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题名: 天然彩色棉类黄酮合成途径相关基因的克隆及表达特性研究
作者: 朱奇朗
答辩日期: 2013-05-17
导师: 李晓波
专业: 有机化学
授予单位: 中国科学院大学
授予地点: 北京
学位: 硕士
关键词: 类黄酮合成途径 ; RT -PCR ; 二氢黄酮醇 -4-还原酶 ; 莽草酸奎尼羟基肉桂酰转移酶 ; 实时荧光定量PCR
摘要:

本研究从彩棉中克隆了F3’H F3’5’HHCT基因,研究了HCT基因的表达特性,同时从不同花色的矮牵牛品种克隆了3DFR基因,并对这几个基因进行了生物信息学分析。本研究为彩棉色彩和品质改良提供候选目的基因,同时为进一步利用这些基因资源进行创新彩棉新色彩提供基础。具体研究结果如下:1) 根据NCBI上已知的F3’HF3’5’HCDS序列来设计引物,以开花后16dDPA16)的新彩棉5号(XC-5)的叶片作为材料,利用反转录PCR技术克隆得到F3’HF3’5’H基因,而HCT基因引物的设计根据棉花基因的数据库,以新彩棉6号(XC-6)的纤维作为材料,利用RT-PCR技术分离得到HCT基因。最后得到了含有酶切位点的CDS序列,通过序列比对得出与已知序列有很高的同源性,XC-6HCT的氨基酸序列与木槿、毛果杨、拟南芥、蓖麻和野草莓氨基酸同源性分别为81.76%73.90%72.58%71.74%73.11%。为进一步利用这些基因资源进行创新彩棉新色彩提供基础。2) 二氢黄酮醇4-还原酶基因(DFR)是花色素合成途径中的一个关键基因。以新疆种植的白、红和蓝色矮牵牛为实验材料,通过同源克隆的方法从花中克隆到三个完整的DFR基因的全长编码序列(CDS),与已知的矮牵牛DFR基因(GeneBank 登录号:X15537)序列的相似性分别为97.79%96.59%97.99%,分别命名为PhDFR1PhDFR2PhDFR3;三个基因编码380个氨基酸,同已知矮牵牛DFR基因编码的蛋白(GeneBank 登录号:CAA33544)的同源性分别是95.53%94.21%95.79%;生物信息学分析表明,三个蛋白均具有NADB-Rossmann 家族中高度保守的NADPH结合位点、底物特异性结合位点。三个矮牵牛品种DFR都不具有信号肽,为亲水蛋白,且定位于细胞质的可能性最高;均具有两个跨膜结构,α-螺旋和β-折叠是三个DFR的主要二级结构元件,并且形成了β-α-β-α-βRossmann折叠,整本上呈对称分布。利用同源建模分析三个DFR蛋白与已知葡萄的DFR晶体结构有很高的相似性。系统进化树分析表明,PhDFR1PhDFR2PhDFR3与已知矮牵牛的DFR蛋白亲缘关系最近。为彩棉色彩改良提供丰富的候选目的基因。3) XC-6为材料进行实时荧光定量PCR实验,结果表明HCT基因在XC-6的不同组织中都有表达,但在叶片中的绝对表达量最低。HCT基因的拷贝数要小于内参基因UBI的拷贝数。

英文摘要:
In this study F3’H, F3’5’H and HCT genes from colored cotton were cloned and the HCT gene expression characteristics was analysized. Meanwhile we cloned three DFR genes color varieties with different colors and analysised these genes by bioinformatics. This research provides the theoretical basis and candidate genes for colored cotton color and quality improvement. The results are as follows: 1) With DPA16 of XC-5 leaves as material the F3’H and F3’5’H gene primers were designed according to the known CDS sequences in NCBI database and the genes were cloned by reverse transcription PCR technology. Meanwhile with DPA16 of XC -5 fibres as material the HCT primer was designed according to the sequence information of the Cotton genome databases and the genes were cloned by reverse transcription PCR technology.In the end we acquired the complete CDS sequences. They have high homology with known the gene sequences from other species. Homologous alignment indicated that the nucleotide similarity of Hibiscus cannabinus, Populus trichocarpa, Arabidopsis thaliana, Ricinus communis and Fragaria vesca is 81.76%, 73.90%, 72.58%, 71.74% and 73.11% with the HCT amino acid residues of XC-6. This research provides the basis for the further use of these gene resources in germplasminnovation of new colored cotton. 2) In this study, through homologous cloning technology, three complete CDSs encoding dihydroflavonol 4-reductase (DFR), which is a key enzyme in the pathway of anthocyanin biosynthesis and were cloned from the petal of petunia hybrid planted in Xinjiang with white, red and blue petal color respectively. The three DFR genes were named PhDFR1, PhDFR2 and PhDFR3. Homologous alignment indicated that the nucleotide similarity of three DFR is 97.79 %,96.59% and 97.99% with another DFR gene from Petunia hybrida ( GeneBank access number: X15537). All DFR cDNAs encoded a poly peptide composed of 380 amino acid residues. The mino acid residues similarity of three DFR is 95.53 %,94.21% and 95.79% with another DFR gene from Petunia hybrida ( GeneBank access number: CAA33544). DFRs of three colored Petunia hybrida varieties contain a highly conserved NADP(H)-binding site and substrate specificity site which belong to NADB-Rossmann superfamily. They were satable proteins without signal peptide and were hydrophilic proteins probably located in cytoplasm. All of them had transmembrane domains. α-helix and random coil were primary secondary structural components of DFR. Forming Rossmann folding of β-α-β-α-β and were symmetrical distributed on the whole.The phylogenetic analysis of DFR proteins from different species revealed that the PhDFR1、PhDFR2 and PhDFR3 had closer relationship with the known Petunia hybrida DFR . The cloning of these three DFR genes provided candidate genes for colored cotton genetic improvement. 3) Real-time fluorescent quantitative PCR experiments were conducted using XC-6 as material. The result: HCT gene was expressed in different tissues but in the minimum amount of relative expression in the leaf. The mRNA copy number of HCT gene is less than that of internal reference gene UBI.
内容类型: 学位论文
URI标识: http://ir.xjipc.cas.cn/handle/365002/2500
Appears in Collections:资源化学研究室_学位论文

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作者单位: 中国科学院新疆理化技术研究所

Recommended Citation:
朱奇朗. 天然彩色棉类黄酮合成途径相关基因的克隆及表达特性研究[D]. 北京. 中国科学院大学. 2013.
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