【摘要】：常用中藥紅花來(lái)源于菊科植物Carthamus tinctorius L的干燥花,具有活血通經(jīng),散瘀止痛的功效。其功效相關(guān)的主要有效成分為黃酮類(lèi),包括查爾酮、二氫黃酮、二氫黃酮醇及黃酮醇等。紅花黃酮類(lèi)成分合成途徑中,查爾酮異構(gòu)酶基因(chalcone isomerase gene. CHI)是紅花醌式查爾酮類(lèi)與其他黃酮類(lèi)成分合成路徑分支上的第一個(gè)關(guān)鍵基因。因此,紅花中CHI表達(dá)與查爾酮及其他類(lèi)型黃酮類(lèi)成分的累積密切相關(guān)。本研究克隆CHI,進(jìn)行開(kāi)花1-7dCHI的表達(dá)譜、CHI的原核表達(dá)分析,檢測(cè)開(kāi)花1-7 d黃酮類(lèi)成分含量,分析紅花生物量-成分含量-基因表達(dá)的關(guān)系,為紅花生物合成關(guān)鍵酶基因表達(dá)與有效成分累積的關(guān)系、紅花黃酮類(lèi)化合物生物合成機(jī)制的研究奠定了基礎(chǔ)。結(jié)果如下：1.查爾酮異構(gòu)酶基因的克隆及生物信息學(xué)分析使用改良Trizol法提取總RNA,通過(guò)PCR擴(kuò)增,克隆得到CHI序列全長(zhǎng)共696 bp,包含一段633 bp的ORF,編碼蛋白由210個(gè)氨基酸組成,blastp比對(duì)發(fā)現(xiàn),CHI編碼蛋白屬查爾酮超基因家族。生物信息學(xué)分析,預(yù)測(cè)得到的CHI編碼蛋白質(zhì)的一級(jí)、二級(jí)和三級(jí)結(jié)構(gòu)；使用MEGA5.1構(gòu)建系統(tǒng)樹(shù),紅花中CHI與菊科茼蒿屬植物相似度最高,達(dá)到90%。為CHI結(jié)構(gòu)、功能及進(jìn)化關(guān)系等研究奠定基礎(chǔ)。2.紅花查爾酮異構(gòu)酶基因的表達(dá)分析通過(guò)標(biāo)準(zhǔn)曲線(xiàn)、擴(kuò)增曲線(xiàn)、溶解曲線(xiàn)考察,建立紅花CHI和內(nèi)參基因Ct60s的real-time PCR表達(dá)方法,得到紅花開(kāi)花1-7d CHI的表達(dá)譜,CHI相對(duì)表達(dá)量呈現(xiàn)四個(gè)階段：1-3 d為緩慢上升期,3-4 d為快速上升拐點(diǎn)期,4-6 d為快速下降期,6-7 d為衰減期。通過(guò)酶切引物設(shè)計(jì)、PCR擴(kuò)增(在目的基因中插入酶切位點(diǎn))、目的基因及載體PET32的酶切和連接(構(gòu)建重組載體)、將重組載體導(dǎo)入宿主Rosatta大腸桿菌并使用IPTG誘導(dǎo)外源基因表達(dá),構(gòu)建了CHI的原核表達(dá)系統(tǒng),實(shí)現(xiàn)外源基因CHI在Rosatta大腸桿菌中表達(dá)的目的,鑒定了CHI的相對(duì)分子量大小為24 KD左右。為研究重組CHI酶學(xué)性質(zhì)、CHI的結(jié)構(gòu)功能研究等奠定基礎(chǔ)。3.紅花生物量-成分含量-基因表達(dá)的分析分別采用CM-5分光測(cè)色計(jì)檢測(cè)紅花各樣品顏色,紫外分光光度計(jì)檢測(cè)紅花各樣品含量,HPLC檢測(cè)紅花各樣品HSYA含量。分析紅花生物量-成分含量-基因表達(dá)的關(guān)系,結(jié)果表明：紅花開(kāi)花1-7 d生物量與每個(gè)花序總黃酮含量、HSYA含量及CHI相對(duì)表達(dá)量呈現(xiàn)相似的變化趨勢(shì),均在1-3/4 d(始花期至盛花期)逐漸增加至最大值,在3/4-7 d(盛花期至衰敗期)逐漸降低。開(kāi)花3-4 d樣品粉末由黃轉(zhuǎn)紅、為紅黃色且顏色鮮艷。采用DPS進(jìn)行總黃酮/HSYA含量與顏色的灰色關(guān)聯(lián)度分析,各樣品透度及黃色度與HSYA含量關(guān)聯(lián)度較大、紅色度與總黃酮含量關(guān)聯(lián)度較大。采用AMOS進(jìn)行總黃酮及HSYA含量與顏色相關(guān)性分析,總黃酮及HSYA含量與黃色度相關(guān)性大于紅色度及透度。綜合本草記載及本研究認(rèn)為：紅花應(yīng)于“夏季盛花期采摘,此時(shí)花朵由黃變紅,管狀花充分展開(kāi),花冠頂端金黃色、中部橘紅色�！贝藭r(shí)采摘紅花所得藥材的成分含量高、顏色紅黃鮮艷、品質(zhì)最好。植物藥材有效成分多為植物的次生代謝產(chǎn)物,是決定藥材質(zhì)量的物質(zhì)基礎(chǔ)；次生代謝產(chǎn)物與個(gè)體發(fā)育程度、組織分化相關(guān),多受外界因素刺激通過(guò)影響生物合成基因表達(dá)而調(diào)控。生物合成功能基因調(diào)控有效成分積累,進(jìn)而影響藥材品質(zhì)�；蚪M學(xué)、基因過(guò)表達(dá)、基因沉默等可為功能基因挖掘、研究基因表達(dá)與有效成分累積關(guān)系等研究提供思路和方法。
[Abstract]:Carthamus tinctorius L, a common Chinese herbal medicine, is derived from the dried flower of Carthamus tinctorius L. It has the effect of promoting blood circulation and dredging menstruation, dispersing blood stasis and relieving pain. Its main effective components are flavonoids, including chalcone, dihydroflavonol, dihydroflavonol and flavonol. Erasegene.CHI) is the first key gene in the branching pathway of safflower quinone chalcones and other flavonoids. Therefore, the expression of CHI in safflower is closely related to the accumulation