红花查尔酮异构酶基因的克隆表达及化学成分累积研究

发布时间：2018-09-12 07:14

【摘要】：常用中药红花来源于菊科植物Carthamus tinctorius L的干燥花,具有活血通经,散瘀止痛的功效。其功效相关的主要有效成分为黄酮类,包括查尔酮、二氢黄酮、二氢黄酮醇及黄酮醇等。红花黄酮类成分合成途径中,查尔酮异构酶基因(chalcone isomerase gene. CHI)是红花醌式查尔酮类与其他黄酮类成分合成路径分支上的第一个关键基因。因此,红花中CHI表达与查尔酮及其他类型黄酮类成分的累积密切相关。本研究克隆CHI,进行开花1-7dCHI的表达谱、CHI的原核表达分析,检测开花1-7 d黄酮类成分含量,分析红花生物量-成分含量-基因表达的关系,为红花生物合成关键酶基因表达与有效成分累积的关系、红花黄酮类化合物生物合成机制的研究奠定了基础。结果如下：1.查尔酮异构酶基因的克隆及生物信息学分析使用改良Trizol法提取总RNA,通过PCR扩增,克隆得到CHI序列全长共696 bp,包含一段633 bp的ORF,编码蛋白由210个氨基酸组成,blastp比对发现,CHI编码蛋白属查尔酮超基因家族。生物信息学分析,预测得到的CHI编码蛋白质的一级、二级和三级结构；使用MEGA5.1构建系统树,红花中CHI与菊科茼蒿属植物相似度最高,达到90%。为CHI结构、功能及进化关系等研究奠定基础。2.红花查尔酮异构酶基因的表达分析通过标准曲线、扩增曲线、溶解曲线考察,建立红花CHI和内参基因Ct60s的real-time PCR表达方法,得到红花开花1-7d CHI的表达谱,CHI相对表达量呈现四个阶段：1-3 d为缓慢上升期,3-4 d为快速上升拐点期,4-6 d为快速下降期,6-7 d为衰减期。通过酶切引物设计、PCR扩增(在目的基因中插入酶切位点)、目的基因及载体PET32的酶切和连接(构建重组载体)、将重组载体导入宿主Rosatta大肠杆菌并使用IPTG诱导外源基因表达,构建了CHI的原核表达系统,实现外源基因CHI在Rosatta大肠杆菌中表达的目的,鉴定了CHI的相对分子量大小为24 KD左右。为研究重组CHI酶学性质、CHI的结构功能研究等奠定基础。3.红花生物量-成分含量-基因表达的分析分别采用CM-5分光测色计检测红花各样品颜色,紫外分光光度计检测红花各样品含量,HPLC检测红花各样品HSYA含量。分析红花生物量-成分含量-基因表达的关系,结果表明：红花开花1-7 d生物量与每个花序总黄酮含量、HSYA含量及CHI相对表达量呈现相似的变化趋势,均在1-3/4 d(始花期至盛花期)逐渐增加至最大值,在3/4-7 d(盛花期至衰败期)逐渐降低。开花3-4 d样品粉末由黄转红、为红黄色且颜色鲜艳。采用DPS进行总黄酮/HSYA含量与颜色的灰色关联度分析,各样品透度及黄色度与HSYA含量关联度较大、红色度与总黄酮含量关联度较大。采用AMOS进行总黄酮及HSYA含量与颜色相关性分析,总黄酮及HSYA含量与黄色度相关性大于红色度及透度。综合本草记载及本研究认为：红花应于“夏季盛花期采摘,此时花朵由黄变红,管状花充分展开,花冠顶端金黄色、中部橘红色。”此时采摘红花所得药材的成分含量高、颜色红黄鲜艳、品质最好。植物药材有效成分多为植物的次生代谢产物,是决定药材质量的物质基础；次生代谢产物与个体发育程度、组织分化相关,多受外界因素刺激通过影响生物合成基因表达而调控。生物合成功能基因调控有效成分积累,进而影响药材品质。基因组学、基因过表达、基因沉默等可为功能基因挖掘、研究基因表达与有效成分累积关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.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S567.219

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