摘要
为挖掘蓖麻耐盐相关基因,以盐生植物蓖麻(Ricinus communis L.)叶片为材料。设计特异性引物,克隆高亲和性钾离子转运蛋白(high-affinity K+transporter,HKT)耐盐基因,并对其序列进行生物信息学分析。结果表明,该基因全长1 596 bp,编码531个氨基酸,推测分子量为59.74 kD,等电点(pI)为9.32。多重序列比对和系统发育树分析表明该蛋白与木薯和橡胶树的同源性最高,分别为69.13%和67.03%,且与HKT1蛋白家族成员同源性较高,为S-G-G-G类型蛋白,推测该蛋白为HKT1类蛋白。二级结构预测蓖麻HKT蛋白有9个跨膜结构域,主要定位于内质网上,是典型的跨膜蛋白。根据蓖麻HKT基因序列设计带有Bam HⅠ和PstⅠ酶切位点的引物扩增出全长基因,用限制性内切酶Bam HⅠ和PstⅠ进行双酶切后与表达载体pCHF-3300连接。本研究成功构建了蓖麻HKT基因的表达载体,为进一步研究该基因的转化及功能验证提供参考。
In order to discover salt tolerant genes in castor,the halophyte castor（Ricinus communis L.） leaves were used as the materials.The specific primers were designed to clone the salt tolerant high-affinity K＋transporter（HKT） gene,and its sequence was analyzed by bioinformatics.The results showed that full-length c DNA of this gene was 1 596 bp,encoding 531 amino acids.The predicted molecular weight was 59.74 kD and the isoelectric point（p I） was 9.32.Multiple sequence alignment and phylogenetic tree analysis indicated that this protein had the highest homology with cassava（69.13%） and rubber tree（67.03%）.In addition,it had high homology with HKT1 protein family members and belonged to S-G-G-G type protein,suggesting that this protein was HKT1-like protein.Secondary structure predicted that castor HKT protein had nine transmembrane domains,mainly located in the endoplasmic reticulum,which was a typical transmembrane protein.Based on the HKT sequence,the specificprimer with Bam H Ⅰ and Pst Ⅰ was designed for the amplification of gene with full length.Restriction enzyme Bam H Ⅰ and Pst Ⅰ were used to connect the expression vector pCHF-3300 after double enzyme digestion.This research successfully constructed the expression vector of HKT gene in castor,which could provide reference for the further study on transformation and function verification of this gene.
作者
丛娇娇
王晓宇
李平
李惠根
张丽雪
张继星
Cong Jiaojiao;Wang Xiaoyu;Li Ping;Li Huigen;Zhang Lixue;Zhang Jixing(College of Life Science, Inner Mongolia University for the Nationalities, Tongliao, 028000;Inner Mongolia Industrial Engineering Research Center of Universities for Castor, Tongliao, 028000;Inner Mongolia Key Laboratory of Castor Breeding, Tongliao, 028000;Inner Mongolia Collaborative Innovation Cultivate Center for Castor, Tongliao, 028000;Horqin Plant Stress Biology Research Institute of Inner Mongolia University for Nationalities, Tongliao, 028000)
出处
《分子植物育种》
CAS
CSCD
北大核心
2018年第14期4648-4657,共10页
Molecular Plant Breeding
基金
国家自然基金项目(31260336
31760399
31701467)
内蒙古自治区蓖麻育种重点实验室开放基金项(MDK2-017036
MDK2017037)
博士科研启动基金(BS398)共同资助
关键词
蓖麻
高亲和钾离子转运蛋白
基因克隆
生物信息学分析
表达载体
Ricinus communis
High-affinity K＋ transporter
Gene cloning
Bioinformatics analysis
Expression vector
