马尾松β-蒎烯合酶基因克隆以及对松材线虫侵染的响应  

Cloning ofβ-pinene Synthase Gene in Pinus massoniana and Its Response to Pine Wood Nematode Infection

作　　者：刘彬 刘青华[1] 周志春[1] 罗柠 解懿妮 陈献志 LIU Bin;LIU Qing-hua;ZHOU Zhi-chun;LUO Ning;XIE Yi-ni;CHEN Xian-zhi(Research Institute of Subtropical Forestry,Chinese Academy of Forestry,Key Laboratory of Tree Breeding of Zhejiang Province,Hangzhou 311400,Zhejiang,China;Natural Resources and Planning Bureau of Linhai City,Zhejiang Province,Linhai 317000,Zhejiang,China)

机构地区：[1]中国林业科学研究院亚热带林业研究所,浙江省林木育种技术研究重点实验室,浙江杭州311400 [2]浙江省临海市自然资源和规划局,浙江临海317000

出　　处：《林业科学研究》2020年第6期1-12,共12页Forest Research

基　　金：“十三五”浙江省林木新品种选育重大专项课题(2016C02056-4);国家自然科学基金项目(31470670);江西省林业厅林业科技创新专项项目(201703);中央级公益性科研院所基本科研业务费专项资金项目(CAFYBB2017ZA01-2)。

摘　　要：[目的]对马尾松β-蒎烯合酶基因PmPinS进行克隆,分析其序列特征和高抗易感马尾松中松材线虫侵染后的表达模式,为解析马尾松抗松材线虫病机制提供理论基础。[方法]采用PCR扩增技术克隆马尾松Pm-PinS的全长编码区序列,进行同源氨基酸序列比对和系统发育分析,利用生物信息学方法分析其基因特征;利用实时定量PCR技术分析马尾松蒎烯合酶基因在接种松材线虫后的高抗和易感马尾松中的表达模式。[结果]克隆获得马尾松蒎烯合酶基因的全长编码区序列,包含1878 bp的完整开放阅读框,编码625个氨基酸,分子量为71.95 kD,二级结构以α-螺旋和β-折叠为主,含有萜类合酶典型的RR(X)8W、DDXXD和NSE/DTE结构域。系统进化分析发现,PmPinS与扭叶松(-)β-蒎烯合酶聚为一支,序列相似性达93.16%。对高抗和易感马尾松PmPinS在松材线虫接种后的表达模式进行分析发现,PmPinS在高抗马尾松中的表达量显著高于易感马尾松,且在接种30 d内呈现上升趋势,而易感马尾松PmPinS在接种1 d后迅速下降,在接种2 a后,PmPinS表达量在高抗马尾松茎部组织特异性高表达。对松材线虫外源施加α-蒎烯、β-蒎烯及其混合溶液,结果表明:α-蒎烯与β-蒎烯可明显抑制松材线虫存活率,且混合溶液抑制作用更明显。[结论]PmPinS为马尾松单萜合酶家族的一员,复杂催化GPP参与β-蒎烯的合成,该基因在高抗马尾松茎部组织特异性高表达,同时其可能的催化产物α-蒎烯与β-蒎烯对松材线虫具有明显的抑制作用,表明其在马尾松抗松材线虫过程中起正向调控作用。[Objective]To cloneβ-pinene synthase gene in Pinus massoniana(PmPinS)and analyze its sequence characteristics and expression pattern after pine wood nematode(PWN)infection in order to provide theoretical basis to probe into the defense mechanism of P.massoniana to pine wood nematode infection.[Method]Based on PCR amplification technology,the full-length coding region sequence of PmPinS was cloned,the homologous amino acid sequence alignment,and the phylogenetic and genetic characteristics were analyzed with bioinformatics method.The expression pattern of PmPinS in resistant and susceptible P.massoniana after inoculation with PWN was analyzed with real-time quantitative PCR.[Result]The full-length coding region sequence of PmPinS was cloned,including a complete open reading frame of 1878 bp,encoding a 625 amino acid,with a molecular weight of 71.95 kD.The secondary structure wasα-helix andβ-fold,containing the typical terpenoid synthase domains including RR(X)8W,DDXXD and NSE/DTE.Phylogenetic analysis revealed that PmPinS and(-)β-pinene synthase in P.contortus were aggregated into one branch with a sequence similarity of 93.16%.The expression of PmPinS in resistant P.massoniana was higher than the susceptible ones after PWN inoculation,which showed an up-regulation trend within the following 30 days after inoculation.However,the expression of PmPinS in susceptible P.massoniana began to fell rapidly 1 day after inoculation.2 years after inoculation,the expression level of PmPinS was highly expressed in the stem tissue of resistant P.massoniana.In addition,exogenous application ofα-pinene,β-pinene and their mixed solutions to PWN showed thatα-pinene andβ-pinene could significantly inhibit the survival rate of PWN,especially for the mixed solution with more inhibition.[Conclusion]PmPinS,as a member of the monoterpene synthase family of P.massoniana,may be involved in the synthesis ofβ-pinene with GPP catalyzed intricately.The PmPinS is highly specific-tissue expressed in the stem of resistant P.massoniana,an

关 键 词：β-蒎烯合酶 马尾松 松材线虫病 抑制作用 

分 类 号：S718.46[农业科学—林学] S763