云杉NBS-LRR基因的克隆与表达分析  

Cloning and Expression Analysis of NBS-LRR Gene from Spruce

作　　者：刘裕峰 刘利娟 杨帅[1] 曾倩[1] 刘广川 刘应高[1] LIU Yufeng;LIU Lijuan;YANG Shuai;ZENG Qian;LIU Guangchuan;LIU Yinggao(College of Forestry, Sichuan Agricultural University, Chengdu 611130, China)

机构地区：[1]四川农业大学林学院,成都611130

出　　处：《西北植物学报》2020年第7期1114-1122,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：四川省教育厅项目(09za068)。

摘　　要：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2616 bp(基因登录号:MK044348),且包含一个2508 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。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 2616 bp with a 2508 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.

关 键 词：云杉 NBS-LRR 基因克隆 表达分析 

分 类 号：Q785[生物学—分子生物学] Q786