丹参质膜H+-ATP4酶基因(SmPHA4)在酿酒酵母中表达及其抗逆功能分析  

Expression of plasma membrane H+-ATP4 gene(SmPHA4)of Salvia miltiorrhiza in Saccharomyces cerevisiae and its anti-stress function analysis

作　　者：刘娜娜[1,2] 杨晓冰 李晔 李秀红 LIU Na-na;YANG Xiao-bing;LI Ye;LI Xiu-hong(Sanmenxia Polytechnic,Sanmenxia 472000,China;College of Applied Engineering,Henan University of Science and Technology(HAUST),Sanmenxia 472000,China;College of Life Sciences,Northwest A&F University,Yangling 712100,China)

机构地区：[1]三门峡职业技术学院,河南三门峡472000 [2]河南科技大学应用工程学院,河南三门峡472000 [3]西北农林科技大学林学院,陕西杨凌712100

出　　处：《生物技术》2022年第3期269-275,324,共8页Biotechnology

基　　金：国家自然科学基金项目(31700265)。

摘　　要：[目的]将丹参质膜H^(+)-ATP4酶基因(SmPHA4)克隆并导入到真核生物模型INVSc1酿酒酵母中,测试SmPHA4基因的酵母转化子在非生物胁迫下的耐受性,以分析该基因(SmPHA4)的功能。[方法]将SmPHA4基因全长通过无缝克隆的方式连入pYES2载体中获得重组质粒pYES2-SmPHA4,并把该重组质粒导入到真核生物INVSc1酿酒酵母中,对SmPHA4基因的酵母转化子在不同温度、盐碱度、氧化剂等条件胁迫下的菌株存活率进行了分析统计。[结果]SmPHA4基因的酵母转化子对极端温度的耐受性有所增强(47℃和-20℃)(P<0.05或P<0.01)。但是在其余几种非生物胁迫下如3 mol/L NaCl、3 mol/L KCl、100 mmol/L H_(2)O_(2)和20%PEG 6000等胁迫条件下均显示出降低的耐受性(P<0.05或P<0.01)。[结论]SmPHA4基因可以增强酵母转化子对特定极端温度的耐受性,但降低了其在试验条件中盐胁迫、氧化胁迫、干旱胁迫因子的耐受性,显示SmPHA4基因与丹参的抗非生物胁迫功能有关。[Objective]The plasma membrane H^(+)-ATP4 enzyme gene of Salvia miltiorrhiza was cloned and introduced into the eukaryotic model INVSc1 Saccharomyces cerevisiae to test the tolerance of yeast transformants of SmPHA4 gene under abiotic stresses to analyze the gene(SmPHA4)function.[Method]In this experiment,the full length of the SmPHA4 gene was seamlessly cloned into the pYES2 vector to obtain the recombinant plasmid pYES2-SmPHA4,and the recombinant plasmid was introduced into the eukaryotic INVSc1 Saccharomyces cerevisiae.The survival rates of the yeast transformant strains under different temperature,salinity,oxidant conditions were analyzed and counted.[Result]The yeast transformants of SmPHA4 gene have enhanced tolerance to extreme temperatures(47℃and-20℃)(P<0.05 or P<0.01).However,The yeast transformants showed reduced tolerance under other abiotic stresses such as 3 mol/L NaCl,3 mol/L KCl,100 mmol/L H_(2)O_(2) and 20%PEG 6000,(P<0.05 or P<0.01).[Conclusion]SmPHA4 gene can enhance the tolerance of yeast transformants to specific extreme temperatures,but reduces their tolerance to salt stress,oxidative stress,and drought stress in conditions of this experiment.It shows that SmPHA4 gene of Salvia miltiorrhiza are related with the function of organisms resistant to abiotic stresses.

关 键 词：丹参 质膜H+-ATP4酶 酿酒酵母 基因表达 功能分析 

分 类 号：Q785[生物学—分子生物学]