of chalcones and other flavonoids. The results are as follows: 1. Cloning of chalcone isomerase gene and its application in bioinformatics analysis Total RNA was extracted by good Trizol method and amplified by PCR. The CHI sequence was 696 bp, including a 633 BP ORF. The coding protein was composed of 210 amino acids. Blastp analysis showed that the CHI coding protein belonged to Chalcone supergene family. GA5.1 constructed a phylogenetic tree, in which the similarity between CHI and Compositae was the highest, reaching 90%. It laid a foundation for the study of the structure, function and evolution of CHI. 2. The expression of chalcone isomerase gene in Safflower was analyzed by standard curve, amplification curve and dissolution curve, and the real-time PCR expression formula of CHI and Ct60s was established. The relative expression of CHI in safflower flower showed four stages: 1-3 D was the slow rising stage, 3-4 D was the fast rising inflection point, 4-6 D was the fast declining stage and 6-7 D was the attenuation stage. The recombinant vector was introduced into the host E.coli Rosatta and induced by IPTG. The prokaryotic expression system of CHI was constructed to realize the expression of CHI in E.coli Rosatta. The relative molecular weight of CHI was identified to be about 24 KD. 3. The analysis of biomass-component content-gene expression of safflower was carried out by CM-5 spectrophotometer, UV spectrophotometer, and HPLC respectively. The relationship between biomass-component content-gene expression of safflower was analyzed. The results showed that safflower was in bloom. The biomass of 1-7 D flower was similar to that of total flavonoids in each inflorescence, HSYA content and relative expression of CHI. The biomass increased to the maximum at 1-3/4 D (from the beginning to the full flowering stage) and decreased gradually at 3/4-7 D (from the full flowering stage to the decay stage). The gray correlation analysis of ketone/HSYA content and color showed that the transmittance and yellowness of each sample had a greater correlation with HSYA content, while the red had a greater correlation with total flavonoids content. This study suggests that safflower should be harvested during the peak flowering season in summer, when the flowers turn yellow to red, the tubular flowers are fully developed, the crown is golden yellow, and the middle is orange red. Biosynthetic genes regulate the accumulation of active ingredients, thereby affecting the quality of medicinal materials. Genomics, gene overexpression, gene silencing and so on can be used for functional gene mining and research. Research on gene expression and accumulation of effective components provides ideas and methods.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S567.219

