本文選題：紅豆草 + 原花青素��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：原花青素又稱(chēng)縮合單寧,其在生物體內(nèi)具有抗紫外線(xiàn)、抗病、抗蟲(chóng)、清除自由基、調(diào)節(jié)種子休眠和萌發(fā)等重要的生理功能,并影響牧草的適口性和可消化性,兼具保健價(jià)值。本研究利用分子生物學(xué)方法,克隆了甘肅紅豆草原花青素合成途徑中的兩個(gè)關(guān)鍵酶基因,即花青素還原酶(Anthocyanidin reductase)和無(wú)色花青素還原酶(Leucoanthocyanidin reductase)的編碼基因BAN和LAR。采用生物信息學(xué)相關(guān)軟件分析,預(yù)測(cè)了二者cDNA序列編碼的蛋白質(zhì)結(jié)構(gòu)和功能,構(gòu)建了含有bar和GFP基因的雙標(biāo)記選擇的植物表達(dá)載體。利用植物轉(zhuǎn)基因技術(shù),研究了紅豆草原花青素生物合成關(guān)鍵酶基因的功能;通過(guò)BAN和LAR表達(dá)的定量分析,結(jié)合原花青素及其組分含量的測(cè)定,研究了甘肅紅豆草原花青素生物合成相關(guān)酶基因的表達(dá)水平與原花青素積累的關(guān)系。本研究的主要研究結(jié)果如下:1)原花青素總量和不溶性原花青素含量在紅豆草葉片中的含量最低,二者在莖和生殖器官的含量較高�？扇苄栽ㄇ嗨卦谌~片中含量最高,而在莖、花、果中的含量較低。葉中原花青素總量、不溶性原花青素和可溶性原花青素含量在營(yíng)養(yǎng)生長(zhǎng)階段高,而在生殖生長(zhǎng)階段低,這與莖中三者含量的季節(jié)變化趨勢(shì)相反。因此,葉片是原花青素合成的關(guān)鍵器官,但不是主要的貯存器官。甘肅紅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。莖和葉花青素含量總體上在生殖生長(zhǎng)階段含量高于營(yíng)養(yǎng)生長(zhǎng)階段,這也說(shuō)明花青素在花色形成或生殖生長(zhǎng)過(guò)程中發(fā)揮著重要的生理作用。2)以甘肅紅豆草葉片總RNA為模板,通過(guò)RT-PCR技術(shù)獲得編碼花青素還原酶的BAN基因,克隆的BAN基因與已知基因序列相似性達(dá)到99.41%,其ORF長(zhǎng)為1020 bp,編碼339個(gè)氨基酸殘基。具有花青素還原酶保守結(jié)構(gòu)域及許多重要功能位點(diǎn),表明可能具有合成原花青素單體的功能�；蛐蛄幸炎�(cè)到GenBank,序列登錄號(hào)為KM924437。以此為靶序列,構(gòu)建攜帶綠色熒光蛋白GFP和抗除草劑bar基因的雙標(biāo)記選擇植物表達(dá)載體,將其命名為CPB-BAN-GFP。3)以甘肅紅豆草葉片總RNA為模板,采用RT-PCR法克隆編碼無(wú)色花青素還原酶LAR基因,與GenBank已報(bào)道LAR基因(登錄號(hào):HM152981)序列相似性達(dá)到98.34%,其ORF長(zhǎng)為1089 bp,編碼362個(gè)氨基酸殘基;其編碼的氨基酸序列與不同屬豆科物種的相似性存在較大的差異�；蛐蛄幸炎�(cè)到GenBank,序列登錄號(hào)為KP013623。以此為靶序列,構(gòu)建攜帶綠色熒光蛋白GFP和抗除草劑bar基因的雙標(biāo)記選擇植物表達(dá)載體,將其命名為CPB-LAR-GFP。4)以甘肅紅豆草葉片總RNA為模板,采用定量RT-PCR方法,編碼原花青素還原酶的BAN和LAR基因具有較強(qiáng)的組織特異性表達(dá)特征,在器官間的表達(dá)量差異較大。其中BAN基因表達(dá)量的高低順序依次為果、葉、蕾、花、莖,LAR基因表達(dá)量的高低順序依次為蕾、果、花、莖、葉。5)直接導(dǎo)入法將植物表達(dá)載體CPB-BAN-GFP和CPB-LAR-GFP分別轉(zhuǎn)化至根癌農(nóng)桿菌LBA4404的感受態(tài)細(xì)胞中。分別侵染了紫花苜蓿下胚軸,經(jīng)共培養(yǎng)和分化培養(yǎng),熒光鑒定,抗除草劑鑒定和PCR鑒定,證明將BAN和LAR基因已整合到紫花苜蓿愈傷組織中。整合有這兩種基因的愈傷組織中原花青素含量都高于對(duì)照。本研究從甘肅紅豆草克隆出原花青素生物合成途徑中的BAN和LAR基因。生物信息學(xué)分析表明,這兩個(gè)基因編碼蛋白在序列、結(jié)構(gòu)、結(jié)構(gòu)域、催化活性位點(diǎn)上都存在較高的保守性,推斷其蛋白功能與其它植物相似。轉(zhuǎn)基因?qū)嶒?yàn)表明,異源表達(dá)BAN和LAR基因提高了紫花苜蓿愈傷組織的原花青素含量。從分子生物學(xué)水平解析了甘肅紅豆草原花青素積累與相關(guān)基因之間的關(guān)系。這些研究豐富了原花青素的基礎(chǔ)研究,為紅豆草及其紫花苜蓿品種改良奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S541.4

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