应用抑制消减杂交技术筛选葡萄冬芽和夏芽生理休眠相关基因

doi:10.3864/j.issn.0578-1752.2014.05.019

摘要/Abstract

摘要： 【目的】葡萄新梢上的芽有冬芽和夏芽两种，根据这两种芽休眠特性的差异，筛选与芽生理休眠相关基因，进而从分子水平研究葡萄芽生理休眠的相关机制。【方法】以‘赤霞珠’葡萄新梢上冬芽和夏芽为材料，夏芽作为驱动方（driver），冬芽作为实验方（tester），采用抑制消减杂交技术（suppression subtractive hybridization，SSH）构建冬芽和夏芽的SSH-cDNA文库，筛选冬芽和夏芽差异表达的基因片段。通过NCBI同源比对，寻找与葡萄芽生理休眠相关的基因，并对差异表达基因进行功能注释和分类。最后，运用Real-time PCR技术对部分候选基因进行检测，以验证其与葡萄芽生理休眠的相关性。【结果】共获得359条有效、高质量的差异基因片段，经CAP3软件聚类拼接，得到106个unique EST。在GenBank中进行BLASTN同源比对，有98条EST与已知蛋白或基因具有不同程度的同源性，占全部EST的92.4%。进行BLASTX同源比对，有54条EST与功能已知蛋白或基因具有不同程度的同源性，占全部EST的51%。已知功能的EST涉及休眠、逆境胁迫、细胞代谢、胞内物质运输等方面，且绝大多数基因信息来源于葡萄、苜蓿、橄榄、油菜、高粱、棉花、大蒜、拟南芥等植物。对差异基因片段进一步的功能分析发现，调控开花的基因占生物学过程的20%；与环境胁迫相关的基因占生物学过程的6%；与细胞代谢相关的基因占生物学过程的20%；与胞内物质运输相关的基因占总生物学过程的6%；其他包括DNA转录、减数分裂、细胞死亡及细胞壁松弛作用等。这些基因的分子功能主要有半胱氨酸型肽链内切酶、甲基转移酶、蛋白激酶、催化活性、结构分子活性、蛋白结合功能、DNA结合功能、ATP结合功能、蛋白质二聚体催化活性及丙酮酸氨基转移酶活性等。经Real-time PCR检测得知，在夏芽萌发过程中，候选基因在冬芽和夏芽中表达量呈不规律变化，且同一基因在相同节位的冬芽和夏芽中表达量均有较大差异。【结论】发现了一些在冬芽中表达频率较高的基因，如编码MADS FLC-like蛋白、钙依赖蛋白激酶、NADP依赖型的苹果酸酶、细胞壁水解酶、细胞壁松弛蛋白、外被体蛋白β亚基、热激蛋白、衰老相关蛋白、细胞色素P450、细胞壁关联蛋白等基因，认为线粒体蛋白基因、未知蛋白基因、开花相关基因、ATP合成酶β亚基基因、MADs FLC-like蛋白基因等与葡萄芽生理休眠具有相关性。

