生菜NRT2/3的生物信息及功能的初步验证  

Biological information of lettuce NRT2/3 and preliminary functional validation

作　　者：钱雨轩 武占会 刘宁 王宝驹 荣子豪 刘伟 梁浩 佟静 QIAN Yu-xuan;WU Zhan-hui;LIU Ning;WANG Bao-ju;RONG Zi-hao;LIU Wei;LIANG Hao;TONG Jing(Beijing Vegetable Research Center,Beijing Academy of Agriculture and Forestry Science/Key Laboratory of Urban Agriculture(North)of Ministry of Agriculture and Rural Affairs/National Key Laboratory of Biological Breeding,Beijing 100097,China;National Vegetable Engineering Technology Research Center,Beijing 100097,China)

机构地区：[1]北京农林科学院蔬菜研究所/农业农村部华北都市农业重点实验室/全国生物育种重点实验室,北京100097 [2]国家蔬菜工程技术研究中心,北京100097

出　　处：《植物营养与肥料学报》2024年第11期2161-2180,共20页Journal of Plant Nutrition and Fertilizers

基　　金：北京市农林科学院青年科研基金资助项目(QNJJ202248);北京市农林科学院蔬菜研究所改革与发展项目(KYCX202406);财政部和农业农村部:国家现代农业产业技术体系资助项目(CARS-24-B-02);现代农业产业技术体系北京市设施蔬菜创新团队项目(BAIC01-2024)。

摘　　要：【目的】硝态氮是旱地植物的主要氮源,NRT2/3是植物吸收硝态氮、调节氮素吸收的重要蛋白家族。本研究对生菜NRT2/3家族成员进行鉴定,并研究各成员基因理化性质、基因结构等生物信息和其在蔬菜生长中的功能。【方法】在NCBI网站(https://www.ncbi.nlm.nih.gov)查询并下载拟南芥、水稻的NRT2/3及已被注释的生菜NRT,通过生物信息学手段鉴定生菜NRT2/3家族成员,并使用DNAMAN软件对生菜和拟南芥的NRT2/3蛋白进行多序列比对,使用MEGA11软件对生菜、水稻和拟南芥的NRT2/3蛋白进行多序列比对,并构建系统发育树。通过在线软件对生菜NRT2/3家族蛋白理化性质、功能结构域、保守基序、亲水性、顺式作用元件、基因结构和染色体定位进行分析,对生菜NRT2/3家族蛋白二级结构、磷酸化位点和跨膜区域进行预测。选用‘意大利’和‘绿雅’两个品种生菜为试材,根据本课题组前期转录组测序结果选取表达量相对较高的NRT2/3基因,进行表达分析:1)将生长至20天的两个生菜品种幼苗定植于培养基质中,用4个不同浓度NO_(3)^(−)(0、0.35、0.7和11.5 mmol/L)浇灌处理,研究NRT2/3表达水平;2)利用TRV-VIGS瞬时沉默,降低该基因在两种生菜中的表达水平,然后将两种生菜移栽于正常NO_(3)^(−)水平的营养液中,生长21天后收获,调查NRT2/3家族TRV-VIGS沉默后生菜的生长状况。【结果】本研究共鉴定出8个生菜NRT2/3家族成员,包括7个LsaNRT2蛋白和一个LsaNRT3.1蛋白,它们不均匀分布在4条染色体上,其蛋白理化性质、功能结构域、跨膜区域、磷酸化位点、顺式作用元件和基因结构等基本符合NRT2/3家族共有的特征。转录组测序结果显示,两个生菜品种的LsaNRT2.4和LsaNRT3.1基因在叶片中的表达水平相对较高。LsaNRT2.4和LsaNRT3.1基因能够在一定程度上响应低浓度NO_(3)^(−)。VIGS沉默后的生菜LsaNRT2.4和LsaNRT3.1表达量均显著下降,【Objectives】Nitrate nitrogen is the primary nitrogen source for dryland plants,NRT2/3 plays a crucial role in plant nitrate absorption and the regulation of nitrogen uptake.We analyzed the members of lettuce NRT2/3 family using bioinformatics,studied their physicochemical properties,and gene structure evaluation,and assessed the functions of select NRT2/3 genes utilizing VIGS technology.【Methods】The NRT2/3 proteins from Arabidopsis and rice,as well as the annotated NRT proteins from lettuce were downloaded from the NCBI website.Bioinformatics methods were utilized to identify the members of the lettuce NRT2/3 family,and a multi-sequence alignment of the NRT2/3 proteins from lettuce and Arabidopsis was conducted using DNAMAN software.Furthermore,a multiple sequence alignment of the NRT2/3 proteins from lettuce,rice,and Arabidopsis was conducted using MEGA11 software,followed by the construction of a phylogenetic tree.Then,their coincidences of the common NRT2/3 proteins were proved in physicochemical properties,functional domains,conserved motifs,hydrophilicity,cis-acting elements,and gene structure,using online software.The chromosome localization,the secondary structure,phosphorylation sites,and transmembrane regions of lettuce NRT2/3 family proteins were predicted.Drawing from our team’s prior transcriptome sequencing findings,the NRT2/3 genes,which had comparatively high expression levels,were targeted for expression analysis:1)The seedlings of the two lettuce varieties grown to 20 days were planted on substrates and irrigated by NO_(3)^(−)0,0.35,0.7,and 11.5 mmol/L solutions for the invetigated of expression levels.2)The expression levels of these genes in the two lettuce varieties were reduced using TRV-VIGS transient silencing method,subsequently,the lettuce seedlings were grown in normal nitrogen nutrient solutions for 21 days,and then invetigated the expression levels,N uptakes and growth indexes.【Results】This study identified a total of 8 lettuce NRT2/3 family members,consisting of 7 LsaN

关 键 词：生菜 硝酸盐 生物信息 NRT VIGS 

分 类 号：S636.2[农业科学—蔬菜学]