韧皮部特异启动子驱动密码子优化的抗菌肽D基因的植物表达载体构建

Construction of plant expression vectors containing antibacterial peptide D gene with citrus preference codons driven by A phloem specific promoter

根据177个GenBank中登录的柑橘编码蛋白密码子用法的分析结果,优化并重新设计和合成了含柑橘偏爱密码子、对柑橘黄龙病有杀灭作用的柞蚕抗菌肽D基因(命名为CAPD),克隆入pUC19克隆载体并经测序验证后,获得了含新抗病基因的重组质粒pUC19-CAPD。用限制性内切酶BamHI和SacI双酶切pUC19-CAPD克隆载体和pBI121植物表达载体的质粒DNA,回收pUC19-CAPD克隆载体中的CAPD基因小片段和pBI121植物表达载体中去掉GUS报告基因的大片段,经连接、转化和鉴定后,构建了由CaMV35S组成型启动子(35SP)驱动CAPD目的基因的新植物表达载体(命名为pHZ05);用限制性内切酶BamHI和HindIII双酶切含笋瓜韧皮部特异启动子(PSP)的pUCm-PSP克隆载体和pHZ05植物表达载体的质粒DNA,分别回收pUCm-PSP克隆载体中的PSP小片段和pHZ05植物表达载体中去掉CAPD目的基因上游35SP的大片段,经连接、转化和鉴定后,构建了由PSP驱动CAPD目的基因的新植物表达载体(命名为pHZ06)。利用细胞感受态法直接将2个由不同启动子驱动的含CAPD目的基因的新重组植物表达载体分别导入根癌农杆菌LBA4404、GV3101、EHA105和发根农杆菌Ri15834等4个农杆菌菌株中,为利用农杆菌介导的遗传转化技术培育抵抗由韧皮部传导的毁灭性和检疫性病害柑橘黄龙病的新种质奠定了基础。

Based on the codon usage for the amino acids of 177 citrus functional proteins deposited in GenBank, a new antibacterial peptide D （A PD） gene with citrus preference codons was designed and chemcially synthesized. The peptide has an ability to kill the phloem conducted pathogens for Huanglongbing, a destructive and widespread disease for citrus. The new gene encoding the same peptide, referred to as CAPD,was cloned into pUC19 to generate a recombinant plasmid pUC19- CAPD, and was verified by sequencing. Both pUC19-CAPD and plant expression vector pBI121 were digested by BamH Ⅰ and Sac Ⅰ, and then the smaller fragment containing CAPD gene from the former and the larger vector fragment without GUS gene from the latter were recovered, ligated, transformed and identified. As a result, a new plant expression vector, referred to as pHZ05, harboring CA PD target gene driven by the constitutive 35S promoter of CaMV （35SP） was constructed. BamH Ⅰand Hind Ⅲ were applied to digest pHZ05 and pUCm-PSP, a plasmid containing phloem specific promoter from Cucubita maxima （PSP）, and then the larger vector fragment without 35SP from the former and the smaller fragment containing PSP promoter from the latter were recovered. After ligation, transformation and verification, another new plant expression vector, refered to as pHZ06, harboring CA PD gene driven by PSP was constructed. The two new vectors, which harbor the same target gene CAPD but driven by different promoters, were then successfully introduced into competent cells of Agrobacterium tumefaciens strains LBA4404, GV3101, EHA105 and A. rhizogenes strains Ri 15834, respectively. The study is helpful for further work to find a new way to develop new germplasms resistant to citrus disease Huanglongbing.