葡萄硫双加氧酶基因VvETHE1克隆及表达分析  

Cloning and Expression Analysis of Sulfur Dioxygenase Gene VvETHE1 in Grapevine(Vitis vinifera)

作　　者：严静 宋珈凝 王玲 王跃进[1,2] 张朝红 YAN Jing;SONG Jia-Ning;WANG Ling;WANG Yue-Jin;ZHANG Chao-Hong(State Key Laboratory of Crop Stress Biology for Arid Areas,College of Horticulture,Northwest A&F University,Yangling 712100,China;Key Laboratory of Biology and Genetic Improvement of Horticultural Crops(Northwest Region),Ministry of Agriculture,Yangling 712100,China)

机构地区：[1]西北农林科技大学园艺学院,旱区作物逆境生物学国家重点实验室,杨凌712100 [2]农业部西北地区园艺作物生物学与种质创制重点实验室,杨凌712100

出　　处：《农业生物技术学报》2020年第12期2141-2150,共10页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2019YFD1001405);西安市科技计划项目(201806114YF02NC10-4)。

摘　　要：硫双加氧酶1(ethylmalonic encephalopathy protein 1,ETHE1)是硫化物氧化成硫酸盐的关键酶,主要参与植物抗逆、激素响应、种子发育与存活。本研究通过逆转录PCR(reverse transcription PCR,RTPCR),从’黑比诺’(’Pinot Noir’)葡萄(Vitis vinifera)中克隆到2个VvETHE1,命名为VvETHE1A(GenBank No.LOC100248855)和VvETHE1B(GenBank No.LOC100262655)。VvETHE1A的ORF长867 bp,编码288个氨基酸;VvETHE1B的ORF长861 bp,编码286个氨基酸。多序列比对显示,VvETHE1都有金属β-内酰胺酶家族的保守结构域。进化分析发现,与VvETHE1B相比,VvETHE1A与枣(Ziziphus jujuba)ETHE1的遗传距离更近。半定量反转录-PCR(semi-quantitative reverse transcription and PCR,sqRT-PCR)分析发现,VvETHE1在各器官中均表达,但表达量有所差异,其中VvETHE1B在根中大量表达。qRT-PCR分析表明,VvETHE1在有核品种和无核品种种子中差异表达,并都在’无核白’(’Thompson Seedless’)花后35~45 d种子中表达量降低。用外源激素处理葡萄叶片发现,VvETHE1A对脱落酸(abscisic acid,ABA)响应最强,乙烯(ethylene,ETH)其次;VvETHE1B强烈响应茉莉酸甲酯(methyljasmonate,MeJA),对ABA和ETH的响应较弱。为进一步分析VvETHE1基因功能,构建其原核表达载体,诱导获得2个分子量约35 k D的重组蛋白,以包涵体形式存在。不同诱导温度和不同诱导时间对VvETHE1s表达有影响,确定最佳表达体系为28℃诱导6 h,进一步通过16℃低温诱导,表达出可溶性融合蛋白。本研究为深入研究ETHE1在葡萄种子败育、激素响应中的调控机制及其生理生化特性提供基础。Sulfur dioxygenase 1(ethylmalonic encephalopathy protein 1,ETHE1),the key enzyme in the oxidation of sulfides into sulfates,is mainly involved in plant stress tolerance,hormone response,seed development and survival.In this study,two VvETHE1 s were cloned from Vitis vinifera.’Pinot Noir’through reverse transcription PCR(RT-PCR),and named VvETHE1 A(GenBank No.LOC100248855)and VvETHE1 B(GenBank No.LOC100262655).The ORF of VvETHE1 A was 867 bp,encoding 288 amino acids;while VvETHE1 B was 861 bp encoding 286 amino acids.Multiple sequence alignment indicated that VvETHE1 s were highly conserved in the domain of metallo-β-lactamase family.Evolutionary analysis showed that the genetic distance between VvETHE1 A and jujube(Ziziphus jujuba)ETHE1 was closer than VvETHE1 B.By semi-quantitative reverse transcription and PCR(sqRT-PCR),tissue-specific expression analysis showed both VvETHE1 A and VvETHE1 B were expressed in all tissues with different levels,among which VvETHE1 B was highly expressed in root.qRT-PCR analysis showed that the expression levels of VvETHE1 A and VvETHE1 B were significantly different in the ovules of Vitis vinifera’Pinot noir’and’Thompson Seedless’.And both of them decreased in the ovules of 35~45 d after full-bloom of Thompson Seedless.VvETHE1 A responded strongly to exogenous abscisic acid(ABA),followed by ethylene(ETH),while VvETHE1 B strongly responded to methyljasmonate(MeJA),weakly to ABA and ETH.Moreover,the prokaryotic expression vectors of VvETHE1s were constructed and 2 fusions of about 35 kD were induced in the form of inclusion body.Decrease of induction temperature and increase of time resulted in high yield of recombinant VvETHE1s.The optimal induction condition was 28℃for 6 h,and more soluble proteins were induced at 16℃.This study provide a basis for further studies on the regulatory mechanism of VvETHE1s in seed abortion and hormone response,and on the physiological and biochemical characteristics of VvETHE1s.

关 键 词：葡萄 VvETHE1 种子败育 激素响应 原核表达 基因表达 

分 类 号：S663.1[农业科学—果树学] Q786[农业科学—园艺学]