Ectopic expression of a novel peach (Prunus persica) CBF
细胞信号转导通路
Chromatin/Epigenetics Resources
Overview of Chromatin / Epigenetics
Chromatin regulation refers to the events affecting chromatin structure and therefore, transcriptional control of gene expression patterns. Epigenetics, specifically, refers to the heritable modifications which result in altered gene expression and are not known to be encoded in DNA. The nucleosome, made up of four histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have more recently been shown to be dynamic proteins, undergoing multiple types of post-translational modifications (PTMs) and interacting with regulatory proteins to control gene expression. Protein acetylation plays a crucial role in regulating chromatin structure and transcriptional activity. Histone hyperacetylation by histone acetyltransferases (HATs) is associated with transcriptional activation, whereas histone deacetylation by histone deacetylases (HDACs) is associated with transcriptional repression. Hyperacetylation can directly affect chromatin structure by neutralizing the positive charge on histone tails and disrupting nucleosome-nucleosome and nucleosomeDNA interactions. In addition, acetylation creates binding sites for bromodomain-containing chromatin regulatory proteins (histone modification readers). Unlike acetylation, methylation does not alter the charge of arginine and lysine residues and is unlikely to directly modulate nucleosomal interactions required for chromatin folding. Methylated arginine and lysine residues are major determinants for formation of active and inactive regions of the genome. Methylation facilitates binding of chromatin regulatory proteins/histone modification readers that contain various methyl-lysine or methyl-arginine binding domains (PHD, chromo, WD40, Tudor, MBT, Ankyrin repeats, PWWP domains). Recruitment of co-activator and co-repressor proteins is dependent on the specific lysine residue that is modified. The modulation of chromatin structure is an essential component in the regulation of transcriptional activation and repression. One strategy by which chromatin structure can be modulated is through disruption of histone-DNA contacts by ATP-dependent chromatin remodelers, such as the NuRD, Polycomb, and SWI/SNF complexes, which have been shown to regulate gene activation/repression, cell growth, the cell cycle, and differentiation. Chromatin structure is also modulated through other PTMs such as phosphorylation of histone proteins, which affects association with DNA-interacting proteins and has been recently identified to play a role in coordinating other histone modifications. Furthermore, methylation of DNA at cytosine residues in mammalian cells affects chromatin folding and is a heritable, epigenetic modification that is critical for proper regulation of gene silencing, genomic imprinting, and development. Three families of mammalian DNA methyl-transferases have been identified, DNMT1/2/3, that play distinct roles in embryonic stem cells and adult somatic cells. In addition to the core histone proteins, a number of histone variants exist that confer different structural properties to nucleosomes and play a number of specific functions such as DNA repair, proper kinetochore assembly and chromosome segregation during mitosis, and regulation of transcription. Chromatin and epigenetic regulation is crucial for proper programming of the genome during development and under stress conditions, as the misregulation of gene expression can lead to diseased states such as cancer.
甘蓝型油菜PEBP基因家族的鉴定与表达分析
Identification and expression analysis of PEBP gene family in oilseed rape
JIAN Hong-Ju**, YANG Bo**, LI Yang-Yang, YANG Hong, LIU Lie-Zhao, XU Xin-Fu, and LI Jia-Na*
College of Agronomy and Biotechnology / Chongqing Engineering Research Center for Rapeseed / Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China
FT 基因编码一个称为“促花素”的可以长距离移 动的信号蛋白, 在开花调控中起着核心调控作用[6]。 该 基 因 属 于 植 物 磷 脂 酰 乙 醇 胺 结 合 蛋 白 PEBP (phosphatidylethanolamine-binding protein)基因, PEBP 基 因 编 码 蛋 白 包 含 一 个 保 守 结 构 域 (InterPro: IPR00891)。进化研究表明, 该蛋白家族分为 3 个亚 家族, 即 FT、TFL1 (TERMINAL FLOWER 1)和 MFT (MOTHER OF FT AND TFL1)。其中, FT 主要是诱 导开花, TFL1 抑制开花, 而 MFT 主要参与种子发 育 和 萌 发 过 程 [7-9]。在 拟 南 芥 中 , PEBP 家 族 包 含 6 个基因成 员, 即 FT、TFL1、TSF (TWIN SISTER OF FT) 、 BFT(BROTHER OF FT AND TFL1) 、 ATC (ARABIDOPSIS THALIANA CENTRORADIALIS) 和 MFT[10]。其中, FT、TSF 和 MFT 促进开花, TFL1、 ATC 和 BFT 抑制开花[11-13]。在拟南芥中, 2 个 FT-like 基因, FT 和 TSF 是开花激活因子, 突变该基因将延 迟开花[11,14]。在长日照环境中, FT 和 TSF 在叶子韧 皮部的伴胞细胞中上调表达[15]。作为 ATC 的旁系同 源基因, TSF 在营养生长期的成熟顶端分生组织的 内表皮中少量积累, 而开花之后, 其表达量显著下 调[16-17]。TFL1 蛋白是一个移动信号, 可从顶端分生 组织的内表皮移动到外表皮[18]。虽然 TFL1 和 FT 在 序 列 上 非 常 相 似 , 都 可 以 与 FD (FLOWERING LOCUS D)互作来调控 FD 依赖的靶基因, 但在调控 开花过程中的作用是相反的[19]。在高盐胁迫中, BFT 可以通过调节光周期来适应外界胁迫[20]。MFT 特异 诱导下胚轴和根的转变, 其突变体表现为在种子萌 发过程中对 ABA 的超敏反应, 而且在萌发的种子中, MFT 的表达直接受到 ABA-INSENSITIVE3 (ABI3) 和 ABI5 的调控, 同时, MFT 通过反馈抑制 ABI5 的 表达来促进胚的发育[21]。
