水稻低亲和性阳离子转运蛋白基因OsLCT3的克隆与功能研究  

Cloning and Functional Analysis of OsLCT3,a Low-affinity Cation Transporter Gene of Rice

作　　者：李兴容 谭志兵 赵燕[3] 李曜魁 赵炳然 唐丽[1,2] LI Xing-rong;TAN Zhi-bing;ZHAO Yan;LI Yao-kui;ZHAO Bing-ran;TANG Li(Longping Branch,College of Biology,Hunan University,Changsha 410125;State Key Laboratory of Hybrid Rice,Hunan Hybrid Rice Research Center,Changsha 410125;College of Bioscience and Technology,Hunan Agricultural University,Changsha 410125)

机构地区：[1]湖南大学生物学院隆平分院,长沙410125 [2]湖南杂交水稻研究中心杂交水稻全国重点实验室,长沙410125 [3]湖南农业大学生物科学技术学院,长沙410125

出　　处：《生物技术通报》2024年第4期97-109,共13页Biotechnology Bulletin

基　　金：湖南省自然科学基金项目(2022JJ30034);国家自然科学基金项目(32201727);湖南省农业科技创新资金项目(2022CX19);科技“揭榜挂帅”湖南省重大科技攻关项目(2022NK1010)。

摘　　要：【目的】部分稻米镉超标严重影响我国粮食质量安全,本研究旨在鉴定对稻米镉积累有调控作用的基因,为阻控稻米镉积累提供新的基因资源。【方法】通过逆转录PCR和RACE技术,克隆了水稻低亲和性阳离子转运蛋白基因家族的一个新成员OsLCT3。通过生物信息学方法对OsLCT3的自然变异进行分析,对OsLCT3蛋白的理化性质进行预测;利用实时荧光定量PCR分析其全生育期表达模式及对镉、锰、铁胁迫的响应;采用融合报告基因定位法探究OsLCT3的亚细胞定位。通过苗期镉胁迫水培以及镉污染土壤的成熟期植株各部位的镉及二价矿质金属元素测定,分析敲除OsLCT3对水稻二价阳离子运输的影响。此外,通过酵母细胞的异源功能互补验证OsLCT3对酵母镉耐受性的影响。【结果】OsLCT3仅存在于部分水稻品种中,编码区全长1263 bp,根据编码区变异其氨基酸序列可划分为5个单倍型,编码的蛋白具有12个跨膜结构域,与小麦、节节麦的LCT亲缘关系较近,与水稻OsLCT_(2)的同源性仅52%,与籼稻、粳稻两个亚种OsLCT_(1)的同源性分别为49%、47%。OsLCT3在生长发育的各时期均在根部高表达,在扬花期的表达最高,且在根部的表达受镉胁迫及过量铁、锰的抑制,蛋白定位于质膜。与野生型相比,oslct3敲除系苗期株高降低,地上部的镉、铁、锌含量降低,根部铁含量升高,其他元素含量不变;根部镉、铁、锌向地上部的转运率降低。大田条件下,oslct3敲除系成熟期的茎叶和糙米镉含量较野生型均显著下降,稻草和糙米锰、铜、铁、锌含量较对照无显著差异。表达OsLCT3导致酵母对镉胁迫更敏感。【结论】OsLCT3参与根部的镉、铁、锌向地上部的运输,正调控稻米镉积累。【Objective】Excessive cadmium(Cd)in some rice grains seriously affect food safety in China.The aim of this study is to identify the novel gene that regulate accumulation of Cd in rice grains and provide the genetic resource for reducing Cd accumulation in rice grains.【Method】OsLCT3,a member of the low-affinity cation transporter family in rice,was cloned by reverse transcription PCR and RACE techniques.Natural variations of OsLCT3 and physicochemical properties of its coding protein were analyzed by bioinformatics methods.Its expression profiles throughout the growth period and under Cd,Mn,Fe stress were analyzed by quantitative real-time PCR(RT-qPCR).Its subcellular localization was explored by investigating localization of OsLCT3-GFP fusion protein in in rice protoplasts.The effect of OsLCT3 knockout on divalent cation transport in rice was analyzed by measuring cadmium and other divalent mineral metal elements in various parts of plants at seedling stage in cadmium-stressed hydroponics and at maturity stage in cadmium-polluted soil.In addition,the effect of OsLCT3 on the yeast tolerance to Cd was verified by heterologous functional complementarity in yeast cells.【Result】OsLCT3 only existed in some indica and japonica rice varieties,with a total coding region of 1263 bp.The amino acid sequences were classified into five haplotypes based on coding region variation,and the encoded proteins had 12 sub-transmembrane structural domains.OsLCT3 was similar to the LCT-like proteins in Triticum aestivum and Aegilops tauschii,whereas it shared only 52%sequence identity with OsLCT_(2),49%and 47%sequence identity with OsLCT_(1) in indica and japonica rice subspecies,respectively.OsLCT3 was highly expressed in the roots at all stages of growth and development.Its expression levels in the roots were suppressed by Cd,excessive iron(Fe)and manganese(Mn)stress.OsLCT3 was localized to the plasma membrane.Compared with the wild type plants,the oslct3 knockout lines showed the reduced plant height,the lower concentratio

关 键 词：水稻 OsLCT3 镉转运 铁转运 锌转运 金属转运蛋白 

分 类 号：S511[农业科学—作物学] Q943.2[生物学—植物学]