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马铃薯小热激蛋白基因sHSP-F的克隆及植物表达载体的构建

Clonging and Construction of a Plant Experessing Vector of Small Heat Shock Protein sHSP-F Gene from Solanum tuberosum
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摘要 热激蛋白(heat shock proteins,HSP)是生物体在不利环境条件因素刺激下应激合成的一组在进化上高度保守的蛋白质。前期转录组测序的结果发现马铃薯Favorita的小热激蛋白(small heat shock proteins,s HSPs)基因(PGSC0003DMG400009255)在接种晚疫病菌(Phytophthora infestans)24 h后表达量显著上调。因此,以马铃薯Favorita为材料,根据PGSC0003DMG400009255基因序列设计引物,并在引物5'端加上Bam HⅠ和SalⅠ酶切位点,从接种P.infestans 24小时后的Favorita的RNA中通过RT-PCR的方法获得PGSC0003DMG400009255的基因片段,并命名为s HSP-F,该基因最大开放阅读框(ORF)为594 bp,编码197个氨基酸。通过酶切连接将s HSP-F连接至表达载体p CAMBIA1301中。通过测序和酶切验证,表明s HSP-F基因成功克隆到表达载体中,该工作为进一步研究该基因的功能提供了基础。 Heat shock protein(HSP) is a group of evolutionarily conserved proteins,stress synthesized under adverse environmental conditions.In a previously transcriptome sequencing analysis,the expression of small heat shock protein(s HSPs) gene(PGSC0003DMG400009255) of potato was upregulated significantly after 24 h of late blight fungus(Phytophthora infestans) inoculation.Here,we obtained the PGSC0003DMG400009255 gene by RT-PCR amplification using the primers having the Bam HⅠ and SalⅠ enzyme loci in the their 5 'end that designed based on the gene sequence,and using the RNA template extracted from the Favorita inoculated with P.infestans of 24 h.We named this gene segment as s HSP-F,which shared the open reading frame(ORF) of 594 bp putatively encoding the protein of 197 amino acids.We ligated this s HSP-F to vector p CAMBIA1301 via restriction endonuclease digestion and ligase connection.Sequencing and enzyme digestion validation revealed that s HSP-F was cloned into the expression vector.These work laid the foundation for next research of gene functional verification.
作者 张帅扬 秦玉芝 熊兴耀 周倩 Zhang Shuaiyang1, Qin Yuzhi2, Xiong Xingyao2,3,4, Zhou Qian1(1. Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests, College of Plant Protection, Hunan Agricultural University, Changsha, 410128; 2. Hunan Provincial Engineering Research Center of Potatoes, Horticulture and Landscape College, Hunan Agricultural Univer- sity, Changsha, 410128; 3. Institute of Vegetable and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081; 4. Pre-State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops, Hunan Agricultural University, Changsha, 410128)
出处 《分子植物育种》 CSCD 北大核心 2017年第4期1327-1331,共5页 Molecular Plant Breeding
基金 国家自然科学基金项目(31371683)资助
关键词 马铃薯 小热激蛋白 基因克隆 表达载体构建 Solanum tuberosum s HSPs Gene clong Construction of plant expression vector
作者简介 通讯作者.zhouqian2617@hunau.edu.cn
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