关键词: 葡萄 , 抑制消减杂交 , 芽生理休眠 , 冬芽 , 夏芽

Abstract: 【Objective】There are two kinds of buds, latent buds and prompt buds, along shoots of grapevine. The aim of this study is to screen the genes associated with the bud endodormancy of grapevine depending on the different characteristics of grape latent buds and prompt buds in dormancy, on the basis of which the mechanism of grapevine bud endodormancy was explored.【Method】The SSH-cDNA library of latent buds and prompt buds of Cabernet Sauvignon was constructed using the technology of suppression subtractive hybridization (SSH) to screen the differential expressed gene fragments between them, during which latent buds were used as driver and prompt buds as tester. These fragment sequences were analyzed based on the NCBI and the gene functions were annotated. Furthermore, real-time PCR was employed to detect the expression profile of part of candidate genes with bud development to confirm the relationship between candidate genes and bud endodormancy. 【Result】Totally 359 effective differentially expressed gene fragments with high quality were obtained and assembled by CAP3 Assembly Program, and 106 unique expressed sequence tags (ESTs) were obtained. After comparing the sequences with BLASTN in the Genbank, it was found that 98 unigenes, accounting for 92.4% of all the unigenes, showed homology with the known genes or proteins. And after analysis with BLASTX, it was found that 54 unigenes, accounting for 51% of all, showed homology with the known proteins or genes, which were involved in the dormancy, cell metabolism, abiotic and biotic stress and intracellular transportation etc. Moreover, most of the gene information came from plants such as grape, alfalfa, olive, rape, sorghum, cotton, garlic, arabidopsis etc. Gene ontology (GO) analysis revealed that these ESTs were involved in the regulation of flower development, response to environmental stress, cell metabolism, intracellular material transport, and they accounted for 20%, 6%, 20%, and 6% of all the biological processes, respectively. The rest part included DNA transcription, male meiosis, cell death and cell wall loosening etc. As for the molecular function, these genes were found to have cysteine-type endopeptidase activity, methyltransferase activity, protein kinase activity, catalytic activity, structural molecule activity, protein binding, DNA binding, ATP binding, protein dimerization activity and pyruvate aminotransferase activity, etc. Real-time PCR detection showed that the expression of candidate genes varied greatly with the bud break of prompt buds and showed a big difference between these two kinds of buds even in the same node. 【Conclusion】 Some genes which had high expression in latent buds were indentified such as MADS flowering FLC-like protein, calcium-dependent protein kinase, NADP-dependent malic enzyme, cell wall-associated hydrolase, cell wall loosening protein, subunit beta of coatomer protein complex, heat shock protein, senescence-associated protein, cytochrome P450, cell wall-associated protein etc, and it showed that mitochondrial protein gene, unnamed protein gene, flower bloom associated gene, ATP synthase subunit beta gene, and MADs FLC-like protein gene played important roles in bud endodormancy of grapevine.

Key words: grapevine , SSH , bud endodormancy , latent buds , prompt buds

闵卓1, 耿万刚1, 房玉林1, 2, 张振文1, 2, 李华1, 2. 应用抑制消减杂交技术筛选葡萄冬芽和夏芽生理休眠相关基因[J]. 中国农业科学, 2014, 47(5): 1029-1040.

MIN Zhuo-1, GENG Wan-Gang-1, FANG Yu-Lin-1, 2 , ZHANG Zhen-Wen-1, 2 , LI Hua-1, 2 . Screening and Identification of Differentially Expressed Endodormant Genes from Latent Buds and Prompt Buds by Suppression Subtractive Hybridization[J]. Scientia Agricultura Sinica, 2014, 47(5): 1029-1040.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.05.019

https://www.chinaagrisci.com/CN/Y2014/V47/I5/1029

参考文献

[1]Gilreath P R, Buchanan D W. Rest prediction [mathematical] model for low-chilling ‘Sungold’ nectarine. Journal American Society for Horticultural Science, 1981, 106: 426-429.

[2]Mathur D D, Couvillon G A, Vines H M. Stratification effects on endogenous gibberellic acid (GA) in peach seeds. Hortscience, 1971, 6: 538-539.

[3]Dun E A, Ferguson B J, Beveridge C A. Apical dominance and shoot branching. Divergent opinions or divergent mechanisms? Plant Physiology, 2006, 142(3): 812-819.

[4]Frewen B E, Chen T H, Howe G T, Davis J, Rohde A, Boerjan W, Bradshaw H D J. Quantitative trait loci and candidate gene mapping of bud set and bud flush in Populus. Genetics, 2000, 154(2): 837-845.

[5]Stupnikova I V, Borovskii G B, Antipina A I, Voinikov V K. Polymorphism of thermo stable proteins in soft wheat seedlings during low-temperature acclimation. Russian Journal of Plant Physiology, 2001, 48(6): 804-810.

[6]Kozarewa I, Ibáñez C, Johansson M, Ögren E, Mozley D, Nylander E, Chono M, Moritz T, Eriksson M E. Alteration of PHYA expression change circadian rhythms and timing of bud set in Populus. Plant Molecular Biology, 2010, 73(1/2): 143-156.

