蜡梅热激蛋白基因 Cp HSP70-1的克隆、亚细胞定位与表达分析
- 西南大学园艺园林学院/重庆市花卉工程技术研究中心/南方山地园艺学教育部重点实验室,重庆400715; 越南农业科学院果树蔬菜研究所,越南河内 ; 西南大学园艺园林学院/重庆市花卉工程技术研究中心/南方山地园艺学教育部重点实验室,重庆,400715
摘要: 在已构建的蜡梅花转录组数据库EST序列分析的基础上,采用 RACE技术克隆获得蜡梅热激蛋白 HSP70的cDNA序列,命名为CpHSP70‐1(GenBank登录号:KR071130).该序列全长2520 bp ,包括1962 bp的完整开放阅读框,编码653个氨基酸蛋白序列,含有3个 HSP70家族典型基序.CpHS P70‐1蛋白与其他真核生物的HSP70蛋白具有较高的同源性,聚类分析显示其属于定位于细胞核/细胞质的蛋白.亚细胞定位结果支持该蛋白分布于细胞核的预测.利用实时荧光定量PCR对 CpHS P70‐1基因的表达特性进行分析,其在各组织中均有不同程度的表达,其中在成熟叶片中表达量最高;在不同花发育阶段呈现持续稳定的表达模式;该基因在低温和高温诱导下表达量提高,而且对高温的响应更为敏感.
Cloning,Subcellular Localization and Expression Analysis of Heat Shock Protein Gene CpHSP70-1 from Chimonanthus praecox (L.)
- 西南大学园艺园林学院/重庆市花卉工程技术研究中心/南方山地园艺学教育部重点实验室,重庆400715; 越南农业科学院果树蔬菜研究所,越南河内 ; 西南大学园艺园林学院/重庆市花卉工程技术研究中心/南方山地园艺学教育部重点实验室,重庆,400715
Abstract: Based on the EST analysis of Winter sweet transcriptome database constructed from Chimonan‐thus praecox flowers ,and using RACE technology ,the full‐length cDNA sequences of heat shock protein HSP70 was obtained by cloning ,named as CpHSP70‐1 (GenBank accession number :KR071130) .The full‐length of CpHSP70‐1 cDNA sequence was 2 520 bp ,with an open reading frame (ORF) of 1 962 bp , encoding a putative polypeptide of 653 amino acid residues and containing 3 signature motifs of the HSP70 family .CpHSP70‐1 protein was shown to have high homology with HSP70 proteins from other eu‐karyotes ,and phylogenetic analysis revealed that it belongs to the subclasses located in the nucleus/cyto‐plasm .Subcellular localization result supported the prediction that this protein is located in the nucleus . Real‐time fluorescence quantitative PCR was performed to analyze the expression characteristics of Cp H‐S P70‐1 gene .It was expressed ,at different levels ,in various tissues ,with the highest expression level found in mature leaves ;and sustained and stable expression was detected at different flower developmental stages .Cp H S P70‐1 gene expression increased under low or high temperature stresses ,and it responded more sensitively to high temperature .