萼脊蘭MADS-box基因的克隆及表達載體構(gòu)建
發(fā)布時間：2019-03-31 15:55
【摘要】：為了研究"ABC"模型的B類PI基因,探究決定花瓣和雄蕊形成的基因特征。采用CTAB法提取萼脊蘭花瓣總RNA,并通過RT-PCR法克隆萼脊蘭PI基因的編碼序列,該序列長度為633 bp,編碼210個氨基酸,與小蘭嶼蝴蝶蘭的氨基酸相似性最高。對PI所表達蛋白質(zhì)的結(jié)構(gòu)、親水性和二級結(jié)構(gòu)進行了分析,結(jié)果顯示,該基因包含MADS-box和K-box保守結(jié)構(gòu)域,屬于MADS-box基因家族;該蛋白分子屬于親水性蛋白,包含56.19%α螺旋、13.81%的延伸鏈以及30%的不規(guī)則折疊,實時熒光定量PCR結(jié)果顯示,PI基因主要在花器官中表達,且表達量高,說明PI基因的表達具有組織特異性。構(gòu)建植物表達載體的結(jié)果顯示,目的基因和帶啟動子的片段已插入到表達載體p CAMBIA1301中,表明成功構(gòu)建植物表達載體1301-PI,為最終獲得新奇花型的萼脊蘭奠定基礎(chǔ)。
[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% 偽-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.
【作者單位】： 鄭州師范學(xué)院生物工程研究所;河南農(nóng)業(yè)職業(yè)學(xué)院;
【基金】：鄭州市科技發(fā)展計劃項目(20150448) 鄭州市重大科技專項(141PZDZX038) 河南省教育廳重點科研項目(14B180036)
【分類號】：Q943.2;S682.31
本文編號：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% 偽-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.
【作者單位】： 鄭州師范學(xué)院生物工程研究所;河南農(nóng)業(yè)職業(yè)學(xué)院;
【基金】：鄭州市科技發(fā)展計劃項目(20150448) 鄭州市重大科技專項(141PZDZX038) 河南省教育廳重點科研項目(14B180036)
【分類號】：Q943.2;S682.31
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