[7]姚骅珊, 张博, 诸葛强. 多年生木本植物季节性休眠的分子机制. 分子植物育种, 2009, 7(5): 985-989.

Yao H S, Zhang B, Zhu G Q. Molecular mechanism of seasonal dormancy in perennial woody plant. Molecular Plant Breeding, 2009, 7(5): 985-989. (in Chinese)

[8]Böhlenius H, Huang T, Charbonnel-Campaa L, Brunner A M, Jansson S, Strauss S H, Nilsson O. CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science, 2006, 312(5776): 1040-1043.

[9]Ruonala R, Rinne P L H, Kangasjärvi J, van der Schoot C. CENL1 expression in the rib meristem affects stem elongation and the transition to dormancy in Populus. The Plant Cell Online, 2008, 20(1): 59-74.

[10]Bielenberg D G, Wang Y, Li Z G, Zhebentyayeva T, Fan S H, Reighard G L, Scorza R, Abbott A G. Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation. Tree Genetics and Genomes, 2008, 4(3): 495-507.

[11]Bielenberg D G, Wang Y, Fan S, Reighard G L, Scorza R, Abbott A G. A deletion affecting several gene candidates is present in the evergrowing peach mutant. Journal of Heredity, 2004, 95(5): 436-444.

[12]Rohde A, Ruttink T, Hostyn V, Sterck L, van Driessche K, Boerjan W. Gene expression during the induction, maintenance, and release of dormancy in apical buds of poplar. Journal of Experimental Botany, 2007, 58(15/16): 4047-4060.

[13]Yamane H, Kashiwa Y, Ooka T, Tao R, Yonemori K. Suppression subtractive hybridization and differential screening reveals endodormancy-associated expression of an SVP/AGL24-type MADS- box gene in lateral vegetative buds of japanese apricot. Journal of the American Society of Horticultural Science, 2008, 133(5): 708-716.

[14]Mazzitelli L, Hancock R D, Haupt S, Walker P G, Pont S D A, Mcnicol J, Cardle L, Morris J, Viola R, Brennan R, Hedley P E, Taylor M A. Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.) buds. Journal of Experimental Botany, 2007, 58(5): 1035-1045.

[15]Horvath D P, Chao W S, Suttle J C, Thimmapuram J, Anderson J V. Transcriptome analysis identifies novel responses and potential regulatory genes involved in seasonal dormancy transitions of leafy spurge (Euphorbia esula L.). BMC Genomics, 2008, 9(1): 536-542.

[16]王倩. 中国野生毛葡萄抗黑痘病抑制消减杂交文库构建及表达序列标签（ESTs）分析[D]. 杨陵: 西北农林科技大学, 2011.

Wang Q. Construction and EST analysis of a suppressive subtraction cDNA library of chinese wild Vitis quinquangularis inoculated with Elsinoe ampeina [D]. Yangling: Northwest A&F University, 2011. (in Chinese)

[17]孙洪波, 王国英, 孙振元, 古润泽, 赵梁军. 应用抑制差减杂交法分离粗枝大叶黄杨幼苗的冷诱导表达基因. 中国农业科学, 2005, 38(1): 135-139.

Sun H B, Wang G Y, Sun Z Y, Gu R Z, Zhao L J. Isolating cold-regulated genes from Euonymus japonicus ‘Zhuzi’ seedlings through suppression subtractive hybridization. Scientia Agricultura Sinica, 2005, 38(1): 135-139. (in Chinese)

[18]周志钦. 马铃薯从休眠到发芽过程差异表达基因的分析. 西南农业大学学报, 2001, 23(3): 213-215.

Zhou Z Q. Analysis of differentially expressed genes from dormancy to sprouting of potato tubers. Journal of Southwest Agricultural University, 2001, 23(3): 213-215. (in Chinese)

[19]王新超, 杨亚军, 陈亮, 马春雷, 姚明哲. 茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析. 茶叶科学, 2010, 30(2): 129-135.

