橡胶树HbHMGR1基因克隆及其愈伤组织转化  

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

作　　者：马晓晓[1] 李哲[2] 戴雪梅[2] 毕政鸿[1] 方家林[2] 黄华孙[2] 李运合[3] 贺永国 

机构地区：[1]海南大学农学院,海口570228 [2]中国热带农业科学院橡胶研究所/农业部橡胶树生物学重点开放实验室,海南儋州571737 [3]中国热带农业科学院南亚热带作物研究所,广东湛江524091 [4]海南大学园艺园林学院,海口570228

出　　处：《南方农业学报》2014年第5期818-823,共6页Journal of Southern Agriculture

基　　金：中国热带农业科学院院本级基本科研业务费专项项目(1630022013029)

摘　　要：【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pCAMBIA2301-35S-HbHMGR1植物表达载体。再用EHA105(携带pCAMBIA2301-35S-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pCAMBIA2301-35S-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。[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.

关 键 词：橡胶树 HbHMGR1基因 易碎愈伤组织 根癌农杆菌 遗传转化 

分 类 号：S794.1[农业科学—林木遗传育种]