能源甜菜BvHIPP24基因的特性及在酵母中的镉耐受功能分析  

BvHIPP24 Characterization of Energy Beet and Its Function Analysis in Yeast Under Cadmium Stress

作　　者：高卓[1,2] 谭义雯 魏多 王锦霞 汪曼 周婉婷 刘大丽[2] 鲁振强[1] Gao Zhuo;Tan Yiwen;Wei Duo;Wang Jinxia;Wang Man;Zhou Wanting;Liu Dali;Lu Zhenqiang(Key Laboratory of Biochemistry and Molecular Biology/College of Life Sciences,Heilongjiang University,Harbin 150080;Key Laboratory of Sugarbeet Genetics and Breeding,Heilongjiang University/College of Advanced Agriculture and Ecological Environment,Harbin 150080)

机构地区：[1]黑龙江大学省高校生化与分子生物学重点实验室/生命科学学院,哈尔滨150080 [2]黑龙江大学省高校甜菜遗传育种重点实验室/现代农业与生态环境学院,哈尔滨150080

出　　处：《中国农学通报》2021年第21期126-133,共8页Chinese Agricultural Science Bulletin

基　　金：黑龙江省自然科学基金项目“BvHIPP24基因在能源甜菜重金属镉污染生物修复中的分子机制研究”(LH2019C057);黑龙江省高校基本科研业务费黑龙江大学专项资金项目“能源甜菜BvMPT11基因的Cd污染生物修复应答机制研究”(KJCX201920);农业部糖料现代产业技术体系建设项目“甜菜养分管理与土壤肥料”(CARS-170204),“甜菜高品质品种改良”(CARS-170111);科技部财政部国家科技资源共享服务平台项目“国家作物种质资源库甜菜分库运行服务”(NCGRC-2021-017);农作物种质资源保护与利用专项“甜菜种质资源的收集、鉴定、编目、繁种与入库(圃)保存”(1921-026);黑龙江省大学生创新创业训练计划项目“未授粉胚珠单倍体培养系在甜菜抗性基因工程中的应用”(201910212083)。

摘　　要：为了进一步研究重金属相关异戊二烯化植物蛋白在重金属逆境下的解毒机制,本研究克隆了能源甜菜BvHIPP24基因并分析了其在镉逆境下酵母中的功能。本研究以能源甜菜为实验材料,利用RTPCR的方法克隆获得能源甜菜BvHIPP24基因,并对BvHIPP24基因进行生物信息学分析;利用酵母表达载体构建pYES2-BvHIPP24重组质粒并转化到酵母InVSc1,在真核酵母细胞中进行BvHIPP24基因应答镉胁迫的研究。生物信息学分析显示BvHIPP24基因的CDS全长444 bp,编码147个氨基酸,氨基酸序列的分子量为16377.34 Da,理论等电点为9.39,为亲水蛋白,不含有跨膜区域,定位于叶绿体。保守结构域分析该基因具有一个HMA结构域。构建进化树分析表明,能源甜菜BvHIPP24蛋白与BvHIPP23蛋白质具有较近的亲缘关系。当施加0.5 mmol/L CdCl_(2)胁迫后,适量表达BvHIPP24的重组菌与对照菌株相比,其生长趋势明显优于后者,并发挥着相对更高的Cd耐受性。这说明,能源甜菜BvHIPP24基因在镉逆境胁迫下发挥着重要的解毒作用,并有可能直接或间接参与细胞重金属离子吸收、转运及代谢平衡等过程。To further study the detoxification mechanism of heavy metal-associated isoprenylated plant proteins under heavy metal stress,the cloning of BvHIPP24 gene and its function analysis in yeast under cadmium stress were conducted.The energy sugar beet was used as material and BvHIPP24 gene was cloned by RT-PCR.BvHIPP24 gene was analyzed by bioinformatics.The recombinant plasmid pYES2-BvHIPP24 was constructed using yeast expression vector and transformed into Saccharomyces cerevisiae InVSc1,and the response of BvHIPP24 gene to cadmium stress was studied in eukaryotic yeast cells.Bioinformatics analysis showed that the CDS of BBvHIPP24 gene was 444 bp,encoding 147 amino acids.Its protein molecular weight was 16377.34 Da,and isoelectric point was 9.39.It was a hydrophilic protein,which did not contain transmembrane region and was located in chloroplasts.Conservative domain analysis showed that the gene had a HMA domain.The analysis of phylogenetic tree showed that BvHIPP24 protein and BvHIPP23 protein in energy sugar beet had a close relationship.Under 0.5 mmol/L CdCl_(2) stress,compared with the control strain,the recombinant bacteria expressing BvHIPP24 had a significantly better growth trend,and had relatively higher Cd tolerance.These results indicate that BvHIPP24 gene in energy sugar beet plays an important role in detoxification under cadmium stress,and might be involved in the process of cell heavy metal ion absorption,transport and metabolic balance directly or indirectly.

关 键 词：能源甜菜 重金属关联异戊二烯化植物蛋白 镉逆境 酵母 功能分析 

分 类 号：S566.3[农业科学—作物学]