盐穗木过氧化氢酶基因的克隆与功能分析  

Cloning and Functional Identification of Catalase Gene in Halostachys caspica

作　　者：彭丹[1] 张霞[1] 张富春[1] 

机构地区：[1]新疆大学生命科学与技术学院、新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《西北植物学报》2013年第10期1933-1939,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家“973”计划前期研究专项资助(2012CB722204)

摘　　要：利用同源基因克隆法,从新疆荒漠盐碱地多年生灌木盐穗木(Halostachys caspica)中克隆获得盐穗木过氧化氢酶基因(HcCAT1)。序列分析表明,HcCAT1基因开放阅读框为1 479bp,编码492个氨基酸,推测编码蛋白质的分子量为56.7kD,等电点为6.84。基因序列比对发现,HcCAT1与多种植物过氧化氢酶基因具有较高的同源性。半定量RT-PCR结果表明,HcCAT1基因受盐胁迫而上调表达。将构建的重组质粒pET32a-HcCAT1转化大肠杆菌BL21(DE3),以IPTG诱导重组蛋白His-HcCAT1的表达;SDS-PAGE和Western印迹检测显示,重组蛋白的分子量大小为77.7kD,其大小与推测的大小一致;在低温诱导下以可溶性形式表达,且表现出一定的过氧化氢酶活性。盐胁迫实验结果显示,在添加400mmol/L NaCl、400mmol/L KCl以及300mmol/L甘露醇的LB培养基中,重组质粒转化菌的生长情况和生长速率明显优于对照,表明HcCAT1可明显提高大肠杆菌的耐盐性。该研究结果将有助于从抗氧化角度认识盐生植物盐穗木的耐盐分子机理。Catalase plays important roles in plant salt tolerance.HcCAT1,a catalase gene from the perennial shrub Halostachys caspica,which distributes in saline-alkali arid land in Xinjiang,was isolated by homologous sequence cloning method.Sequence analysis showed that the ORF of HcCAT1 was 1 479 bp,encodes 492 amino acids with a molecular mass of 56.7 kD and a pI of 6.84.HcCAT1 shared high similarity with those from other plant species.Semi-quantitative PCR results showed that the expression of HcCAT1 gene was up-regulated by salt stress.The recombinant plasmid pET32a-HcCAT1 was constructed and transformed into E.coli BL21（DE3）,The fusion protein His-HcCAT1 was expressed by IPTG induction.SDS-PAGE and Western blotting showed that His-HcCAT1 was 77.7 kD,same size to the expected.Under low temperature,His-HcCAT1 was expressed in soluble form,and had certain catalase activity.Salt stress experiments showed that E.coli carrying the recombinant plasmid pET32a-HcCAT1 exhibited better growth phenotypes and higher growth rates than the control bacteria in the LB medium supplemented with 400 mmol/L NaCl,400 mmol/L KCl and 300 mmol/L mannitol,demonstrating that HcCAT1 could enhance the salt stress tolerance for E.coli cells.These results will help to understand the molecular mechanism that H.caspica for salt stress and oxidative stress.

关 键 词：盐穗木 过氧化氢酶 原核表达 耐盐性 功能鉴定 

分 类 号：Q785[生物学—分子生物学]