procedures described in this experiment were conducted under an experimental licence from the Irish Department of Health in accordance with the cruelty to Animals Act1876 and the European Communities(Amendments of the Cruelty to Animals Act1976)Regulations.Experimental dietsThe experiment was designed as a complete randomised block design and comprised four dietary treatments.Thedietary treatments were as follows:(1)control diet(0ppm COS),(2)control diet plus5to10kDa COS,(3)control diet plus10to50kDa COS and(4)control diet plus50to100kDa COS.The COS was sourced from Kitto Life Co.Ltd(Kyungki-do,Seoul,Korea)and was supplemented in the experimental diets at a concentration of250ppm.The diets were fed for 8days ad libitium,after which time the pigs were humanely sacriﬁced.The diets were formulated to have similar diges-tible energy(16MJ/kg)and standardised ileal digestible (SID)lysine(14g/kg)contents.All amino acids requirements were met relative to SID lysine(National Research Council, 1998).The ingredient composition and chemical analysis of the dietary treatments are presented in Table1.Animals and managementA total of28piglets(progeny of large white3(large white3landrace sows))were selected from a commercial pig unit at24days of age.The piglets had a weaning weight of9.1kg(s.d.50.80)and were blocked on the basis of litter of origin and live weight(n57).The piglets were individu-ally housed in fully slated pens(1.7m31.2m).They were individually fed and had ad libitum access to feed and water. The house temperature was thermostatically controlled at 308C throughout the experiment.This study was not a growth performance study but some performance data were recorded.The piglets were weighed at the beginning of the experiment(day0)and at the end of the experiment(day8). Food was available up to theﬁnal weighing and all remaining food was weighed back for the purpose of cal-culating feed efﬁciency.Pigs were observed for clinical signs of diarrhoea and a scoring system was applied to indicate the presence and severity of this as described by Pierce et al. (2006).Faeces scores were assigned daily for individual pigs from day0and continued until day8.The following faeces scoring system was used:15hard faeces,25slightly soft faeces in the pen,35soft,partially formed faeces,45loose, semi-liquid faeces and55watery,mucous-like faeces.Gut morphological analysisThe piglets were humanely sacriﬁced on day8by a lethal injection of Euthatal(pentobarbitone sodium BP–Merial Animal Ltd,Sandringham House,Essex,UK)at a rate of1ml/ 1.4kg BW.On removal of the digestive tract,sections of the duodenum(10cm from the stomach),the jejunum(60cm from stomach)and the ileum(15cm from caecum)were excised andﬁxed in10%phosphate-buffered formalin.The preserved segments were prepared using standard parafﬁn-embedding techniques.The samples were sectioned at5m m Table1Composition and chemical analysis of experimental diets (as-fed basis)Items Starter diet* Ingredient(g/kg)Whey permeate125.0 Wheat444.2 Soya bean meal142.5 Whey protein isolate130.0 Full-fat soybean80.0 Soya oil65.0 Vitamins and minerals 5.0 Lysine HCL 4.5 DL-methionine 1.6L-threonine 2.2 Analysis(g/kg,unless otherwise stated)DM892.5 CP(N36.25)224.2 GE(MJ/kg)18.2 Ash43.7 NDF110.3 Lysine-16.5 Methionine and cysteine-9.9 Threonine-10.7 Tryptophan- 2.5 Calcium-8.0 Phosphorous- 6.0 DM5dry matter;GE5gross energy.Starter diet provided(mg/kg completed diet):Cu,175;Fe,140;Mn,47;Zn, 120;I,0.6;Se,0.3;retinol,1.8;cholecalciferol,0.025;alpha-tocopherol,67; phytylmenaquinone,4;cyanocobalamin,0.01;riboﬂavin,2;nicotinic acid,12; pantothenic acid,10;choline chloride,250;thiamine,2;pyridoxine,0.015.*COS was included in dietary treatments T2–T4at a rate of250mg/kg.-Calculated for tabulated nutritional composition(Sauvant et al.,2004).Chitooligosaccharide in piglet diets1621thickness and stained with haemotoxylin and eosin(Pierce et al.,2006).Villous height and crypt depth were measured on the stained sections(43objective)using a light micro-scopeﬁtted with an image analyser(Image Pro Plus,Media Cybernetics,Buckinghamshire,UK).Measurements of15well oriented and intact villi and crypts were taken for each seg-ment.Villous height was measured from the crypt–villous junction to the tip.Crypt depth was measured from the crypt–villous junction to the base.Results were expressed as the mean villous height or crypt depth in micrometres. Intestinal microﬂoraFor microbial analysis,digesta samples(,1061g)were aseptically recovered from the proximal colon of each pig immediately post slaughter.Digesta samples were stored in sterile containers(Sarstedt,Wexford,Ireland),placed on ice and transported to the laboratory within2h.A1.0g sample was removed from the digesta sample,serially diluted (1:10)in9.0ml aliquots of maximum recovery diluents (Oxoid,Basingstoke,UK)and spread plated(0.1ml aliquots) onto selective agars,as follows:Lactobacillus spp.were isolated on de Man,Rogosa and Sharp(MRS)agar(Oxoid) with an overnight(18to24h)incubation at378C in an atmosphere enriched with5%CO2,as recommended by the manufacturers(Oxoid).The Escherichia coli species were isolated on MacConkey agar(Oxoid)following aerobic incubation at378C for18to24h(O’Doherty et al.,2010). Target colonies of Lactobacilli and E.coli were identiﬁed by Gram stains and colony morphology(Salanitro et al.,1977). The API50CHL(BioMerieux,Biomerieux,Craponne,France) kit was used to conﬁrm suspect Lactobacilli spp.Suspect E. coli colonies were conﬁrmed with API20E(BioMerieux, France).This API system identiﬁes the suspect colonies by measuring their ability to produce cytochrome oxidase. Typical colonies of each bacteria on each agar were counted, log transformed and the numbers of bacteria were expressed per gram of digesta after being serially diluted.Volatile fatty acid(VFA)analysisSamples of digesta from individual pigs were taken from the caecum and the proximal colon to measure the VFA concentration and molar proportions of VFAs.The VFA con-centrations in the digesta were determined using gas liquid chromatography according to the method described by Pierce et al.