甘蓝型油菜雄性不育基因BnaX.MS1.a的克隆及其amiRNA载体的构建

Molecular cloning of male sterile gene BnaX. MS1. a in Brassica napus and construction of a vector with MS1 amiRNA

根据拟南芥MS1基因已知保守序列设计特异性引物,分离克隆甘蓝型油菜中与拟南芥MS1基因同源的基因片段。应用PCR-Walking技术获得甘蓝型油菜核雄性不育基因BnaX.MS1.a,其全长3 424 bp。BnaX.MS1.a基因与拟南芥MS1基因全长序列同源性为60.3%,编码序列同源性为89.5%。根据基因序列比对结果推测开放阅读框长2 004 bp,编码667个氨基酸,分子量为76.5 kD,等电点为8.01。BnaX.MS1.a基因编码的氨基酸序列与已知拟南芥MS1基因编码的氨基酸序列同源性达93%。构建BnaX.MS1.a基因的amiRNA载体转化甘蓝型油菜,旨在研发核不育基因工程油菜。

MS1 gene is a tapetum-specific expressed gene related with male sterility in Arabidopsis thaliana.According to the coding sequences of Arabidopsis MS1 gene,a series of primers were designed to clone the homologous sequences in Brassica napus.PCR-Walking method was used to clone the full sequence of BnaX.MS1.a gene of Brassica napus.The total length of BnaX.MS1.a gene cloned here was 3 424 bp.The MS1 from Brassica napus shared a similarity of 89.5% in encoding sequence and 60.3% in full sequence with that from Arabidopsis thaliana.According to the results,it was deduced that it had an open reading frame（ORF） of 2 004 bp,encoding a protein of 667 amino acid,with protein molecular weight of 26 kD and isoelectric point of 8.01.The similarity between MS1 gene from Brassica napus and that from Arabidopsis thaliana was 93% in amino acid sequence.A plant expressed vector with the artificial miRNA was constructed for further studies on male sterility of Brassica napus.