蔓花生PEPC基因家族的生物信息学分析
热带亚热带植物学报2018, 26(2): 107 ~ 115Journal of Tropical and Subtropical Botany蔓花生PEPC基因家族的生物信息学分析涂嘉琦, 甘璐, 冯兰兰, 袁良兵, 黎茵*(广东省热带亚热带植物资源重点实验室，中山大学生命科学学院，广州510275)摘要：为了解花生中磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)的功能，对二倍体祖先种野生蔓花生(Arachis duranensis)基因组数据库进行分析，发现存在9个AdPEPC基因家族成员，这些基因的序列长度为3 584~12 956 bp，开放阅读框(ORF)长度为702~3 168 bp，分布在3、5、7、8、9、10号染色体上。
蔓花生AdPEPC家族蛋白的氨基酸序列中均含有HCO3-结合位点和PEP结合位点等保守结构域，根据序列特征可分为植物型、细菌型和序列较短的PEPC等3类, 同类蛋白序列的同源性较高，基因结构中的内含子与外显子的数目也较相似。
基因表达分析表明，多数成员在花或茎中的表达量较高，AdPEPC1;2和AdPEPC4;2在茎中的表达量最高，其他家族成员尤其是AdPEPC2、AdPEPC1;5和AdPEPC1;3在花中的表达量明显高于其他组织，AdPEPC1;5基因在叶中不表达。
AdPEPC3在根、茎、叶和花中均不表达，推测该基因为假基因。
这为深入研究AdPEPC家族基因的功能奠定了基础。
关键词：花生；磷酸烯醇式丙酮酸羧化酶；基因家族；生物信息doi: 10.11926/jtsb.3804Bioinformatics Analysis of PEPC Gene Family in Arachis duranensisTU Jia-qi, GAN Lu, FENG Lan-lan, YUAN Liang-bing, LI Yin*(Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University,Guangzhou 510275, China)Abstract: To understand the function of phosphoenolpyruvate carboxylase (PEPC) in peanut, PEPC sequences of the diploid progenitor Arachis duranensis genome database were analyzed. Nine gene family members were found, and their sequence lengths ranged from 3 584to12 956 bp with 702-3 168 bp open reading frame (ORF), distributed in the 3, 5, 7, 8, 9, and 10th chromosomes. In the amino acid sequences of AdPEPC proteins, there were conserved domains, such as HCO3- binding sites and PEP binding sites, et al. According to the sequence characteristics, AdPEPC proteins could be divided into 3 groups as plant type, bacterium type and short sequence type. Protein homologies of the same type were high, and the number of intron and exon in gene structures was similar. Gene expression analysis showed that the expressions of most members were high in flowers or stems. AdPEPC1;2 and AdPEPC4;2 had the highest expression in stem. Other family members, especially AdPEPC2, AdPEPC1;5and AdPEPC1;3, expressions were significantly higher in flower than other tissues. AdPEPC1;5 expression was not detected in leaf. No expression of AdPEPC3 was detected in root, stem, leaf and flower, and it was presumed to be pseudogenes based on its gene structure. These would lay a foundation for further study on the functions of AdPEPC family genes.Key words: Arachis duranensis; Phosphoenolpyruvate carboxylase; Gene family; Bioinformation收稿日期: 2017-08-16 接受日期: 2017-11-21基金项目: 国家自然科学基金项目(31171625)；广东省自然科学基金项目(10151027501000058)；广州市科技计划项目(201510010270)资助This work was supported by the National Natural Science Foundation of China (Grant No. 31171625), the Natural Science Foundation of Guangdong Province (Grant No. 10151027501000058), and the Science and Technology Program of Guangzhou (Grant No. 201510010270).作者简介: 涂嘉琦(1992~)，女，硕士研究生，研究方向为植物生理与分子生物学。
4种葫芦科植物对酞酸酯的胁迫反应与吸收
福建农林大学学报（自然科学版）Journal of Fujian Agriculture and Forestr^^ University (Natural Science Edition)第46卷第6期 2017年11月4种葫芦科植物对酞酸酯的胁迫反应与吸收张明\游广永\崔军2,欧阳琰1(1.环境保护部南京环境科学研究所，江苏南京210042;2.盐城师范学院江苏省盐土生物资源研究重点实验室，江苏盐城224002)摘要：通过水培方法，研究了 4种不同葫芦科植物对酞酸酯的胁迫反应与吸收作用.结果表明，黄瓜、南瓜、丝瓜在一系列 P A E s浓度胁迫下其常见生理指标变化差异不大，在低浓度时对某些指标还具有促进作用，在一定范围内对P A E s具有较好 的耐受能力.对D B P的吸收作用，黄瓜和南瓜根部含量远大于冬瓜和丝瓜（P<0.05);除冬瓜外，其余三种植物的地上部 D B P含量均大于根部的含量（P<0.05).对D EH P的吸收作用，4种植物的根部含量均远髙于各自地上部的；冬瓜和黄瓜根 部含量明显大于南瓜和丝瓜(P<0.05);南瓜地上部的含量显著大于其余三种植物地上部的含量（P<0.05).4种葫芦科植物 对D B P和D EH P的吸收能力强于一些常见的叶菜类植物.黄瓜和南瓜比其余两种葫芦科植物对D B P和D EH P均有更强的 吸收能力，是P A E s修复的潜力植物，较适于在酞酸酯污染土壤中种植.关键词：酞酸酯；植物修复；葫芦科；胁迫反应中图分类号：S154.1 文献标识码： A 文章编号：1671-5470(2017)06-0618-05D O I:10.13323/ki.j.fafu( nat.sci.) .2017.06.004Stress response and absorption of 4 Cucurbitaceae plants to PAEsZHANG Ming1,YOU Guangyong1,CUI Jun2,OUYANG Yan1(1.Nanjing Institute of Environmental Sciences，Ministry of Environmental Protection，Nanjing，Jiangsu 210042，China；2.Jiangsu Provincial Key Laboratory of Bioresources in Coastal Saline Soils，Yancheng Teachers' University，Yancheng，Jiangsu 224002，China)A b s t r a c t: Changes in the growth and physiological characteristics of 4 different Cucurbitaceae plants grown in phthalate esters (PAEs) contaminated water was studied under laborator^^ simulation testing. The results showed that