(2007).A1-g sample was diluted with distilled water (2.53weight of sample)and centrifuged at14003g for4min(Sorvall GLC–2B laboratory centrifuge,Dupont, Wilmington,DE,USA).Then,1ml of the subsequent super-natant and1m l of internal standard(0.5g3-methyl-n-valeric acid in1l of0.15mol/l oxalic acid)were mixed with3ml of distilled water.Following centrifugation to remove the precipitate,the sample wasﬁltered through Whatman 0.45m m polyethersulphone membraneﬁlters into a chromato-graphic sample vial.A1-m l sample was injected into a model 3800Varian gas chromatograph with a25m30.53mm i.d. megabore column(coating CP-Wax58(FFAP)–CB(no. CP7614))(Varian,Middelburg,the Netherlands).RNA extraction and complementary DNA(cDNA)synthesis Tissue samples were collected from the mesenteric side of the colon,rinsed with ice-cold sterile phosphate-buffered saline(Oxoid)and stripped of overlying smooth muscle cells. Approximately1to2g of the porcine colon tissue was cut into small pieces and placed in tubes containing15ml of RNAlater(Applied Biosystems,Foster City,CA,USA)and immediately stored at2208C pending RNA extraction.Total RNA was extracted from colon tissue samples(25mg)using a GenElute Mammalian Total RNA Miniprep Kit(RTN70, Sigma-Aldrich,St Louis,MO,USA)according to the manu-facturer’s instructions.To eliminate possible genomic DNA contamination,total RNA samples were subjected to DNAse I(AMPD1,Sigma-Aldrich)treatment according to the man-ufacturer’s protocol.Then RNA puriﬁcation was performed using a phenol–chloroform extraction method(Chomczynski and Sacchi,2006).The total RNA was quantiﬁed using a NanoDrop-ND1000Spectrophotometer(Thermo Fisher Scien-tiﬁc,Wilmington,DE,USA)and the purity was assessed by determining the ratio of the absorbance at260and280nm. All total RNA samples had260/280nm ratios above1.8.In addition,RNA integrity was veriﬁed by visualisation of the18 and28S ribosomal RNA bands stained with ethidium bromide after gel electrophoresis on1.2%agarose gels(Egel,Invitro-gen Inc.,Carlsbad,CA,USA).Total RNA(1m g)was reverse transcribed(RT)using the RevertAid H minusﬁrst strand cDNA synthesis kit(Fermentas GmbH,St Leon-Rot,Germany)with oligo dT primers.Theﬁnal RT product was adjusted to a volume of120m l using nuclease-free water.Real-time quantitative PCRAll primers for the selected cytokines,genes such as Inter-leukin-1a(IL-1a),IL-6,IL-10,tumour necrosis factor(TNF-a) and the reference genes b-actin(ACTB),b2-microglobin (B2M),glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and peptidylprolyl isomerise A(PPIA)are presented in Table2. Ampliﬁcation was carried out in a reaction volume of20m l containing10m l SYBR Green Fast PCR Mastermix(Applied Biosystem),forward and reverse primer mix(1m l),8m l DEPC treated water and1m l of template cDNA.Quantitative real-time PCR was carried out using an ABI PRISM7500Fast sequence detection system for96-well plates(Applied Biosys-tem).The thermal cycling conditions were as follows:an initial denaturation step at958C for10min,40cycles of958C for15s, followed by608C for1min.Dissociation analyses of the PCR product were performed to conﬁrm the speciﬁcity of the resulting PCR products.All samples were run in triplicate.The cycle threshold value(C t)is deﬁned as the fractional cycle number at whichﬂuorescence passes theﬁxed threshold.The mean C t values of triplicates of each sample were used for calculations.Normalisation of quantitative PCR dataNormalisation of the C t values obtained from real-time PCR was performed by(i)transforming the raw C t values into relative quantities using the formula,relative quantities5 (PCR efﬁciency)D C t,where D C t is the change in the C t valuesWalsh,Sweeney,Bahar,Flynn and O’Doherty 1622of the sample relative to the highest expression (minimum C t value),(ii)using geNorm,a normalisation factor was obtained from the relative quantities of four most stable housekeeping genes (GAPDH,B2M,ACTB and PPIA)and (iii)the normalised fold change or the relative abundance of each of the target genes was calculated by dividing their relative quantities by the normalisation factor.Statistical analysisThe experimental data were analysed as a randomised block design using the GLM procedure of SAS (2004).The individualpig served as the experimental unit.Food intake was inclu-ded as a covariate in the model for villous height,crypt depth and villous height to crypt depth ratio in the digestive tract.The microbial counts were log transformed.The data were checked for normality using the Proc Univariate function of SAS.The means were separated using the Tukey–Kramer