桑树Na^+/H^+逆向转运蛋白基因(MnNHX1)的克隆与耐盐力表达  

Cloning and Expression to Salt Stress of Na^+/H^+ Antiporter Gene(MnNHX1) in Mulberry Tree

作　　者：边晨凯[1] 龙定沛[1] 刘雪琴[1] 魏从进[1] 龚加红 赵爱春[1] 

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室农业部蚕桑功能基因组与生物技术重点实验室,重庆400716

出　　处：《林业科学》2015年第8期16-25,共10页Scientia Silvae Sinicae

基　　金：国家大学生创新创业训练项目(201210635005);国家蚕桑产业技术体系项目(CARS-22-ZJ0102);国家农业部公益性行业(农业)科研专项(201403064)

摘　　要：【目的】研究川桑液泡膜型Na+/H+逆向转运蛋白(NHX)基因的功能,探究桑树耐盐机制,为植物抗逆基因工程筛选提供优良的候选基因。【方法】以川桑基因组数据库为基础,基于同源基因序列的保守性,以川桑叶片cDNA为模板克隆川桑液泡膜型NHX基因;利用生物学软件和在线公共平台,分析所得基因编码的蛋白质序列及功能结构域,并构建系统进化树,分析与其他物种的亲缘关系。采用荧光定量PCR方法研究在NaCl胁迫条件下不同时间段‘湖桑32号’根、茎、叶等不同组织中桑树NHX1表达量的变化情况;通过构建超量表达载体,将其转化到拟南芥中,分析转基因拟南芥在Na Cl胁迫环境中的种子发芽数,根长、侧根生长情况和幼苗成活率,并对转基因拟南芥连续浇灌含高浓度Na Cl的营养液,研究过量表达NHX基因对拟南芥的影响。【结果】本研究得到1个液泡膜型NHX基因,命名为MnNHX1(Gen Bank登录号:KJ720637);该基因ORF长度为1 644bp,编码547个氨基酸残基,具有Na+/H+交换泵,且在其上游含有抑制剂氨氯吡嗪脒结合位点(LFFIYLLPPI)以及糖基化位点等结构域,TMHMM在线程序预测Mn NHX1具有12个明显的跨膜结构区;系统进化树分析结果显示,Mn NHX1具有较高的保守性,先与源于蔷薇科的桃聚合,与桑树形态学和基因组进化分析分类结果一致。荧光定量PCR试验表明,在无Na Cl胁迫条件下桑树NHX1在‘湖桑32号’根、茎、叶中均有表达;在Na Cl胁迫处理12 h后,根、茎中桑树NHX1的表达量显著增加,而后回落;而在胁迫处理24 h后,叶中桑树NHX1的表达量显著提高,随后回落。过量表达Mn NHX1的转基因拟南芥在Na Cl胁迫环境中,种子发芽率低于野生型,而根长和侧根生长情况以及幼苗成活率都优于野生型;连续浇灌含高浓度Na Cl营养液的转基因拟南芥生长状态更为优良。【结论】MnNHX1为优良的植物耐盐基因,在桑树中为组成型表Objective] To study the function of Na ＋ /H ＋antiporter （ NHX） in vacuolar membrane from mulberry tree Morus notabilis,and to explore the mechanism of salt tolerance in mulberry,and to provide an excellent candidate gene for the screening of plant resistance gene engineering. [Method]In this study,a Na ＋ /H ＋antiporter gene named as MnNHX1 was identified based on the M. notabilis genomic database and other homologous sequences. The MnNHX1 was cloned using the cDNA from M. notabilis leaves as template. The analysis of the primary structure and functional domains from MnNHX1 was completed by the bioinformatics analysis. The phylogenetic tree was generated to analyse the relationships between mulberry NHX1 and other species. Quantitative PCR was conducted to analyse the expression profiles of mulberry NHX1 in different tissues of M. multicaulis‘Husang No. 32’and treatment time under NaCl stress. The overexpression vector was constructed and transformed into Arabidopsis thaliana. The seed germination rate,the growth of roots and the survival rate of seedlings of the transgenic A. thaliana were analyzed under NaCl stress. Furthermore,the transgenic A. thaliana was continuously irrigated with the nutrient solution containing high concentration of NaCl to study the functional effects of MnNHX1 gene in the transgenic A. thaliana. [Result]We cloned a Na ＋ /H ＋ antiporter gene designated as MnNHX1（GenBank accession No. KJ720637）. The open reading frame （ORF） of MnNHX1 is 1 644 bp and encodes a protein of 547 amino acid with a Na ＋ /H ＋ exchange pump. At the upstream of this pump,there are some domains such as inhibitors amiloride binding sites （ LFFIYLLPPI） and glycosylation sites. The analysis of the online program of TMHMM showed that MnNHX1 have 12 obvious transmembrane region. Phylogenetic analysis showed that MnNHX1 was firstly clustered with Prunus persica from the Rosaceae family,which is consistent with morphological classification and genomic phylogenetic analysis of mulberry. Q

关 键 词：桑树 Na + /H +逆向转运蛋白 非生物胁迫 耐盐性 基因功能 

分 类 号：S718.46[农业科学—林学]