physiological indexes of Cucu-mis sativus，Cucurbita moschata and Luffa cylindrical changed little under PAEs stress，and PAEs even enhanced some physiological indexes at low concentration. C.sativus，C.moschata and L.cylindrical had good tolerance to PAEs in certain range. DBP contents in roots were higher in C.sativus and C.moschata than Benincasa hispida and L.cylindrical( P<0.05). Apart from B.hispida，DBP contents were higher in aboveground parts than root for 3 other kinds of Cucurbitaceae ( P < 0.05). DEHP contents were higher in aboveground parts than roots for all 4 plants (P<0.05). DEHP contents were higher in the roots of B.hispida and C.sativus than C. moschata and L.cylindrical(P<0.05)，while DEHP content was highest in the aboveground parts of C.moschata(P<0.05). The absorbability of DBP and DEHP for all 4 Cucurbitaceae plants were stronger than common leafy plants. Although，absorbabilities of C. sativus and C.moschata were stronger than L.cylindrical and B.hispida，so they were potential to be applied to remediate PAEs contaminated soil.K e y w o r d s：phthalate acid ester；plant remediation；Cucurbitaceae；stress response酞酸酯（phthalic acid esters,PAEs),又称邻苯二甲酸酯,是环境激素类的有机化合物[l],广泛用于各 类塑料制品、包装材料、医疗用品及化妆品等.塑料产品的生产、使用、丢弃和处置过程伴随着PAEs的大量 释放，从而污染了大气、水体和土壤环境[2].在自然条件下,PAEs具有较强的反应活性，容易被降解，水溶 性低，脂溶性高⑴，但土壤理化性质的差异导致PAEs在土壤中呈现特殊的环境行为[2],以及土壤独特的 结构体系，导致PAEs在其中大量富集，并影响到土壤环境质量和农产品质量，威胁到环境安全[3].我国土壤总体上均已遭受PAEs不同程度的污染，含量一般在g •kg_1至m g •kg_1数量级[4].土壤中的 PAEs通过挥发、淋溶、植物吸收等不同途径进入大气、水体、植物等自然介质中[5,6],对生态系统的结构和收稿日期：2017-02-15 修回日期：2017-03-20基金项目：江苏省自然科学基金项目（B K20130421);国家环境保护公益性行业科研专项（201409055).作者简介：张明（1982-),男，助理研究员.研究方向：生态修复与保护■Email:Z ha11gmi11g@.通讯作者崔军（ 1982-),男，博士，副教授.研 究方向：生态学、土壤生物地球化学及生物炭研究.Email:jscj2004@ .第6期张明等:4种葫芦科植物对酞酸酯的胁迫反应与吸收• 619 •功能的稳定性构成潜在危害，引发全球性环境污染和人类健康风险[7].土壤PAE S通过食物链延伸或生产 生活中的直接接触进入人体[8]，会干扰人体正常内分泌，扰乱生殖系统和生长发育功能[9].此外，长时间暴 露于某些PAES化合物将会影响机体免疫功能，产生“致突、致畸和致癌效应”[|()].因此,开展区域土壤 PAES植物修复技术研究，不仅有利于制定PAES污染土壤的修复治理措施,而且对保障生态环境与人类 健康具有重要意义.目前国内外对酞酸酯污染物与植物之间的相互作用研究，更多地侧重于植物吸收修复方面，对酞酸 酯污染物如何影响植物生长的报道较少[11]，对葫芦科植物的胁迫性影响更是鲜有报道.本试验以4种葫 芦科植物为材料,初步探讨了不同葫芦科植物在对酞酸酯的胁迫反应和吸收作用，旨在为酞酸酯污染土地 的农作物栽培和土壤修复提供依据.1材料与方法1.1植物培养受试植物为丝瓜(雅绿六号）、南瓜(蜜本三号）、冬瓜（铁柱168)及黄瓜（抗病二号）.在胁迫试验中，种子发芽后于1/4 Hoagland水培液中育苗两周，待长出两片真叶后，转移至装有100 m L的1/4 Hoagland 水培液中.分别从500、2 500、5000和10 000 m g •L_|的DBP和DEHP混合甲醇溶液中取0.1 mL加入到 100 mL水培液中,配成总PAEs浓度分别为1、5、10和20 m g •L_1的加标水培液.每种植物每个浓度4个重复.空白对照(CK)加入等量的甲醇.每3天更换1次营养液并重新加入相应浓度的PAES.22天后收获植物，测定 植物相关生理指标.在植物吸收试验中，DBP和DEHP的处理浓度均为10 m g •L-1，其余与胁迫试验一致. 1.2植物根系形态测定根系形态特征使用加拿大Regent Instruments公司生产的WinRHIZO根系分析系统进行测定.将根系 样品放置在30X40 cm树脂玻璃槽内，并注水至3~4 mm深，使根系充分散开，双面光源扫描根系，经专用 数字化软件（WinRHIZO2008a)分析后获得根长、根表面积、根体积、平均根直径等形态指标.1.3植物PAEs测定准确称取植物样品0.5 g(精确至0.000 1g)于离心管中，加入回收率指示物.加入20 mL提取液(丙酮 :正己烧=1/1，v/v)，振摇数下，超声提取20 min，13 000 r •min_1离心10 min，合并上清液于鸡心瓶中.样 品抽滤后旋蒸至约0.5 mL.浓缩后样品过层析柱净化(填料由下至上为12 cm中性硅胶，6 cm中性氧化铝，1cm无水硫酸钠）.加入20 mL正己烷淋洗并弃去，加入70 mL二氯甲烷/正己烷（v/v= 3/7)淋洗并弃去，加入40 mL洗脱液丙酮/正己烷(v/v=1/4)并收集于鸡心瓶中.旋蒸至0.5 mL后将样品转移到2 mL进样 瓶中，用适量正己烷润洗鸡心瓶.氮吹至小于1mL后，加入内标，准确定容至1mL，用涡旋器搅匀后保存 至-20 T；冰箱中待GC-MS检测.2结果与分析2.1 PAEs胁迫对葫芦科植物生长的影响本研究主要关注不同PAEs污染水平下不同属葫芦科植物的生长状况.不同处理下3种葫芦科植物的 生物量比较见表1.从表1可以看出，黄瓜的根部生物量随着PAEs处理浓度增大呈现低浓度增大,高浓度 减小的趋势，低浓度和高浓度下生物量有显著性差异（P<0.05).黄瓜地上部生物量随处理浓度增大变化 不大（除20 m g •L-1处理），但各处理间没有显著性差异.南瓜根部和地上部随处理浓度增大而减小，但各 处理间差异不显著.添加PAEs处理后，丝瓜的根部和地上部生物量与对照相比均有所增大（除10 m g •L-1处理的根部），但各处理间也没有显著性差异.3种葫芦科植物的根系形态数据见表2〜表4.可见，黄瓜、南瓜、丝瓜的总根长、根表面积、根体积都随 着处理浓度增大而减小,部分数据在低浓度处理时比对照略有增大，但由于标准差较大导致不同处理之间 没有显著性差异.在实验过程中发现，几种植物的根系在PAEs的胁迫下有变粗的现象，这点从根系平均直 径的数据也能看出，PAEs处理的根系平均直径比对照的有所增加,但各处理间没有显著性差异（除20 m g •L-1处理下的丝瓜外）.• 620 •福建农林大学学报（自然科学版)第46卷Table 1表1不同处理下3种葫芦科植物的生物量11Biomass of 3 Cucurbitaceae plants under different concentrations of PAEs(g•株-I,D W)处理部位黄瓜南瓜丝瓜CK根部0.108 6±0.009 8ab0.157 6±0.017 0a0.144 4±0.013 7a地上部0.822 3±0.176 5a 1.389 5±0.350 8a0.949 7±0.063 4a P A E s/(m g • _L_i)1根部0.130 7±0.015 1a/0.170 8±0.039 5a地上部0.799 3±0.013 9a/0.993 4±0.015 8a 5根部0.112 3±0.015 1ab/0.146 6±0.013 7a地上部0.837 1±0.153 6a/ 1.036 5±0.139 1a 10根部0.1215±0.007 4a0.150 6±0.015 2a0.131 6±0.009 5a地上部0.811 4±0.029 1a0.863 0±0.033 2a0.968 8±0.050 6a 20根部0.080 9±0.015 2b0.130 2±0.018 0a0.189 8±0.061 9a地上部0.604 5±0.134 7a0.832 3±0.083 9a0.998 9±0.030 7a n表中不同字母表示同一指标不同浓度处理间存在显著差异.表2丝瓜根系形态特征11Table 2 Root morphology of指标CKP A E s/(m g • _L-1)151020总根长/cm1507.24±314.56a1282.33±347.74a1274.49±147.81a1288.07±205.94a1145.68±72.60a 根表面积/cm2211.92±43.23a191.55±48.96a190.91±14.87a192.79±29.62a201.64±18.33a 平均直径/cm0.45±0.04a0.48±0.02a0.48±0.03a0.48±0.03a0.56±0.04b 根长/根体积/(c m• cm_-3)640.03±107.13b561.97±51.92b559.36±58.13b562.59±70.03b413.61±71.66a 根体积/cm3 2.38±0.57a 2.28±0.56a 2.28±0.16a 2.30±0.38a 2.82±0.43a "表中不同字母表示同一指标不同浓度处理间存在显著差异;数据均为4个平行试验.