马尾松PmCBL3基因的克隆及其表达分析

【目的】为明确马尾松(Pinus massoniana)CBL基因结构特征及干旱胁迫下的表达特性,探讨该基因在马尾松抗旱调控过程中的生物学功能。【方法】以马尾松优良家系为试材,采用RT-PCR和RACE方法克隆马尾松CBL基因全长cDNA,运用生物信息学方法,分析其蛋白质性质和亲缘关系,利用荧光定量PCR分析干旱胁迫下的基因表达特性。【结果】获得马尾松的一个CBL同源基因,命名为PmCBL3,该基因cDNA序列全长1 035 bp,包括681 bp的完整开放阅读框,编码226个氨基酸。PmCBL3蛋白含4个EF-hand功能域,且相邻EF-hand功能域之间的氨基酸数目非常保守,属于EFh家族蛋白。系统进化分析表明:该蛋白与其他植物CBL同源性达62%～98%,其中与北美云杉(Picea sitchensis)CBL亲缘关系最近。qRT-PCR结果显示,PmCBL3在马尾松根中表达量最高,茎和叶次之; 干旱第15天表达量最大,随干旱胁迫加剧,表达量逐渐降低。【结论】PmCBL3基因属于CBLs家族,与北美云杉亲缘关系最近。该基因响应干旱胁迫,推测其可能参与马尾松干旱逆境的应答调控。

Cloning and expression analysis of PmCBL 3 from Pinus massoniana

[Objective] CBLs participates in plants response to abiotic stress.The objectives of this study were to investigate biological function of CBL,and to provide a candidate gene and theoretical basis for the drought tolerant breeding for Pinus massoniana,by cloning the CBL from P.massoniana,and analyzing its sequence features,evolutionary relationships and expression characteristics.[Method] The full-length cDNA of CBL had been isolated from P.massoniana by RT-PCR and RACE,and bioinformatics analysis was conducted to analyze the properties and phylogenetic tree.In addition,the expression profile of CBL was analyzed by qRT-PCR under drought stress.[Result] A full-length cDNA,designated PmCBL3,was obtained.Sequence analysis revealed that PmCBL3 was 1 035 bp in length,including a 681 bp open reading frame that encodes 226 amino acids.PmCBL3 comprised four EF-Hand functional domains,and the distance between the EF-hand motifs was conservative.Evolutionary analysis further revealed that PmCBL3 had the closest relationship with Picea sitchensis,and CBL exhibited 62％-98％ homologies with other plants.qRT-PCR results showed that PmCBL3 expression was the highest in the roots,then was in the stems and leaves.PmCBL3 expression was in the peak at 15 d after treatment,but decreased with drought stress aggravation.[Conclusion] The results indicated that PmCBL3 belonged to the member of CBLs family genes,and had the closest relationship with CBL of P.sitchensis.PmCBL3 responded drought stress,which suggested that it was involved in drought stress regulation