毛竹PeDWF4基因克隆及表达模式分析  

Cloning and Expression Pattern Analysis of PeDWF4 Gene in Moso Bamboo(Phyllostachys edulis)

作　　者：王思宁[1] 孙化雨 李利超 杨意宏 徐浩 赵韩生[1] 高志民[1] WANG Si-ning;SUN Hua-yu;LI Li-chao;YANG Yi-hong;XU Hao;ZHAO Han-sheng;GAO Zhi-min(State Forestry Administration Key Open Laboratory on the Science and Technology of Bamboo and Rattan,Institute of Gene Science for Bamboo and Rattan Resources,International Center for Bamboo and Rattan,Beijing 100102,China;Hebei Agricultural University,Baoding 071001,Hebei,China)

机构地区：[1]国家林业局竹藤科学与技术重点开放实验室国际竹藤中心竹藤资源基因科学研究所,北京100102 [2]河北农业大学,河北保定071001

出　　处：《林业科学研究》2018年第5期50-56,共7页Forest Research

基　　金：林业公益性行业科研专项经费项目"毛竹核心种质重测序及竹壁发育关键基因研究"(201504106);国家科技支撑计划课题研究任务"竹藤资源收集保存与优质基因资源筛选"(2015BAD04B0101)

摘　　要：[目的]通过对毛竹PeDWF4基因结构特点和表达特征的研究,揭示其在响应逆境胁迫过程中的作用。[方法]采用同源序列比对的方法,从毛竹基因组数据库中获得DWF4同源基因信息并克隆,通过生物信息学方法分析该基因的结构、理化特征,以及基因编码蛋白的保守结构域、进化关系等,应用RT-PCR技术分析基因在毛竹不同组织中的表达情况,使用实时荧光定量PCR技术分别分析高盐、干旱、低温和强光等胁迫条件下该基因在叶片中的表达模式。[结果]从毛竹中克隆获得1个DWF4同源基因PeDWF4,编码区长度为1 503 bp,对应的基因组序列为6 149 bp,包含8个外显子和7个内含子,内含子完全符合GT-AG剪接原则。PeDWF4编码1个500 aa的碱性蛋白,属于细胞色素P450家族的单加氧酶。组织特异性表达分析表明,PeDWF4在毛竹不同组织中均检测到表达,其中,叶片中表达丰度最高,其次是根,茎、叶鞘和笋中的表达量较低。在Na Cl(400 mmol·L-1)和干旱胁迫条件下,叶片中PeDWF4的表达均先受到诱导,后受到抑制,其中,Na Cl处理下,表达量在2 h时达到最高(为对照的3. 5倍),6 h时最低(为对照的20%);干旱处理下,表达量在1 h时达到最高(为对照的2倍),8 h时最低(为对照的60%)。强光(1 200μmol·m-2·s-1)和低温(4℃)胁迫均诱导PeDWF4的表达,其中,强光处理2 h时表达量达到最高(为对照的4. 5倍),随后降低,8 h时仍为对照的2倍;低温处理下,叶片中PeDWF4表达量在1 h时达到最高(约为对照的3倍),随后持续下降,8 h时仍为对照的2倍。[结论]从毛竹中克隆了BL生物合成关键限速酶基因PeDWF4,该基因在毛竹中呈现组成型表达,在叶片中的表达受到Na Cl、干旱、低温和强光等非生物胁迫的影响,基因表达的变化表明PeDWF4可能有助于毛竹适应逆境胁迫。[Objective]This study aims to provide reference for revealing the role of steroid 22-alpha hydroxylase in moso bamboo（ PeDWF4） in the response to abiotic stresses,based on the analysis of its gene structural characteristics and expression patterns. [Method]The method of homologous sequence comparison was used to isolate the homologous gene of DWF4 in moso bamboo with the information in BambooGDB. Bioinformatic method was used to an-alyze the gene structure,the basic physical and chemical characteristics,the conservative domains in the protein encoded by the gene,and the evolutionary relationships,etc. Besides,RT-PCR was applied for the gene expression analysis in different bamboo tissues and real-time fluorescent quantitative PCR（ qRT-PCR） was conducted to find the expression patterns of the gene in leaf blades under the stresses of high salt,drought,low temperature and high light,respectively. [Result]PeDWF4,a homologous gene of DWF4,was isolated from moso bamboo,whose open reading frame is 1 503 bp,and the corresponding genome sequence is 6 149 bp containing 8 exons and 7 introns.The introns completely conformed to the principle of GT-AG splicing. PeDWF4 encoded an alkaline protein with 500 aa,belonging to the single oxygenases of cytochrome P450 family. Tissue specific expression analysis showed that PeDWF4 was detected in all tissues of bamboo roots,stems,fully expanded leaf blades,not fully expanded leaf blades,leaf sheaths and shoots with different levels,among which the highest one was in leaf blades,followed by roots,while those in stems,sheaths and shoots were relatively lower. In accordance with the transcriptome data,PeDWF4 had the highest expression level in leaf blades. Both under NaCl（ 400 mmol·L^-1） and drought stresses,the expression of PeDWF4 in leaf blade showed a trend of induction and then suppression. Under NaCl stress,it was upregulated to 3. 5 times of the control after 2 hours,then decreased gradually with the prolonged processing and reached to nearly only 20% of the control

关 键 词：毛竹 油菜素内酯 DWF4 非生物胁迫 基因表达 

分 类 号：S718.46[农业科学—林学]