表3南瓜根系形态特征Table 3 Root morphology7of C. moschata指标CKP A E s/(m g ■10■L-1)20总根长/cm1635.05±143.33a1501.08±295.08a1377.41±233.05a 根表面积/cm2208.58±9.03a206.59±39.70a183.84±24.21a 平均直径/cm0.41±0.02a0.44±0.01a0.43±0.03a 根长/根体积/(c m•cm-3)771.57±76.55a662.88±44.04a705.82±114.53a 根体积/cm3 2.12±0.06a 2.26±0.43a 1.96±0.26a "表中不同字母表示同一指标不同浓度处理间存在显著差异;数据均为4个平行试验.表4黄瓜根系形态特征11Table 4 Root morphology7of B.hispida指标CKP A Es/(m g•L-1)151020总根长/cm 2 288.16±47.85a 2 282.38±161.17a 2 139.26±184.66a 2 200.80±263.06a 2 126.13±183.11a根表面积/cm2268.31±10.26a276.44±26.46a254.15±24.84a275.34±37.25a245.13±23.70a平均直径/cm0.37±0.01a0.39±0.03a0.38±0.02a0.40±0.01a0.37±0.04a根长/根体积/(c m•cm-3 )915.01±31.06a869.64±152.87a894.65±87.02a806.24±35.55a962.23±202.20a根体积/cm3 2.50±0.14a 2.68±0.45a 2.41±0.30a 2.74±0.42a 2.27±0.40a n表中不同字母表示同一指标不同浓度处理间存在显著差异.2.2葫芦科植物对酞酸酯的吸收作用4种植物对DBP和DEHP的吸收情况分别见图1和图2.可以看出,4种葫芦科植物的根部和地上部 都检测到DBP和DEHP.对DBP而言，黄瓜和南瓜根部含量分别达到99.89和106.27 m g •kg-1,远大于冬 瓜(23.21 m g •kg-1)和丝瓜（2.79 m g •kg-1)(P<0.05).除冬瓜外，其余三种植物的地上部DBP含量均大 于根部的（_P<0.05),其中黄瓜为 174.05 m g •kg-1,南瓜为 126.4 m g •kg-1,丝瓜为 69.42 m g •kg-1.对 DE-H P而言,4种植物的根部含量均远高于各自地上部的.冬瓜和黄瓜分别为149.67和152.15 m g •kg-1,明显 大于南瓜（89.63 m g •kg-1)和丝瓜(78.05 m g •kg-1)(P<0.05).南瓜地上部的含量为14.27 m g •kg-1,与其第6期张明等:4种葫芦科植物对酞酸酯的胁迫反应与吸收• 621 •余三种植物地上部的含量有显著性差异（尸<〇.〇5).图 1 4种葫芦科植物根部和地上部D B P 含量 图2 4种葫芦科植物根部和地上部D E H P 含量Fig.1DBP content in the root and aboveground part of 4Fig.2 DEHP content in the root and aboveground part of 4Cucurbitaceae plantsCucurbitaceae plants从总体上看，DEHP 主要富集在植物的根部，而DBP 则在根部和地上部皆有富集.DBP 的辛醇一水分 配系数（log [ow )为4.45,小于DEHP 的7.50.研究表明，疏水性有机污染物（log [ow >3.0)被根表面强烈吸 附，不易向上迁移，而亲水性有机污染物（log 尺胃<0.5)不易被根吸收或较难通过植物的细胞膜，而中等亲水性有机污染物（log ^Q W = 0.5~3.0)则较易被植物吸收转运.在本研究中，DBP 可以较大量地被转移至地 上部，但DEHP 则主要被阻隔在根部而较难往地上部转运.表5和表6分别是本研究与其他研究者结果的比较.可以发现，4种葫芦科植物对DBP 和DEHP 的吸 收能力强于一些常见的叶菜类植物.综合来看，4种植物中，黄瓜和南瓜比其余两种葫芦科植物对DBP 和 DEHP 均有较强的吸收能力，是PAEs 修复的潜力植物.表5本研究样本与其他研究植物对P A E s 的吸收能力比较（根部）^Table 5Comparisons of root absorbability to PAEs between Cucurbitaceae plants and other vegetables植物处理/(p g •mL_1 )含量/(p g •.g-1)栽种方式处理时间参考文献DBPDEHP DBP DEHP 冬瓜 5.00523.21149.61水培22 d 本研究黄瓜 5.00599.89152.15水培22 d 本研究南瓜 5.005106.2789.63水培22 d 本研究丝瓜 5.005 2.7978.05水培22 d 本研究辣椒 5.00517.040.00土培3个月Yin et al[12]白菜10.00/0.22/水培42 d Liao et al[13]蕹菜 4.45/0.556-1.25/土培39 d Cai et al[14]小白菜10.00/0.14/水培42 dLiao et al[15]表6本研究样本与其他叶菜类植物对P A E s 的吸收能力比较（地上部）Table 6 Comparisons of absorbability of aboveground part to PAEs between Cucurbitaceae plants and other vegetables植物处理/(^g •mL_1 )含量/(p g •'g-1)栽种方式处理时间参考文献DBPDEHP DBP DEHP 冬瓜 5.005 3.19 3.64水培22 d 本研究黄瓜 5.005174.05 6.95水培22 d 本研究南瓜 5.005126.414.27水培22 d 本研究丝瓜 5.00569.42 3.75水培22 d 本研究辣椒 5.00561.440.00土培 3 个月Yin et al[12]白菜10.00/ 1.36/水培42 d Liao et al[13]蕹菜 4.45/0.322~0.715/土培39 d Cai et al[14]小白菜10.00/无法检出/水培42 dLiao et al[15]3结论3种葫芦科植物黄瓜、南瓜、丝瓜在一系列PAEs 浓度胁迫下其常见生理指标（生物量、根系指标等）变化差异不大，在低浓度时对某些指标还具有促进作用，可见几种葫芦科植物在一定范围内对PAEs 具有• 622 •福建农林大学学报（自然科学版)第46卷较好的耐受能力.对DBP的吸收作用,黄瓜和南瓜根部含量远大于冬瓜和丝瓜;除冬瓜外，其余3种植物的地上部DBP 含量均大于根部的含量.对DEHP的吸收作用,4种植物的根部含量均远高于各自地上部的；冬瓜和黄瓜 根部含量明显大于南瓜和丝瓜;南瓜地上部的含量显著大于其余3种植物地上部的含量.参考文献[1] STALES C A, PETERSON D R, PARKERTON T F, et al. The environmental fate of phthalate esters：A literature review[J].Chemosphere, 1997,35(4) ：667-749.[2] HE L Z, GIELEN G, BOLAN N S, et al. Contamination and remediation of phthalic acid esters in agricultural soils in China：A review[J]. Agronomy for Sustainable Development, 2015,35(2) ：519-534.[3] CAI Q Y, MO C H, WU Q T, et al. The status of soil contamination by semivolatile organic chemicals (SVOCs) in China：Areview[J]. Science of the Total Environment, 2008,389(2-3) ：209-224.[4]崔学慧，李炳华，陈鸿汉，等.中国土壤与沉积物中邻苯二甲酸酯污染水平及其吸附研究进展[J].生态环境学报,2010,19(2) ：472-479.[5] COUSINS I T, JONES K C. Air-soil exchange of semi-volatile organic compounds (SO Cs) in the U K[J]. Environmental Pollution, 1998,102(1)：105-118.[6] ZENG F, LIN Y J, CUI K, et al. Atmospheric deposition of phthalate esters in a subtropical c ity[J]. Atmospheric Environment, 2010,44(6)：834-840.[7] GUO Y, WU Q, KANNAN K. Phthalate metabolites in urine from China, and implications for human exposures[J]. Environment International, 2011,37(5)： 893 - 898.[8] WANG J, LUO Y M, TENG Y, et al. Soil contamination by phthalate esters in Chinese intensive vegetable production systemswith different modes of use of plastic film[J]. Environmental Pollution, 2013,180C(3) ：265-273.[9] SUN T R, CANG L, WANG Q, et al. Roles of abiotic losses, microbes, plant roots, and root exudates on phytoremediation ofPAHs in a baren s o il[J]. Journal of Hazardous Materials, 2010,176( 1-3) ：919-925.[10] LATINI G. Monitoring phthalate exposure in hum ans[J]. Clinica Chimica Acta, 2005,361(1-2) ：20-29.[11]杨子江，饶刚顺，肖立中，等.酞酸酯污染胁迫对2个水稻品种生长和生理特性的影响[J].广东农业科学,2013,40(7):1-3.[12] YIN R, LIN X G, WANG S G, et al. Effect of DBP/DEHP in vegetable planted soil on the quality of capsicum fru it[J].Chemosphere, 2003,50:801-805.[13] LIAO C S, YEN J H, WANG Y S. 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荔枝LcMYB1异源表达促进矮牵牛和番茄花色苷的积累
