秋茄KcTIP1基因克隆及其对植物耐盐性作用的研究  

Molecular cloning and salt-tolerance of gene KcTIP1 from Kandelia candel

作　　者：鲁彦君[1] 韩彦莎[1] 李妮亚[2] 侯佩臣[3] 黄旭新[1] 邓澍荣[1] 赵瑞[1] 沈昕[1] 陈少良[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]海南师范大学生物学院,海南海口571158 [3]国家农业信息化工程技术研究中心,北京100097

出　　处：《西北农林科技大学学报（自然科学版）》2013年第7期162-171,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31160150;31270654;31170570);中央高校基本科研业务费专项(JC2011-2;TD-2012-04);北京市自然科学基金项目(6112017);北京市优秀博士学位论文指导教师专项(YB20081002201);高等学校学科创新引智计划项目(111Project;B13007)

摘　　要：【目的】从秋茄中克隆液泡膜水通道蛋白KcTIP1基因,并研究其对植物耐盐性的作用机制。【方法】以秋茄幼嫩叶片为材料,通过PCR扩增克隆KcTIP1基因并构建其表达载体,采用根癌农杆菌介导法,用含重组质粒的农杆菌转化烟草植株,通过卡那霉素筛选和转基因烟草基因组PCR、RT-PCR鉴定,获得转基因再生植株;并选取其中2个阳性株系和野生型株系进行盐处理,测定野生型烟草(对照)与盐处理烟草根、茎、叶鲜质量,光合参数,叶片组织Na+、K+浓度以及根尖Na+、K+的流速。【结果】成功克隆得到了KcTIP1基因,对其编码蛋白氨基酸序列进行分析表明,秋茄KcTIP1包含MIP家族的特征序列SGGHVNPAVT和2个保守的NPA(Asn-Pro-Ala)位点。RT-PCR检测结果显示,KcTIP1基因在转基因烟草株系中成功表达。盐胁迫后,转基因烟草植株根、茎、叶鲜质量,叶片净光合速率,蒸腾速率,叶片组织Na+、K+积累和根尖Na+、K+内流幅度均高于野生型烟草。【结论】KcTIP1具有与其他水通道蛋白类似的蛋白结构和酶学功能,KcTIP1基因过表达可以提高转基因烟草的抗盐能力。【Objective】 The study investigated the roles of KcTIP1 cloned from Kandelia candel in salt tolerance of plants.【Method】 Using young leaves of Kandelia candel as material,the KcTIP1 gene was amplified by PCR and its expression vector was constructed.Then it was integrated into tobacco plants via Agrobacterium-mediated transformation systems.Transgenic tobacco plants were selected by kanamycin and verified by genomic PCR and RT-PCR analysis.Two transgenic lines and one wild-type were treated with NaCl.Fresh weights of roots,stems and leaf tissues,net photosynthetic rates and transpiration rates were measured in KcTIP1-transgenic and wild-type tobaccos.In addition,Na＋ and K＋ concentrations in leaves were quantified and steady fluxes of Na＋ and K＋ in young roots were measured.【Result】 KcTIP1 gene was successfully cloned,and protein amino acid sequence analysis indicated that KcTIP1 contained the signature sequence SGGHVNPAVT of MIP family and two highly conserved sites of NPA（Asn-Pro-Ala） protein.RT-PCR analysis showed that the KcTIP1 gene was successfully over-expressed in transgenic lines.After salt treatment,the fresh weights,net photosynthetic rates and transpiration rates were significantly higher in KcTIP1-transgenic lines than in the wild-type plants.Na＋ and K＋ concentrations in leaf tissues were highly accumulated and higher influxes of Na＋ and K＋ in root of KcTIP1-transgenic type were observed,compared to the wild type.【Conclusion】 The protein structure and zymologic function of KcTIP1 gene were similar to that of other aquaporins.The KcTIP1 over-expression enhanced the salt tolerance of tobacco plants.

关 键 词：秋茄 烟草 KcTIP1基因 耐盐性 

分 类 号：S796[农业科学—林木遗传育种] Q785[农业科学—林学]