盐碱胁迫诱导的猴樟酵母cDNA文库构建及CbP5CS上游调控因子筛选  

Construction of cDNA Library of Cinnamomun bodinieri Induced by Saline-alkali Stress and Screening of CbP5CS Upstream Regulators

作　　者：韩浩章[1] 张丽华[1] 李素华[1] 赵荣 王芳[1] 王晓立[1] HAN Hao-zhang;ZHANG Li-hua;LI Su-hua;ZHAO Rong;WANG Fang;WANG Xiao-li(Suqian University,Suqian 223800)

机构地区：[1]宿迁学院,宿迁223800

出　　处：《生物技术通报》2023年第9期236-245,共10页Biotechnology Bulletin

基　　金：江苏省自然科学基金面上项目(BK20201481),宿迁学院重点建设学科项目(2021ZDJS04),宿迁学院创新团队项目(2021td05)。

摘　　要：盐碱土壤环境是限制猴樟引种推广的主要因素,脯氨酸是植物适应盐碱胁迫过程中主要的渗透调节物质,筛选并鉴定出调控脯氨酸合成的转录因子对于猴樟耐盐碱分子机理研究具有重要意义。以猴樟实生苗为材料,在水培培养基础上,采用0 mmol/L和10 mmol/L的Na_(2)CO_(3)溶液分别进行处理,选取处理6 h、48 h的根系组织,提取总RNA,构建盐碱胁迫诱导的猴樟根系酵母c DNA文库;以猴樟根系总DNA为模板,克隆CbP5CS启动子序列,采用Y1H酵母单杂交技术筛选出与CbP5CS启动子存在互作的蛋白,并通过高通量测序技术鉴定出调控猴樟脯氨酸合成的转录因子。结果表明,所构建的cDNA文库库容为4.88×10^(7)CFU/mL,总克隆数为9.76×10^(7)CFU,文库插入片段平均长度在1000 bp左右,cDNA片段重组率为100%,符合酵母杂交试验的要求。克隆出的CbP5CS启动子序列长2012 bp,成功构建出诱饵质粒pHIS2-CbP5CS,利用共转化方法从文库中筛选到31个与CbP5CS互作的EST序列。经酵母回转验证,31个EST序列均与CbP5CS存在互作。经NGS测序和BLAST比对搜索,获得6个转录因子基因,分别为锌指CCCH结构域蛋白25异构体x1、AtbHLH104、转录因子TFIIIC、RING/FYVE/PHD锌指超家族蛋白、GATA型锌指转录因子家族蛋白、AtbHLH96。以上结果为进一步研究植物脯氨酸代谢响应盐碱胁迫的分子机制奠定基础。The saline-alkali soil is a major factor limiting the introduction and popularization of Cinnamomun bodinieri.Proline is one of the most important compatible solutes that plants accumulate for osmotic adjustment in response to saline-alkali stress.Therefore,it is of great significance to identify the key transcription factors that regulate the biosynthesis of proline and illustrate the underlying molecular mechanisms.In the present study,the seedlings were treated with 0 and 10 mmol/L Na_(2)CO_(3) solution on the basis of hydroponic culture,respectively.Total RNA was extracted from the root tissues at 6 and 48 h after treatment to construct the yeast cDNA library induced by saline-alkali stress.The promoter sequence of CbP5CS was cloned using total DNA of C.bodinieri root system as template.The titer of the library was 4.88×10^(7)CFU/mL,and the total clone number was 9.76×10^(7)CFU.The average size of the inserts was 1000 bp,and the recombination efficiency was 100%,which met the requirements of yeast hybridization test.The length of the cloned promoter of CbP5CS was 2012 bp,and the decoy plasmid pHIS2-CbP5CS was constructed successfully.Then the promoter region of CbP5CS was cloned and the CbP5CS promoter interacting proteins was screened by yeast one hybrid and high-throughput sequencing.The 31 unique CbP5CS-interatcting ESTs were identified from the library by co-transformation,32 EST sequences were interacted with CbP5CS verified via yeast rotation.Six of them were annotated as transcription factors,including GW020491(zinc finger CCCH domain-containing protein 25 isoform X1),GW028183(AtbHLH104),GW000650(transcription factor TFIIIC),GW007525(RING/FYVE/PHD zinc finger superfamily protein),GW015686(GATA type zinc finger transcription factor family protein),GW027120(AtbHLH96).These results provide a basis for further study on the molecular mechanism of plant proline metabolism in response to saline-alkali stress.

关 键 词：盐碱胁迫 酵母cDNA文库 CbP5CS 猴樟 上游调控因子 转录因子 

分 类 号：S792.23[农业科学—林木遗传育种]