矮牵牛花香基因BSMT3的cDNA克隆与表达特征分析

cDNA Cloning and Expression Characteristics of Floral Scent Gene BSMT3 of Petunia hybrida

以矮牵牛花瓣总RNA为模板,RT-PCR方法克隆得到其BSMTcDNA,全长1100bp,编码357aa,与已有报道phBSMT1(GenBank No.AA0501)和phBSMT2(AAO45013)的同源性分别达到97.76%和98.6%,该序列在Gen-Bank登录号为DQ494491,命名为phBSMT3.构建的BSMT3 cDNA的原核表达质粒pGBSMT转化BL21(DE3)E.coli,获得的重组菌株经0.01mol/LIPTG诱导后得到正确表达的GST-BSMT融合蛋白带,以纯化的重组表达蛋白免疫家兔获得其兔抗免疫血清.免疫组化研究结果证实,矮牵牛花香基因BSMT在花瓣、花管中的表达量较其它组织的表达量高.

A new cDNA （ GenBank DQ494491 ） was cloned from Petunia hybrida by RT-PCR using the total RNA of its petals as template. The sequence was 1 100 bp in length and encoded a mature peptide with 357 amino acids. This cDNA named as phBSMT3 shared 97.76% and 98.6% homologies to phBSMT1 （AAO4501） and phBSMT2 （AAO45013） reported for P. hybrida. The recombinant expression strain, pGBSMT/BL21, was constructed by sub-cloning the cDNA under the promoter of vector pGEX4T-1 and the identified recombinant plasmid pGBSMT was transformed into B1221 （ DE3 ） E. coll. This strain was expressed by the induction of 0.01mol/L IPTG, and the fusion expression product GST-BSMT was purified and used as the antigen to immunize rabbits in order to prepare target polyclonal antibody. The results of immuno-histochemical analyses showed that the BSMT3 gene was expressed largely in the petals and tubes of P. hybrida when compared with other tissues, such as leaves, stems and calyces. Fig 5, Ref 24