中国野生山葡萄抗寒基因文库构建及序列分析

中国野生山葡萄抗寒基因cDNA文库构建及EST序列分析

CONSTRUCTION OF CDNA LIBRARY OF CHINESE WILD VITIS

AMURENSIS WITH RESISTANCE TO COLD-STRESS AND

ANALYSIS OF EXPRESSED SEQUENCE TAGS

作者：刘　楠　　导师：张剑侠

西北农林科技大学　　园艺植物种质资源学2011届硕士

摘　要

温度是影响植物生长发育的一个重要的非生物因素，寒害常对植物造成巨大伤害。由于生产上广泛栽培的欧洲葡萄（Vitis vinifera）抗寒性差，因此研究葡萄的抗寒性和克隆抗寒基因，对于葡萄抗寒育种研究具有重要的意义。中国野生葡萄资源丰富，原产中国的山葡萄（V. amurensis）为葡萄属植物中最抗寒的种，其中株系黑龙江实生表现出较强抗寒性。本研究以中国野生山葡萄黑龙江实生为试材，通过SMART^TM技术构建中国野生山葡萄抗寒基因cDNA文库，并应用生物信息学对所获得的EST序列进行了分析，这将为进一步克隆抗寒性相关基因全长及进行功能表达分析奠定基础。具体研究结果如下：

1.　采集4℃冷处理0、2、6、12、24、48小时的山葡萄黑龙江实生叶片，采用改进SDS/酚法分别提取总RNA，等量混合，通过SMART^TM技术构建山葡萄抗寒基因cDNA文库。经检测，原始文库的滴度为2.67×10⁶pfu/mL，重组率为98.5%。扩增文库的滴度为1.53×10⁹pfu/mL，重组率为98.2%。

2.　经随机挑取单克隆测序，获得227条质量好的EST序列，已登录GenBank数据库（登录号为JG294234-JG294243，JG392010-JG392092，JK006548- JK006681）。利用NCBI的BlastX程序分析，有46条序列与未知蛋白同源性高，占20.3%，181条序列与12类已知功能的基因同源性较高，占总EST的79.7%。其中有38条EST序列参与抗逆反应，包括防御相关蛋白，如几丁质酶，病程相关蛋白PR10，亲免素，脱落胁迫成熟蛋白，耐盐蛋白，金属硫蛋白；与信号传导相关，如乙烯反应因子5，钙调素，G蛋白；逆境胁迫诱导蛋白，如脱水诱导早期应答基因，热稳定蛋白，聚泛素，小泛素相关蛋白，F-box家族，DnaJ蛋白，过敏诱导反应蛋白，臭氧反应相关蛋白；调控基因表达的转录因子，如MYB49转录因子，AP2转录因子，BZIP转录因子，RD22蛋白等。这些物质可能在葡萄抗寒过程中起一定作用。

关键词　山葡萄　冷胁迫　cDNA文库　表达序列标签

Abstract

Temperature is an important non-biological factor of plant growth and development. Plants are often damaged by chilling. Vitisvinifera,the main varieties currently cultivated in grape production are weak in cold-resistance,thus the study of cold hardiness of grape and cloning cold tolerance genes is good for cold for the new grape hardy varieties cultivation. In China,the wild grape resources are rich,and according to previous researches,Chinese wild Vitisamurensis is the highest resistant to cold stress in Vitis L. The test materials used in this study is Heilongjiang Seedling,which is a clone of Chinese wild Vitisamurensis,and performs highly resistant to cold weather. Construct the cDNA library through utilizing SMART^TM cDNA Library Construction Kit. Expression and characteristics analysis of EST sequence based on Bioinformatics method provide theory basis for molecular mechanism studies at the cloning of cold tolerance genes and functional expression analysis. The results are as follows:

Total RNA was extracted from the leaves of Heilongjiang Seedling at 0h,2h,6h ,12h,24h,48h at 4℃respectively with SDS/phenol method modified partially,and pooled equal amounts of RNA. The titer of primary library was 2.67×10⁶ pfu/mL,and the recombination rate was 98.5% . The titer of amplified library was 1.53×10⁹ pfu/mL,and the recombination rate was 98.2% .

Randomly choosing clones,227 sequences with high quality were obtained,and　all of them have submitted to GenBank database,The GenBank accession number are as follows,JG294234-JG294243，JG392010-JG392092，JK006548-JK006681. Using Blastx program of NCBI for homologue search,there are 46 EST sequences　matched to the genes whose protein function were unclassified,20.3%;181 EST sequences showed high homologue to 12 clusters of gene with known function,79.7%. 38 EST sequences were related to stress resistance. These genes included that defense related-protein,such as endochitinase,pathogenesis-related protein 10,immunophilin,abscisic stress ripening protein,salt-tolerance protein,metallothionein-like protein;relating to Signal transduction,such as ethylene response factor 5,calmodulin,GTP binding protein;Stress induced protein,such as early responsive to dehydration,heat stable protein,polyubiquitin,SUMO protein,F-box family protein,DnaJ protein,hypersensitive-induced reaction protein,ozone-responsive stress-related protein-like;Transcriptional regulators,such as MYB49,AP2 domain class transcription factor,BZIP domain class transcription factor,RD22-like protein. These substances　may play a role in cold-resistance response in grapes.

Key words　Vitis amurensis　Cold-stress　cDNA library　Expressed sequence