Cloning And Expression Analysis Of A Rhamnosyltransferase Gene Cm1,2Rhat From Citrus Maxima Cv.Liangping

Citrus grandis L.)Osbeck [C.Maxima(Burm.)Merr.] is part of Rutaceae Citrus plant and often referred to aspomelo et.al. Citrus bitterness has been confirmed from both the role of flavonoids naringin and limonin compounds. Naringin and limonin have higher content in grapefruit fruit.Naringin is the mainly flavanone in pomelo, which can reduce the level of plasma total cholesterol and low density lipoprotein cholesterol (atherosclerosis main pathogenic factors). In the food industry, naringin can be used as a natural pigment, flavor improver and bitter agent in food and beverage production, and a raw materials of synthetic high sweetness, non-toxic, low energy of the new sweetener, two hydrogen naringin and neohesperidin chalcone. The Naringin is widely used. The research of plant secondary metabolism enzymatic reaction mechanism is to separate and clone its key enzymes. While the specific plant secondary metabolite synthesis and accumulation depends on its synthesis pathway in the rate-limiting enzyme in plant secondary metabolites, its biosynthetic pathways often located in branch forks or downstream of metabolic pathways. Rhamnosyltransferase as the key enzymes in the process of synthesis of naringin in the main citrus bitter substance has been widely concerned in recent years.Based on the previous studies, Rhamnosyltransferase glycosyltransferase gene Cm1,2RhaT was amplified from Citrus maxima cv.Liangping. Cm1,2RhaT prokaryotic expression vector and the plant expression vector had been constructed one after another. The main findings were as follows:1.The amplification of Rhamnosyltransferase glycosyltransferase gene Cm1,2RhaT and characterization of the encoded protein According to the nucleotide sequences of Rhamnosyltransferase glycosyltransferase gene (RhaT) published on GenBank, a pair of specific primers was designed and synthesized. The whole coding sequence of RhaT gene was obtained by RT-PCR from total RNA in young leaves of Citrus maxima cv.Liangping adult tree as template. The properties and structural features of the protein encoded by gene Cm1,2RhaT were analyzed by way of bioinformatics,the results showed it encoded453amino acids with molecular weight of about51.2KD, isoelectric Point of6.2,14phosphorylation sites, no signal peptide. Cm1,2RhaT protein secondary structure was composed of a-helix (42.04%), extended strand (16.15%), and random coil (41.81%). The encoded protein had Glycosyltransferase_GTB_type family characteristics on the basin of conserved domain analysis in NCBI.2. The construction of the prokaryotic expression vector Cm1,2RhaT and expression system optimizationThe fusion expression vector pET28a-CmRhaT was constructed by the target gene cloned into the prokaryotic expression vector pET28a(+),which was then transformed into E.coli BL21(DE3).The objective fusion protein with the size of about56kD was induced and expressed via optimal conditions(4h,37℃and1.0mmol·L-1IPTG). The soluble analysis of this fusion protein revealed that it exists in the form of inclusion bodies.3. Purification of the protein, renaturation of the protein and Rhamnosyltransferase enzyme activity assayRecombinant protein exists in the form of inclusion body, which dissolved in8M urea, according to Merck company’s protein purification system operation guide for the purification, slightly improved and purified by dialysis refolding. We obtained the fusion protein after isolation and purification.Its concentration and purity is0.526mg.ml-1and95%.The natural substances in grapefruit peel and leaf were used to determine the enzyme activity which showed that the naringin content in the leaves is higher than in fruit, though the data obtained are not able to calculate the enzyme activity.4. Cm1,2RhaT gene expression analysisThe SYBR Green I Real-time qRT-PCR was employed to analyze the expression of Cm1,2RhaT in Citrus maxima cv.Liangping and other four citrus varieties (Citus reticulate cv.Unshiu,Citrus limon L.Bum F.Femminello,Citus maxima cv.Shatian,Citrus sinensis cv.(L.)Xuecheng) in different periods.Real-time fluorescence qPCR(RT-PCR) showed that the expression of Cm1,2RhaT in the LiangpingYou’s leaves are much higher than that of fruits. The expression level of Cm1,2RhaT was reduced gradually with the fruit developing from thirty to eighty days after flowering. The gene is expressed volatility mode in other species of citrus. Five species are expressed basically the same expression trend.