Identification And Function Analysis Of MaUFGTs From Mulberry

Mulberry(Morus alba L.) is a perennial dicotyledonous woody plant. The mulberry leaves are the optimal nature source for domesticated silkworm(Bombyx mori L.); Mulberry fruit is one of new type of green fruit andattracts people for its delicious fruit which has a good taste, juicy flesh as well as considerable nutritional value. Mulberry has some extensive applications, such as nutrition and health, Chinese medicine resources, ecological protection, and so on.Flavonoids are a kind of important secondary metabolites in plants, which often exist in many food-borne plants in the form of binding state or free state. Quercetin is one of the most typical flavonoids. The UDP-glucose: flavonoid 3-O-glucosyltransferase gene(UFGT) catalyzes the transfer of the glucosyl from uridine- 5 ’- two phosphoric acid disodium salt(UDP)- glucose to quercetin to form quercetin 3- β- D- glucoside. Studing the expression and function of its encoding gene has great significance for understanding of flavonoid synthesis and accumulation mechanism. In the previous research, the group cloned and identified the key enzyme genes in the biosynthesis pathway of anthocyanins from mulberry trees, but there is no analysis about MaUFGT function. In this research, the fruits of ‘M. atropurpurea cv. Jialing No.40’, a new mulberry cultivated variety for obtaining mulberry fruits and were grown in the mulberry Garden of Southwest University, were used as the research objects. We studied the function of the gene in the anthocyanin biosynthesis pathway by using the methods of molecular biology. To lay the foundation for breeding and utilization of new varieties of mulberry fruit and regulating the anthocyanins biosynthesis by means of genetic engineering. The main results of this study were showed as followed: 1、Expression analysis of MaUFGT gene in various tissuesThe expression levels of MaUFGT in different tissues and at different growth stages of mulberry was analyzed with qRT-PCR(quantitative real-time fluorescent PCR). With the development and maturation of the fruit, the expression of MaUFGT1, MaUFGT2 and MaUFGT3 showed an upward trend. Their expression generally dropped first and then increased, and was followed by another drop and another rise at different leaf positions. They were expressed abundantly in the cortex and stipule, but scantily in the epidermis, xylem and petiole. Of the three members of the MaUFGT family, MaUFGT2 always had the highest expression, therefore, is speculated to be the major gene. 2、Molecular cloning of MaUFGT2 and bioinformatics analysis of MaUFGTs gene from mulberryWe have identified three UFGT genes from morus notabilis genome database by bioinformatic analysis. What’s more, we cloned the major gene named MaUFGT2. The gene sequence is submitted to NCBI for login, the accession number is: Gen Bank: KP455729.1. A full-length 1 386 bp cDNA of the MaUFGT2 gene, which encodes 461 amino acids, was cloned from the ripe fruit of mulberry ‘Jialing 40’. Its calculated molecular mass was about 51 kDa, The deduced protein has a pI of 7.77. Sequence alignment showed that the encoded proteins were not highly conservative between different species. 3、Prokaryotic expression and Functional Analysis of MaUFGT2 GeneMaUFGT2 was inserted into the vector pET-28a(+) and then expressed in E.coli BL21(DE3). The recombinant protein pET-28a(+)-UFGT2 was expressed successfully under IPTG(final concentration 0.4 mmol/L) at 28℃. SDS-PAGE results showed that the obtained MaUFGT2 protein was nearly 52 kDa in size and was abundantly expressed in inclusion bodies and scarcely as a soluble protein. Using for ultrasonic crushing methods to lysis the cell, then inclusion bodies was dissolved with 8mol/L urea. Purifing expression protein with the Ni-NTA resin, the experimental results showed that high purity of the recombinant protein has been obtained. The purified protein was analysised by Western bolt. Finally, the MaUFGT2 protein with biological activity was obtained by sufficiently decrease the urea concentration in dialysate and by the method of dialysis refolding. High performance liquid chromatography(HPLC) of the enzymatic activity of the purified and renaturated MaUFGT2 protein showed that the recombinant protein could catalyze UDP-glucose to quercetin to form the quercetin 3-β-D-glucoside,thus confirming that MaUFGT2 possessed a glycosyltransferase function.