甘肃红豆草原花青素合成途径的两个关键酶基因的克隆和功能分析

发布时间：2018-04-11 01:19

本文选题：红豆草 + 原花青素　；参考：《甘肃农业大学》2016年博士论文

【摘要】：原花青素又称缩合单宁,其在生物体内具有抗紫外线、抗病、抗虫、清除自由基、调节种子休眠和萌发等重要的生理功能,并影响牧草的适口性和可消化性,兼具保健价值。本研究利用分子生物学方法,克隆了甘肃红豆草原花青素合成途径中的两个关键酶基因,即花青素还原酶(Anthocyanidin reductase)和无色花青素还原酶(Leucoanthocyanidin reductase)的编码基因BAN和LAR。采用生物信息学相关软件分析,预测了二者cDNA序列编码的蛋白质结构和功能,构建了含有bar和GFP基因的双标记选择的植物表达载体。利用植物转基因技术,研究了红豆草原花青素生物合成关键酶基因的功能;通过BAN和LAR表达的定量分析,结合原花青素及其组分含量的测定,研究了甘肃红豆草原花青素生物合成相关酶基因的表达水平与原花青素积累的关系。本研究的主要研究结果如下:1)原花青素总量和不溶性原花青素含量在红豆草叶片中的含量最低,二者在茎和生殖器官的含量较高。可溶性原花青素在叶片中含量最高,而在茎、花、果中的含量较低。叶中原花青素总量、不溶性原花青素和可溶性原花青素含量在营养生长阶段高,而在生殖生长阶段低,这与茎中三者含量的季节变化趋势相反。因此,叶片是原花青素合成的关键器官,但不是主要的贮存器官。甘肃红豆草花中的花青素含量在生殖器官花蕾、花和果中含量最高。茎和叶花青素含量总体上在生殖生长阶段含量高于营养生长阶段,这也说明花青素在花色形成或生殖生长过程中发挥着重要的生理作用。2)以甘肃红豆草叶片总RNA为模板,通过RT-PCR技术获得编码花青素还原酶的BAN基因,克隆的BAN基因与已知基因序列相似性达到99.41%,其ORF长为1020 bp,编码339个氨基酸残基。具有花青素还原酶保守结构域及许多重要功能位点,表明可能具有合成原花青素单体的功能。基因序列已注册到GenBank,序列登录号为KM924437。以此为靶序列,构建携带绿色荧光蛋白GFP和抗除草剂bar基因的双标记选择植物表达载体,将其命名为CPB-BAN-GFP。3)以甘肃红豆草叶片总RNA为模板,采用RT-PCR法克隆编码无色花青素还原酶LAR基因,与GenBank已报道LAR基因(登录号:HM152981)序列相似性达到98.34%,其ORF长为1089 bp,编码362个氨基酸残基;其编码的氨基酸序列与不同属豆科物种的相似性存在较大的差异。基因序列已注册到GenBank,序列登录号为KP013623。以此为靶序列,构建携带绿色荧光蛋白GFP和抗除草剂bar基因的双标记选择植物表达载体,将其命名为CPB-LAR-GFP。4)以甘肃红豆草叶片总RNA为模板,采用定量RT-PCR方法,编码原花青素还原酶的BAN和LAR基因具有较强的组织特异性表达特征,在器官间的表达量差异较大。其中BAN基因表达量的高低顺序依次为果、叶、蕾、花、茎,LAR基因表达量的高低顺序依次为蕾、果、花、茎、叶。5)直接导入法将植物表达载体CPB-BAN-GFP和CPB-LAR-GFP分别转化至根癌农杆菌LBA4404的感受态细胞中。分别侵染了紫花苜蓿下胚轴,经共培养和分化培养,荧光鉴定,抗除草剂鉴定和PCR鉴定,证明将BAN和LAR基因已整合到紫花苜蓿愈伤组织中。整合有这两种基因的愈伤组织中原花青素含量都高于对照。本研究从甘肃红豆草克隆出原花青素生物合成途径中的BAN和LAR基因。生物信息学分析表明,这两个基因编码蛋白在序列、结构、结构域、催化活性位点上都存在较高的保守性,推断其蛋白功能与其它植物相似。转基因实验表明,异源表达BAN和LAR基因提高了紫花苜蓿愈伤组织的原花青素含量。从分子生物学水平解析了甘肃红豆草原花青素积累与相关基因之间的关系。这些研究丰富了原花青素的基础研究,为红豆草及其紫花苜蓿品种改良奠定了基础。
[Abstract]:Proanthocyanidins also called condensed tannins, which have anti ultraviolet, disease resistance, insect resistance in vivo, scavenging free radicals, regulation of seed dormancy and germination and other important physiological functions, and the effects of forage palatability and digestibility, with health care value. This study using molecular biology method, cloned two key enzymes Gansu Grassland Red gene involved in anthocyanin synthesis, namely anthocyanin reductase (Anthocyanidin reductase) and leucoanthocyanins (Leucoanthocyanidin reductase) reductase genes encoding BAN and LAR. by bioinformatics analysis software, the prediction of protein structure and function of the two cDNA sequences encoding the construct containing bar and GFP gene double marker selection the plant expression vector. The use of transgenic technology, research on the key enzymes of anthocyanin biosynthesis genes in Red Steppe; quantitative by BAN and the expression of LAR According to analysis, determination of proanthocyanidin content and composition, study the relationship between the expression level of enzymes related to accumulation of Gansu red bean grassland of anthocyanin biosynthesis genes and procyanidins. The main results of this study are as follows: 1) the total amount of proanthocyanidins and insoluble proanthocyanidins content in bean leaves in high content was the lowest. Two in the stem and reproductive organs. The content of soluble proanthocyanidins highest in leaves, and flowers, fruit content in stem, the lower leaf procyanidins. Total insoluble proanthocyanidins and soluble proanthocyanidin content in the vegetative growth stage and reproductive stage in high, low, the seasonal variation trend of the three this stem was the opposite. Therefore, the blade is a key organ of