利用RNA干涉技术及微束激光转化法培育抗病毒马铃薯  

The Development of Virus Resistance Transgenic Potato Plants Using RNAi Technology and Laser Microbeam Puncture Technique

作　　者：郭志鸿[1] 张金文[1] 陈正华 王兰岚 

机构地区：[1]甘肃农业大学农学院,甘肃兰州730070 [2]亚盛集团博士后科研工作站北京分站,北京100101

出　　处：《激光生物学报》2006年第5期525-531,共7页Acta Laser Biology Sinica

基　　金：国家自然科学基金(30270843)

摘　　要：针对马铃薯生产中危害严重、分布广泛并且具有强烈协生作用的马铃薯X病毒和马铃薯Y病毒,开展了抗病毒病的育种研究.采用RT-PCR技术分别克隆了267 bp的编码马铃薯X病毒外壳蛋白（PVX-cp）基因的相对保守区段X和294 bp的编码马铃薯Y病毒外壳蛋白（PVY-cp）基因的相对保守区段Y两个片段,将其按顺序连接,形成融合基因XY,然后分别将融合基因XY正向和反向插入到载体pHANNIBAL中,得到了载体pHIV,再用NotI对pHIV进行酶切,将产生的大片段插入到载体pART27中,得到同时以PVX-cp基因和PVY-cp基因为靶标的XY的RNAi载体pARIV.采用微束激光穿刺转化技术转化马铃薯品种Favorita的愈伤组织,得到了14株表型正常的转基因植株,转基因植株的southern blot分析表明,导入的外源融合基因拷贝数介于2～4之间.攻毒实验表明,其中11株转基因植株对PVX、PVY°以及PVX和PVY°混合侵染均表现免疫,而其它3株的病毒浓度也低于对照.这一结果将为利用RNAi干涉技术开展植物抗多种病毒病的育种提供重要依据,验证了所构建的RNA干涉（RNA interference RNAi）载体具有良好的功能,同时又一次证明微束激光穿刺法是一种有效的遗传转化手段.The present research aims to develop transgenic potato plants resistant to PVX and PVY. RT-PCR was employed to clone a 267 bp fragment X from PVX-cp gene and a 294 bp fragment Y from PVY-cp gene. The two cloned fragments were ligated to form a fused gene XY. Then the fused gene XY was inserted in both sense and antisense orientation into vector pHANNIBAL to get a vector named pHIV. A NotⅠ fragment from pHIV containing a complete reading framework of an inverted repeat of fused gene XY was inserted to pART27 to get a vector pARIV which aimed to silence PVX-cp gene and PVY-cp gene simultaneously. Potato callus from elite potato cuhivar favorita was transformed via laser microbeam puncture technique and 14 transgenic plants were regenerated. Southern blot analysis shows that there are 2 - 4 copies of fused XY gene in the transgenic plants. Virus infection indicates that 11 of the transgenic plants are immune to the infection of PVX、PVY° or PVX and PVY°, while the other three can be infected but virus concentration is much lower than that of untransformed plants infected by the same virus or viruses. This research would be helpful to develop transgenic plants resistant to multiple viruses using RNAi technology. It also suggests that laser microbeam puncture technique is an efficient method to deliver RNAi vector into plants.

关 键 词：RNA干涉 马铃薯X病毒 马铃薯Y病毒 融合基因 微束激光穿刺技术 

分 类 号：Q945.4[生物学—植物学] Q6-33