油菜BnSGS3基因克隆及转基因拟南芥病毒抗性

Brassica napus BnSGS3 gene sequence analysis,vector construction and resistance identification of transgenic Arabidopsis thaliana to viruses

为阐明甘蓝型油菜BnSGS3抗病毒功能,用5′-RACE和巢氏PCR方法从油菜中克隆了BnSGS3。该基因全长2638bp,包含4个内含子和1个1824bp的完整开放阅读框。序列对比发现,BnSGS3与其他SGS3基因同源性在58.9%～97.5%之间。构建BnSGS3过表达载体（BnSGS3-Ov）和干扰表达载体（BnSGS3-Si）,通过浸花（Floral-dip）法转化拟南芥,经除草剂筛选及PCR检测,获得2种转基因拟南芥各30株。拟南芥接种黄瓜花叶病毒（CMV）的抗性鉴定显示,转BnSGS3-Ov植株发病轻,株型基本正常,后期能正常开花结籽,对CMV表现中抗;而转BnSGS3-Si植株发病重,不能正常开花结籽。接种油菜花叶病毒（YoMV）后,2种转基因和非转基因拟南芥均对YoMV表现感病,说明BnSGS3对CMV有一定抗性,但对YoMV无明显抗性。

To understand the anti- virus function of SGS3 gene in Brassica napus,gene Bn SGS3 was cloned from rapeseed cultivar Zhongshuang 6 by 5＇- RACE and nested PCR. Sequence analysis showed that Bn SGS3 includes 2 638 bp,4 introns and a 1 824 bp complete ORF. DNAMAN comparison result showed that the similarities between Bn SGS3 and other SGS3 genes were 58. 9% to 97. 5%. Over- expressing vector Bn SGS3- Ov and interfering vector Bn SGS3- Si were constructed and transformed into Arabidopsis thaliana Columbia ecotype by floral-dip method. 30 transgenic plants of each vector were obtained verified by herbicide spray and PCR identification.After cucumber mosaic virus（ CMV） inoculation,Bn SGS3- Ov transgenic Arabidopsis plants showed moderate resistance to CMV with slighter symptoms than those from Bn SGS3- Si transgenic plants. These Bn SGS3- Ov plants could grow and flower normally. But Bn SGS3- Si plants were seriously infected by CMV with abnormal plants and no flower. Youcai mosaic virus（ Yo MV） inoculation result showed that both transgenic and wild type plants were susceptive. It suggested that Bn SGS3 gene conferred moderate resistance to CMV,but no obvious resistance to YoMV.