毛白杨PtPCP-like基因的克隆及其遗传转化初报  

Cloning and genetic transformation of PtPCP-like from Populus tomentosa

作　　者：郭斌[1] 刘军梅[1] 李英[1] 陈仲[1] 李昊[1] 叶梅霞[1] 王佳[1] 安新民[1] 

机构地区：[1]北京林业大学林木育种国家工程实验室,林木花卉遗传育种教育部重点实验室,国家林业局树木花卉育种与生物工程重点开放实验室

出　　处：《北京林业大学学报》2012年第5期31-36,共6页Journal of Beijing Forestry University

基　　金：国家自然科学基金项目(31170631);“973”国家重点基础研究发展计划项目(2012CB114505);“863”国家高技术研究发展计划项目(2011AA100201)

摘　　要：利用滤纸吸附噬菌体-PCR法,从毛白杨花芽cDNA文库中分离克隆了PtPCP-like基因cDNA全长序列,测序表明克隆得到的该序列全长595bp,包含一个完整的开放阅读框,编码91个氨基酸;经BLAST分析发现,该基因包含与拟南芥花粉外被蛋白基因相似的序列,命名为PtPCP-like。采用RT-PCR技术检测PtPCP-like基因在各个组织部位的表达模式,结果显示在毛白杨的根和雄花芽中表达丰度最高,而在雌花芽部位表达丰度最低。并且构建35S∶∶PtPCP-like植物表达载体,采用农杆菌介导法将PtPCP-like基因导入烟草中,获得了一批阳性转化植株。采用qRT-PCR技术检测PtPCP-like基因在各个转基因烟草中的表达模式,结果显示在转基因烟草中各个株系均比野生型表达量高,且不同株系间相对表达量差异显著。To understand the genetic and molecular mechanisms of floral development in Populus tomentosa,a full-length cDNA sequence of PtPCP-like gene was isolated from a male floral bud cDNA library of P. tomentosa,with the method of bacteriophage absorption by filter paper-PCR. The sequencing results indicated that the 595 bp sequence contained a complete open reading frame,which encoded 91 amino acid residuals. BLAST analysis revealed that the gene had a similar sequence domain with Arabidopsis pollen coat protein genes,therefore it was named as PtPCP-like. RT-PCR showed that PtPCP-like had higher expression abundance in male poplar floral buds and roots,and a lower abundance in female floral buds. A plant expression vector carrying 35S∶ ∶ PtPCP-like was constructed and transformed into tobacco and poplar via Agrobacterium mediated method,a batch of positive transformation plants were obtained. Expression pattern of PtPCP-like in different tobacco lines was examined using qRT-PCR. Results showed that PtPCP-like was expressed higher in all transgenic lines than in wide type,and significant expression differences were seen among different transgenic lines.

关 键 词：毛白杨 烟草 QRT-PCR PtPCP—like 

分 类 号：S718.46[农业科学—林学]