Wang X C, Yang Y J, Chen L, Ma C L, Yao M Z. Construction and preliminary analysis of the suppression subtractive hybridization cDNA libraries between dormant and sprouting buds of tea plant (Camellia sinensis). Journal of Tea Science, 2010, 30(2): 129-135. (in Chinese)

[20]李婉莎, 刘德团, 杨永平, 胡向阳. 番茄茎腺毛差异表达序列分离与分析. 植物分类与资源学报, 2011, 33(6): 660-666.

Li W S, Liu D T, Yang Y P, Hu X Y. Isolation and analysis of differential expressed ESTs from stem trichomes of Lycopersicon esculentum (Solanaceae). Plant Diversity and Resources, 2011, 33(6): 660-666. (in Chinese)

[21]丁勇, 常玮, 张石宝, 胡虹. 热胁迫下中甸角蒿叶片SSH文库的构建及初步分析. 植物分类与资源学报, 2012, 34(1): 47-55.

Ding Y, Chang W, Zhang S B, Hu H. Construction of leave library by SSH and preliminary analysis of genes responsible for heat stress in Incarvillea zhongdiannensis. Plant Diversity and Resources, 2012, 34(1): 47-55. (in Chinese)

[22]李娟娟, 陈福龙, 李鑫, 王蕾, 贾俊忠, 陈远良, 陈芳, 高剑峰. 黄瓜芽黄突变体抑制消减杂交文库的构建及初步分析. 西北植物学报, 2010, 30(5) : 0905-0910.

Li J J, Chen F L, Li X, Wang L, Jia J Z Chen Y L, Chen F, Gao J F. Construction and analyses of SSH cDNA libraries of a cucumber mutant of yellow virescent. Acta Botanica Boreali-Occidentalia Sinica, 2010, 30(5): 0905-0910. (in Chinese)

[23]樊奇, 王凭青, 杨青川, 宋文静, 邓腊梅, 张宝云. 抑制消减杂交法分离紫花苜蓿幼苗铝胁迫诱导表达的cDNA. 生物技术通报, 2011, 1: 95-98.

Fan Q, Wang P Q, Yang Q C, Song W J, Deng L M, Zhang B Y. Isolation and cloning of cDNA induced by aluminum stress in alfalfa seedling using suppression subtractive hybridization. Biotechnology Bulletin, 2011, 1: 95-98. (in Chinese)

[24]颜克亮, 陈波, 姚家玲, 周新安, 张会芳, 魏文辉. 大豆种子形态建成期与成熟期正反抑制消减文库构建及差异表达基因分析. 武汉大学学报: 理学版, 2008, 54(2): 202-208.

Yan K L, Chen B, Yao J L, Zhou X A, Zhang H F, Wei W H. Analysis of differentially expressed genes at pattern formation and maturation stages of soybean seed. Journal of Wuhan University: Natural Science Edition, 2008, 54(2): 202-208. (in Chinese)

[25]Yooyongwech S, Horigane A K, Yoshida M, Yamaguchi M, Sekozawa Y, Sugaya S, Gemma H. Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy. Physiologia Plantarum, 2008, 134(3): 522-533.

[26]郭新红, 姜孝成, 潘晓玲, 戴玉池, 姜维明, 陈良碧. 用抑制消减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段. 植物生理学报, 2001, 27(5) : 401-406.

Guo X H, Jiang X C, Pan X L, Dai Y C, Jiang W M, Chen L B. Isolation and cloning of cDNA related with the genes of halonxylon ammodendron (Mey.) Bge. Seedlings induced by osmotic stress by suppression subtractive hybridization. Plant Physiology Journal, 2001, 27(5): 401-406. (in Chinese)

[27]唐永洽, 于拴仓, 朱月林, 张凤兰, 余阳俊, 赵岫云, 张德双. 大白菜霜霉菌诱导抑制性消减杂交cDNA文库的构建和分析. 植物生理学通讯, 2010, 46(5): 453-458.

Tang Y Q, Yu S C, Zhu Y L, Zhang F L, Yu Y J, Zhao Y Y, Zhang D S. Construction and analysis of suppression subtractive hybridization cDNA library in Chinese cabbage (Brassica rapa ssp. pekinensis) leaves induced by Peronospora parasitica. Plant Physiology Communications, 2010, 46(5): 453-458. (in Chinese)

[28]院海英, 徐芳, 吕新华, 崔百明, 黄先忠. 小拟南芥高盐胁迫诱导cDNA文库的构建与测序分析. 石河子大学学报: 自然科学版, 2012, 30(1): 1-4.

