板栗外生菌根诱导基因CmNRT3的表达及功能研究  

Expression and function of ectomycorrhizal induced gene CmNRT3 in Chinese chestnut

作　　者：任艳艳 李子平 何玉吉 张昊琳 王韵清 张卿[1] 肖婷婷 李虎臣 曹庆芹[1] Huchen;CAO Qingqin(College of Plant Science and Technology,Beijing University of Agriculture/Beijing Key Laboratory for Agricultural Application and New Technique,Beijing 102206,China)

机构地区：[1]北京农学院植物科学技术学院·农业应用新技术北京市重点实验室,北京102206

出　　处：《果树学报》2024年第10期2014-2024,共11页Journal of Fruit Science

基　　金：北京市基金市教委联合项目(KZ20231002035)。

摘　　要：【目的】外生菌根是板栗获取土壤氮素的重要途径,但目前外生菌根对提高板栗氮素吸收和利用的分子机制尚不明确。探明板栗外生菌根诱导上调的硝酸盐转运蛋白基因CmNRT3的序列特征、表达模式及相关功能,将为外生菌根促氮吸收提供理论依据。【方法】通过转录组数据分析、荧光定量PCR、瞬时转化体系及酵母互补等方法研究CmNRT3基因的表达特征和生理功能。【结果】CmNRT3基因在板栗外生菌根中显著上调表达。在未接种板栗及苜蓿转基因根系的表皮细胞中检测到CmNRT3启动子驱动的GUS信号,而在苜蓿的丛枝菌根中,GUS信号主要存在于含有丛枝的皮层细胞中。亚细胞定位结果显示CmNRT3定位于细胞膜及苜蓿含有丛枝的细胞膜上。酵母互补试验表明,CmNRT3转运蛋白不能互补硝酸盐转运缺陷型酵母的功能。【结论】CmNRT3受外生菌根诱导表达,定位于细胞膜上。CmNRT3启动子驱动的GUS信号在含丛枝的苜蓿根系皮层细胞中强烈表达,但不具备硝酸盐吸收或转运功能。该研究为进一步揭示板栗外生菌根促氮吸收提供了理论基础。【Objective】Ectomycorrhizae is an important microbial that enables chestnut trees to obtain limited soil nutrients,such as nitrogen.The molecular mechanism involved in nitrogen absorption and utilization by ectomycorrhizae has not yet been clarified in chestnut.In this study,the highly induced nitrate transporter gene CmNRT3 of Chinese chestnut in response to ectomycorrhizal symbiosis was identified and its spatial expression pattern and protein function were further analyzed.【Methods】The plant materials used were Chinese chestnut Jingshuhong,which produced healthy,pest-free fruits,and Medicago truncatula ecotype A17 and Nicotiana benthamiana,which were propagated in our laboratory.The ectomycorrhizal fungus selected was Scleroderma citrinum,obtained by propagation on P20 medium.The arbuscular mycorrhizal fungus used was spores of Rhizophagus irregularis,provided by the arbuscular mycorrhizal fungi germplasm resource center of Beijing Academy of Agriculture and Forestry Sciences.△ynt-Leu-yeast lacking of nitrate transportation ability was provided by Yuan Lixing's research group(China Agricultural University),and cultured in YNB yeast medium.To investigate which chestnut NRTs were involved in ectomycorrhizal symbiosis,the excavation and expression analysis of Chinese chestnut NRT gene family in both control and ectomycorrhizae colonized roots were conducted.The CmNRTs were identified using protein sequence BLAST of Arabidopsis and rice NRTs against the chestnut genome,and MEGA7.0.14 software for gene sequence alignment and evolutionary analysis was employed.The high induction of CmNRT3 in chestnut ectomycorrhizal roots was confirmed using quantitative real-time PCR(BIO-RAD,USA).Primers used were designed by Primer 3(v.0.4.0)(http://bioinfo.ut.ee/primer3-0.4.0/).Primer sequences of CmNRT3 were CmNRT3-F:5'-GTCTAGCTGTAACTTGTTATGGA-3',CmNRT3-R:5'-CTGGCAAACTCTGGTTTAGA-3'.Primer sequences of CmACTIN as a reference gene were CmACTIN-F:5'-GTGGCGGTTCAACCATGTTC-3',CmACTINR:5'-GGATGGACCACTCTCATCGT-3'.Further,a 1.6

关 键 词：板栗 外生菌根 CmNRT3 硝酸盐吸收转运 基因功能 

分 类 号：S664.2[农业科学—果树学]