云杉NBS-LRR基因的克隆与表达分析

为深入研究NBS-LRR基因在川西云杉(Picea balfouriana)抗落针病过程中的分子作用机制,该研究根据GenBank数据库中其他植物NBS-LRR基因保守序列设计引物,利用RT-PCR技术,克隆云杉NBS-LRR基因全长cDNA序列(PbNBS-LRR),分析该基因及其编码蛋白的相关信息并进行基因表达研究。结果表明:(1)成功获得PbNBS-LRR基因的全长2 616 bp(基因登录号:MK044348),且包含一个2 508 bp的完整阅读框(ORF),共编码836个氨基酸,其氨基酸序列具有NBS-LRR类抗病基因典型的NB-ARC结构域和LRR结构域。(2)云杉PbNBS-LRR与北美云杉(Picea sitchensis)NBS-LRR类抗病蛋白相似性最高,达到98%;分子进化分析进一步表明,PbNBS-LRR与北美云杉NBS-LRR亲缘关系最近,其次为糖松(Pinus lambertiana)和火炬松(Pinus taeda)。(3)qRT-PCR分析表明,NBS-LRR基因在川西云杉、粗枝云杉(Picea asperata)和丽江云杉(Picea likiangensis)的根、树干韧皮部、嫩枝及针叶中均有表达,在川西云杉和粗枝云杉的根部以及丽江云杉的树干韧皮部中表达量最高;在落针病病原菌侵染川西云杉和粗枝云杉的初期(5月)以及丽江云杉的后期(9月),NBS-LRR基因的表达量最高,分别为对照的1.73倍、2.11倍和90.49倍,表明NBS-LRR基因参与了云杉落针病的防御反应。

Cloning and Expression Analysis of NBS LRR Gene from Spruce

In this study, the primers were designed based on the conserved sequences of other plant NBS LRR genes in GenBank. The full length cDNA (PbNBS LRR) sequence of NBS LRR gene in Picea balfouriana was cloned by RT PCR. Analysis of the information of PbNBS LRR and its encoding protein were carried out and the expression characteristics of the gene in spruce was studied. The results showed that: (1) PbNBS LRR gene is 2 616 bp with a 2 508 bp ORF, encoding 836 amino acids, which has the typical NB ARC domain and LRR domain of NBS LRR resistance genes, and gene accession number is MK044348. (2) At the amino acids level, the similarity of the PbNBS LRR protein to the corresponding sequence in Picea sitchensis NBS LRR protein is 98%. Molecular evolution analysis further indicated that PbNBS LRR was closely related to the NBS LRR of P. sitchensis. (3) Quantitative real time PCR analysis showed that the expression of NBS LRR was observed in all tissues of P. balfouriana, P. asperata, and P. likiangensis, and the highest expression level was found in the roots of P. balfouriana and P. asperata and the trunk phloem of P. likiangensis. In addition, the expression of NBS LRR was up regulated by infection with Lophodermium piceae. The highest expression of NBS LRR gene in P. balfouriana and P. asperata was found in May and P. likiangensis was found in Steptember (1.73 fold, 2.11 fold and 90.49 fold higher than that in control, respectively), which indicated that the NBS LRR gene is involved in the defense response of the needle cast disease in spruce.