摘要
【目的】内根-贝壳杉烯氧化酶(KO)是赤霉素(GAs)生物合成途径关键酶,也是植物GAs生物合成抑制剂多效唑的靶酶,若GAs生物合成途径中在KO基因位点发生阻塞,会影响植株的正常生长。本研究克隆山核桃CcKO基因(Caca0733S0131)及其启动子并进行表达分析,以利于进一步探究山核桃CcKO基因在植物生长和发育过程中,尤其是与株高调控相关的生物学功能,从而助力山核桃优新种质的创制。【方法】以山核桃体细胞胚为试验材料,采用同源重组及PCR扩增技术,克隆获得山核桃KO的基因及启动子序列,并进一步构建了具有强启动子的35S∷CcKO∷GFP过表达载体和CcKOpro∷GUS启动子表达载体;利用BLAST在线工具得到该基因编码的氨基酸序列,并对其进行生物信息学分析和同源性分析;进一步通过农杆菌介导法将启动子表达载体及过表达载体转入核桃体细胞胚并进行植株再生试验,分别获得阳性再生植株,从而深入分析山核桃CcKO基因的生物学功能。【结果】通过克隆,获得1条山核桃CcKO开放阅读框,其全长为1563 bp,共编码520个氨基酸,相对分子量为59.076 kDa。氨基酸同源性分析表明,山核桃CcKO含有细胞色素P450核心功能域FXXGXRXCXG和氨基端的跨膜结构域。BLAST结果表明,山核桃CcKO氨基酸序列与核桃JrKO氨基酸序列同源性为96%,与白梨、苹果、板栗、欧洲栓皮栎的氨基酸同源性分别为71%、79%、77%、76%。对核桃体细胞胚进行遗传转化:启动子表达经GUS染色和PCR验证表明,CcKOpro∷GUS启动子表达载体被成功转入核桃体细胞胚中,且山核桃CcKO基因主要定位于维管束中;过量表达经荧光检测显示及PCR验证表明,35S∷CcKO∷GFP过表达载体被成功转入核桃体细胞胚中;表型分析结果显示,阳性再生植株株高显著高于对照,且山核桃CcKO基因的表达丰度越高,其株高越高;实时荧光定量PCR及相关分析表明,该基因表达具有
【Objective】Ent-kaurene oxidase(KO)is the key enzyme of gibberellins(GAs)biosynthetic pathway and the target enzyme of the plant GAs biosynthesis inhibitor paclobutrazol.Blocking of KO gene in the GAs biosynthetic pathway will affect the normal growth of plants.In this research,we cloned and analyzed the expression of CcKO(Caca0733S0131)gene and its promoter in Carya cathayensis,which is beneficial to further explore the function of CcKO in plant growth and development,especially the biological function related to plant height regulation,so as to help the creation of superior new germplasm of C.cathayensis.【Method】The somatic embryos of C.cathayensis were used to clone the sequence of KO gene and its promoter by homologous recombination and PCR amplification.35S∷CcKO∷GFP overexpression vectors and CcKOpro∷GUS expression vectors were further constructed.The amino acid sequence of this gene was obtained by BLAST online tool,and bioinformatics analysis and homology analysis were performed as well.Furthermore,the overexpression vector and promoter expression vector were transformed into Juglans regia somatic embryos which was mediated by Agrobacterium,and the plant regeneration experiments were carried out to obtain the positive regenerated plants to further analyze the biological functions of CcKO.【Result】A C.cathayensis CcKO open reading frame(ORF)was obtained,which was 1563 bp and encoding 520 amino acids and molecular weight was 59.076 kDa.Amino acids homology analysis exhibited that CcKO contained a core functional domain of cytochrome P450 FXXGXRXCXG and transmembrane region near the N-terminus.BLAST analysis indicated that the amino acid sequence of CcKO was 96%homologous with JrKO,while 71%,79%,77%and 76%homologous with the amino acid sequences of Pyrus bretschneideri,Malus domestica,Castanea mollissima and Quercus suber.Heterologous genetic transformation of J.regia somatic embryos:The expression vector of CcKOpro∷GUS was successfully transferred into walnut somatic embryos by GUS staining
作者
梁璧
张佳琦
任飞
胡恒康
徐川梅
胡渊渊
黄有军
娄和强
张启香
Liang Bi;Zhang Jiaqi;Ren Fei;Hu Hengkang;Xu Chuanmei;Hu Yuanyuan;Huang Youjun;Lou Heqiang;Zhang Qixiang(State Key Laboratory of Subtropical Silviculture College of Forestry and Biotechnology,Zhejiang A&F University,Hangzhou 311300)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2020年第10期70-82,共13页
Scientia Silvae Sinicae
基金
国家自然科学基金项目(31670682,31800563,31600547,31971672)
浙江省自然科学基金项目(LY18C150002)
浙江省农业(果品)新品种选育重大科技专项(2016C02052-13)。
关键词
山核桃
核桃
内根-贝壳杉烯氧化酶
启动子
遗传转化
基因表达
Carya cathayensis
Juglans regia
ent-kaurene oxidase gene
promoter
genetic transformation
gene expression
