发菜固氮酶nifH基因克隆及其干旱胁迫下的差异表达  

Cloning of nifHfrom N.flagelliforme and Its Differential Expression Pattern under Drought Stress

作　　者：吴诗杰 王玲霞[1] 刘阳[1] 严奉坤[1] 丁苗苗[1] 李晓旭[1] 梁文裕[1] 

机构地区：[1]宁夏大学生命科学学院,银川750021

出　　处：《西北农业学报》2017年第11期1639-1647,共9页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：宁夏自然科学基金(NZ1608)~~

摘　　要：通过对发菜固氮酶H亚基基因(nifH)全长进行克隆、原核表达和生物信息学分析,采用qRT-PCR技术,分析不同干旱胁迫下发菜固氮酶基因nifH在转录水平的表达变化。结果表明,根据特异性引物克隆获得长度为894bp的nifH,GenBank登陆号为BankIt1901364(KU886163)。将nifH在大肠杆菌中表达,获得约36ku的外源蛋白。生物信息学分析表明,发菜nifH与已报道的多种蓝藻的nifH及推导的氨基酸序列具有较高的相似性,nifH二级结构和三级结构主要由α螺旋、β-折叠、随机卷曲和β-转角构成。随藻体含水量的逐渐降低,发菜nifH在转录水平上的表达量逐渐增加,固氮酶活性呈现先增加后下降的趋势。研究结果为进一步研究发菜固氮酶基因的分子结构和发菜响应干旱胁迫的固氮机制及氮代谢过程奠定基础。N.flagelliformeis a kind of terrestrial nitrogen-fixing cyanobacterium,which is distribute in arid or semiarid steppes and has important ecological value.Full length of nifH was cloned by specific primer,nifH was expressed in E.coli,DNA sequence and its encoded protein was analyzed by bioinformatics methods,differential expression in transcriptional level was also tested by qRTPCR.The results indicated that full length of nifH is 894 bp（GenBank access number is BankIt1901364（KU886163）.Heterologous protein（36 ku）was expressed in E.coli.The nucleotide sequence and the encoded amino acid sequence of nifH were highly homologous to other cyanobacteria species.The secondary structure and tertiary structure were made up ofα-helix,β-sheet,random coil andβ-turn.In addition,expression level of nifH gradually increased in transcriptional level with the decrease of water content in colonies of N.flagelliforme,and changes of nitrogenase activities incresed firstly and then decreased under drought stress.The results laid a foundation for further research on structure of nitrogenase gene and molecular mechanism of nitrogen-fixing for N.flagelliforme in response for drought stress.

关 键 词：发菜 固氮酶 基因克隆 生物信息学 差异表达 

分 类 号：Q943.2[生物学—植物学]