梨黑斑病抗病相关基因PpEMS1的克隆与分析

doi:10.13560/j.cnki.biotech.bull.1985.2019-0240

摘要/Abstract

摘要： 克隆梨抗病相关基因,并对其在梨黑斑病抗病防御中的作用进行初步分析。基于同源基因克隆方法从“黄冠梨”中克隆获得一个预测为受体样蛋白激“酶EMS1的基因,暂命名为PpEMS1。利用生物信息学、Southern Blot、亚细胞定位和实时荧光定量PCR技术对该基因进行分析。结果表明,该基因CDS全长3 894 bp,编码1 296个氨基酸,开放阅读框3 891 bp。Southern Blot分析表明,在“黄冠梨”、“黄花梨”、“翠玉梨”中,该基因都以单拷贝形式存在,在“爱宕梨”中以多拷贝形式存在。亚细胞定位结果显示,融合蛋白在细胞膜和细胞核均有表达。荧光定量PCR分析表明,接种黑斑病菌1、2、3、4、5、6、7和8 d后,在“黄花梨”和“爱宕梨”中该基因表达均呈现先上升后下降的趋势。推测该基因可能与梨黑斑病抗性相关。

关键词: 梨, PpEMS1, Southern Blot, 荧光定量PCR, 亚细胞定位

Abstract: This study aims to clone the gene related to pear disease resistance and to preliminary analyze the effect of the gene on resistance and defense of pear black spot. Based on homologous gene cloning method,a gene predicted to be the receptor protein kinase EMS1 was cloned from the ‘Huang-guan’ pear,temporarily named as PpEMS1. The gene then was analyzed by bioinformatics,Southern Blot,subcellular localization and quantitative real-time PCR. As result,its CDS was 3 894 bp,containing an open reading frame of 3 891 bp and encoding 1 296 amino acids. Southern blot analysis indicated that this gene existed as a single copy in ‘Huang-guan’ pear,‘Huang-hua’ pear and ‘Cui-yu’ pear,while it existed as multiple copies in ‘Ai-dang’ pear. Subcellular localization demonstrated that the fusion protein was expressed in both cytomembrane and nucleus. The results from the quantitative real-time PCR revealed that the expression of the PpEMS1 gene increased first and then decreased in both ‘Huang-hua’ and ‘Ai-dang’ pears and was induced at 1 d,2 d,3 d,4 d,5 d,6 d,7 d and 8 d after inoculating black spot pathogen. It is inferred that the PpEMS1 gene may be associated with the pear black spot resistance.

Key words: pear, PpEMS1, southern blot, quantitative real-time PCR, subcellular localization

段敏杰, 伊洪伟, 王进, 武峥. 梨黑斑病抗病相关基因PpEMS1的克隆与分析[J]. 生物技术通报, 2019, 35(11): 16-21.

DUAN Min-jie, YI Hong-wei, WANG Jin, WU Zheng. Cloning and Expression Analysis of the Pear Black Spot Resistant Gene PpEMS1[J]. Biotechnology Bulletin, 2019, 35(11): 16-21.

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