Test.Probability values of ,0.05were used as the criterion of statistical signiﬁcance.All results are presented in the tables as least square means 6standard error of the means (s.e.).ResultsPerformance and faecal scoringThe average faecal scores of the pigs are presented in Table 3.The supplementation of different MW of COS had no signiﬁcant effect on the growth performance of the pig during the 8-day experimental period (P .0.05).However,the inclusion of COS at all MW in the diet signiﬁcantly reduced faecal scores com-pared with the control treatment (P ,0.01).MicrobiologyThe effect of COS supplementation at different MW on selected microbial populations in the colon of the pig is shown in Table 3.Pigs offered diets containing 5to 10kDa COS had a lower E.coli number compared with the control (P ,0.05)and the 50to 100kDa COS (P ,0.05)treatments.The 10to 50kDa treatment had a neumerically lower E.coli number compared with the control group (P 50.09).Pigs offered diets containing 5to 10kDa COS had a signiﬁcantly lower population of lacto-bacilli in the colon compared with the control group (P ,0.05)and the 50to 100kDa COS diet (P ,0.01).Pigs offered 50to 100kDa COS had a higher lactobacilli number than pigs offered 10to 50kDa COS (P ,0.05).The supplementation of different MW of COS had no signiﬁcant dietary effect on the lacto-bacilli :E.coli ratio in the colon of the pig.Table 2Porcine-speciﬁc primers used for real-time PCR 1.Forward primer sequence (50-30)Gene 2.Reverse primer sequence (50-30)T m (8C)IL-6 1.AGACAAAGCCACCACCCCTAA59.82.CTCGTTCTGTGACTGCAGCAGCTTATC 62.7IL-8 1.TGCACTTACTCTTGCCAGAGAACTG 61.92.CAAACTGGCTGTTGCCTTCTT 61.7IL-10 1.GCCTTCGGCCCAGTGAA 57.62.AGAGACCCGGTCAGCAACAA 59.4TNF-a 1.TGGCCCCTTGAGCATCA55.22.CGGGCTTATCTGAGGTTTGAGA 60.3GAPDH 1.CAGCAATGCCTCCTGTACCA 62.22.ACGATGCCGAAGTTGTCATG 62.1B2M 1.CGGAAAGCCAAATTACCTGAAC 59.02.TCTCCCCGTTTTTCAGCAAAT 60.0ACTB 1.CAAATGCTTCTAGGCGGACTGT 59.02.TCTCATTTTCTGCGCAAGTTAGG 60.0PPIA1.CGGGTCCTGGCATCTTGT58.02.TGGCAGTGCAAATGAAAAACTG56.5IL 5interleukin;TNF 5tumour necrosis factor;GAPDH 5glyceraldehyde-3-phosphate dehydrogenase;B2M 5b 2-microglobin;ACTB 5genes b -actin;PPIA 5peptidylprolyl isomerise A.Primers were designed using Primer Express TM software and were synthesisedby MWG Biotech (Milton Keynes,UK).Table 3Effect of COS supplementation at different MW on faecal scoring,selected microbial populations in the proximal colon and the total VFA concentration and the proportions of VFAs in the caecum of the weaned pig (least square means and s.e.;n 57)Dietary treatmentsControl 5to 10kDa 10to 50kDa50to 100kDas.e.SigniﬁcanceFaeces scoring Days 0to 84.06b 3.31a 3.44a 3.38a 0.124**Proximal colonic bacterial population (log cfu/g of digesta)Escherichia coli5.94b 4.34a 4.71a 5.81b 0.477*Lactobacilli spp.7.39bc 6.24a 6.56ab 7.56c 0.347*VFA concentrations in the caecum Total VFA (mmol/g of digesta)95.8770.25103.00116.7913.006ns Acetic acid 67.36b 45.77a 70.30b 79.20b 8.543*Propionic acid 19.8416.5123.3927.05 3.616ns Isobutyric acid 0.770.490.810.790.157ns Butyric acid 6.45 6.14 6.817.51 1.575ns Isovaleric acid 0.67b 0.45a 0.68b 0.83b 0.092*Valeric acid0.790.881.011.410.282nsCOS 5chitooligosaccharide;MW 5molecular weight;VFA 5volatile fatty acid.Probability of signiﬁcance:*P ,0.05;**P ,0.01;ns,P ,0.05.Means with the same superscript alphabets within rows are not signiﬁcantly different (P .0.05).Chitooligosaccharide in piglet diets1623Volatile fatty acidsThe effects of COS supplementation at different MW on the VFA concentrations in the caecum are shown in Table3.The supplementation of different MW of COS had a signiﬁcant effect on the concentrations of acetic acid(P,0.05)and isovaleric acid(P,0.05)in the caecum.Pigs fed5to10kDa COS had lower levels of acetic acid and isovaleric acid compared with the control(P,0.05),10to50kDa COS (P,0.05)and50to100kDa COS(P,005).There was no signiﬁcant effect of MW on VFA concentrations(P.0.05)in the proximal colon(data not shown).Gut morphologyThe effects of varying COS MW on villous height,crypt depth and the villous height:crypt depth ratios in the gastro-intestinal tract are shown in Table4.Pigs fed the10to 50kDa COS had a higher villous height in the duodenum and the jejunum compared with the control group(P,0.05), 5to10kDa COS(P,0.01)and50to100kDa COS diets (P,0.05).There was no effect of dietary treatment on crypt depth in the duodenum(P.0.05).Pigs offered the10to 50kDa COS had a higher villous height:crypt ratio in the duodenum and the jejunum compared with the control group(P,0.05)and the5to10kDa COS diet(P,0.01).Cytokine gene expression analysisThe effects of COS supplementation on the immune response in colon tissues of the pig are shown in Table5.The supplementation of different MW of COS had no signiﬁcant effect on the expression of the cytokines TNF-a,IL-6,IL-8 and IL-10(P.0.05)in the gastro-intestinal tract of the pig. DiscussionThe hypothesis of the current experiment is that the biolo-gical properties of COS may be inﬂuenced by its MW and COS will enhance selected indices of health in weaned pig-lets.It was demonstrated in the current study that the lower MW of5to10kDa possessed antibacterial activity while the higher MW of10to50kDa was optimum for enhancing intestinal structure.Dietary supplementation of COS at the low MW of5to 10kDa decreased both lactobacilli and E.coli counts,while the10to50kDa COS numerically decreased E.coli popula-tions in the colon of the pig.In a study by Liu et al.