黄瓜S-腺苷-L-高半胱氨酸水解酶全长DNA的克隆及表达分析

【目的】克隆黄瓜S-腺苷-L-高半胱氨酸水解酶基因的cDNA，并进行生物信息学和表达分析，为研究该基因的功能奠定基础。【方法】通过对乙烯利诱导黄瓜茎尖SSH文库的筛选，采用RT-PCR 和电子克隆技术，获得黄瓜CsSAHH基因的cDNA全长序列(NCBI编号：HQ444960, CsSAHH)。运用半定量RT-PCR，分析CsSAHH基因在乙烯利诱导后的茎尖和雌雄花不同部位的表达。通过生物信息学方法预测CsSAHH基因的蛋白结构。【结果】黄瓜CsSAHH基因的cDNA全长1 545 bp，编码485个氨基酸，其理论上的等电点pI＝5.66，分子量MW＝53.1 kD。CsSAHH基因在黄瓜茎尖受乙烯利诱导增强表达，在黄瓜雄花中的雄蕊表达较弱。CsSAHH理化性质表明，该蛋白无明显的信号肽；蛋白二级结构主要由loop 环和α螺旋构成，含少量的β折叠，预测发现该蛋白分别在85—99 氨基酸残基和262—279氨基酸残基处各有1个S-腺苷-L-高半胱氨酸水解酶活性功能区。CsSAHH基因氨基酸序列与苜蓿的同源性为63%，与水稻、玉米、拟南芥等作物同源性较低。【结论】成功克隆黄瓜CsSAHH基因cDNA序列，在85—99 氨基酸残基和262—279氨基酸残基处各有1个S-腺苷-L-高半胱氨酸水解酶活性功能区。该基因在茎尖受乙烯利诱导增强表达，在雄蕊中表达较弱。在未处理的雌雄花芽发育不同阶段，雌花芽中表达强于幼果和雄花芽。

Cloning and Expression Analysis of S-Adenosyl-L-Homocysteine Hydrolase in Cucumber(Cucumis stavius L.)

【Objective】 The cDNA sequence of cucumber S-adenosyl-L-homocysteine hydrolase(CsSAHH) gene was cloned and the gene expression patterns and protein structure were analyzed by bioinformatics methods to study the function of the gene.【Method】 Based on selecting SSH of stem tips treated by ethrel,the cDNA sequence of cucumber SAHH gene(CsSAHH,GenBank accession,HQ444960) was obtained by RT-PCR and in silico cloning.The mRNA expressions of the gene in stem tips treated by ethrel and different tissues of male and female flowers were detected through semi-quantitative RT-PCR.And the protein structure was analyzed by bioinformatics methods.【Result】 The cDNA sequence of the gene was 1 545 bp,contained a predicted protein sequence of 485 amino acids,pI=5.66,MW=53.1 kD.The semi-quantitative RT-PCR results revealed that CsSAHH mRNA expression was increased by ethrel in stem tips and was lower in stamen than that in the other flower tissues.The physical and chemical properties of cucumber SAHH indicated that this protein has no clear signal peptid.The secondary structure of cucumber SAHH was mainly made of loops,α helixes and few β sheets.There were two S-adenosyl-L-homocysteine hydrolase active domains between positions 85-99 and 262-279.The speculated amino acids sequence of cucumber SAHH shared 63% homologies with Medicago truncatula and had low homologies with Oryza sativa,Zea mays and Arabidopsis thaliana.【Conclusion】 The full length of the CsSAHH cDNA were obtained.There are two S-adenosyl-L-homocysteine hydrolase active domains between positions 85-99 and 262-279.The CsSAHH mRNA expression was induced by ethrel in stem tips,and lowest in stamen.The expression of CsSAHH was stronger in female flower buds than small fruits and male flower buds at different development stages of untreated cucumber flowers.