热带作物学报2021, 42(5): 1290 1296 Chinese Journal of Tropical Crops收稿日期 2020-06-09；修回日期 2020-07-21基金项目 长江师范学院校级科研项目（No. 2016KYQD19，No. 2016KYQD20，No. 2016XJQN07）。
作者简介 杜丽娜（1987—），女，博士，副教授，研究方向：园艺植物生物技术。
*通信作者（Corresponding author ）：赖 彪（LAI Biao ），E-mail ：*******************。
荔枝LcMYB1异源表达促进矮牵牛和番茄花色苷的积累杜丽娜1,2，陈春帆1，苏 睿1，谭春艳1，赖 彪1,2*1. 长江师范学院现代农业与生物工程学院，重庆涪陵 408100；2. 长江师范学院武陵山片区绿色发展协同创新中心，重庆涪陵 408100摘 要：为分析荔枝LcMYB1的功能，以白色‘W115’矮牵牛和‘Micro-Tom ’番茄为材料，利用农杆菌介导法将LcMYB1在矮牵牛和番茄中异源表达，观测转基因植株表型。
结果表明：与‘W115’相比，转基因矮牵牛的叶片和花瓣中积累了花色苷，且矮牵牛花色苷生物合成结构基因PhCHS 和PhDFR 、调控基因PhAN1的表达显著上调；与野生型‘Micro-Tom ’番茄相比，转基因番茄的叶片和花药中积累了花色苷，且相应组织花色苷生物合成结构基因SlDFR 、调控基因SlAN1和SlJAF13的表达显著上调，虽然果实中没有积累花色苷，但SlAN1和SlJAF13表达也显著上调。
LcMYB1在矮牵牛和番茄中异源表达时通过上调花色苷生物合成关键结构基因和调控基因bHLH 的表达诱导花色苷积累。
因此，荔枝LcMYB1是花色苷生物合成中的关键转录因子，具备异源转化利用的潜力。
关键词：LcMYB1；矮牵牛；番茄；花色苷 中图分类号：S667.1 文献标识码：AEctopic Expressed LcMYB1 Induced Anthocyanin Biosynthesis in Petunia and TomatoDU Lina 1, CHEN Chunfan 1, SU Rui 1, TAN Chunyan 1, LAI Biao 1,2*1. School of Advanced Agriculture, Bioengineering, Yangtze Normal University, Fuling, Chongqing 408100, China;2.Correlative Innovation Centre for Green Development in Wulingshan Region, Yangtze Normal University, Fuling, Chongqing 408100, ChinaAbstract: In order to better understand the function of LcMYB1 and provide theoretical support for further utilization, LcMYB1 was transformed into both petunia and tomato. White flower ‘W115’ petunia and ‘Micro-Tom’ tomato were used as materials in LcMYB1 ectopic expressed assays. Anthocyanin contents and related gene expressions were ana-lyzed in transgenic plants. Ectopic expression of LcMYB1 in ‘W115’ resulted in anthocyanin production in vegetative and floral tissues such as leaves and petals, probably by transcriptional activation of anthocyanin biosynthetic genes such as PhCHS and PhDFR and endogenous anthocyanin regulatory gene PhAN1. However, the transgenic tomato only produced anthocyanin in anthers and leaves but not in tomato fruits and petals. The results suggested that LcMYB1 could enhance anthocyanin production in vegetative and floral tissues of both petunia and tomato by activating anthocyanin biosynthesis and regulatory genes. LcMYB1 is an important transcriptional regulatory factor in anthocyanin biosynthesis of plants and is potentially used in ectopic transformation. Keywords: LcMYB1; petunia; tomato; anthocyanin DOI: 10.3969/j.issn.1000-2561.2021.05.013色泽是花卉和果蔬的重要品质组成之一。
玉米ECT家族成员的全基因组分析及非生物胁迫下的响应表达
核农学报2024，38（6）：1035~1047Journal of Nuclear Agricultural Sciences玉米ECT家族成员的全基因组分析及非生物胁迫下的响应表达徐可丽周美君郭怡婷吉虹菁黄敏李曼菲杜何为 *刘睿 *（长江大学生命科学学院，湖北荆州434000）摘要：ECT结构域蛋白家族作为一种重要的转录后调控因子，在调控m6A修饰中发挥重要功能，在植物正常生长发育和响应逆境胁迫时的基因表达调控中具有重要作用。
为了研究ECT结构域蛋白家族在玉米（Zea mays L.）非生物胁迫下的生长发育与胁迫响应中的功能，本研究采用生物信息学方法鉴定到玉米ECT家族成员22个，并分析其序列和结构特征、染色体分布、启动子顺式作用元件、GO富集、蛋白质互作网络和系统发育进化关系，通过实时荧光定量PCR（qRT-PCR）分析了玉米ZmECTs在冷胁迫以及不同激素处理下的表达模式。
结果表明，玉米ECT家族成员分布在10条染色体上，编码的蛋白质所含氨基酸残基数目为119~748 aa，相对分子量（MW）范围为13 412.17~81 823.70 Da，等电点（pI）范围为5.43~8.82，大部分蛋白定位在细胞核。
在ZmECT家族中共鉴定到10个motif。
顺式作用元件分析结果表明，ZmECT家族成员启动子包含多个与胁迫、激素和生长发育相关的响应元件。
GO富集分析结果显示，ZmECT家族成员可能参与mRNA的剪接和维持RNA稳定性。
蛋白质互作网络预测，ZmECT6是该家族蛋白的核心成员。
系统进化树显示，ZmECT家族成员分为4组。
qRT-PCR结果显示，经不同激素处理后，ZmECT家族成员表现出复杂的响应模式，部分ZmECT家族成员响应冷胁迫。
本研究结果为玉米ECT基因后续的生物学功能解析及分子机制研究提供了参考。
关键词：玉米（Zea mays L.）； ECT家族；生物信息学；非生物胁迫；响应表达DOI：10.11869/j.issn.1000‑8551.2024.06.1035RNA结合蛋白（RNA binding proteins， RBPs）作为一种重要的转录后调控因子，能与RNA结合形成核糖核蛋白复合物，从而在真核生物细胞的RNA代谢过程中发挥重要的调控作用［1］。
γ-射线照射梨试管苗诱导产生多倍体变异
园 艺 学 报 2009,36(2):257-260Acta Horticulturae Sinicaγ-射线照射梨试管苗诱导产生多倍体变异孙清荣13,孙洪雁1,祝恩元2,李林光1(1山东省果树研究所,农业部泰安温带果树资源重点野外科学观测试验站,山东省果树生物技术育种重点实验室,山东泰安271000;2山东省科学技术厅,济南250014)摘 要:用γ-射线照射梨试管苗,照射后对其顶芽、侧芽和叶片3种外植体分别接种,经过继代和筛选,获得了形态变异明显的变异无性系,其变异表现为茎变粗,节间变短,叶长/叶宽比变小,叶柄变短。
经染色体计数鉴定,这些变异中既有三倍体也有四倍体。
多倍体的气孔长度及比叶重较对照二倍体显著增大。
