水曲柳CUC1基因克隆及瞬时表达分析  

Cloning and Transient Expression Analysis of CUC1 Gene from Fraxinus mandshurica Rupr.

作　　者：崔靖弘 于磊[1,2] 梁楠松 宋婷婷[1,2] 吕义品 纪欣童 徐亮 赵福江 詹亚光[1,2] CUI Jing-hong;YU Lei;LIANG Nan-song;SONG Ting-ting;LYU Yi-pin;JI Xin-tong;XU Liang;ZHAO Fu-jiang;ZHAN Ya-guang(Key Laboratory of Saline-alkali Vegetation Ecology Restoration,Ministry of Education,Northeast Forestry University,Harbin 150040,Heilongjiang,China;Heilongjiang Touyan Innovation Team Program(Tree Genetics and Breeding Innovation Team),Harbin 150040,Heilongjiang,China;Linjiang Forestry Bureau,Jilin Province,Linjiang 134600,Jilin,China)

机构地区：[1]东北林业大学东北盐碱植被恢复与重建教育部重点实验室,黑龙江哈尔滨150040 [2]黑龙江省头雁创新团队计划(林木遗传育种创新研究团队),黑龙江哈尔滨150040 [3]吉林省临江林业局,吉林临江134600

出　　处：《林业科学研究》2022年第1期82-91,共10页Forest Research

基　　金：黑龙江省应用技术研究与开发计划项目(GA19B201)。

摘　　要：[目的]本研究克隆了水曲柳(Fraxinus mandshurica Rupr.)CUC1基因,分析其表达特性,为该基因在水曲柳再生的调控研究中奠定基础。[方法]从水曲柳苗克隆FmCUC1基因,利用生物信息学软件分析Fm-CUC1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;利用农杆菌介导法将FmCUC1基因转入洋葱内表皮细胞进行亚细胞定位,通过实时荧光定量分析FmCUC1基因在根、茎、叶、顶芽的组织表达特异性及FmCUC1基因在下胚轴芽再生与种子萌发过程中的差异表达;同时对水曲柳幼苗喷施IAA、6-BA、BR激素处理,分析FmCUC1基因在不同激素信号诱导下的表达模式;利用农杆菌介导法将FmCUC1基因瞬时转化水曲柳72 h后,分析其通路相关基因表达情况。[结果]克隆得到FmCUC1基因全长807 bp,编码269个氨基酸,FmCUC1为稳定疏水蛋白,含有保守的NAC-domain蛋白结构域;FmCUC1蛋白与同科的木犀榄蛋白序列相似性为86.17%,亲缘关系较近;FmCUC1蛋白定位于细胞核上。qRT-PCR分析表明:FmCUC1基因在水曲柳的顶芽中表达量最高;在下胚轴芽再生过程中,FmCUC1基因在芽点产生与丛枝形成期均高表达;在水曲柳种子萌发过程中,FmCUC1基因分别在第4天和第8天分别达到两个峰值,为第0天的8.56倍和8.46倍。外源喷施IAA、6-BA、BR激素处理结果显示,FmCUC1基因表达量与对照相比均呈现上调表达,在IAA、BR处理72 h后均达到最高值,分别为对照的45.72倍和20.36倍;在6-BA处理48 h达到峰值,为对照59.40倍。利用农杆菌介导水曲柳FmCUC1基因瞬时过表达72 h后,该基因表达量显著上调,且其下游STM基因的表达量也明显上升。[结论]FmCUC1基因属于NAC家族转录因子,参与水曲柳芽再生过程,响应了IAA、6-BA、BR植物激素信号诱导,过表达FmCUC1基因可激活其下游STM基因的表达,进而有利于顶端分生组织的形成;为进一步研究CUC1基因在水曲柳芽再生过程中的作用�[Objective]To clone the CUC1 gene of Fraxinus mandshurica and analyze its expression characteristics,so as to lay a foundation for the regulation of the gene in the regeneration of F.mandshurica[Method]The Fm-CUC1 gene was cloned from F.mandshurica seedlings.The nucleotide sequence of FmCUC1 gene and the amino acid sequence of its coding protein were analyzed by bioinformatics software,and the phylogenetic tree was constructed.The FmCUC1 gene was transferred into onion inner epidermis cells by Agrobacterium tumefaciens infection method for subcellular localization.The tissue surface of FmCUC1 gene in root,stem,leaf and apical bud was analyzed by real-time fluorescence quantitative analysis.At the same time,the seedlings of F.mandshurica were sprayed with IAA,6-BA and BR hormones to analyze the expression pattern of FmCUC1 gene induced by different hormone signals;the FmCUC1 gene was transiently transformed into F.mandshurica by A.tumefaciens for 72 hours.[Result]The full length of FmCUC1 gene was 807 bp,encoding 269 amino acids.FmCUC1 was a stable hydrophobic protein with a conserved NAC domain protein domain.The sequence similarity of FmCUC1 protein and Olea europaea subsp.europaea protein was 86.17%,which was close to each other.FmCUC1 protein was located in the nucleus.q-RT PCR analysis showed that the expression of FmCUC1 gene was the highest in the terminal bud of F.mandshurica;during hypocotyl bud regeneration,the expression of FmCUC1 gene was high in both bud point formation and clump formation;during seed germination,the expression of FmCUC1 gene reached two peaks on the 4th and 8th day,which were 8.56 and 8.46 times of that on the first day.The results of exogenous spraying IAA,6-BA and BR showed that the expression of FmCUC1 gene was up-regulated compared with the control,and reached the highest value after 72 h of IAA and BR treatment,which were 45.72 times and 20.36 times of the control,respectively,and reached the peak value after 48 h of 6-BA treatment,which was 59.40 times of the control.Agrobacter

关 键 词：水曲柳 FmCUC1基因 生物信息学分析 瞬时转化 表达模式分析 

分 类 号：S792.41[农业科学—林木遗传育种]