蝴蝶兰MADS-Box基因克隆及植物表达载体的构建

【目的】克隆蝴蝶兰MADS-Box基因并构建其正义和反义植物表达载体,为其功能研究奠定基础。【方法】以蝴蝶兰杂交品种(Phalaenopsis hybrid cv.Jiuhbao Red Rose)为试验材料,采用RT-PCR和RACE技术从花葶中克隆MADS-Box基因,并构建正义和反义植物表达载体。【结果】克隆获得一个蝴蝶兰MADS-Box基因,命名为DtpsMADS1(GeneBank登录号JQ065097)。该基因cDNA全长960 bp,包含37 bp的5'非编码区、185 bp的3'非编码区和一个738 bp的编码区;该基因编码245个氨基酸。生物信息学分析结果表明,该基因编码的蛋白质为碱性亲水性蛋白,具有62.45%的α-螺旋,8.16%的延伸链和29.39%的不规则折叠。序列比对和系统进化分析结果表明,DtpsMADS1与蝴蝶兰ORAP13的亲缘关系最近,同源性达99.0%,与石斛和蕙兰的同源性分别为83.0%和82.0%,属于MADS家族A类亚家族。将DtpsMADS1基因连接到植物表达载体pBI121上,构建获得正、反义植物表达载体pBI121-DtpsMADS1-S和pBI121-DtpsMADS1-A。【结论】成功克隆的蝴蝶兰DtpsMADS1基因属于MADS家族A类亚家族,具有明显的保守性和特异性,可为蝴蝶兰DtpsMADS1基因功能的鉴定及蝴蝶兰的遗传改良奠定基础。

Cloning and vector construction of MADS-Box gene in Phalaenopsishybrid

[Objective]Cloning as well as sense and antisense plant expression vector of MADS-Box gene in Phalaenopsis hybrid were conducted to provide references for researching its functions. [Method]The MADS-Box gene was cloned from scape of Phalaenopsis hybrid cv. Jiuhbao Red Rose using RT-PCR and RACE. Sense and antisense plant expression vector were also constructed. [Result]The cloned MADS-Box gene from scape was named DtpsMADS1(GenBank accession No. JQ065097). The full length cDNA of DtpsMADS1 was 960 bp, containing 37 bp of a 5'-untranslated region (5'-UTR),185 bp of 3'-UTR,and 738 bp of an opening reading frame (ORF). The gene encoded 245 predicted amino acids. The bioinfor-mation analysis results showed that the gene encoding protein was a basic hydrophilic protein and contained 62.45%ofα-heli-cal domains, 8.16%of extended strand and 29.39%of random coil. Sequence comparison and phylogenetic analysis revealed that DtpsMADS1 shared 99.0%homology with ORAP13 of Phalaenopsis amabilis, 83.0%and 82.0%homology with Dendrobi-um nobile and Cymbidium orchid, respectively. DtpsMADS1 belonged to A-class subfamily of MADS family. Sense and anti-sense plant expression vector pBI121-DtpsMADS1-S and pBI121-DtpsMADS1-A were constructed when DtpsMADS1 was connected to plant expression vector pBI121. [Conclusion]The DtpsMADS1 gene of Phalaenopsis hybrid was cloned suc-cessfully. The sense and antisense plant expression vector were constructed successfully and could be used for DtpsMADS1 genetic function identification and genetic improvement of Phalaenopsis hybrid.