沙棘转录因子HrWRKY53基因分离及其参与抵御绕实蝇的作用分析  

Isolation of Transcription Factor HRWRKY53 Gene from Hippophae rhamnoides and Analysis of Its Role in Resisting Rhagoletis batava obseuriosa

作　　者：姚莹 曹金峰[2] 陆世曈 丁文彬 张一文 魏建荣[1] 刘建凤[1] Yao Ying;Cao Jinfeng;Lu Shitong;Ding Wenbin;Zhang Yiwen;Wei Jianrong;Liu Jianfeng(College of Life Science,Hebei University Institute of Life Science and Green Development,Hebei University,Baoding 071002;Hebei Key Laboratory of Crop Salt-Alkali Stress Tolerance Evaluation and Genetic Improvement,Cangzhou Academy of Agriculture and Forestry Sciences,Cangzhou 061001)

机构地区：[1]河北大学生命科学学院,河北大学生命科学与绿色发展研究院,保定071002 [2]河北省农作物耐盐碱评价与遗传改良重点实验室,沧州市农林科学院,沧州061001

出　　处：《林业科学》2022年第5期121-130,共10页Scientia Silvae Sinicae

基　　金：河北省自然科学基金项目(C2020201001)。

摘　　要：【目的】明确HrWRKY53基因的生物信息学及其参与沙棘抗绕实蝇功能,为解析沙棘等胡颓子科植物抗虫机制及分子育种奠定基础。【方法】基于绕实蝇危害前后沙棘果实转录组数据,挖掘HrWRKY基因家族成员中参与绕实蝇危害的关键差异表达基因HrWRKY53,进一步分析该基因的二级结构、亲疏水性及预测磷酸化位点等。利用BLASTP检索同源性高的其他物种蛋白序列进行比对并构建系统发育树;利用qRT-PCR检测该基因的组织表达特异性,绕实蝇危害前后以及脱落酸(Abscisic acid,ABA)、茉莉酸甲酯(Methyl jasmonate,MeJA)、乙烯(Ethylene,ETH)处理下的该基因表达模式;并进一步分析响应绕实蝇危害的茉莉酸(Jasmonic acid,JA)信号途径上关键基因HrJA2、HrLOX1、HrAOS和HrAOC的表达水平,分析其与HrWRKY53基因表达的相关性。【结果】基于沙棘转录组获得HrWRKY53的开放阅读框(Open reading frame,ORF)全长为1302 bp,编码433个氨基酸残基的蛋白,该基因含有2个WRKY保守结构域和1个C_(2)H_(2)型锌指结构,属于第Ⅰ类WRKY转录因子家族;二级结构预测其编码的蛋白主要由无规则卷曲构成,含有141个磷酸化位点,推测可能与磷酸化有密切的关系,亲疏水性预测表明该蛋白属于疏水性蛋白;系统发育树分析显示该蛋白与蔷薇科野草莓FvWRKY25同源性最高。qRT-PCR显示该基因具有明显的组织表达特异性,其在沙棘果实中的表达量最高;绕实蝇危害与机械损伤后该基因的表达量均显著上升,激素ABA、MeJA、ET处理后,基因的表达量也呈明显上升的趋势,其中MeJA诱导后,该基因的表达量上升最显著;另外,基于转录组数据及荧光定量数据对JA信号通路的关键差异表达基因分析发现,其通路上的HrJA2基因表达量提高最显著,且HrWRKY53与HrJA2基因表达量变化呈明显的正相关。【结论】沙棘HrWRKY53属于第Ⅰ类WRKY转录因子家族,且与蔷薇科草莓FvWRKY25同源�【Objective】This study aims to clarify the bioinformatics of HrWRKY53 gene and its role in sea-buckthorn resisting Rhagoletis batava obseuriosa(RBO),so as to lay a foundation for the analysis of insect resistance mechanism and molecular breeding of Elaeagnaceae such as Hippophae rhamnoides.【Method】Based on the transcriptome data of sea-buckthorn fruits before and after RBO infestation,the key differentially expressed gene HrWRKY53 involved in the damage of sea-buckthorn were explored from members of HrWRKY gene family.Bioinformatics analysis was carried out to analyze the secondary structure and hydrophobicity and to predict the phosphorylation sites.In the NCBI website,the protein sequences with high homology were searched by BLASTP for sequence alignment,and the phylogenetic tree was constructed.qRT-PCR was used to detect the tissue expression specificity of the gene and the gene expression pattern before and after RBO infestation and under the treatment of abscisic acid(ABA),methyl jasmonate(MeJA)and ethylene(ET).The expression levels of HrJA2,HrLOX1,HrAOS and HrAOC in the JA signal pathway in response to the damage of RBO were analyzed,and their correlation with the expression of HrWRKY53 was was analyzed.【Result】The open reading frame(ORF)of HrWRKY53 gene obtained from sea-buckthorn transcriptome was 1302 bp,which encoded 433 amino acid residues.HrWRKY53 protein included two WRKY conserved domains and C2H2 zinc finger structures,and belonged to the family of type I WRKY transcription factors.The secondary structure prediction showed that HrWRKY53 was mainly composed of random curl and contained 141 threonine phosphorylation sites,which could be closely related to phosphorylation.The hydrophobicity prediction showed that the protein belonged to a hydrophobic protein.Phylogenetic tree analysis displayed that the gene had the highest homology with FvWRKY25.qRT-PCR showed that the gene had obvious tissue expression specificity,and its expression was the highest in sea-buckthorn fruit.The gene expressi

关 键 词：沙棘 绕实蝇 HrWRKY转录因子家族 HrWRKY53 激素处理 

分 类 号：S718.46[农业科学—林学]