Screening, Cloning And Characterizing Of The Anthocyanidin-3-O-Galactosyl Transferase Gene In Crataegus Pinnatifida

Anthocyanins are water-soluble plant pigments that are widely distributed in various plant fruits, and gain more attention because of their biological activities such as anti-oxidation, anti-cancer, cardiovascular protection, anti-viral and antibacterial etc. Due to the instability of anthocyanins, it is difficult to obtain anthocyanin monomer by chemical separation and purification, which leads to the high price of reference anthocyanin. Also, some kinds of anthocyanins cannot be isolated from plants by chemical separation for their very low abundance in plants. So it is urgent to develop an efficient method for producing anthocyanin, and the emerging biosynthesis technology provides us a new tool for the production of active ingredients. At present, multiple studies related to the biosynthesis of anthocyanin have been performed in several plants such as rice, corn, Arabidopsis thaliana, apples, grapes etc. According to our preliminary data, two anthocyanins, named by cyanidin-3-O-β-galactoside and cyanidin-3-O-a-arabinoside respectively, have been isolated from the fruits of Crataegus pinnatifida, but the procedures of extraction and separation are time-consuming and inefficient.In this study, the strategy combined chemical analysis with the molecular biology was used to screen key enzyme genes for the synthesis of anthocyanins in hawthorn fruit. This study will enhance our understanding the synthesis of anthocyanins in hawthorn fruit and provide candidate genes to producing anthocyanins by synthetic biology.The dissertation is made up of four chapters.Chapter 1:Literatue reviewThe related literature has been summarized and analyzed from multiple aspects, including chemical component of hawthorn and the separation, chemical analysis, pharmacological activities, and biosynthesis of anthocyanin.Chapter 2:Quantification of the polyphenol and triterpene acid across different developmental stages of hawthorn fruits.The sample preparation method of hawthorn fruits was optimized by means of orthogonal design. The high performance liquid chromatography (HPLC) method was established to determine the seven major ingredients in hawthorn fruits, namely procyanidin B2, epicatechin, chlorogenic acid, hyperoside, isoquercitrin, cyanidin-3-O-galactoside and ursolic acid. The contents of the seven compounds and their changes across different developmental stages were analyzed. Results showed that the rang of epicatechin, procyanidin B2, hyperoside, isoquercitrin, chlorogenic acid, ursolic acid and cyanidin-3-O-galactoside were 4.932-19.315 mg/g, 2.218-14.549 mg/g,0.066-0.184 mg/g,0.053-0.117 mg/g,0.532-1.834 mg/g, 2.163-5.942 mg/g,0.005-0.188 mg/g, respectively. The content of cyanidin-3-O-galactoside in hawthorn fruits increased significantly during different mature stages of fruits, with the maximum found at the mature period. The contents of epicatechin and procyanidin B2 in hawthorn fruitsgradually reduced with maturing hawthorn fruit, which showed a different pattern from the cyanidin-3-O-galactoside,Chapter 3:The screen of key genes in the biosynthesis pathway of anthocyanin in hawthorn fruit.In this paper, the modified cetyl trimethyl ammonium bromide (CTAB) method was used to isolate RNA from hawthorn fruits at different developmental stage. The de novo transcriptome sequencing of the qualified RNA was obtained. The relative expression level of genes involved in the biosynthesis of anthocyanin were analyzed by the real-time quantitative PCR across different developmental stages of hawthorn fruits. Significant positive correlations were found between the expression level of several genes including Cp4CL2, Cp4CL3, Cp4CL4, Cp4CL5, CpCHI, CpF3H, CpF3’5’H, CpLDOX and Cp3OGT gene and the content of Cy-3-O-Gal, respectively. Our findings suggested that these above genes played a key role in the biosynthesis of anthocyanin in hawthorn fruit.Chapter 4:Cloning and analysis of Cp3OGT from hawthorn fruit.One cDNA sequence of 3GT gene named Cp3OGT was cloned and its bioinformatics analysis and gene expression analysis were carried out. Then, the Cp3OGT ORF was constructed into the pET28a(+), namely pET28a-Cp3OGT, which was imported to E.coli. The protein identified by SDS-PAGE was used in vitro enzymatic reaction, whose products were identified by LC-MS. It suggested that the Cp3OGT can catalyze the formation of Cy-3-O-Gal via cyanidin.In summary, an HPLC method was established to determine the contents of seven major ingredients in hawthorn fruit, and the content variations of the seven compounds across different developmental stages were analyzed. The relative expression level of genes in the biosynthesis pathway of anthocyanins was analyzed across different developmental stages of hawthorn fruit, and the key genes were predicted according to the correlations between the gene expression pattern and the content of major ingredients. Furthermore, the Cp3OGT gene was cloned and its en2ymtic activity was investigated, which provided better understanding on the biosynthetic pathaway of anthocyanins and laid a basis for further synthetic biology of anthocyanins.