马铃薯病毒诱导应答基因抑制消减杂交文库构建及分析

doi:10.11686/cyxb2017063

草业学报 ›› 2017, Vol. 26 ›› Issue (12): 152-159.DOI: 10.11686/cyxb2017063

马铃薯病毒诱导应答基因抑制消减杂交文库构建及分析

李忠旺, 陈玉梁, 欧巧明, 叶春雷, 裴怀弟, 刘新星, 王红梅, 罗俊杰^*

甘肃省农业科学院生物技术研究所,甘肃兰州 730070

收稿日期:2017-02-20 修回日期:2017-04-19 出版日期:2017-12-20 发布日期:2017-12-20
通讯作者: E-mail:hnsljjie@163.com
作者简介:李忠旺(1980-),男,甘肃山丹人,助理研究员。E-mail:lizhongwang33@163.com
基金资助:
甘肃省农科院青年创新基金(2013GAAS29),甘肃省农业科学院创新团队(2015GAAS02),甘肃省农业科学院农业科技创新重大项目(2016GAAS59-02)和国家自然科学基金项目(31460388)资助

Construction and analysis of a suppression subtractive hybridization library for the potato virus induced response gene

LI Zhong-Wang, CHEN Yu-Liang, OU Qiao-Ming, YE Chun-Lei, PEI Huai-Di, LIU Xin-Xing, WANG Hong-Mei, LUO Jun-Jie^*

Biotechnology Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, China

Received:2017-02-20 Revised:2017-04-19 Online:2017-12-20 Published:2017-12-20
Contact: E-mail:hnsljjie@163.com

摘要/Abstract

摘要： 马铃薯病毒积累引起的种薯退化是马铃薯生产中造成产量和品质下降的重要原因之一。本研究以马铃薯病毒病携带植株叶片cDNA为试验组(Tester)、脱毒种苗叶片cDNA为驱动组(Driver),采用抑制消减杂交(suppression subtractive hybridization, SSH)技术构建了马铃薯病毒诱导应答基因的cDNA文库;为验证文库构建效果,从文库中随机挑取了98个阳性克隆经PCR验证后测序,获得了45条高质量的有效非重复序列;经与GenBank进行同源比对后发现,其中14条非重复序列属于马铃薯病毒基因序列,22条与已知基因序列同源性较高,9条无同源参考基因;选取文库中出现频率较高的2个ESTs(expressed sequence tag,表达序列标签)用qRT-PCR技术分析发现,其表达量受马铃薯病毒侵染的诱导。结果表明,该SSH文库构建较为成功,为进一步筛选与马铃薯病毒致病、防御相关的应答基因,解析马铃薯与病毒互作的分子机理,利用生物技术手段培育抗病毒马铃薯奠定了基础。

Abstract: Potato virus accumulation is one of the important reasons for the decline of yield and quality in potato production. In order to screen and clone the potato virus induced response gene, we constructed a suppression subtractive hybridization (SSH) library using cDNAs from potato virus disease carrying plant leaf as the tester, and those cDNAs from a virus-free seedling leaf as the driver. To verify the effect of library, 98 randomly selected positive clones were identified by PCR from the library and sequenced, and 45 high quality non repeat sequences were obtained. By blast analysis in GenBank, we found that 14 of them were sequences belonging to the potato virus gene, 22 of them had high homology with known genes, and 9 of them had no homologous reference gene. Two frequently found ESTs in the library were analyzed using quantitative real time PCR, and their expression was induced by potato virus. The results therefore showed that the SSH library was constructed successfully. This work has thus laid a foundation for further screening for potato virus related pathogenicity, and induction of corresponding defense response genes. This may lead to analysis of the molecular mechanisms of interaction between the potato plant and the virus, and use of biotechnology to cultivate virus resistant potato varieties.

李忠旺, 陈玉梁, 欧巧明, 叶春雷, 裴怀弟, 刘新星, 王红梅, 罗俊杰. 马铃薯病毒诱导应答基因抑制消减杂交文库构建及分析[J]. 草业学报, 2017, 26(12): 152-159.

LI Zhong-Wang, CHEN Yu-Liang, OU Qiao-Ming, YE Chun-Lei, PEI Huai-Di, LIU Xin-Xing, WANG Hong-Mei, LUO Jun-Jie. Construction and analysis of a suppression subtractive hybridization library for the potato virus induced response gene[J]. Acta Prataculturae Sinica, 2017, 26(12): 152-159.

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马铃薯病毒诱导应答基因抑制消减杂交文库构建及分析

Construction and analysis of a suppression subtractive hybridization library for the potato virus induced response gene

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