马铃薯卷叶病毒侵染激活花色苷生物合成的分子机制

项目名称： 马铃薯卷叶病毒侵染激活花色苷生物合成的分子机制

项目编号： No.30860033

项目类型： 地区科学基金项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 卢其能

作者单位： 宜春学院

项目金额： 26万元

中文摘要： 马铃薯卷叶病毒（PLRV）侵染某些马铃薯品种时会引起顶叶红化现象，这是花色苷非正常合成和积累的结果。利用RT-PCR方法，以保守序列为引物，从有明显PLRV侵染症状的马铃薯中克隆到长度为5882bp的PLRV基因组序列，为了研究基因组的转录活性，构建了3个PLRV基因组表达载体，用农杆菌侵染法进行遗传转化，其中获得的Desiree转化体中有2株叶片发生了明显变化，叶片卷曲，表现出顶叶红化现象，同时检测到与PLRV基因组相融合的GFP在叶片中表达时所产生的绿色荧光。根据PLRV全基因组序列分别设计ORF0、ORF1、ORF2、ORF3、ORF4、ORF5的特异引物，克隆到6个ORF，分别构建表达载体，用农杆菌侵染法筛选到了各个ORF序列的转化体，尽管在转化体的叶片中也检测到了与各个ORF序列相融合的GFP表达时产生的绿色荧光点，但植株和叶片颜色无明显变化，没有表现出顶叶红化现象和明显卷叶现象；这说明单个ORF的表达可能不足以引起马铃薯花色苷的生物合成和积累，因此，PLRV侵染激活花色苷生物合成可能涉及到多个ORF的表达和相互作用。此外，还研究了花色苷合成相关基因的表达及其他相关内容。

中文关键词： 马铃薯卷叶病毒；花色苷；基因克隆；遗传转化；表达分析。

英文摘要： The leaves turn reddish or purple to be infected by PLRV (potato leaf-roll virus) in some varieties of potato, these symptoms are caused by abnormal biosynthesis and accumulation of anthocyanins.The genome sequence of PLRV was isolated from the leaves of potato which showed symptoms of virus infection by RT-PCR with the primers designed in conserved sequence. The length of the genome sequence of PLRV is 5882bp.There were 3 different plant expression vectors constructed to investigate the transcriptional activity of the genome itself of PLRV. Agrobacterium AGL1were transformated by plant expression vectors, and then were introduced into Solanum tuberosum cv. Desiree by Agrobacterium infection. There were 2 transformants selected for their turned red leaves and symptoms of PLRV infection.It was detected which green fluorescence generated in the leaves of transformants , and this showed the together expression of the PLRV genome fusion with GFP gene. The sequences of ORF0,ORF1, ORF2,ORF3,ORF4 and ORF5 were isolated from the genome of PLRV with specific primers by PCR.The plant expression vectors with different ORF sequences were constructed, and then were introduced into Desiree by Agrobacterium LBA4404 infection, respectively. The transformants were selected, there were the dot of green fluorescence generated in the leaves, and no turned red leaves and symptoms of virus infection. These showed that the expression of a single ORF gene may not be sufficient to cause potato anthocyanin biosynthesis and accumulation.It may involve the expression and interaction of multiple ORF gene that anthocynin biosynthesis was activated by PLRV infection.In addition, the correlative genes of anthocynin biosynthesis and other related content were investigated.

英文关键词： potato leaf-roll virus；anthocyanins；gene cloning；genetic transformation； expression analysis.