(2008), COS supplementation at different concentrations reduced E. coli concentrations in the caecum of the weanling pig.E.coli is considered to be one of the most important causes of post-weaning diarrhoea in weaned pigs;therefore,a reduction inTable4Effect of COS supplementation at different MW on villous height,crypt depth and the villous height:crypt depth ratio in the gastro-intestinal tract of the weaned pig(least square means and s.e.)Dietary treatments Control5to10kDa10to50kDa50to100kDa s.e.Signiﬁcance Covariate(intake) Villous height(m m)Duodenum284.0a256.0a326.3b266.2a17.38*ns Jejunum271.6a270.7a316.5b260.8a16.15*ns Ileum239.8268.3251.5242.915.07ns ns Crypt depth(m m)Duodenum305.7330.2280.1311.718.98ns ns Jejunum294.1298.4281.6268.420.93ns ns Ileum207.5242.8228.2239.811.29ns ns Villous:crypt depth ratioDuodenum 1.0a0.8a 1.2b0.9a0.08*ns Jejunum0.9a0.9a 1.2b 1.0ab0.06*ns Ileum 1.2 1.1 1.1 1.00.06ns nsCOS5chitooligosaccharide;MW5molecular weight.Probability of signiﬁcance:*P,0.05;**P,0.01;ns,P,0.05.Means with the same superscript alphabets within rows are not signiﬁcantly different(P.0.05).Table5Effect of COS supplementation at different MW on the immune response in unchallenged proximal colon tissues(leastsquare means of fold change in normalised relative gene expression with their s.e.;n57animals)Dietary treatments Control5to10kDa10to50kDa50to100kDa s.e.SigniﬁcanceColonTNF-a0.3660.3530.3760.3620.0568nsIL-60.2480.3590.3180.3220.0645nsIL-80.3850.5440.3700.3580.0797nsIL-100.3640.3420.3110.3070.0614ns COS5chitooligosaccharide;MW5molecular weight;TNF5tumour necrosis factor;IL5interleukin.Probability of signiﬁcance:*P,0.05;**P,0.01;ns,P,0.05.Walsh,Sweeney,Bahar,Flynn and O’Doherty1624E.coli populations may reduce the incidence of diarrhoea in post-weaned pigs(Fairbrother et al.,2005).Although many species of E.coli are commensal,high levels of speciﬁc E.coli (like ETEC)will increase the risk of disease.Unfortunately, ETEC numbers were not measured in the current study.In the current study,the faecal score was decreased in pigs fed the COS diets compared with the control.These results suggest that the supplementation of the5to10kDa and10 to50kDa COS reduces E.coli populations in the colon, resulting in a lower faecal score in the post-weaning period. The50to100kDa COS led to a reduced diarrhoea score but no reduction in E.coli populations;therefore,this MW of COS may be working as a bulking agent to affect the faecal score.The50to100kDa COS may retard the rate of passage through the intestine and may have the ability to absorb water.In the current study,it was demonstrated that supple-mentation of5to10kDa COS had the strongest antimicrobial effect against both lactobacilli and E.coli.This is in agreement with other studies in which low MW COS(5to10kDa)were shown to possess strong antibacterial properties compared with higher MW COS and the antibacterial properties of COS increased at a low MW of,5kDa against Gram-negative such as E.coli(Zheng and Zhu,2003;Kittur et al.,2005).In a study by Liu et al.(2010),COS supplementation decreased E.coli populations compared with the control in the caecum of weaned pigs,while Jeon et al.(2001)observed a anti-microbial effect of COS against Gram-positive bacteria such as Lactobacilli under in-vitro conditions.To explain COS antibacterial activity,two mechanisms have been proposed.Theﬁrst mechanism is that the posi-tively charged COS reacts with negatively charged molecules at the microbial cell surface,thereby altering cell perme-ability(Chung and Chen,2008).Therefore,COS may interact with the membrane of the cell to alter cell permeability. However,as evident from the current study,this activity may differ with varying MW as the50to100kDa group had no inhibitive effect on the selected microbial populations,while the MWs of5to10kDa and10to50kDa COS had the strongest inhibitive effect.The other antibacterial mechan-ism is the binding of COS with DNA to inhibit RNA synthesis (Liu et al.,2004).It has been proposed that COS penetrates the nuclei of the bacteria and interferes with RNA and pro-tein synthesis.It is noteworthy that all the COS samples used in the current study were soluble in aqueous solutions.Kim and Rajapakse(2005)found that COS with a MW of .30kDa were not effective as antibacterial agents due to their poor solubility in aqueous solutions at a neutral pH. Volatile fatty acids are the major end products of bacterial metabolism in the large intestine(Macfarlane and Macfarlane, 2003).Both protein and carbohydrate fermentation contribute to the production of acetic acid;however,branched-chain fatty acids such as isovaleric acid are produced from protein fermentation(Mackie et al.,1998).In the current study,the5 to10kDa group had the lowest selected microbial populations while also reducing isovaleric acid and acetic acid concen-trations in the caecum.The shift in the production of the fermentation end products is reﬂected in the reduction of the selected microbial populations.The quantity of VFA produced depends on the amount and composition of the substrate and on the type of microbes present in the large intestine (van Beers-Schreurs et al.,1998).Reduced VFA concentrations indicate that lower amounts of substrate