中国南瓜CmHSP70基因的克隆及表达分析

Cloning and Expression Analysis of CmHSP70 Gene in Cucurbita moschata

为了探究中国南瓜(Cucurbita moschata)热激蛋白70基因的功能,该研究采用转录组测序和RT-PCR方法,从中国南瓜中分离获得3个HSP70基因的开放阅读框(ORF)全长,分别命名为CmHSP70-1、CmHSP70-2和CmHSP70-3,三者的序列长度分别为1 998、1 941和2 118 bp,编码666、647和706个氨基酸。蛋白序列分析显示,3个CmHSP70蛋白均属于亲水性蛋白,都含有典型的NBD和SBD保守结构域;CmHSP70-1蛋白含有信号肽和跨膜结构,亚细胞主要定位于内质网;CmHSP70-2蛋白和CmHSP70-3蛋白不含信号肽和跨膜结构,亚细胞主要定位于细胞质和叶绿体。同源比对和进化分析发现,3个CmHSP70蛋白与苦瓜、甜瓜和黄瓜的HSP70蛋白的相似性最高且遗传距离最近。qRT-PCR分析显示,42℃处理能够诱导3个CmHSP70基因在根、茎、嫩叶和成熟叶中表达,并且表达量存在明显的组织特异性,成熟叶中基因的表达量最高;高温条件下,成熟叶中3个CmHSP70基因均能够在短时间内(处理0~2 h)响应热胁迫而大量表达,尤其是CmHSP70-2基因,推测该基因可能在中国南瓜热胁迫过程发挥重要的调节作用。该研究结果为深入了解HSP70基因功能以及阐明中国南瓜的耐热机制提供理论依据。

In order to investigate the function of heat shock protein gene in Cucurbita moschata,we isolated the open reading frames(ORFs)of three HSP 70 genes from C.moschata using transcriptome sequencing and RT-PCR methods and named CmHSP 70-1,CmHSP 70-2 and CmHSP 70-3.Their sequence length were 1 998,1 941 and 2 118 bp,encoding 666,647 and 706 amino acids,respectively.Protein sequence analysis showed that all three CmHSP70 were hydrophilic proteins with typical conserved domains of NBD and SBD.CmHSP70-1 contained signal peptide and transmembrane structure,and it was mainly located in endoplasmic reticulum.CmHSP70-2 and CmHSP70-3 did not contain signal peptide and transmembrane structure,and they were mainly located in cytoplasm and chloroplast,respectively.Homologous comparison and evolutionary analysis revealed that three CmHSP70 had the highest consistency with the HSP70 of Momordica charantia,Cucumis melo and Cucumis sativus,and the genetic distance was the closest.The qRT-PCR analysis showed that three CmHSP70 could be induced in roots,stems,immature leaves and mature leaves by 42℃,and the expression level had obvious tissue specificity,with the highest in mature leaves.Under high temperature conditions,three CmHSP70 in mature leaves could be expressed in response to heat stress in a short time(0-2 h),especially CmHSP70-2.It is speculated that CmHSP70-2 may play an important regulatory role in the process of heat stress in C.moschata.The results provided a theoretical basis for further understanding the function of HSP70 and elucidating the thermotolerance mechanism of C.moschata.