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張霞;;羥基紅花黃色素A的研究新進(jìn)展[J];中國(guó)基層醫(yī)藥;2015年21期

2 劉娟;李建偉;喬玉峰;牛天增;李明花;;紅花臨床應(yīng)用研究進(jìn)展[J];醫(yī)學(xué)研究與教育;2015年03期

3 劉秀明;楊文婷;張雪萌;焦重達(dá);姚娜;楊美英;官麗莉;李海燕;李校X;;紅花查爾酮異構(gòu)酶基因全長(zhǎng)cDNA的克隆及表達(dá)分析[J];西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版);2015年07期

4 溫昕;邵鐵梅;柴錫慶;安勝軍;;基于組織培養(yǎng)的紅花生物反應(yīng)器研究進(jìn)展[J];作物雜志;2015年01期

5 丁麗麗;段陳平;李芳;呂重寧;岳剛;路金才;;紅花不同采收期及不同部位中羥基紅花黃色素A及山奈素的含量變化[J];沈陽(yáng)藥科大學(xué)學(xué)報(bào);2015年01期

6 宋玉龍;石明輝;賈月梅;賈曉光;陳剛;努爾艾力;郭寶林;;我國(guó)不同產(chǎn)地紅花的質(zhì)量分析[J];吉林中醫(yī)藥;2014年12期

7 劉雅新;劉珊珊;譚勇;陳文;;新疆不同產(chǎn)地紅花質(zhì)量評(píng)價(jià)[J];中成藥;2014年01期

8 李淑嬌;唐于平;沈娟;李建萍;郭建明;段金廒;;藥對(duì)研究(Ⅷ)——丹參-紅花藥對(duì)[J];中國(guó)中藥雜志;2013年24期

9 扈曉佳;殷莎;袁婷婷;王玉玨;黃珍茹;陸陽(yáng);;紅花的化學(xué)成分及其藥理活性研究進(jìn)展[J];藥學(xué)實(shí)踐雜志;2013年03期

10 趙啟明;李范;李萍;;花青素生物合成關(guān)鍵酶的研究進(jìn)展[J];生物技術(shù)通報(bào);2012年12期

相關(guān)博士學(xué)位論文 前4條

1 劉飛;紅花黃酮類(lèi)化合物生物合成途徑關(guān)鍵酶基因的克隆與功能驗(yàn)證[D];第二軍醫(yī)大學(xué);2014年

2 曹世江;紅花FAD2基因家族的克隆及高油酸紅花分子機(jī)制研究[D];東北師范大學(xué);2013年

3 姜建雙;紅花化學(xué)成分及生物活性研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2008年

4 李鳳霞;查爾酮異構(gòu)酶基因過(guò)表達(dá)對(duì)新疆雪蓮類(lèi)黃酮生物合成的調(diào)控[D];中國(guó)科學(xué)院研究生院（植物研究所）;2006年

相關(guān)碩士學(xué)位論文 前2條

1 康亞蘭;紅花查爾酮合成酶基因的表達(dá)及紅花黃色素類(lèi)成分積累的分析[D];成都中醫(yī)藥大學(xué);2014年

2 王阿麗;紅花紅色苷的研究[D];重慶大學(xué);2006年

，

本文編號(hào)：2238287