were fermented as a result of a lower microbial activity in the caecum(Htoo et al.,2007).Villous height is generally reduced and crypt depth is increased,which may explain the increased occurrence of diarrhoea and reduced growth after weaning(Pluske et al., 1996).The inclusion of10to50kDa COS in the present study was found to increase the villous height and villous:crypt depth ratio in the duodenum and also in the jejunum com-pared with the control group.Very little data have been published on the effects of COS MW on gut morphology in weaned piglets;thus,the exact mechanism for the increase in villous height and villous:crypt depth ratio is unclear.It may be hypothesised that low MW COS has the potential to promote intestinal morphology through cell proliferation. The COS has been shown to inﬂuence colonic cell prolifera-tion,crypt depth and crypt circumference in mice(Torzsas et al.,1996).A study carried out by Liu et al.(2008),on different con-centrations of COS,demonstrated that200mg/kg of COS increased villous height and villous:crypt ratio in the jeju-num and ileum(Liu et al.,2008).The possible explanation for this improved intestinal structure was that COS is com-posed of N-acetyl glucosamine(Kim and Rajapakse,2005), which may bind to certain types of bacteria and possibly interfere with their adhesion to the gut tissue of host animals (Ofek et al.,2003;Liu et al.,2008).This result is in agree-ment with Moura˜o et al.(2006),who reported that an increase in villi length in the ileum of weaned rabbits was correlated to a lower intestinal microﬂora.A decrease in bacteria load has been shown to increase the proliferation of epithelial cells,which leads to an improved intestinal mor-phology and increased villous height(Moura˜o et al.,2006). In the present study,in pigs fed the lower MW of5to10kDa COS,a strong antimicrobial effect on both Lactobacilli and E.coli populations was observed,with no effect on villous structure,while the higher MW of10to50kDa resulted in a reduction in E.coli numbers in comparison with the control and was optimum for improving villous integrity.There were no effects of COS supplementation in colon tissue on any of the cytokines analysed.This overall lack of an effect on these inﬂammatory cytokines implies that COS inclusion in the diet had no effects on immune gene expression of the pigs.Mori et al.(1997)also demonstrated that chitin and its derivatives do not stimulate the production of IL-6,IL-1and TNF-a byﬁbroblasts.In our study,no dif-ferences were observed on growth performance between days0and8post-weaning.In conclusion,MW is an important factor to consider when investigating the biological properties of COS.On the basis of the current study,the lower MW of5to10kDa possessed antibacterial activity while the higher MW of10to50kDaChitooligosaccharide in piglet diets1625。

石榴赤霉素合成相关基因PgKO的克隆及表达分析

西北植物学报,２０１８,３８(４):０６３１－０６３６A c t aB o t ．B o r e a l ．ＧO c c i d e n t ．S i n．㊀㊀文章编号:１０００Ｇ４０２５(２０１８)０４Ｇ０６３１Ｇ０６㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀d o i :１０．７６０６/j．i s s n ．１０００Ｇ４０２５．２０１８．０４．０６３１收稿日期:２０１８Ｇ０１Ｇ１０;修改稿收到日期:２０１８Ｇ０４Ｇ１３基金项目:国家自然科学基金(３０９００９７１);安徽农业大学研究生创新基金(２０１８y js Ｇ８)作者简介:黄㊀蓉(１９９３－),女,在读硕士研究生,主要研究果树资源与生物技术育种.E Ｇm a i l :１３７３２５８３３７７＠１６３．c o m∗通信作者:张水明,博士,副教授,主要从事园艺植物种质资源与生物技术育种研究.E Ｇm a i l :z h a n gs h m ８９３＠s o h u ．c o m 石榴赤霉素合成相关基因P gK O 的克隆及表达分析黄㊀蓉,刘㊀娜,王㊀琦,熊㊀枫,张水明∗(安徽农业大学园艺学院,合肥２３００３６)摘㊀要:该研究以红玉石籽 (P u n i c a g r a n a t u m c v ．H o n g y u s h i z i )石榴为试验材料,采用R T ＧP C R 和R A C E 技术,从石榴花中克隆得到赤霉素合成关键酶内根贝壳杉烯氧化酶(K O )基因,命名为P gK O (G e n B a n k 登录号为MG ２０８０１７).P g K O 全长c D N A 为１７２９b p ,包含１５４２b p 开放阅读框(O R F ),编码５１３个氨基酸,相对分子量为１４１．１６k D ,理论等电点为４．９.多重序列对比显示,P gK O 与苹果M d K O ㊁白梨P c K O ㊁葡萄V v K O 的相似性分别为６１．２８％㊁６４．５６％和７３％,并且具有C Y P ４５０结构域(脯氨酸富含域㊁氧还原位点和血红素结合域).系统进化树分析表明,P g K O 与其他物种的起源相同,并与桉树E g K O 的亲缘关系最近.荧光定量P C R 发现,在石榴蕾期和盛花期,P g K O 基因的表达量均为筒状花高于钟状花,且P g K O 基因在两种花型的子房㊁花药㊁花萼中均有表达,但筒状花中子房的表达量最高,花萼的表达量最低;钟状花中花药的表达量最高,子房的表达量最低.关键词:石榴;内根Ｇ贝壳杉烯氧化酶(K O );基因克隆;基因表达中图分类号:Q ７８５;Q ７８６;S ６６５．４文献标志码:AC l o n i n g a n dE x p r e s s i o nA n a l y s i s o fG i b b e r e l l i nB i o s yn t h e s i s ＧR e l a t e d G e n e P g K O i nP o m e gr a n a t e HU A N G R o n g ,L I U N a ,WA N G Q i ,X I O N GF e n g ,Z H A N GS h u i m i n g∗(C o l l e g e o fH o r t i c u l t u r e ,A n h u iA g r i c u l t u r a lU n i v e r s i t y,H e f e i ２３００３６,C h i n a )A b s t r a c t :I nt h i ss t u d y ,P gK O ,t h e g i b b e r e l l i nb i o s y n t h e s i s Ｇr e l a t e d g e n e ,w a sc l o n e df r o m f l o w e r so f p o m e g r a n a t eu s i n g R T ＧP C Ra n dR A C E t e c h n o l o g y m e t h o d s ,w i t h P u n i c a g r a n a t u m c v ．H o n g yu s h i z i a s t h e e x p e r i m e n t a lm a t e r i a l ．I t s a c c e s s i o n o fG e n B a n k i sMG ２０８０１７．T h e c D N Af u l l Ｇl e n g t h s e q u e n c e o f P gＧK O w a s １７２９b p ,w i t h１５４２b p o p e nr e a d i n g f l a m ee n c o d i n g ５１３a m i n oa c i d s ．T h e p r o t e i n m o l e c u l a r w e i g h t a n d i s o e l e c t r i c p o i n tw e r e １４１．１６k Da n d４．９r e s p e c t i v e l y ．M u l t i p l e a l i g n m e n t s h o w e d t h a tP g K O h a d ６１．２８％,６４．５６％a n d７３％s i m i l a r i t y w i t h M a l u s ˑd o m e s t i c a M d K O ,P yr u s c o m m u n i s P c K Oa n d V i t i s v i n i fe r a V v K Or e s p e c t i v e l y ,a l s oi n c l u d e d C Y P ４５０d o m a i ne m b r a c i n gp r o l i n e Ｇr i c hd o m a i n ,r e d i o s i t e s a n d h e m e Ｇb i n d i n g d o m a i n ．