proanthocyanidin biosynthesis, but not the main storage organ. The anthocyanin content of Gansu red bean flower in the reproductive organs of bud, flower and fruit bearing The highest amount of stem and leaf. The anthocyanin content in the overall reproductive growth stage was higher than that in the vegetative growth stage, which also shows that the play an important physiological role of.2 in the variety of anthocyanin formation or reproductive growth process) to Gansu red bean leaf total RNA as template, the BAN gene was RT-PCR encoding anthocyanidin reductase, similarity up to 99.41% BAN genes with known gene sequences cloned, the length of ORF is 1020 BP, encoding 339 amino acid residues with a conserved domain. Anthocyanin reductase and many important functional sites that may have the original anthocyanin synthesis of functional monomer. The gene sequence has been registered to the GenBank sequence, the accession number is KM924437. as the target sequence, carrying green fluorescent protein GFP and herbicide resistant bar gene double marker selection plant expression vector, named CPB-BAN-GFP.3) to Gansu red bean leaf total RNA As a template, using RT-PCR method to clone encoding leucoanthocyanidin reductase LAR gene, and GenBank has been reported LAR gene (accession number: HM152981) the sequence similarity is 98.34%, the length of ORF is 1089 BP, encoding 362 amino acid residues; its encoding amino acid sequence and different genera of different leguminous species similarity. Registered to the GenBank gene sequence, sequence accession number KP013623. as the target sequence, carrying green fluorescent protein GFP and herbicide resistant bar gene double marker selection plant expression vector, named CPB-LAR-GFP.4) to Gansu red bean leaf total RNA as template, using the quantitative method of RT-PCR, BAN and LAR gene encoding the original anthocyanidin reductase has the features of strong expression of tissue specific expression in large differences between organs. The BAN gene expression level order of fruit, leaves, buds, flowers, stems, LAR gene The level of expression in order of bud, fruit, flowers, stems, leaves,.5) method directly into the plant expression vector CPB-BAN-GFP and CPB-LAR-GFP competent cells were transformed into Agrobacterium tumefaciens LBA4404. Alfalfa hypocotyls were infected by co culture and differentiation culture, fluorescent identification, herbicide resistance identification and identification of PCR, prove that the BAN and LAR gene had been integrated into the alfalfa callus. Integrated with callus of procyanidins content of these two genes were higher than that of control. The study of BAN and LAR genes in the biosynthetic pathway of Proanthocyanidins from Gansu sainfoin clones. Bioinformatics analysis showed that the two genes encoding in the protein sequence, structure, domain, are highly conserved catalytic site, infer its protein function similar to other plants. The transgenic experiments showed that the heterologous expression of BAN and LAR gene increased purple The content of proanthocyanidins in callus of alfalfa was analyzed. The relationship between anthocyanin accumulation and related genes in Gansu red bean steppe was analyzed from molecular biology level. These studies enriched the basic research of proanthocyanidins, and laid a foundation for the improvement of ormosia and its alfalfa varieties.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S541.4

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