萼脊兰MADS-box基因的克隆及表达载体构建
发布时间：2019-03-31 15:55
【摘要】：为了研究"ABC"模型的B类PI基因,探究决定花瓣和雄蕊形成的基因特征。采用CTAB法提取萼脊兰花瓣总RNA,并通过RT-PCR法克隆萼脊兰PI基因的编码序列,该序列长度为633 bp,编码210个氨基酸,与小兰屿蝴蝶兰的氨基酸相似性最高。对PI所表达蛋白质的结构、亲水性和二级结构进行了分析,结果显示,该基因包含MADS-box和K-box保守结构域,属于MADS-box基因家族;该蛋白分子属于亲水性蛋白,包含56.19%α螺旋、13.81%的延伸链以及30%的不规则折叠,实时荧光定量PCR结果显示,PI基因主要在花器官中表达,且表达量高,说明PI基因的表达具有组织特异性。构建植物表达载体的结果显示,目的基因和带启动子的片段已插入到表达载体p CAMBIA1301中,表明成功构建植物表达载体1301-PI,为最终获得新奇花型的萼脊兰奠定基础。
[Abstract]:In order to study the class B PI gene of the "ABC" model, the genetic characteristics that determine the formation of petals and stamens were explored. The total RNA, of Calyx Ridge Orchid flap was extracted by CTAB method and the coding sequence of PI gene was cloned by RT-PCR method. The length of PI gene was 633 bp, encoding 210 amino acids, which had the highest amino acid similarity with that of Phalaenopsis xiaolanyu. The structure, hydrophilicity and secondary structure of the protein expressed by PI were analyzed. The results showed that the gene contained the conserved domains of MADS-box and K-box and belonged to the MADS-box gene family. The protein is a hydrophilic protein, which contains 56.19% 伪-helix, 13.81% extended chain and 30% irregular folding. The real-time fluorescence quantitative PCR results show that PI gene is mainly expressed in flower organs, and the expression level is high. The results showed that the expression of PI gene had tissue specificity. The results of construction of plant expression vector showed that the target gene and promoter fragment had been inserted into the expression vector p-CAMBIA1301, which indicated that the plant expression vector 1301-Pi was successfully constructed, which laid a foundation for obtaining a novel flower-shaped calyx chiropractic orchid.
【作者单位】： 郑州师范学院生物工程研究所;河南农业职业学院;
【基金】：郑州市科技发展计划项目(20150448) 郑州市重大科技专项(141PZDZX038) 河南省教育厅重点科研项目(14B180036)
【分类号】：Q943.2;S682.31
本文编号：2451056
[Abstract]:In order to study the class B PI gene of the "ABC" model, the genetic characteristics that determine the formation of petals and stamens were explored. The total RNA, of Calyx Ridge Orchid flap was extracted by CTAB method and the coding sequence of PI gene was cloned by RT-PCR method. The length of PI gene was 633 bp, encoding 210 amino acids, which had the highest amino acid similarity with that of Phalaenopsis xiaolanyu. The structure, hydrophilicity and secondary structure of the protein expressed by PI were analyzed. The results showed that the gene contained the conserved domains of MADS-box and K-box and belonged to the MADS-box gene family. The protein is a hydrophilic protein, which contains 56.19% 伪-helix, 13.81% extended chain and 30% irregular folding. The real-time fluorescence quantitative PCR results show that PI gene is mainly expressed in flower organs, and the expression level is high. The results showed that the expression of PI gene had tissue specificity. The results of construction of plant expression vector showed that the target gene and promoter fragment had been inserted into the expression vector p-CAMBIA1301, which indicated that the plant expression vector 1301-Pi was successfully constructed, which laid a foundation for obtaining a novel flower-shaped calyx chiropractic orchid.
【作者单位】： 郑州师范学院生物工程研究所;河南农业职业学院;
【基金】：郑州市科技发展计划项目(20150448) 郑州市重大科技专项(141PZDZX038) 河南省教育厅重点科研项目(14B180036)
【分类号】：Q943.2;S682.31
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