四价抗马铃薯病毒植物表达载体构建及其对烟草的转化

Construction of potato tetravalent anti virus plant expression vector and its tobacco transformation

关键词:  马铃薯病毒， 融合基因， 载体构建， 遗传转化， 转基因烟草

Key words:  potato virus, fusion gene, vector construction, genetic transformation, transgenic tobacco

摘要:

该研究为了培育兼抗4种病毒的马铃薯品种，采用RT PCR技术对PVX、PVS、PVY和PLRV的外壳蛋白（CP）基因进行克隆与分析，获得了大小分别为670、800、700、600 bp的CP基因序列，将获得的CP基因序列与NCBI中已报道的序列进行比对分析，其同源性都在96%以上。根据所克隆的CP基因对靶标片段进行筛选，获得了大小约300 bp的靶标片段PVX rh、PVS rh、PVY rh和PLRV rh，同时利用 Overlap PCR技术将4种病毒的靶标片段进行拼接，得到了长度约为1 200 bp的融合片段XSYV rh，与预期目标片段XSYV yxz的相似性达100%。利用DNA重组技术将融合片段XSYV rh克隆到pGM T载体上构建成克隆载体pGM T XSYV rh，用SpeⅠ和SacⅠ对克隆载体pGM T XSYV rh和植物表达载体pART27进行同步双酶切，用T4 DNA连接酶将XSYV rh片段连接到载体pART27上，成功构建了同时含4种病毒CP基因片段的植物表达载体pART27 XSYV rh。采用直接转化法将植物表达载体导入根癌农杆菌LBA4404中，并利用农杆菌介导法对烟草品种T12试管苗进行遗传转化，转化后的烟草植株经PCR检测，有40株转化植株可扩增出目的条带，表明XSYV rh融合基因已成功转入烟草基因组中。

Abstract:

In order to simultaneously foster anti four virus potato varieties(PVX, PVS, PVY and PLRV), four different viral coat protein (CP) gene PVX CP (670 bp), PVS CP(800 bp), PVY CP(700 bp) and PLRV CP(600 bp) were obtained via RT PCR and sequenced to confirm respectively. And the comparison of the sequences that we obtained and the already reported sequences from NCBI database showed that all four viral CP genessequences were more than 90% homologous; then around 300 bp size conservative gene fragments PVX rh, PVS rh, PVY rh, PLRV rh were selected from their respective viral CP genesand four specific bands which were consistent with the fragment size were obtained via PCR amplification. The fusion sequence XSYV rh which is around 1 200 bp long was created with PVX rh, PVS rh, PVY rh and PLRV rh gene fragments via Overlap PCR technique. With the help of DNA recombination technique, we integrated XSYV rh sequence into pGM T vector and created cloning vector pGM T XSYV rh. The cloning vector pGM T XSYV rh and the plant expression vector pART27 were treated with incision enzyme Spe Ⅰ and Sac Ⅰ; then under the effect of T4 DNA ligase, XSYV rh sequence was integrated with pART27 expression vector; at last pART27 XSYV rh plant expression vector was successfully constructed. The pART27 XSYV rh plant expression vector was introduced into Agrobacterium tumefaciens LBA4404, and subsequently introduced into tobacco (T12) assisted by the agrobacterium mediated transformation. PCR results showed that there were 40 transgenic plants with targeted gene integrated into their genomes.