关键词:梨;试管苗;γ-射线;多倍体中图分类号:S66112 文献标识码:A 文章编号:05132353X(2009)022*******Polyplo i d I nducti on i n Pear in vitro Trea tm en t w ith Gamma2raysS UN Q ing2r ong13,S UN Hong2yan1,ZHU En2yuan2,and L IL in2guang1(1Shandong Institute of Po m ology,the S tate A griculture M inistry Experi m ent S tation of Te m perate Fruits Ger m plas m Key O utdoor O bservation in Taiπan,Shandong Fruits B iotechnology B reeding Key L aboratory,Taiπan,Shandong271000,China;2D epart m ent of Science and Technology of Shandong Province,J inan250014,China)Abstract:In vitro shoots of pear cultivar‘Fertility’(Pyrus co mm unis L.)were irradiated with ga mma2 rays.Three different exp lants of shoot ti p,lateral bud and young leaf fr om irradiated shoots were cultured separately.Shoots fr om3different exp lants were subcultured and selected.Mor phol ogical mutants with thicker ste m,shorter internode,s maller leaf index and shorter peti ole were obtained.Chr omos ome counting confir med that the mutants were tri p l oids and tetrap l oids.The lengths of st omatal guard cells and s pecific leaf mass of polyp l oids were significantly increased than that of di p l oids.Key words:pear;in vitro shoots;ga mma2ray;polyp l oid随着果树组织培养技术的成熟和完善,离体组织培养和诱发突变相结合已成为一项很有前途的育种研究项目,组织培养可提供更广泛的处理材料,如器官、组织和细胞,比原位活体(in vivo)更适于诱变,而且辐射诱变育种与离体培养结合能大大降低嵌合体的比例,可在有限的空间内对大量诱变群体进行选择,提高后代筛选和鉴定效率(刘进平和郑成木,2004)。
费尔干猪毛菜病程相关蛋白SfPR1a基因的异源表达增强了烟草对干旱、盐及叶斑病的抗性
作物学报ACTA AGRONOMICA SINICA 2020, 46(4): 503 512 / ISSN 0496-3490; CN 11-1809/S; CODEN TSHPA9E-mail: zwxb301@DOI: 10.3724/SP.J.1006.2020.94082费尔干猪毛菜病程相关蛋白SfPR1a基因的异源表达增强了烟草对干旱、盐及叶斑病的抗性衡友强游西龙王艳*新疆大学生命科学与技术学院 / 新疆生物资源与基因工程重点实验室, 新疆乌鲁木齐 830046摘要: 为明确一年生草本盐生植物费尔干猪毛菜(Salsola ferganica Drob.)病程相关蛋白基因SfPR1a (GenBank登录号为JQ670917)是否参与了植物对逆境胁迫的响应, 采用qRT-PCR检测了该基因在不同组织部位和脱落酸(ABA)、茉莉酸(JAs)、乙烯合成直接前体(ACC)等相关激素胁迫及NaCl处理下的表达规律, 同时对转基因烟草在盐、旱及丁香假单胞菌等胁迫下的抗性进行了鉴定。
结果显示, SfPR1a基因在费尔干猪毛菜根中的表达量显著高于茎叶中, 且受到ABA、JAs、ACC、NaCl的积极诱导; 干旱胁迫下, 转基因烟草的丙二醛(MDA)含量显著低于野生型烟草, 显示出较强的抗旱表型; 盐胁迫下, 异源表达SfPR1a的转基因烟草幼苗生长显著优于野生型烟草; 丁香假单胞菌攻毒后的转基因烟草叶片呈现严重的坏死反应, 但植株的整体抗性表型显著优于野生型烟草; 亚细胞定位结果显示该蛋白定位于植物细胞质外体空间。
以上结果表明, 费尔干猪毛菜病程相关蛋白SfPR1a基因参与了植物对非生物及生物胁迫的抗性。
关键词:病程相关蛋白基因SfPR1a; 表达规律; 转基因烟草; 抗性功能; 亚细胞定位Pathogenesis-related protein gene SfPR1a from Salsola ferganica enhances theresistances to drought, salt and leaf spot disease in transgenic tobaccoHENG You-Qiang, YOU Xi-Long, and WANG Yan*Xinjiang Key Laboratory of Biological resources and Genetic Engineering / College of Life Science and Technology, Xinjiang University, Urumqi830046, Xinjiang, ChinaAbstract: In order to investigate whether SfPR1a, a pathogenesis-related protein gene from an annual halophytic species Salsolaferganica Drob., was involved in the response to plant defense, qRT-PCR was employed to detect its expression patterns underabscisic acid (ABA), jasmonic acid (JAs), ethylene synthesis direct precursor (ACC), and NaCl treatments. We also identifiedresistances to salt, drought and Pseudomonas syringae tomato (Ps tDC3000) of transgenic tobacco were identified. The expressionof SfPR1a gene in roots was significantly higher than that in shoots, and positively induced by ABA, JAs, ACC, and NaCl treat-ments. The malondialdehyde (MDA) content of transgenic tobacco was significantly lower than that of wild-type tobacco, show-ing a strong resistance to drought. The ectopic expression of SfPR1a gene improved plant growth under salt stress. After infectionof P. syringae, transgenic tobacco leaves showed serious necrosis reaction, but the overall resistance phenotype of the plants wassignificantly better than that of WT. Subcellular localization analysis showed SfPR1a was localized in the plant cell apoplasticspace. The above results indicated that the SfPR1a gene is involved in plant resistance to abiotic and biotic stresses.Keywords: pathogenesis-related protein gene SfPR1a; expression pattern; transgenic tobacco; resistance function; subcel-lular localization盐、碱、干旱和病原微生物等外界胁迫已经成为影响农作物产量的关键因素。
bHLH_转录因子在植物耐冷基因工程中的应用进展
河南农业科学，2023，52（11）：1⁃9Journal of Henan Agricultural Sciencesdoi:10.15933/ki.1004-3268.2023.11.001bHLH 转录因子在植物耐冷基因工程中的应用进展齐学礼1，李莹2，李春盈3，韩留鹏1，赵明忠1，张建周3（1.河南省作物分子育种研究院，河南郑州450002；2.《河南农业大学学报》编辑部，河南郑州450002；3.河南省农业科学院小麦研究所，河南郑州450002）摘要：植物在生长发育过程中经常遭遇低温胁迫，影响其生长发育、地理分布，降低产量、品质。
bHLH （Basic helix⁃loop⁃helix ）是植物中第二大转录因子家族，在植物抵御低温胁迫反应中具有重要的调控作用。
阐述了植物bHLH 转录因子的基本结构，综述了类似MYC （Avian myelocytoma virus ）的bHLH 转录因子ICE ［Inducer of CBF （C⁃repeat binding factor ）expression ］和其他bHLH 转录因子在植物耐冷基因工程中的应用进展，以期为bHLH 转录因子在植物耐冷遗传改良、育种中的应用提供参考。
关键词：植物；bHLH 转录因子；耐冷；基因工程中图分类号：Q943.2文献标志码：A文章编号：1004-3268（2023）11-0001-09收稿日期：2023-09-28基金项目：国家小麦产业技术体系项目（CARS-3-7）作者简介：齐学礼（1982-），男，河北晋州人，副研究员，博士，主要从事小麦遗传育种工作。
E-mail ：******************。
李莹为同等贡献作者通信作者：张建周（1974-），男，河南长葛人，副研究员，主要从事小麦育种与示范推广工作。