P h y l o g e n e t i c t r e e a n a l y s i s i n d i c a t e d t h a t P g K Os h a r e d t h e s a m e o r i gi nw i t h o t h e r s p e c i e s ,a n dh a dt h ec l o s e s tr e l a t i o n s h i p w i t h E u c a l y pt u s g r a n d i s E g K O．R e a l t i m e q u a n t i t a t i v e P C Rs h o w e d t h a t n om a t t e r t h e b u d s t a g e o r f l o w e r i n g s t a g e ,P gK O e x p r e s s i o n i n c y l i n d r i c Ｇs h a p e d f l o w e r s w e r eh i g h e r t h a n t h a t i n t r u m p e t Ｇs h a p e d f l o w e r s ．S o m e t i m e s P gK O e x p r e s s i o nw a s d e t e c t e d i no v a r y ,a n Ｇt h e r a n d c a l y x ．I n c y l i n d r i c Ｇs h a p e d f l o w e r s ,t h e e x p r e s s i o n i no v a r y i s t h e h i g h e s t ,w h i l e l o w e s t i n c a l yx ;I n t r u m p e t Ｇs h a p e d f l o w e r s ,t h e e x p r e s s i o n i na n t h e r i s t h eh i g h e s t ,w h i l e l o w e s t i no v a r y．K e y wo r d s :p o m e g r a n a t e ;e n t Ｇk a u r e n e o x i d a s e (K O );g e n e c l o n i n g ;g e n e e x p r e s s i o n㊀㊀石榴花为两性花即雌雄同株,根据雌蕊发育状况分为完全花与不完全花.完全花是葫芦状花或筒状花,俗称大屁股果花 ;不完全花的萼筒呈喇叭形,称钟状花㊁尖屁股幌花 [１Ｇ２].其中,不完全花呈萼筒钟状花内虽可见雌蕊,但已明显退化,子房瘦小,胚珠发育不良,雌蕊萎缩,虽可开花但不结实或结实很少,并且雄蕊过度发育导致坐果极低[３].雌蕊败育已经成为影响石榴产量和经济效益的关键因素之一.如今大量研究表明植株花芽分化与植物激素有关[４].其中赤霉素不仅有利于不完全花的发育,影响植物的花芽分化,而且有利于幼果的坐果和生长发育[５].有关学者在对蓖麻㊁油茶㊁荔枝㊁龙眼㊁光皮树成花的研究中均发现赤霉素对花芽分化具有显著的促进作用[６Ｇ７].内根Ｇ贝壳杉烯氧化酶(K O )是赤霉素生物合成的关键调控对象之一,同时是赤霉素内质网及细胞质基质中生物合成的关键酶[８],受N A D P H 调控,具有一定高度保守的血红素特征结合域P ４５０酶[９].近几年人们大多是从生理方面来探寻石榴雌蕊退化败育的原因,而从分子角度的研究报道很少.因此,本研究以石榴红玉石籽为实验材料,运用R T ＧP C R 和R A C E 技术,克隆得到P gK O ,并采用实时荧光定量P C R ,对该基因在不同花型的不同花期㊁同一花期不同花型３个组织中的表达水平进行分析,为石榴雌蕊退化败育等原因提供参考,也为进一步研究P gK O 的功能奠定基础.１㊀材料和方法１．１㊀实验材料所用实验材料取自安徽农业大学农业园石榴种质资源圃;分别在蕾期(４月２０日)和盛花期(６月１５日),采取同一品种红玉石籽的筒状花和钟状花,以及盛花期(６月１５日)时这两种花型的子房㊁花药㊁花萼组织,浸于液氮中带回实验室,随后储存于－８０ħ冰箱中备用.１．２㊀方㊀法１．２．１㊀基因全长序列的克隆㊀以红玉石籽的花为材料,用R N A p r e p Pu r eP l a n tK i t 植物总R N A 提取试剂盒(天根公司)提取石榴花总R N A .根据G e n B a n k 中已登录的其他多个物种的保守序列,设计兼并引物K O ＧF １/K O ＧR １(表１)进行中间片段的扩增.在获取的中间片断基础上,继续设计引物K O Ｇ５ᶄG S P １/K O Ｇ５ᶄG S P ２和K O Ｇ３ᶄG S P １/K O Ｇ３ᶄG S P ２(表１),按照S MA R T e r T MR A C Ec D N A A m Ｇpl i f i c a t i o n K i t (C l o n t e c h )操作步骤,通过巢式P C R ,分别进行K O 基因５ᶄ和３ᶄ端序列的扩增.最后设计引物K O ＧF /K O ＧR ,将３部分序列进行拼接,使用K O D p l u s 高保真酶全长扩增,连接到p G E M ＧT e a s y 载体,送上海生工公司测序.１．２．２㊀生物学信息分析㊀序列比对和同源性分析使用C l u s t a l x ㊁G E N E D O C 和D N AMA N 软件;运用E x P A S y ＧP r o t pa r a m T o o l 在线工具分析其蛋白质理化性质;运用P s o r t (h t t p ://p s o r t ．h g c ．j p/f o r m．h t m l)进行亚细胞定位分析;构建进化系统发育树使用S P S S 和M E G A 软件;运用P B I LL Y O N ＧG E R L A N D 数据库(h t t p ://n p s a p b i l ．i b c p ．f r /c gi b Ｇi n /n p s a _a u t o m a t ．p l p a g e＝/N P S A /n ps a _h n n ．h t Ｇm l)进行氨基酸二级结构预测.１．２．３㊀荧光定量P C R 分析㊀根据获得的P g K O 全长序列,使用P r i m e r p r e m i e r５．０设计荧光定量P C R 引物(表２).采用T r i z o l 方法提取花㊁子房㊁花药和花萼的R N A ,分别取４μg 经DN a s e I 处理后表２㊀荧光定量P C R 扩增所需要的引物及其序列T a b l e ２㊀R e a l t i m e ＧP C Ra m p l i f i c a t i o n r e qu i r e d p r i m e r s a n d s e qu e n c e s 引物P r i m e r序列(５ᶄң３ᶄ)S e qu e n c e K O ＧR T ＧF C C C T C C C C A T C A A A C C C A A T A C C K O ＧR T ＧR C G C C G C T G C C T C T G C C C C C G C T A a c t i n ＧP F A G T C C T C T T C C A G C C A T C T C a c t i n ＧP RC A C T G A G C A C A A T G T T T C C A表１㊀扩增所需要的引物T a b l e １㊀T h e p r i m e r su s e d i na m pl i f i c a t i o n 引物P r i m e r 序列(５ᶄң３ᶄ)S e qu e n c e 用途U s a ge U P M L o n gC T A A T A C G A C T C A C T A T A G G G C AA G C A G T G G T A T C A A C G C A G A G T 通用引物U n i v e r s a l p r i m e r s N U PK O Ｇ３ᶄG S P １K O Ｇ３ᶄG S P ２A A G C A G T G G T A T C A A C G C A G A G T A A A T G A A C T T T T C G G G C T T G C G T T A T C A G A G A G C A G A A G A A A C G G A P gK O ３ᶄ端扩增A m p l i f i c a t i o n f o r ３ᶄr a c e o f P gK O K O Ｇ５ᶄG S P １K O Ｇ５ᶄG S P ２A G G T C T C T A T T A T C G C C T C C C A G T T C G G T C C T C T G T G A G C G T C T T T P g K O ５ᶄ端扩增A m p l i f i c a t i o n f o r ５ᶄr a c e o f P g K O K O ＧF １K O ＧR ２T T G A C A G A A C A G G G A G A A G C A G T T T C A T C C T A T T C T T T C C G C A GP g K O 全长扩增A m p l i f i c a t i o n f o r f u l l l e n g t ho f P gK O ２３６西㊀北㊀植㊀物㊀学㊀报㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀３８卷的R N A,利用MM L V反转录酶进行反转录,获得c D N A模板.使用A B IS T E P O N E荧光定量P C R 仪进行荧光定量P C R分析,反应体系按T a K a R a S Y B R P r e m i x E X T a q试剂盒步骤进行操作: S Y B R T M P r e m i xE x T a q１０μL,正㊁反向引物(１０μm o l L－１)各０．８μL,R O X R e f e r e n c eD y e５０ˑ０．４μL,c D N A模板２．０μL,d d H２O６．０μL.反应程序为:９５ħ预变性３０s,９５ħ变性５s,６０ħ退火３０s,７２ħ延伸１m i n,共４０个循环.以石榴的内参基因a c t i n作为内参,使用２－ΔΔC t法,求得待测样品的相对表达量.２㊀结果与分析２．１㊀石榴P g K O基因全长的克隆与序列分析根据已经公布的苹果㊁白梨㊁橡胶树等物种的K O序列,设计兼并引物K OＧF１/K OＧR１进行扩增,经回收㊁转化和阳性菌落P C R检测后送至上海生工测序,得到一个长为２６８b p的核酸片段(图１,A).用基因特异性引物K OＧ５ᶄG S P１和U P M L o n g作为第１轮引物,以此P C R产物为模板,K OＧ５ᶄG S P２与N U P作为引物进行第２轮扩增,获得K O基因的５ᶄ端序列,测序得到一个７９２b p片段(图１,B),同理得到长为６６９b p的片段的５ᶄ序列(图１,C).最后将获得的石榴K O基因中间片段㊁３ᶄ端序列和５ᶄ端序列进行拼接,获得全长为１７２９b p的P g K O基因序列(图３).该基因的开放阅读框为１５４２b p,编码５１３个氨基酸.２．２㊀石榴P g K O氨基酸序列与进化分析将克隆的P g K O与N C B I公布的其他植物的K O 氨基序列进行比对,结果(图２)发现,P g K O与其他物种具有一定的相似性,且具有C Y P４５０结构域:脯氨酸富含域(p r o l i n eＧr i c hd o m a i n)㊁氧还原位点(r e d i o s i t e s)和血红素结合域(h e m eＧb i n d i n g d o m a i n).为了确定P g K O与其他物种K O的亲缘关系,利用M E G A５．０软件,将P g K O与桉树㊁苹果㊁葡萄等１３个物种的K O氨基酸序列进行系统进化分析.