Yuan H Y, Xu F, Lü X H, Cui B M, Huang X Z. Construction and sequencing analysis of cDNA library induced by high salt stress in Olimarabidopsis pumila. Journal of Shihezi University: Natural Science Edition, 2012, 30(1): 1-4. (in Chinese)

[29]Pacey-Miller T, Scott K, Ablett E, Tingey S, Ching A, Henry R. Genes associated with the end of dormancy in grapes. Functional & Integrative Genomics, 2003, 3(4): 144-152.

[30]马海燕. 葡萄生长过程中内源激素含量变化的研究[D]. 杨陵: 西北农林科技大学, 2007.

Ma H Y. Study on changes of endogenous hormones in the process of grape growing [D]. Yangling: Northwest A&F University, 2007. (in Chinese)

[31]张今今, 王跃进, 王西平, 杨克强, 杨进孝. 葡萄总RNA提取方法的研究. 果树学报, 2003, 20(3): 178-181.

Zhang J J, Wang Y J, Wang X P, Yang K Q, Yang J X. An improved method for rapidly extracting total RNA from Vitis. Journal of Fruit Science, 2003, 20(3): 178-181. (in Chinese)

[32]张今今. 中国葡萄属野生种抗白粉病基因cDNA克隆及序列分析[D]. 杨陵: 西北农林科技大学. 2003.

Zhang J J. cDNA cloning and sequence analysis of powdery mildew resistance gene in chinese wild Vitis species[D]. Yangling: Northwest A&F University, 2003. (in Chinese)

[33]王新超, 马春雷, 杨亚军. 多年生植物的芽休眠及调控机理研究进展. 应用与环境生物学报, 2011, 17(4): 589-595.

Wang X C, Ma C L, Yang Y J. Progress in research on bud dormancy and its regulation mechanisms in perennial plants. Chinese Journal of Applied and Environmental Biology, 2011, 17(4): 589-595. (in Chinese)

[34]Horvath D. Common mechanisms regulate flowering and dormancy. Plant Science, 2009, 177(6): 523-531.

[35]Hemming M N, Trevaskis B. Make hay when the sun shines: The role of MADS-box genes in temperature-dependant seasonal flowering responses. Plant Science, 2011, 180(3): 447-453.

[36]Lee J H, Yoo S J, Park S H, Hwang I, Lee J S, Ahn J H. Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. Genes & Development, 2007, 21(4): 397-402.

[37]Jiménez S, Li Z, Reighard G L, Bielenberg D G, Judd M J, Meyer D H, Meekings J S, Richardson A C, Walton E F. An FTIR study of the induction and release of kiwifruit buds from dormancy. Journal of the Science of Food and Agriculture, 2010, 90(6): 1071-1080.

[38]Mazzitelli L, Hancock R D, Haupt S, Walker P G, Pont S D A, Mcnicol J, Cardle L, Morris J, Viola R, Brennan R, Hedley P E, Taylor M A. Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.) buds. Journal of Experimental Botany, 2007, 58(5): 1035-1045.

[39]Zhang J Z, Li Z M, Mei L, Yao J L, Hu C G. PtFLC homolog from trifoliate orange (Poncirus trifoliata) is regulated by alternative splicing and experiences seasonal fluctuation in expression level. Planta, 2009, 229(4): 847-859.

[40]Do?ramaci M, Horvath D P, Chao W S, Foley M E, Christoffers M J, Anderson J V. Low temperatures impact dormancy status, flowering competence, and transcript profiles in crown buds of leafy spurge. Plant Molecular Biology, 2010, 73 (1/2): 207-226.

[41]Screekantan L, Mathiason K, Grimplet J, Schlauch K, Dickerson J A, Fennell A Y. Differential floral development and gene expression in grapevines during long and short photoperiods suggests a role for floral genes in dormancy transitioning. Plant Molecular Biology, 2010, 73 (1/2): 191-205.