13个甘蓝型油菜GPATs编码基因的酵母遗传互补功能鉴定  

Functional Identification of 13 Brassica napus GPATs Encoding Genes by Genetic Complementation in Yeast(Saccharomyces cerevisiae)

作　　者：段芊芊 林怡馨 丁硕 魏琳燕 甘毅[1] 郑月萍 刘宏波 DUAN Qian-Qian;LIN Yi-Xin;DING Shuo;WEI Lin-Yan;GAN Yi;ZHENG Yue-Ping;LIU Hong-Bo(School of Agriculture and Food Science,Zhejiang A&F University,Hangzhou 311300,China;School of Forestry and Biotechnology,Zhejiang A&F University,Hangzhou 311300,China)

机构地区：[1]浙江农林大学农业与食品科学学院,杭州311300 [2]浙江农林大学林业与生物技术学院,杭州311300

出　　处：《农业生物技术学报》2020年第7期1156-1164,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31301272;31370287);农业部引进国际先进农业科学技术计划(948计划)(2014-Z30);国家重点基础研究发展计划课题(2015CB150206)

摘　　要：3-磷酸甘油酰基转移酶(glycerol-3-phosphate acyltransferases,GPAT)是催化植物甘油脂从头合成第1步酰化反应的限速酶。分离和鉴定甘蓝型油菜(Brasscia napus)的GPAT编码基因将有助于深入解析种子中甘油脂的合成代谢机制。本研究通过反转录PCR(reverse transcription-PCR,RT-PCR)扩增获得13个甘蓝型油菜中具有酰基转移酶保守结构域的候选编码基因,构建至酵母(Saccharomyces cerevisiae)异源表达载体yADH1-pYES2-Kan V2(外源基因表达受葡萄糖诱导,受半乳糖抑制)。重组酵母表达载体酶切和测序验证结果显示,克隆的13个基因片段大小和序列与预期相符;通过氨基酸序列比对分析,发现克隆基因编码的氨基酸序列分别含有1~2个跨膜结构域和4个酰基转移酶保守序列。进一步利用本实验室创建的高效条件致死型酵母双敲除突变体菌株ZAFU1筛选体系(BY4742,gat1Δgat2Δ+[pGAL1::AtGPAT1 LEU2])进行遗传互补验证,结果显示,在葡萄糖诱导条件下,BnGPAT1-1、BnGPAT4-1、BnGPAT4-2和BnGPAT9-1基因的异源表达能恢复ZAFU1的生长,表明其具有类似酵母酰基转移酶编码基因YKR067w(命名为GAT1)的功能。本研究结果可为甘蓝型油菜含油量的遗传改良提供参考依据。Glycerol-3-phosphate acyltransferases(GPAT)are rate limited enzymes that catalyze the initial step of de novo glycerolipid biosynthesis in plants.Thus,isolation and identification of GPAT encoding genes from Brassica napus will help to in-depth uncover the molecular mechanisms of glycerolipid biosynthesis and metabolism in oilseeds.In this study,13 candidate genes,possessing conserved acyltransferase domains,were amplified by reverse transcription-PCR(RT-PCR)from B.napus,and constructed into yeast(Saccharomyces cerevisiae)heterologous expression vector yADH1-pYES2-Kan V2(exogenous gene expression was induced by glucose,inhibited by galactose).Verification of digestion and sequencing of the recombinant yeast expression vector showed that the sizes and sequences of 13 gene fragments were as expected.According to alignment of amino acid sequences,1~2 transmembrane domains and 4 conserved acyltransferase domains were in these BnGPATs,respectively.Then,yeast conditional lethal double knockout mutant strain ZAFU1(BY4742,gat1Δgat2Δ+[pGAL1::AtGPAT1 LEU2])was used to conduct genetic complementation verification.It was found that the growth of ZAFU1 could be rescued with heterologous expression of BnGPAT1-1,BnGPAT4-1,BnGPAT4-2 and BnGPAT9-1 induced by glucose,which indicated that the enzymes encoded by these genes might mimic the function of the yeast acyltransferase encoding gene YKR067w(named GAT1).The results of this study could provide reference data for the genetic improvement of oil content in B.napus.

关 键 词：甘蓝型油菜 3-磷酸甘油酰基转移酶(GPAT) 酵母遗传互补 

分 类 号：S312[农业科学—作物栽培与耕作技术]