海马齿小热激蛋白SpHSP18.1基因的克隆及盐胁迫表达分析  

Molecular clone of Sesuvium portulacastrum small heat shock protein SpHSP18.1 gene and its expression profiling under salt stress

作　　者：许天委[1] 张晓楠[2] 刘煜[2] 叶绵源[2] 张世杰[2] 刘然[2] 刘盛琨 张颖[2] 

机构地区：[1]琼台师范高等专科学校数理系,海口571127 [2]海南师范大学生命科学学院,海口571158

出　　处：《应用与环境生物学报》2015年第5期882-886,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31360173);海南省自然科学基金项目(312091);海南师范大学2015年度"大学生创新创业训练计划"项目资助~~

摘　　要：为探究海马齿高盐环境耐受性的分子机制,根据海马齿c DNA文库序列设计引物,采用RACE(Rapid amplification of cDNA ends)技术克隆得到海马齿小分子热激蛋白Sp Hsp18.1基因序列.Blast分析表明,该c DNA序列(968 bp)含完整开放阅读框(ORF)全长为489 bp,编码162个氨基酸.等电点(p I)为6.19,分子量(Mr)大小为1860 5.90.Sp HSP18.1编码氨基酸序列具有分子伴侣蛋白特有的ACD保守区域(Alpha crystallin domain),与拟南芥、西瓜等植物的细胞质I型小热激蛋白具较高同源性(>75%).将Sp Hsp18.1基因克隆到PUCm-T原核表达载体上,得到重组菌株,通过过量表达探究其耐热胁迫能力.结果表明,在37℃下重组菌株与野生型菌株的生长曲线基本相似,但在高温(45℃)下,重组菌株较对照组菌株存活率有显著提高.荧光实时定量PCR分析表明,在不同浓度海水培养条件下,海马齿根部均表达Sp Hsp18.1基因,且随着海水浓度的增加,该基因的表达量呈现上升的趋势.说明Sp Hsp18.1基因在海马齿应答高盐胁迫过程中起到一定的作用.For analyzing the molecular mechanism of Sesuvium portulacastrum under salt stress,the small hot shock protein gene Hsp18.1（968 bp） was cloned by using RACE technique.The primers were designed based on the cDNA library sequences.The result showed that SpHspl8.1 cDNA coded 162 amino acids with the full length of 968 bp.The pI of the encoded protein was 6.19 and the Mr was 18 605.90.The amino acid sequence of SpHspl8.1 had the Alpha crystal in domain belonging to chaperonin family protein.Blast analysis found high homology（ 〉75%） with the cytoplasm type I small heat shock proteins of plants including Arabidopsis and watermelon.To verify the possible function of SpHspl8.1 protein,the PUCm-T vector was chosen and the recombinant SpHspl8.1 was overexpressed in Escherihia coli.The results showed that the growth of the transformed cells was similar to that of wild type at 37 ℃,but the recombinant gene significantly improved cell viability in thermal stress conditions（45 ℃）.The results of real time PCR analysis showed expression of SpHspl8.1 gene on the roots of Sesuvium portulacastrum under salt stress in each treatment.At the same time,the gene expression quantity increased with the increase of the concentration of sea water.These results suggested that SpHspl8.1 protein may play a role in the response of Sesuvium portulacastrum to heavy salt stress.

关 键 词：海马齿 小分子热激蛋白 分子克隆 盐胁迫 

分 类 号：Q943.2[生物学—植物学]