结果(图４)显示,P g K O与桉树E g K O的亲缘关系最近,聚为一枝,而与咖啡C a K O的亲缘关系最远.２．３㊀石榴P g K O蛋白结构特征分析从疏水性分析结果(图５)可以看出,P g K O蛋白的前部和后部有较强亲水区,中部有明显疏水区.在４６７氨基酸处有最大值３．００２,在第１６１氨基酸P c K O．西洋梨(J N１０６４６４．１);M d K O．苹果(A Y５６３５４９．２); V v K O．葡萄(J Q０８６５５３．１);E g K O．桉树(X M_０１８８７６５７１．１); T c K O．可可(X M_０１８１２５９８０．１);方框内为C Y P４５０结构域图２㊀P g K O蛋白及其他物种K O氨基酸多重序列比对P c K O．P y r u s c o m m u n i s(J N１０６４６４．１);M d K O．M a l u s x d o m e s t i c a (A Y５６３５４９．２);V v K O．V i t i s v i n i f e r a(J Q０８６５５３．１);E g K O．E u c a l y p t u s g r a n d i s(X M_０１８８７６５７１．１);T c K O．T h e o b r o m a c a c a o(X M_０１８１２５９８０．１);C Y P４５０m o t i f s a r c i n t h e b o x e sF i g．２㊀M u l t i p l e a l i g n m e n t o f t h e a m i n o a c i ds e q u e n c e s o fP g K Oa n do t h e rK OsM．D L２０００;A．中间片断;B．５ᶄＧR A C E;C．３ᶄＧR A C E图１㊀P g K O基因扩增结果M．D L２０００;A．I n t e r m e d i a t e f r a g m e n t;B．５ᶄＧR A C E;C．３ᶄＧR A C EF i g．１㊀T h e a m p l i f i c a t i o n r e s u l t s o f P g K O３３６４期㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀黄㊀蓉,等:石榴赤霉素合成相关基因P g K O的克隆及表达分析A T G为起始密码子;T A A为终止密码子;方框内序列为开放阅读框图３㊀石榴P g K O基因核苷酸序列A T Gi s t h e i n i t i a t i o n c o d o n;T A Ai s t h e t e r m i n a t o r c o d o n;T h e s e q u e n c e i ns q u a r e i s o p e n r e a d i n g f r a m eF i g．３㊀N u c l e o t i d e s e q u e n c e o f P g K O g e n e i n p o m e g r a n a t e处有最小值－３．５１１,在N端具有跨膜的疏水序列. P g K O基因合成的蛋白分子式为C２６１９H４１５９N７１３O７４５S２３,相对分子质量为１４１．１６k D,总共原子数目为８２５９,理论等电点为４．９.该蛋白中含量最多的氨基酸是L e u(L)为１０．３％,其次是A l a(A)为８．４％,含量最少的氨基酸是C y s(C)和T r p(W)均为１．６％.总带负电荷残基(A s p＋G l u)为６０,总带正电荷残基(A r g＋L y s)为６６;脂肪族氨基酸指数为９２．８１,总平均疏水指数(G R A V Y)为－０．２１２,该蛋白为疏水性蛋白.P s o r t进行亚细胞定位结果表明,P g K O蛋白位于内质网的可能性为４４．４％;位于液泡的可能性为２２．２％;位于线粒体和高尔基体的可能性都为１１．１％,因此推测该白蛋白可能位于内质网中.２．４㊀２种花型P g K O基因的表达分析２．４．１㊀不同花期P g K O基因的表达㊀以品种红玉石籽为材料,对筒状花和钟状花在不同花期中的P g K O基因表达水平进行检测.结果(图６)表明:盛花期时,钟状花的表达量约为０．９７,筒状花的表达量约１．２,是钟状花的１．２倍;蕾期时,钟状花的表达量约为０．５,筒状花的表达量约１．１,为钟状花的２．２倍.２．４．２㊀不同组织P g K O基因的表达㊀为探讨P gＧK O基因在石榴２种花型在盛花期时不同组织中的表达水平,对筒状花和钟状花的子房㊁花药㊁花萼中的P g K O相对表达量进行检测.结果(图７)表明:钟状花的子房表达量最低约为０．９５,其次是花萼约为１．３９,表达量最高是花药约为１．４１,分别是子房的１．４８倍㊁花萼的１．０１倍;筒状花的花萼表达量最低约为０．９７,其次是花药约为１．１,表达量最高的是子房约为１．２,分别是花萼的１．２倍㊁花药的１．０倍.４３６西㊀北㊀植㊀物㊀学㊀报㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀３８卷P c K O．白梨(J N １０６４６４);M d K O．苹果(A A S ６８０１７．２);P p K O．巴旦木(A F ４９５７２８．１)．F gK O．草莓(A A R １８４０７．２);C s K O．柑橘(X M _００６４９２４９６．２);M c K O．苦瓜(A D E ０６６６９．１);C m K O．南瓜(A A G ４１７７６．１);E g K O．桉树(X P _０１００２８７１８．１);P gK O．石榴(M G ２０８０１７);V v K O．葡萄(J Q ０８６５５３．１);R c K O．蓖麻(X P _００２５１０２８８．１);P s K O．桃(A A P ６９９８８．１);C a K O．咖啡(A C Q ９９３７４．２);分支点数字表示基于５００次重复该节点的自展支持率;标尺代表遗传距离图４㊀石榴与不同物种K O 蛋白系统进化树P c K O．P y r u s c o m m u n i s (J N １０６４６４);M d K O．M a l u s ˑd o m e s t i c a (A A S ６８０１７．２);P p K O．P r u n u s p e r s i c a (A F ４９５７２８．１)．F g K O．F r a g a r i a g r a n d i fl o r a (A A R １８４０７．２);C s K O．C i t r u s s i n e n s i s (X M _００６４９２４９６．２);M c K O ．M o m o r d i c a c h a r a n t i a (A D E ０６６６９．１);C m K O．C u c u r b i t am a x i m a (A A G ４１７７６．１);E g K O．E u c a l y pt u s gr a n d i s (X P _０１００２８７１８．１);P g K O．P u n i c a g r a n a t u m (M G ２０８０１７);V v K O ．V i t i s v i n i fe r a (J Q ０８６５５３．１);R c K O ．R i c i n u s c o m m u n i s (X P _００２５１０２８８．１);P s K O ．P i s u m s a t i v u m (A A P ６９９８８．１);C a K O．C o f fe a a r a b i c a (A C Q ９９３７４．２);T h en o d e s i n t h ef ig u r e sh o wt h eb o o t s t r a p v a l u e s b a s e do n５００r e p li c a t i o n s ．T h e s c a l e r e pr e s e n t s t h e g e n e t i c d i s t a n c e F i g ．４㊀P h y l o g e n e t i c t r e e c o n s t r u c t e db y am i n o a c i d s e q u e n c e s o fK O p r o t e i n i n p o m e gr a n a t e a n do t h e r p l a n ts 图５㊀P gK O 的疏水性和亲水性预测F i g ．５㊀P r e d i c a t i o no f h y d r o p h o b i c i t y/h y d r o p h i l i c i t y o fP gKO 图中不同小写字母表示花型间差异显著(P ＜０．０５)图６㊀石榴不同花期不同花型P gK O 基因表达D i f f e r e n t n o r m a l l e t t e r sm e a ns i gn i f i c a n t d i f f e r e n c e a m o n g fl o w e r s (P ＜０．０５)F i g ．６㊀T h e r e l a t i v e e x p r e s s i o no f P g K O i nd i f f e r e n t f l o w e r p a t t e r n s a t d i f f e r e n t s t a g e s o f p o m e gr a n a t e．图中不同小写字母表示组织间差异显著(P ＜０．０５)图７㊀石榴同一花期不同花型不同组织P gK O 基因表达D i f f e r e n t n o r m a l l e t t e r sm e a ns i gn i f i c a n t d i f f e r e n c e a m o n g ti s s u e s (P ＜０．０５)F i g ．７㊀T h e r e l a t i v e e x p r e s s i o no f P g K O i nd i f f e r e n t f l o w e r p a t t e r n s a n dd i f f e r e n t t i s s u e s a t t h e s a m es t a g e o f p o m e gr a n a t e ３㊀讨㊀论石榴果实中含有丰富的抗氧化剂和多酚类物质,具有较高的营养保健价值[１０],但其产量和经济效益严重受到了雌蕊败育的影响.有关研究表明赤霉素对不完全花的发育起关键作用,且高质量分数的G A ３有利于坐果和生长发育[５].对番茄 I n d i a n R i v e r 品种[１１]和 M i c r o ＧT o m 品种[１２]施加G A ３可促使花柱的伸长,促进雌蕊发育,从而提高授粉率和坐果率;程华等[１３]通过研究植物激素对板栗花芽分化的影响,发现较高的赤霉素能够促进雌性化.同时赤霉素还能调节雄蕊的生长发育[１４],赤霉素中相５３６４期㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀黄㊀蓉,等:石榴赤霉素合成相关基因P g K O 的克隆及表达分析关受体调节D E L L A蛋白泛素化等过程,因此拟南芥三重突变体(g i d l a㊁g i d l b㊁g i d l c)表现为花丝生长矮小,伸长受到阻碍,而抑制雄蕊的发育,促进雌蕊的生长[１５].G A４＋７可以通过调节拟南芥花序中雄蕊的生长发育,缩短雄蕊和雌蕊之间生长的长度差值,从而解决授粉困难坐果率低等问题[１６]. S a k a t a[１７]研究发现减少G A３含量可以阻碍花粉的发育,抑制雄蕊的生长,提高水稻的结实率.因此可以通过增加石榴中赤霉素的含量来调节雌蕊和雄蕊的生长发育比率,从而提高果实的授粉率和坐果率,提高产量和经济效益.近年来,随着市场对石榴的需求增大,为了探寻石榴雌蕊退化败育的原因,人们大多从生理角度进行研究,例如营养物质和过氧化物酶等方面.但是关于石榴雌蕊败育形成原因在分子方面的研究报道很少.这些生理方面的研究只能从外部环境改变石榴的坐果率,并不能从基因上解决石榴坐果率低等问题.因此本研究从分子角度出发,克隆得到石榴中赤霉素合成关键基因P g K O,并通过荧光定量P C R表明P g K O与石榴的花芽分化和雌雄蕊发育有关.所以研究赤霉素合成关键基因可以初步从分子角度解决石榴雌蕊败育坐果率低等问题,提高石榴的经济产量.同时可为探讨赤霉素对石榴筒状花和钟状花的形成影响奠定基础,对整个石榴产业具有重大意义.参考文献:[１]㊀汪小飞,向其柏,尤传楷,等．石榴品种分类研究[J]．果树学报,２００７,２４(１):９４Ｇ９７．WA N G XF,X I A N G QB,Y O U C K,e t a l．A d v a n c e s i nr eＧs e a r c ho nc l a s s i f i c a t i o no f p o m e g r a n a t e[J]．J o u r n a l o f F r u i t S c i e n c e,２００７,２４(１):９４Ｇ９７．[２]㊀王㊀珊,蔡斌华,章㊀镇,等．果梅完全花与不完全花的差异蛋白分析[J]．植物遗传资源学报,２００８,９(４):４６５Ｇ４６８．WA N GS,C A I BH,Z H A N GZ,e t a l．A n a l y s i s o f d i f f e r e n t i a l p r o t e i n s i n p e r f e c ta n di m p e r f e c tf l o w e r so fJ a p a n e s ea p r i c o t (P r u n u sm u m e S i e b．e t Z u c c．)[J]．J o u r n a l o f P l a n tG e n e t i c R e s 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Performanceof‘Coscia’pear(Pyruscommunis)onninerootstocksinthenorthofIsrael

