罗伦隐球酵母诱导樱桃番茄果实抑制差减杂交文库构建及分析

姜峰; 陈集双; 郑晓冬

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罗伦隐球酵母诱导樱桃番茄果实抑制差减杂交文库构建及分析

首发时间：2009-03-16

姜峰 ¹ 陈集双 ² 郑晓冬 ³

1、浙江大学生物系统工程与食品科学学院
2、浙江理工大学生物工程研究所
3、浙江大学

摘要：【目的】研究番茄果实在生防菌罗伦隐球酵母Cryptococcus laurentii处理后差异表达的基因，以其了解生防酵母诱导番茄果实抗性的分子机理。【方法】以樱桃番茄果实为材料，应用抑制差减杂交技术，构建生防酵母罗伦隐球酵母处理后的番茄果实cDNA文库。经筛选得到阳性克隆并测序，对测序结果进行功能注释和分类。【结果】成功构建了罗伦隐球酵母诱导的番茄果实抑制差减杂交文库。通过筛选，共获得约180个阳性克隆。经测序，获得162条高质量EST。对EST序列进行聚类后，共获得50条非重复序列，与GenBank进行BLASTx和BLASTn同源对比，其中35条非重复序列与已知基因同源性很高，占全部非重复序列的70%。已知功能的EST涉及植物的基础代谢、信号识别与转导和抗性相关等功能。随机选出4个差异表达的基因对其表达的特性用RT-PCR进行分析，结果进一步证明了SSH的可靠性。【结论】通过对功能已知的基因分析，推测促分裂原活化蛋白激酶，丝氨酸蛋白酶、GTP结合蛋白、甘露醇脱氢酶和几种病程相关蛋白等可能参与了生防菌与番茄果实之间的互作过程。同时在生防菌处理番茄果实后差异表达的部分基因，在其他生物和非生物胁迫下也有表达，可以认为正是生防菌诱导了这些基因的上调表达，才增强了处理后番茄果实对采后病害的抗性。

关键词：樱桃番茄果实罗伦隐球酵母采后生物防治抑制差减杂交

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Construction and Analysis of a Tomato Fruit Suppression Subtractive Hybridization Library Induced by Biological Control Yeast Cryptococcus laurentii

Jiang Feng ¹ Chen Ji-shuang ² Zheng Xiaodong ¹

1、Zhejiang University
2、Institute of Biochemical,Zhejiang Sci-Tech University, Hangzhou

Abstract：【Objective】The objective of this paper is to study the gene expression profile of tomato fruit induced by Cryptococcus laurentii and the induced resistant mechanism to the biocontrol yeast at the molecular level. 【Method】Subtractive suppression hybridization (SSH) was used to construct a cDNA library enriched in cherry tomato after treated by Cryptococcus laurentii. After sequencing the positive clones picked up randomly, the qualified expressed sequence tags (ESTs) were annotated and classified. 【Result】A high quality SSH cDNA library was constructed successfully and 180 positive clones were obtained in the SSH library. All the positive clones One were sequenced, and then 162 qualified ESTs were obtained. The ESTs clustering was analyzed and 50 unigenes were acquired. ESTs similarity analysis of these unigenes was performed with the Blastx and Blastn by comparing sequences in non-redundance database from GenBank. As a result, 35 unigenes accounted for 70% of all the unigenes showed high homology with the function known genes or ESTs. The function-known ESTs related to energy metabolism, signal transduction, transcription regulation and defense response. Four cDNA clones were selected for temporal expression analysis using RT-PCR.【Conclusion】Through analysing the genes with known function, mitogen-activated protein kinase, serine/threonine protein kinase, GTP-binding protein, glutathione-S-transferase, mannitol dehydrogenas and PRs, etc., were supposed to involve in the process of the interaction between tomato fruit and Cryptococcus laurentii. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated under some biotic and abiotic stress are also up-regulated after the application of biological control yeast to cherry tomato fruit. The expression of these proteins might increase the fruit resistance towards postharvest pathogen infection and damage.

Keywords： Cherry tomato fruit Postharvest biocontrol Cryptococcus laurentii Subtractive suppression hybridization (SSH)

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姜峰，陈集双，郑晓冬. 罗伦隐球酵母诱导樱桃番茄果实抑制差减杂交文库构建及分析[EB/OL]. 北京：中国科技论文在线 [2009-03-16]. https://www.paper.edu.cn/releasepaper/content/200903-563.

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论文编号	200903-563
论文题目	罗伦隐球酵母诱导樱桃番茄果实抑制差减杂交文库构建及分析
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