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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (9): 48-54.

所属专题： 生物技术

• 农学 农业基础科学 • 上一篇    下一篇

高粱广谱抗蚜基因SSH文库的构建及其序列分析

齐金凤   

  • 收稿日期:2011-08-01 修回日期:2012-01-14 出版日期:2012-03-25 发布日期:2012-03-25
  • 基金资助:

    沈阳农业大学校青年基金

Construction and Sequence Analysis of SSH cDNA Library form Aphid-resistant Sorghum

  • Received:2011-08-01 Revised:2012-01-14 Online:2012-03-25 Published:2012-03-25

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摘要：

为了从分子水平上解析高粱广谱抗蚜基因在高粱与蚜虫互作中的分子遗传基础和作用机理，为发掘并利用抗蚜基因提供可靠的信息支持。以高粱抗蚜品种‘河农16’与感蚜品种‘千三’为实验材料，构建抑制性消减杂交(SSH)文库，使差异抗蚜基因得到富集，对有效差异基因序列进行序列分析。随机挑选SSH文库中的200个阳性克隆进行测序和分析，得到18条ORF序列，同NCBI数据库进行Blastx在线比对和相似性分析，发现这些序列与叶绿体放氧增强相关蛋白、腺苷酸环化相关蛋白、核糖体蛋白、衰老联合蛋白高度相关。不具有ORF的序列，通过NCBI利用Blastn和Blastx进行序列同源性比对和相似性分析，发现66条与已知多种植物抗病序列有较高的同源性，如核糖体蛋白、转运膜蛋白、NBS-LRR抗病蛋白家族-1、锌指蛋白、ATP合酶、NADH脱氢酶、反转录转座子、腺苷酸环化相关蛋白、bZIP转录因子等，还有部分序列与高粱抗条锈病基因高度相似。这些同源基因涉及次生代谢、能量代谢、膜运输、细胞信号传递和转录调控等多种过程。成功构建高粱抗蚜基因SSH文库，表明SSH技术在高粱抗蚜机制研究中具有可行性，研究结果为进一步高粱抗蚜基因功能研究奠定基础。

关键词: 转基因植物, 转基因植物, 发展前景, 应用

Abstract:

The aim was to study broad-spectrum anti-aphid gene in sorghum and sorghum aphid interactions in the molecular genetic basis and mechanism, and to explore and use the information of aphid resistance genes. In this study, ‘Henong16’ with aphid-resistance genes and ‘Qian3’ susceptible to sorghum aphid were used as the tester and the driver and vice versa to construct SSH library, so that the aphid resistance genes were enriched effectively, then the differential gene sequence was analyzed. 200 positive clones were randomly selected and sequenced. 18 open reading frame (ORF) sequences were found through the NCBI ORF Finder online prediction. Then the 18 ORF sequences were blasted and analyzed using online Blastx. The results showed that the 18 ORF sequences were highly related with putative oxygen-evolving enhancer protein in chloroplast, adenylate cyclase-associated protein, ribosomal protein, putative senescence-associated protein. The contigs without ORF were blasted and analyzed using online Blastx and Blastn, and 66 ESTs had highly homologous to genes sequences with known disease resistance in many plants, such as ribosomal protein, transport membrane protein, NBS-LRR disease resistance protein family-1, zinc finger protein, ATP synthase, NADH dehydrogenase, retrotransposon, Adenylyl cyclase-associated protein, and bZIP transcription factor etc.. These homologous genes involved in secondary metabolism, energy metabolism, membrane transport, signal transduction and transcription regulation and so on. The SSH library of sorghum aphid resistance gene was constructed successfully, which indicated that the SSH technique in the mechanism of aphid resistance of sorghum was a feasibility study. The results laid the foundation for further studying on sorghum aphid-resistance gene function.