粗枝云杉病程相关蛋白基因PaPR10的克隆及表达分析  

Cloning and Expression Analysis of a Pathogenesis Related Protein Gene PaPR10 in Picea asperata

作　　者：梁芳 刘利娟 李成松 赵炜栋 刘应高[1] 杨春琳[1] LIANG Fang;LIU Lijuan;LI Chengsong;ZHAO Weidong;LIU Yinggao;YANG Chunlin*(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》2022年第9期1450-1459,共10页Acta Botanica Boreali-Occidentalia Sinica

基　　金：四川省教育厅项目(09ZA068)。

摘　　要：为了探究粗枝云杉(Picea asperata)病程相关蛋白PR10的核糖核酸酶活性,该研究以粗枝云杉一年生针叶为材料,以响应云杉落针病菌(Lophodermium piceae)侵染而显著上调的一个PR10基因(PaPR10)序列为研究对象,设计特异性引物,采用RT-PCR技术克隆获得PaPR10基因的cDNA序列全长,使用生物信息学软件预测该基因编码蛋白的序列特征,将该基因进行原核表达和纯化,利用底物法检测纯化蛋白的核糖核酸酶活性,为解析PR10基因的抗菌活性机理奠定基础。结果表明:(1)成功克隆到PaPR10基因(GenBank登录号为OM743228)的ORF长456 bp,编码151个氨基酸序列,相对分子量为16.52 kD,理论等电点为5.73。(2)PaPR10蛋白无信号肽、不含跨膜区、定位在细胞质中,具有典型的病程相关蛋白Bet_v_1家族保守结构域和一个富含甘氨酸环(P-Loop)的保守结构域,但PaPR10蛋白的P-Loop结构域存在一个碱基突变;PaPR10蛋白与北美云杉等多种裸子植物和部分苔藓植物的PR10蛋白相似性较高。(3)IPTG诱导下,PaPR10蛋白以可溶性蛋白和包涵体的形式并存,且以终浓度为0.8 mmol/L的IPTG在30℃下诱导10 h时表达量最佳,但纯化后的PaPR10可溶性蛋白没有核糖核酸酶活性。研究发现,PaPR10蛋白不具备核糖核酸酶活性。The purpose of this study is to lay a foundation for subsequent studies of antibacterial activity of pathogenesis-related protein PR10 through exploring the ribonuclease activity of a PR10 gene in Picea asperata.This study cloned the full length of cDNA sequence of a PaPR10 gene using the annual needles of P.asperata as material by RT-PCR technique.The specific primers were designed based on the sequence of PaPR10 which was significantly up-regulated in response to the infection of spruce needle-falling pathogen(Lophodermium piceae).In addition,the sequence characteristics of the PaPR10 protein were predicted by bioinformatics software.The PaPR10 gene was prokaryotic expressed and purified,and the ribonuclease activity of the purified protein was detected by substrate method.The results showed:(1)the ORF of PaPR10 gene(GenBank accession number:OM743228)was 456 bp,and encoded 151 amino acids with a relative molecular weight of 16.52 kD and a theoretical isoelectric point of 5.73. (2) The PaPR10 protein had no signal peptide, no transmembrane region and was in the cytoplasm. It had a typical con-served domain of pathogenesis related protein Bet_v_1 family, a glycine-rich P-Loop conserved domain. However, there was a base mutation in the P-Loop domain of PaPR10 protein. The results of phylogenetic analysis showed that PaPR10 protein had high similarity with some PR10 proteins in gymnosperms such as Picea crassifolia and some bryophytes. (3) The PaPR10 protein coexisted in the form of soluble and inclu-sion bodies with the induction of IPTG. In addition, the expression was the best when the final concentra-tion of IPTG was 0.8 mmol/L at 30 ℃ for 10 h, but the purified PaPR10 soluble protein had no ribonucle-ase activity. The PaPR10 protein does not have ribonuclease activity.

关 键 词：粗枝云杉 病程相关蛋白 克隆表达 生物信息学分析 核糖核酸酶活性 

分 类 号：Q785[生物学—分子生物学] Q786