马尾松PmPIN1基因的克隆及功能分析  

Cloning and Functional Analysis of PmPIN1 Gene from Pinus massoniana

作　　者：武星 胡兴峰 陈佩珍[1] 孙晓波 吴帆 季孔庶[1] Wu Xing;Hu Xingfeng;Chen Peizhen;Sun Xiaobo;Wu Fan;Ji Kongshu(Key Laboratory of Forest Genetics&Biotechnology of Ministry of Education Co-Innovation Center for Sustainable Forestry in Southern China Nanjing Forestry University,Nanjing 210037)

机构地区：[1]南京林业大学,林木遗传与生物技术省部共建教育部重点实验室,南方现代林业协同创新中心,南京210037

出　　处：《林业科学》2020年第3期184-192,共9页Scientia Silvae Sinicae

基　　金：“十三五”国家重点研发计划项目课题(2017YFD0600304);江苏高校优势学科建设工程(PAPD)项目。

摘　　要：【目的】PIN1基因通过调控生长素极性运输的方式在植物根系生长发育中发挥作用。对马尾松PmPIN1基因进行全长克隆和功能分析,有助于在分子水平揭示马尾松的生根机制。【方法】运用PCR和RACE技术成功克隆马尾松PmPIN1基因cDNA序列;利用生物信息学软件分析PmPIN1基因的核苷酸序列及其编码蛋白的氨基酸序列,并构建系统进化树;通过实时荧光定量分析PmPIN1基因在10年生马尾松幼根、1年生枝条、新叶、花中的表达量差异;利用农杆菌侵染法将PmPIN1基因转入烟草获得转基因植株,比较转基因烟草与转pCAMBIA-1302空白载体烟草之间的表型差异;用激光共聚焦显微镜观察PmPIN1-GFP荧光蛋白在转基因烟草根中的表达模式;酶联免疫法测定野生型和转基因烟草的根、根茎结合处及叶中的生长素含量;用不同浓度的生长素极性运输抑制剂1-N-萘基邻氨甲酰苯甲酸(NPA)处理野生型与转基因烟草,分析烟草根、根茎结合处、叶中的生长素含量变化;利用PEG介导法将16318-hGFP-PmPIN1重组载体转化至拟南芥原生质体中进行亚细胞定位分析。【结果】PmPIN1基因全长2914 bp,包含2085 bp的ORF,编码的蛋白由695个氨基酸组成,N端与C端均有膜运输蛋白。系统进化树显示马尾松与油松在发育树同一支上。通过实时荧光定量发现PmPIN1在不同组织中的表达量差异达到极显著,1年生枝条与幼根中表达量较高,花中最低。农杆菌介导法转化烟草获得的转基因烟草,较转空载烟草株高增长,生根量增加且根系变长。激光共聚焦显微镜观察得出转基因烟草根部中间有明显绿色荧光富集现象。酶联免疫法测定结果表明转基因烟草根、根茎结合处生长素含量高于野生型,且叶较根和根茎结合处减少。不同浓度NPA处理后,野生型烟草根中生长素含量变化达极显著,且生长素含量随NPA处理浓度的增加而减少;处理浓度为10 nm【Objective】The PIN1 gene plays a role in the growth and development of plant roots by regulating the polar transport of auxin.In this study,the full-length cloning and functional analysis of the PmPIN1 gene of Pinus massoniana was carried out in order to reveal the molecular mechanism of rooting for cutting propagation of P.massoniana.【Method】The cDNA sequence of PmPIN1 gene of P.massoniana was successfully cloned by PCR and RACE.Bioinformatics software was used to analyze the nucleic acid sequence,amino acid sequence and construct a phylogenetic tree of PmPIN1 gene.The expression of PmPIN1 gene in young roots,annual branch,young leaf and flowers of 10-year-old P.massoniana was analyzed by real-time polymerase chain reaction.The PmPIN1 gene was overexpressed in tobacco(Nicotiana tabacum)by Agrobacterium infection method to obtain transgenic plants,and the phenotypic differences between transgenic plants and pCAMBIA-1302 blank vector were compared,and PmPIN1-GFP fluorescent protein was observed in transgenic tobacco roots by laser scanning confocal microscope.The expression of auxin in roots,rhizomes and leaves of transgenic tobacco and wild tobacco was determined by enzyme-linked immunosorbent assay(ELISA).Treatment of wild-type and transgenic tobacco with different concentrations of auxin polar transport inhibitor(1-N-naphthylphthalamic acid,NPA),the changes of auxin content in tobacco roots,rhizome junctions and leaves were analyzed.16318-hGFP-PmPIN1 recombinant vector was transferred to Arabidopsis thaliana protoplasts by PEG-mediated method for subcellular localization analysis.【Result】The PmPIN1 gene is 2914 bp in length and contains a 2085 bp ORF,which encodes a protein consisting of 695 amino acids,with a membrane transport protein at the N-terminus and C-terminus.The phylogenetic tree showed that P.massoniana and P.tabulaeformis are on the same branch of the development tree.The difference in expression of PmPIN1 in different tissues was found to be extremely significant by real-time polymeras

关 键 词：马尾松 PmPIN1基因 转基因 功能分析 

分 类 号：S718.46[农业科学—林学]