胞囊线虫侵染后小粒黑豆抑制消减杂交cDNA文库的构建及EST分析

doi:10.3864/j.issn.0578-1752.2012.06.008

摘要/Abstract

摘要： 【目的】研究胞囊线虫侵染后大豆根部早期基因表达情况，从分子水平探索大豆的抗病机制。【方法】以抗大豆胞囊线虫（Heterodera glycines）3号生理小种的小粒黑豆（Glycine max）为材料，利用抑制消减杂交（suppression subtractive hybridization，SSH）技术，构建大豆胞囊线虫诱导大豆根部早期表达的SSH-cDNA 文库，并结合反向Northern斑点杂交筛选阳性克隆。【结果】获得280个表达序列标签（EST），经 CAP3 软件聚类拼接，得到166个unique EST，在GenBank进行Blastn与Blastx同源比对，有119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83%，已知基因功能的EST涉及信号识别和传导、能量与物质新陈代谢、膜及转运、木质素和类黄酮的生物合成、抗病防御、细胞保护和转录调控。【结论】本研究在大豆胞囊线虫侵染早期发现了表达频率较高的基因如过氧化氢酶、泛肽素、几丁质酶、脂肪氧合酶、水通道蛋白、成熟调控蛋白、金属硫蛋白、脂膜嵌入蛋白、细胞色素P450和3-磷酸甘油醛脱氢酶等，并推测这些基因在大豆与胞囊线虫的非亲和互作过程中起重要作用。

关键词: 大豆胞囊线虫, 大豆, 抑制消减杂交, 表达序列标签

Abstract: 【Objective】The objective of this study is to research into the early stage root gene expression profile of G. max induced by H. glycines and to explore the resistant mechanism to the pathogen at molecular level. 【Method】SSH-cDNA libraries enriched with differentially expressed ESTs were constructed from SCN-challenged root tissues at pre-penetration and early infection stages from Xiaoli black bean (G. max) by suppression subtractive hybridization and reverse Northern blot. 【Result】 One hundred and sixty-six unique ESTs were identified and analyzed with Blastx and Blastn by comparing sequences in the GenBank. One hundred and ninteen unigenes accounted for 83% of all the unigenes showed high homology with the function known genes or proteins. The function-known ESTs were annotated into functional categories including signal recognition and transduction, energy and material metabolism, stress responses, transcriptional regulation, protein synthesis and/or modification, transport functions, cellular architecture.【Conclusion】Discreet gene tag clusters primarily including catalase, ubiquitin, chitinase, lipoxygenase, aquaporin, ripening related protein, metallothionein, plasma membrane intrinsic protein, cytochrome P450, glyceraldehyde-3-phosphate dehydrogenase were abundant in the SCN-infected roots. It is speculated that those genes played an important function in the incompatible interaction between Xiaoli black bean and H. glycines.

Key words: Heterodera glycines, Glycine max, suppression subtractive hybridization, EST

王芳, 段玉玺, 陈立杰, 王媛媛, 朱晓峰, 王东. 胞囊线虫侵染后小粒黑豆抑制消减杂交cDNA文库的构建及EST分析[J]. 中国农业科学, 2012, 45(6): 1106-1115.

WANG Fang, DUAN Yu-Xi, CHEN Li-Jie, WANG Yuan-Yuan, ZHU Xiao-Feng, WANG Dong. Construction of SSH-cDNA Libraries and EST Analysis in Roots of Xiaoli Black Bean in Response to Heterodera glycines Parasitization[J]. Scientia Agricultura Sinica, 2012, 45(6): 1106-1115.

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