过表达胡杨PePKS5提高拟南芥耐盐性  

Overexpression of Populus euphratica PePKS5 enhancing salt tolerance of Arabidopsis thaliana

作　　者：温馨 殷可欣 马思圆 张小萌 赵楠 周晓阳 赵瑞 陈少良 Wen Xin;Yin Kexin;Ma Siyuan;Zhang Xiaomeng;Zhao Nan;Zhou Xiaoyang;Zhao Rui;Chen Shaoliang(Key Laboratory of Forest and Flower Genetics and Breeding of Ministry of Education,School of Biological Science and Biotechnology,Beijing Forestry University,Beijing 100083,China;Public Analysis and Testing Center,Beijing Forestry University,Beijing 100083,China)

机构地区：[1]北京林业大学生物科学与技术学院,林木、花卉遗传育种教育部重点实验室,北京100083 [2]北京林业大学实验室与设备管理处,北京100083

出　　处：《北京林业大学学报》2024年第2期62-74,共13页Journal of Beijing Forestry University

基　　金：国家自然科学基金项目(32071730、31770643);高等学校学科创新引智计划项目(111 Project、B130007);北京市自然科学基金项目(6182030、6172024);中央高校基本科研业务费专项(2019ZY25)。

摘　　要：【目的】盐胁迫是影响植物生长发育的不利环境因子。蛋白激酶PKS5作为植物盐超敏感信号转导途径的重要组分,在植物响应盐胁迫过程中具有重要调节功能。本文旨在生理水平和分子水平上,探究胡杨PePKS5基因在植物耐受盐胁迫过程中的调节作用。【方法】克隆胡杨PePKS5基因,并在拟南芥中过表达,获得T3代转基因拟南芥纯合体。观察盐胁迫下转基因拟南芥株系的耐盐表型,测定其过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶活性及盐胁迫响应基因的表达量。利用非损伤微测技术测定转基因拟南芥株系根尖Na^(+)、K^(+)动态离子流,利用激光共聚焦显微镜观测转基因拟南芥株系根尖Na^(+)和H_(2)O_(2)含量。测定盐处理后转基因拟南芥土培幼苗的叶绿素荧光参数、光合参数等生理指标,揭示PePKS5基因在盐胁迫下对拟南芥的生理调节作用。构建亚细胞定位载体,通过瞬时转化烟草,对PePKS5蛋白进行亚细胞定位观测。【结果】(1)PePKS5基因的CDS序列编码417个氨基酸,PePKS5氨基酸序列与毛白杨PtPKS5相似度最高,并且亲缘关系最近。(2)PePKS5定位于细胞质和细胞核中。(3)NaCl处理6 h后,胡杨叶片PePKS5基因表达量上调,72 h后逐渐恢复至正常水平。(4)盐胁迫下过表达PePKS5基因的拟南芥株系(PePKS5-OE10和PePKS5-OE15)的生存率和根长均明显高于野生型(WT)和转空载体(VC)株系。(5)与WT和VC株系相比,PePKS5-OE10和PePKS5-OE15株系的POD和CAT活性增强,APX1和CAT2基因的表达量均升高。(6)盐胁迫下PePKS5-OE10和PePKS5-OE15株系的Na^(+)外排和K^(+)的内流明显高于WT和VC株系,并且在根尖中积累的Na^(+)浓度和H_(2)O_(2)浓度明显低于WT和VC株系。(7)盐胁迫下PePKS5-OE10和PePKS5-OE15株系的叶绿素相对含量、PSⅡ最大光量子效率、实际光合量子产量和相对电子传递速率均高于WT和VC株系。(8)盐胁迫下PePKS5-OE10和PePKS5-OE15的净�[Objective]Salt stress is an adverse environmental factor affecting plant growth and development.As an important component of plant salt overly sensitive signal transduction pathway,protein kinase PKS5 plays an important regulatory role in plant response to salt stress.The aim of this study was to investigate the regulation mechanism of PePKS5 gene in plant tolerance to salt stress at physiological and molecular levels.[Method]The PePKS5 gene from Populus euphratica was cloned and overexpressed in Arabidopsis thaliana to obtain a T3 generation transgenic A.thaliana homozygous.We observed the salt tolerance phenotype of transgenic A.thaliana lines under salt stress,measured their peroxidase(POD),catalase(CAT),and superoxide dismutase activities,as well as the expression levels of salt stress responsive genes.Non-invasive micro measurement technology was used to measure the dynamic ion flow of Na^(+)and K^(+)in the root tips of transgenic A.thaliana lines,and the Na^(+)and H_(2)O_(2)contents in the root tips of transgenic A.thaliana lines were observed using laser confocal microscopy.We measured the physiological indicators such as chlorophyll fluorescence parameters and photosynthetic parameters of transgenic A.thaliana seedlings cultured in soil after salt treatment,and revealed the physiological regulatory effect of PePKS5 gene on A.thaliana under salt stress.A subcellular localization vector was constructed and the subcellular localization of PePKS5 protein was observed by transient transformation of tobacco.[Result](1)The CDS sequence of PePKS5 gene encoded 417 amino acids,and the amino acid sequence of PePKS5 was the most similar to PtPKS5 of Populus tomentosa.(2)PePKS5 was localized in cytoplasm and nucleus.(3)The expression of PePKS5 gene in leaves of P.euphratica was up-regulated after 6 h of NaCl treatment,and gradually recovered to normal level after 72 h.(4)Under salt stress,the survival rate and root length of PePKS5 gene overexpressing A.thaliana lines(PePKS5-OE10 and PePKS5-OE15)were significantly hi

关 键 词：胡杨 蛋白激酶 盐胁迫 PKS5 离子平衡 活性氧 

分 类 号：S792.11[农业科学—林木遗传育种]