利用杜仲漆酶基因(EuLAC1)创制抗灰霉病番茄新种质  

Creating New Tomato(Solanum lycopersicum)Germplasm Resistance to Gray Mold Using Eucommia ulmoides Laccase 1(EuLAC1)Gene

作　　者：杨金玉 王作日 赵德刚[1,3,4] 赵懿琛 YANG Jin-Yu;WANG Zuo-Ri;ZHAO De-Gang;ZHAO Yi-Chen(College of Life Sciences/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Guizhou University,Guiyang 550025,China;College of Tea Sciences,Guizhou University,Guiyang 550025,China;National-Local Joint Engineering Research Center of Karst Region Plant Resources Utilization&Breeding,Guiyang 550025,China;Guizhou Key Laboratory of Agricultural Biotechnology/Plants Conservation Technology Center,Guizhou Academy of Agricultural Sciences,Guiyang 550025,China)

机构地区：[1]贵州大学生命科学学院/山地植物资源保护与种质创新教育部重点实验室,贵阳550025 [2]贵州大学茶学院,贵阳550025 [3]喀斯特山区植物资源利用与育种国家地方联合工程研究中心,贵阳550025 [4]贵州省农业科学院农业生物技术重点实验室/植物保育技术应用工程研究中心,贵阳550025

出　　处：《农业生物技术学报》2025年第3期523-535,共13页Journal of Agricultural Biotechnology

基　　金：中央引导地方科技发展资金(黔科合中引地[2023]009);国家自然科学基金(31870285,31660076);贵大人基合字(2021)82号。

摘　　要：漆酶(laccase, LAC)是一种糖蛋白氧化酶,影响木质素的合成,调节植物体内酚类物质含量,并参与植物对病虫害的防御机制。为获得抗灰霉病能力强且符合转基因安全的番茄(Solanum lycopersicum)新种质,本研究基于实验室前期构建的载体pGM626-Act1,设计并成功构建了1个由果实特异性E8启动子调控FLP基因的基因删除系统,同时构建了Act1启动子驱动杜仲漆酶1 (Eucommia ulmoides laccase 1,EuLAC1)基因的植物表达载体pGM626-Act1-EuLAC1。采用农杆菌(Agrobacterium tumefaciens)介导的转化技术,对番茄品种'Micro-Tom'进行遗传转化,最终成功获得了5株过表达EuLAC1的阳性番茄转基因植株。抗性分析结果表明,接种灰霉菌(Botrytis cinerea)后,过表达EuLAC1基因的番茄植株在发病时间方面与野生型及转空载体的番茄植株相比呈现出明显延迟。此外,过表达EuLAC1基因的番茄植株病斑直径显著小于对照组(野生型和转空载体的番茄植株)(P<0.05),表明EuLAC1基因的过表达能够明显增强番茄植株对灰霉病的抗性。过表达EuLAC1基因还可提高番茄植株中保护性酶的活性以及病程相关蛋白(pathogenesis related protein, PR)基因的表达水平。对转基因番茄果实中外源基因的删除分析发现,在40个番茄果实中有22个未检测到外源基因,外源基因删除效率为55%。本研究为开发满足转基因安全要求的抗病番茄新种质提供了一种新的技术手段。Laccase(LAC)is a glycoprotein oxidase that influences lignin synthesis,regulates the levels of phenolic compounds in plants,and participates in plant defense mechanisms against pests and diseases.In order to obtain a new tomato(Solanum lycopersicum)germplasm with high resistance to Botrytis cinerea and in compliance with transgenic safety,in this study,based on the pGM626-Act1 vector constructed in the laboratory in the previous stage,a gene-deleter system with the fruit-specific E8 promoter driving the FLP gene and the Act1 promoter driving the Eucommia ulmoides laccase 1(EuLAC1)gene in a plant expression vector was designed and constructed.Agrobacterium-mediated transformation technology was used to genetically transform the tomato variety'Micro-Tom'and 5 positive tomato transgenic plants overexpressing EuLAC1 were successfully obtained.The results of resistance analysis showed that the onset time of EuLAC1 overexpressing tomato plants was significantly delayed after inoculation with B.cinerea,which was in sharp contrast with wild type and empty vector control plants.Additionally,the lesion diameter in tomato plants overexpressing the EuLAC1 gene was significantly smaller than that in the control group(P<0.05),which comprised the wild type and empty vector tomato plants.The result showed that overexpressing the EuLAC1 gene in tomato plants markedly improved their resistance to B.cinerea.Overexpression of the EuLAC1 gene also increased the activity of protective enzymes and the expression level of disease process-related protein(PR)genes in tomato plants.Deletion analysis of exogenous genes in transgenic tomato fruits revealed that no exogenous genes were detected in 22 out of 40 tomato fruits,with an exogenous gene deletion efficiency of 55%.This study provides a new technical method for the development of new disease-resistant tomato germplasm that meets transgenic safety standards.

关 键 词：杜仲漆酶(EuLAC1) 番茄 遗传转化 灰霉病 抗病性分析 

分 类 号：S436.412[农业科学—农业昆虫与害虫防治]