E-mail ：***********************Progress on Application of bHLH Transcription Factors in Cold Tolerance Genetic Engineering of PlantsQI Xueli 1，LI Ying 2，LI Chunying 3，HAN Liupeng 1，ZHAO Mingzhong 1，ZHANG Jianzhou 3（1.Crops Molecular Breeding Academy of Henan ，Zhengzhou 450002，China ；2.Editorial Department of Journal of HenanAgricultural University ，Zhengzhou 450002，China ；3.Wheat Research Institute ，Henan Academy of AgriculturalSciences ，Zhengzhou 450002，China ）Abstract ：Plants often encounter cold stress ，which influences the growth and geographical distribution ，and decreases yield and quality of plants.bHLH （basic helix⁃loop⁃helix ）family is the second largest transcription factor family in plant ，which plays an important role in regulation of tolerance to cold stress.This paper elaborated the structure and the application of MYC （avian myelocytoma virus ）⁃like bHLH transcription factor ICE ［inducer of CBF （C⁃repeat binding factor ）expression ］and other bHLH transcription factors in plant cold tolerance genetic engineering ，so as to provide some references for the utilization of bHLH transcription factors in cold tolerance genetic improvement and breeding.Key words ：Plant ；bHLH transcription factor ；Cold tolerance ；Genetic engineering 低温是植物生长过程中经常遭遇的主要非生物胁迫因子之一，影响植物的地理分布和生长发育，降低产量和品质[1⁃2]。
甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析
DOI: 10.3724/SP.J.1006.2022.14023甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析黄成梁晓梅戴成文静易斌涂金星沈金雄傅廷栋马朝芝*华中农业大学作物遗传改良国家重点实验室/ 国家油菜工程技术研究中心，湖北武汉430070摘要：天冬氨酸蛋白酶(AP)属于四大蛋白水解酶之一，在蛋白质加工、信号转导和胁迫反应中发挥着重要作用。
甘蓝型油菜是我国重要的油料作物，利用蛋白质同源性分析，在甘蓝型油菜中鉴定出154个APs编码基因，分别编码典型、非典型和珠心类天冬氨酸蛋白酶。
基因结构分析结果表明，多数BnAPs基因包含1~4个外显子。
同一类型的天冬氨酸蛋白酶成员之间蛋白质基序(Motif)分布相似。
共线性分析表明，甘蓝型油菜与白菜、甘蓝和拟南芥存在大量的同源基因，约89%的BnAPs 基因来自于全基因组复制事件。
转录水平检测结果表明，BnAPs基因家族成员在各个组织中均有表达，其中BnAP30.A05.1/A05.2/C05.1/C05.2、BnAP36.A04/C08、BnAP39.A06/C03在授粉后的柱头显著提高。
BnAPs基因启动子区域顺式元件分析结果表明，逆境相关的顺式调控元件被显著富集；进一步利用qRT-PCR验证了这些富含逆境相关顺式调控元件的基因在逆境(ABA、NaCl或4℃)处理后的表达水平显著变化，推测这些BnAPs基因可能参与甘蓝型油菜对逆境的响应。
进一步和拟南芥同源基因组织表达模式进行比较后发现，大约有24%的BnAPs与其同源AtAPs具有相同的表达模式。
本研究为进一步解析天冬氨酸蛋白酶家族在甘蓝型油菜中的生物学功能奠定了基础。
关键词: 甘蓝型油菜；天冬氨酸蛋白酶；共线性分析；表达模式；qRT-PCRGenome wide analysis of BnAPs gene family in Brassica napusHUANG Cheng, LIANG Xiao-Mei, DAI Cheng, WEN Jing, YI Bin, TU Jin-Xing, SHEN Jin-Xiong, FU Ting-Dong, and MA Chao-Zhi*National Key Laboratory of Crop Genetic Improvement / National Engineering Research Center of Rapeseed, Huazhong Agricultural University, Wuhan 430070, Hubei, ChinaAbstract: Aspartate protease (AP) is one of the four major proteolytic enzymes and plays an important role in protein processing, signal transduction, and stress response. Brassica napus is an important oil crop in China. We identified 154 APs coding genes by protein homology analysis, which encoded typical, atypical, and nucellar aspartate proteases, respectively. Gene structure analysis showed that most BnAPs genes contained 1–4 exons. The motif distribution of the same type of aspartic protease was similar. Collinearity analysis revealed that there was a large number of homologous genes between Brassica napus and Brassica rape, Brassica oleracea and Arabidopsis thaliana, and about 89% of BnAPs genes came from genome-wide replication events. Transcriptional analysis demonstrated that BnAPs gene family was expressed in all tissues. The stigma of BnAP30.A05.1/ A05.2/C05.1/C05.2, BnAP36.A04/C08, and BnAP39.A06/C03increased significantly after pollination. Cis-element analysis in the promoter region of BnAPs gene presented that stress-related cis regulatory elements were significantly enriched. We further verify本研究由国家重点研发计划项目(2016YFD100803)资助。
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ORIGINALARTICLEEctopicexpressionofanovelpeach(Prunuspersica)CBFtranscriptionfactorinapple(Malus3domestica)resultsinshort-dayinduceddormancyandincreasedcoldhardiness
MichaelWisniewski•JohnNorelli•
CaroleBassett•TimothyArtlip•DumitruMacarisin
Received:27September2010/Accepted:10January2011/Publishedonline:28January2011ÓSpringer-Verlag(outsidetheUSA)2011