RaphaelA.Sterna,*,IsraelDoronbaMIGAL,GalileeTechnologyCenter,P.O.Box831,Kiryat-Shmona11016,Israel

bExtensionService,MinistryofAgriculture,Kiryat-Shmona10200,Israel

1.Introduction‘Spadona’and‘Coscia’arethemainpearcultivarsgrowninthewarmclimateofIsrael.BothofthemaregraftedonthesameclonalquinceA(EMA)(Cydoniaoblonga)rootstock.Whiletheperfor-manceof‘Spadona’issatisfactoryfortreevigor,cumulativeyieldandfruitquality,‘Coscia’suffersfrompoorvegetativegrowth,leadingtoalowyieldofsmallfruit(Sternetal.,2003;Flaishmanetal.,2001).Thus,theadvantageofthedwarﬁngrootstockforthemorevigorous‘Spadona’becomesadisadvantagefortheweaker‘Coscia’.Asconsumerspreferlargepears(Sternetal.,2002,2007b;Flaishmanetal.,2005),fruitsizehasbecomeanimportantmarketingconsideration,andtheeconomicbeneﬁtsoftreatmentscapableofimprovingaveragefruitsizearepotentiallyveryhigh(Looney,1983).Althoughplantgrowthregulatorsofthecytokininfamilyhavebeenshowntoimprovefruitsize(Sternetal.,2003;Flaishmanetal.,2005),amorefundamentalapproachcouldbethechoiceoftheappropriaterootstockforeachcultivar,thereby

affectingtreevigourandconsequentlyfruitsize(Westwood,1993;Jackson,2003;Webster,2003).Thelackofarootstockadaptedtodifferentsoilconditionsandtodifferentgraftcultivarsiswidelyacknowledgedinpearculture.QuincerootstocksaswellasPyrusrootstocks(seedlingorclonal)havetheiradvantagesanddisadvantages.Ineachcase,site-speciﬁcecologicalcharacteristics,speciﬁccultivarresponseandproduc-tionobjectivesmustbeconsideredbeforedecidingonthebeststrategy(LombardandWestwood,1987;Masseron,1989).Forexample,theuseofquincerootstockisalmostuniversallysatisfactoryfor‘Coscia’cultivationinthetemperateclimateofEurope,whereasinthehotanddryweatherofIsrael,itisverypoor(Gur,2000;Guretal.,1968,1978;Sternetal.,2007a).Theselectionofclonalquince(Cydoniaoblonga),suchasEMA,EMCandBA29inEurope,orclonalPyruscommunisL.,suchas‘OldHome’Â‘Farmingdale’(OHF)intheUSAorBPinSouthAfrica,assubstitutesforpearseedlingrootstock,haveclearlyimprovedtheprecocity,productivityandqualityofsomeEuropeanpearcultivars(Westwood,1993;Jackson,2003;Webster,2003;IglesiasandAsin,2005;Carreraetal.,2005).Anoticeableimprovementin‘Coscia’performancewasobser-vedina10-yeartrial,conductedinthewarmestpear-growing

ScientiaHorticulturae119(2009)252–256ARTICLEINFOArticlehistory:Received12March2008Receivedinrevisedform22July2008Accepted1August2008Keywords:PearRootstockYieldFruitsizePyruscommunisLABSTRACTThevegetativeandreproductiveperformancesof‘Coscia’pear(PyruscommunisL.)grownonninerootstocks[OHF69,OHF97,OHF217,OHF333,OHF513andBP1(P.communis),clonalseedling(DavisAxB)ofPyrusbetulifoliaandquinceBA29andEMA(Cydoniaoblonga)]werecomparedduringa9-yearperiod.ThetrialwasconductedattheExperimentalStation‘AvneiEitan’innorthernIsrael(elevation400mabovesealevel,a.s.l.),onawell-drainedsoilwithpH7.1.TreeswereplantedinDecember1998,spacedat4.0mÂ2.0mandtrainedwithacentralaxis.ThemostvigoroustreeswereonP.betulifoliaseedlings,followedbythefourOHFrootstock(69,97,333,513)andBP1(withnosigniﬁcantdifferencebetweenthem).AlltheaboverootstocksdemonstratedgreatervigorthanOHF217andquinceBA29orEMA.ThehighestcumulativeyieldspertreewereharvestedfromtreesonthefourOHFrootstocks(69,97,333,513)andP.betulifoliafollowedbytheBP1.Thetwoquincerootstocks,aswellasOHF217,hadthelowestcumulativeyieldandthelowestyieldoflargefruit.Apositivecorrelationwasfoundbetweenthevigorofthetree,asaffectedbytherootstock,andboththetotalyieldandthefruitsize.Weconcludethatinawarmclimate,yieldefﬁciencyisnottheonlyparameterthatshouldbetakenintoaccount,andbuildingastrongtreeforaweakcultivaristheﬁrstrequirementforestablishinganorchard.ß2008ElsevierB.V.Allrightsreserved.

*Correspondingauthor.Tel.:+97246953508;fax:+97246944980.E-mailaddress:Rafﬁ@migal.org.il(R.A.Stern).

ContentslistsavailableatScienceDirectScientiaHorticulturae

journalhomepage:www.elsevier.com/locate/scihorti

0304-4238/$–seefrontmatterß2008ElsevierB.V.Allrightsreserved.doi:10.1016/j.scienta.2008.08.002regioninnorthernIsrael(theHullavalley—100ma.s.l.),withtreesgraftedonP.betulifoliaascomparedtothoseonquinceA(EMA)rootstock(Sternetal.,2007a).Theimprovementwasexpressedashigheryieldsandlargerfruit,aswellasprecocityinfruitbearing.However,thequestionaroseastowhetherthesameresultwouldbeapplicabletoorchardsgrownonthesurroundinghillswithasomewhatcoolerclimate,andwhetherthevigourcontributedbytheP.betulifoliarootstockmightnotbeexcessiveandupsetthedesiredbalancebetweengrowthandproductivity.Theobjectiveofthisstudywasthereforetodeterminethebestrootstockfor‘Coscia’cultivatedinawarmclimateathigheraltitude.Withtheincreaseinglobalwarming,theﬁndingsinthisstudymightwellbeofawiderrelevance.