李素 陈芳 殷缘 田广宇 陈晓璇 王天真 范诺 ABDUL WAHID BALOCH 张荣沭

摘 要：為筛选林木抗性基因，培育高抗转基因林木，以山新杨（Populus davidiana × P. alba var.pyramidlis）为试材，采用RT-PCR技术克隆获得1 560 bp的PdPapGH12（Potri.004G109200.1）基因cDNA，生物信息学分析表明该基因编码519个氨基酸，该蛋白属于亲水性酸性蛋白。依据亚细胞定位预测结果推测其在胞内发挥水解的作用。蛋白结构分析发现PdPapGH12与4个杨树品种的9条GHs序列具有高度一致性。qRT-PCR分析显示，PdPapGH12在茎基、老叶和根中表达量更高。并发现200 mmol/L NaCl、pH为10的Na2CO3溶液和聚乙二醇6000（PEG 6000;质量分数30%）胁迫48 h其在茎尖、叶和根中均上调表达;5种植物致病病原真菌胁迫48 h均使其在茎尖的表达下调。只有核盘菌使其在叶中表达显著上调;尖孢镰刀菌、链格孢菌和立枯丝核菌使其在根内表达显著上调。受激素诱导48 h，ABA（100 μmol/L）仅使其在根部表达上调;SA（100 μmol/L）仅使其在叶中表达上调，而JA（100 μmol/L）使其在叶和根中的表达均上调。结果表明PdPapGH12参与植物抵抗逆境胁迫和激素诱导的信号途径。

关键词：糖苷水解酶;非生物胁迫;生物胁迫;激素诱导;qRT-PCR

中图分类号：Q786      文献标识码：A   文章编号：1006-8023（2021）04-0011-11

Cloning of PdPapGH12 Gene of Shanxin Poplar

（Populus davidiana × P. alba var. pyramidlis） and Its Tissue

Expression in Response to Stress

LI Su1， CHEN Fang1， YIN Yuan1， TIAN Guangyu1， CHEN Xiaoxuan1， WANG Tianzhen1，

FAN Nuo1， ABDUL WAHID BALOCH2， ZHANG Rongshu1*

（1.College of Landscape Architecture， Northeast Forestry University， Harbin 150040， China;

2.Department of Plant Breeding and Genetics， Sindh Agriculture University， Tandojam 70060， Pakistan）

Abstract：In order to screen for the resistance genes of trees and cultivate transgenic trees with high resistance， Shanxin Poplar （Populus davidiana × P. alba var. pyramidlis） was used as the test material， and the 1 560 bp cDNA of the PdPapGH12 （Potri.004G109200.1） gene was cloned by RT-PCR. Bioinformatics analysis showed that PdPapGH12 encoded a protein of 519 amino acids， and the PdPapGH12 protein was a hydrophilic acidic protein. According to the prediction results of the subcellular localization of PdPapGH12， it might play a role in the hydrolysis reactions in the cytoplast. Protein structure analysis showed that PdPapGH12 shared high similarity with nine other GHs of four poplar varieties. RT-qPCR analysis showed that PdPapGH12 was relatively highly expressed in the stem base， old leaves and roots. Salt （NaCl， 200 mmol/L）， alkali （Na2CO3 solution， pH=10） and PEG 6000 （Polyethylene glycol 6000; 30%） treatments for 48 hours could induce the up-regulation of PdPapGH12 in the apex， mature leaf and root. PdPapGH12 expression in the apex was down-regulated by the inducing of five plant pathogenic fungi for 48 hours. Only Sclerotinia sclerotiorum induction significantly upregulated the expression of PdPapGH12 in mature leaves. Fusarium oxysporum， Alternaria alternate and Rhizoctonia solani induction significantly increased PdPapGH12 expression in the roots. After 48 hours of phytohormone induction， ABA （100 μmol/L） only upregulated PdPapGH12 expression in the roots. SA （100 μmol/L） only upregulated PdPapGH12 expression in mature leaves， while JA （100 μmol/L） upregulated PdPapGH12 expression in both the mature leaves and roots. This study demonstrated that PdPapGH12 had a role in poplar's resistance against environmental stresses and responses to phytohormone induction.

Keywords：Glycoside hydrolase; abiotic stress; biotic stress; hormone induce; RT-qPCR

收稿日期：2021-03-13

基金项目：2019年国家级本科生创新训练项目（201910225087）;国家自然科学基金资助项目（31370642）

*通信作者：张荣沭，博士，副教授。研究方向为园林植物种质资源及植物病害生物防治。E-mail：  zrs6504@sina.com

引文格式：李素， 陈芳， 殷缘， 等. 山新杨PdPapGH12基因克隆及其响应胁迫的组织表达[J]. 森林工程，2021，37（4）：11-21.

LI S， CHEN F， YIN Y， et al. Cloning of PdPapGH12 Gene of Shanxin Poplar （Populus davidiana × P. alba var. pyramidlis） and its tissue expression in response to stress[J]. Forest Engineering，2021，37（4）：11-21.

0 引言

我国实施退耕还林政策以来，植树造林工程、天然林保护工程等相关事项愈发受到行业重视[1]。杨树因其物种资源丰富，分布广泛，具有生长迅速、繁殖率高、树干笔直和枝繁叶茂遮阴效果好等特点，作为绿化树种能起到水土保持、防风固沙和改善环境的作用;又因其生长期短、出材率高还可作为经济树种起到促进地方经济发展的作用[2-4]。……