小立碗藓PpFLS2基因序列分析及敲除载体的构建  

Sequence Analysis and Construction of Knockout Vector for PpFLS2 of Physcomitrella patens

作　　者：吴美勤 闫慧清 姜山 Wu Meiqin;Yan Huiqing;Jiang Shan(School of Life Sciences,Guizhou Normal University,Guiyang,550001;School of International Education,Guizhou Normal University,Guiyang,550001)

机构地区：[1]贵州师范大学生命科学学院,贵阳550001 [2]贵州师范大学国际教育学院,贵阳550001

出　　处：《分子植物育种》2023年第14期4666-4673,共8页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31260426,31560508,32060611)资助。

摘　　要：为了从分子水平上揭示小立碗藓PpFLS2基因的结构特点以及是否参与了由灰霉菌病原体相关分子模式(PAMP)引起的小立碗藓防御反应,对PpFLS2基因做了生物信息学分析并构建PpFLS2-PTN182敲除载体,为研究其功能和早期登陆植物防卫的进化提供依据。利用生物信息学手段,对PpFLS2基因序列和结构进行了分析和预测。提取小立碗藓DNA,PCR扩增PpFLS2基因的上下游片段,使用SalⅠ、Eco R V,SphⅠ、Bam HⅠ酶分别酶切上下游目的片段,同时酶切PTN182,经T_(4)-DNA酶连接,转入大肠杆菌感受态,筛选出阳性克隆,经酶切验证并测序,获得了完整的PpFLS2-PTN182敲除载体。生物信息学分析显示,PpFLS2的DNA序列全长2541 bp,有一个外显子,一个开放阅读框;该蛋白质属于不稳定、亲水性蛋白;蛋白的二级结构存在39.3%的α-螺旋,2.63%的β-转角,11.86%的β-折叠,46.21%的无规则卷曲;系统进化分析结果表明,可能小立碗藓的PpFLS2同源基因在进化过程中发生了功能的较大分化。经PCR检测、酶切验证和测序分析,表明PpFLS2-PTN182敲除载体构建成功。通过生物信息学分析和敲除载体构建成功,研究结果可为后续对PpFLS2的功能研究提供理论基础。In order to reveal the structural characteristics of the PpFLS2 of Physcomitrella patens at the molecular level,we carried out t he sequence analysis and construction of knockout vector for PpFLS2 of Physcomitrella patens,providing the basis for the defense response of Physcomitrella patens caused by the Botrytis cinerea pathogen-associated molecular pattern(PAMP),the sequence and structure of PpFLS2 were analyzed and predicted by bioinformatics.A complete PpFLS2-PTN182 knockout vector was obtained by extracting the DNA of Physcomitrella patens,amplifying the upstream and downstream fragments of PpFLS2 by PCR,using SalⅠ,EcoR V and SphⅠ,BamHⅠ to digest the upstream and downstream respectively,digesting the PTN182,connecting them with T_(4)-DNA,transferring to Escherichia coli.Finally,the knockout PpFLS2-PTN182 was verified through screening positive clones,identifying by digestion and sequencing.Bioinformatics analysis showed that the full-length DNA sequence of PpFLS2 was 2541 bp,with an exon and an open reading frame;this protein belongs to unstable and hydrophilic protein;there are 39.3% α-helix,2.63% β-turn,11.86% extended strand and 46.21% random coil in the secondary structure of the protein;the result of the phylogenetic analysis showed that the PpFLS2 homologous gene of Physcomitrella patens had great functional differentiation in the evolution process.PCR detection,digestion verification and sequencing analysis showed that PpFLS2-PTN182 knockout vector was successfully constructed.Through bioinformatics analysis and successful construction of knockout vectors,the research results can provide a theoretical basis for the subsequent functional research of PpFLS2.

关 键 词：小立碗藓 PpFLS2 生物信息学 载体构建 

分 类 号：Q943.2[生物学—植物学]