摘要
人参果是以营养器官繁殖为主的多年生草本植物,连年种植易造成多种病毒病积累。为了寻求一种能同时抵御多种病毒病的方法,本试验以危害人参果的3种主要病毒为研究对象,根据GenBank中烟草花叶病毒(TMV)P126基因、马铃薯M病毒(PVM)CP基因和黄瓜花叶病毒(CMV)2b蛋白基因序列,利用E-RNAi网站提供的分析软件,筛选出含有较多有效小干扰RNA[siRNA,19 nt]的长dsRNA(Long dsRNA)片段作为RNAi的靶序列。用重叠延伸PCR方法将三个靶标片段融合在一起,构建了一种具有内含子(intro)的反向重复结构和嵌合基因的植物RNAi表达载体pCEIHFR。将pCEIHFR通过冻融法导入农杆菌LBA4404并转化人参果,经过草甘膦筛选和PCR检测,确认其中的24株为转基因阳性植株。
Pepino（Solanum muricatum） is perennial herb with vegetative propagation. It is easy to be infected by many viruses due to continuous planting. In order to find a new method for defending viral diseases, the purpose of this experiment was to study three kinds of harmful viruses which were common in pepino. Based on the genes sequence of Tobacco mosaic virus （ TMV ） P126, potato virus M （ PVM ） CP and Cucumber mosaic virus （ CMV ） 2b in GenBank, the RNAi design website E-RNAi was used to search for Long dsRNA fragment that containing the more effective small interfering RNA [ siRNA, long 19nt] fragment as the RNAi target sequences. The splicing-by-overlap extension PCR was used to fuse three target sequences in together, and at the same time to construct a inverted repetitive structure with pdk （pyruvate orthophosphate dikinase） intro of plant RNAi expression vector pCEIHFR which was supposed to form intron splicing hpRNA （ihpRNA） after transcription. The recombinant plant-expression vector pCEIHFR was introduced into Agrobacterium tumefaciens LBA4404 by direct transferring, and then the target cDNA with inverted repeat was introduced into pepino. Twenty four transgenic plants were confirmed by glyphosate resistant selection and PCR testing.
出处
《核农学报》
CAS
CSCD
北大核心
2014年第1期44-51,共8页
Journal of Nuclear Agricultural Sciences
基金
甘肃省科技支撑计划(1011NKCA077)