AbstractLow,non-freezingtemperaturesand/orshortdaylength(SD)regulatescoldacclimationanddormancyinfruittrees.Regardingcoldacclimation,C-repeatbindingfactor(CBF/DREB)transcriptionalactivatorgeneshavethewell-documentedabilitytoinducetheexpressionofasuiteofgenesassociatedwithincreasedcoldtolerance.Weisolatedafull-lengthcDNAofapeachCBFgene,desig-natedPpCBF1(GenBankAccessionHM992943),andconstitutivelyexpresseditusinganenhanced35Spromoterinapple.Unexpectedly,constitutiveoverexpressionofthePpCBF1inappleresultedinstrongsensitivitytoshortdaylength.GrowthcessationandleafsenescencewereinducedintransgeniclinesexposedtoSDandoptimalgrowthtemperaturesof25°Covera4-weekperiod.Fol-lowing1–4weeksofSDand25°CtreeswerereturnedtoLDand25°Cinthegreenhouse.Control(untransformed)plantscontinuedtogrowwhiletransgeniclinesreceivingtwoormoreweeksofSDremaineddormantandbegantodropleaves.ConstitutiveoverexpressionofthePpCBF1inappleresultedina4–6°Cincreaseinfreezingtoleranceinboththenon-acclimatedandacclimatedstates,respec-tively,comparedwithuntransformedM.26trees.ThisisthefirstinstancethatconstitutiveoverexpressionofaCBFgenehasresultedinSD-inductionofdormancyandtoourknowledgethefirsttimeapplehasbeenshowntostronglyrespondtoshortdaylengthasaresultoftheinsertionofatransgene.
KeywordsColdacclimationÁLeafsenescenceÁAP2/ERFÁDormancyÁTransgenicapple
AbbreviationsAP2Apetala2CBForCBF/DREBC-repeatbindingfactorERFEthyleneresponsefactorDRE/CRT/LTREDehydrationresponsiveelement/C-repeat/lowtemperatureresponsiveelementCORCold-regulatedgenesRT-qPCRReversetranscription,quantitativereal-timePCRSDShortdaylengthLDLongdaylengthLTLowtemperatureHTHightemperature
IntroductionTheabilitytocoldacclimateandundergoaperiodofdor-mancyisessentialtotemperatewoodyplantsinordertosurvivefreezingwintertemperatures.Seasonalregulationofgrowthandfreezingtoleranceismarkedbyphenologicaleventssuchasgrowthcessation,budset,theonsetofdor-mancy,coldacclimation,deacclimation,andbudburst(I-ban˜ezetal.2010;Kalbereretal.2006).Coldacclimationresultsinandisgovernedbyadistinctsetofchangesingeneexpression(Chinnusamyetal.2006;WellingandPalva2006;Thomashowetal.2001).Thesameistruefordormancy(RohdeandBhalerao2007).Inwoodyplants,theprocessesofdormancyandcoldacclimationoverlapandbothcanbeinducedbylow,non-freezingtemperatures
M.Wisniewski(&)ÁJ.NorelliÁC.BassettÁT.ArtlipÁD.MacarisinU.S.DepartmentofAgriculture,AgriculturalResearchService(USDA-ARS),TheAppalachianFruitResearchStation,Kearneysville,WV25430,USAe-mail:michael.wisniewski@ars.usda.gov
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Planta(2011)233:971–983DOI10.1007/s00425-011-1358-3(LT)andshortdaylength(SD)(WellingandPalva2006;Kalbereretal.2006;Wisniewskietal.2003;Weiser1970;SakaiandLarcher1987).TheextenttowhicheachprocessisregulatedbyeitherLTorSD,however,isspeciesspecific(Heide2008).Interestingly,adistinctcontrastexistswithintheRosaceaeregardingenvironmentalcontrolofgrowthcessationanddormancy.Inapple(Malus9domestica)andpear(Pyruscommunis),bothinthetribePyreae,growthcessationandtheonsetofdormancyarerelativelyunre-sponsivetoSDandinsteadrequireLT(HeideandPrestrud2005)whileinpeach(Prunuspersica)andotherPrunusspeciesofthetribeAmygdaleae,growthcessationanddormancyareinducedmainlybySDandenhancedbyLT(Heide2008).Plantphenologyisstronglycontrolledbyclimateandassuchhasbecomeastrongbioindicatorofongoingclimatechange(GordoandSanz2010).Despitetherecognizedroleoftemperatureintheonsetandreleaseofdormancy,andincoldacclimationanddeacclimation,theimpactthatclimatechangewillhaveonthephenologyoffruittreesisnotknown.Increasingepisodesofdevastatingspringfrosts,however,maybemorecommon(Guetal.2008)andinareviewofseveralstudies,BallandHill(2009)indicatedthatelevatedatmosphericCO2concentrationscanhavea
negativeimpactonacclimationandenhancevulnerabilitytofrostdamage.Therefore,thereisaneedtobetterunderstandthemolecularcontrolofdormancyandcoldacclimationintrees(WellingandPalva2006).CBFproteinsbelongtotheCBF/DREbinding(DREB)sub-familyoftheApetala2-ethyleneresponsivefactor(AP2/ERF)superfamilyoftranscriptionfactors(Nakanoetal.2006)thatbindtoacis-element(DRE/CRT/LTRE)containingaconservedCCGAcoresequence(Bakeretal.1994;Yamaguchi-ShinozakiandShinozaki1994).InArabidopsis,LT-inducibleCBFgeneshavebeendemon-stratedtoregulatealargenumberofcold-regulated(COR)genes,whoseproductsarethoughttocontributetofreezingtolerance.TheroleofCBFincoldresponseandacclima-tionhasbeenwelldocumentedinbothherbaceous(Thomashowetal.2001)andwoodyplants(WellingandPalva2006).Inrelationtowoodyplants,CBFtranscriptionfactorshavebeenisolatedandshowntobecold-responsiveinbirch(Betulapendula)(WellingandPalva2008),poplar(Benedictetal.2006),eucalyptus(Navarroetal.2009;ElKayaletal.2006),grape(Xiaoetal.2006),sweetcherry(Prunusavium)(Kitashibaetal.2004),citrus(Champetal.2007),blueberry(Vacciniumcorymbosum)(Polashocketal.2010),anddwarfapple(Malusbaccata)(Yangetal.2010).IncomparisonwithherbaceousplantstheregulationofCBFinwoodyplantsappearstobemorecomplex.TheexpressionpatternofspecificCBFgeneswithinaspeciescanbedifferentinannualversusperennialtissues