橡胶树HbHMGR1基因克隆及其愈伤组织转化

目的]克隆橡胶树HbHMGR1基因,转化橡胶树易碎愈伤组织,为橡胶树遗传转化和品种改良打下基础.方法]根据已经报道的HbHMGR1基因序列（NCBI登录号X54659.1）设计特异引物,通过RT-PCR克隆HbHMGR1基因,构建pCAMBIA2301-35 S-HbHMGR1植物表达载体.再用EHA105（携带pCAMBIA2301-35 S-HbHMGR1,内含NPTⅡ和uidA基因）侵染橡胶树易碎愈伤组织,数月筛选后对抗性易碎愈伤组织系进行GUS染色和分子检测.结果]成功克隆了HbHMGR1基因,双酶切重组质粒、重组质粒的PCR扩增结果证明成功地构建了该基因的植物表达载体pCAMBI-A2301-35S-HbHMGR1.侵染后抗性橡胶树易碎胚性愈伤组织GUS检测为蓝色,且分子检测呈现阳性,共获得15个抗性易碎愈伤组织系.结论]橡胶树HbHMGR1基因在愈伤组织阶段开始表达,有效提高了HbHMGR1基因在橡胶中的表达量.

Gene cloning of HbHMGR1 and genetic transformation of callus in Hevea brasiliensis

Objective]HbHMGR1 gene was cloned and the plant expression vector pCAMBIA2301-35S-HbHMGR1 was constructed to provide references for genetic transformation and improvement of Ⅱ.brasiliensis.Method]According to the reported sequence of HbHMGR1（NCBI accession No.X54659.1）,a pair of special primers was designed.HbHMGR1 gene was cloned successfully by RT-PCR and the vector pCAMBIA2301-35S-HbHMGR1 was constructed.Then EHA105 harboring vector pCAMBIA2301-35S-HbHMGR1 which included NPTII and uidA was used to infect friable callus of H.brasiliensis.The resistant callus was detected using GUS staining and RT-PCR after months of screening.Result]HbHMGR1 gene was cloned successfully and it was used to construct the vector of pCAMBIA2301-35S-HbHMGR1.The results of pCAMBIA2301-35S-HbHMGR1 vector by double restriction enzyme digestion and PCR showed that the recombinant vector was constructed successfully.GUS detection of resistant friable embryogenic callus showed the blue color and its molecular detection was positive.Fifteen resistant callus lines were obtained successfully.Conclusion]The target gene HbHMGR1 expressed during callus growth stage,which improved expression of HbHMGR1 gene in rubber.