Cloning, Expression Of Citrus Maxima Limonins Glycosyltransferase Gene And Construction Of Plant Expression Vector

Limonoids is one of the main reasons causing orange bitterness, by which citrus fruit on the fresh and processing quality was adversely affected. In addition, limonoids have many pharmacological activities like anti-tumor, anti-inflammatory, analgesic, insect antifeedant and so on. As limonoids possessed these functions, its demand was increasing; however, its intake was limited for bitter existence. Limonoids glycoside has water-soluble, no bitter taste and other characteristics, and its biologi-cal activity are similar to limonoids. Therefore, as an alternative food additive, Limonoids glycoside has a promising market prospect.Based on the previous studies, Limonoids glycosyltransferase gene CmLGT was amplified from Citrus maxima cv.Liangping. CmLGT prokaryotic expression vector and the plant expression vector had been constructed one after another. The main findings were as follows:1. The amplification of limonoids glycosyltransferase gene CmLGT and characterization of the encoded protein According to the nucleotide sequences of limonoids glycosyltransferase gene (LGT) published on GenBank, a pair of specific primers was designed and synthesized. The whole coding sequence of LGT 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 CmLGT were analyzed by way of bioinformatics, the results showed it encoded 511 amino acids with molecular weight of about 57.467 KD, isoelectric Point of 6.01, 21 phosphorylation sites, no signal peptide. CmLGT protein secondary structure was composed ofα-helix (28.18%), extended strand (22.11%), and random coil (49.71%). The encoded protein had GT family characteristics on the basin of conserved domain analysis in NCBI.2. The construction of the prokaryotic expression vector CmLGT and expression system optimization The fusion expression vector pET28a-CmLGT was constructed by the target gene cloned into the prokaryotic expression vector pET-28a(+), which was then transformed into E.coli BL21(DE3).The objective fusion protein with the size of about 62 kD was induced and expressed via optimal conditions(4 h,37℃and 1.0 mmol·L-1 IPTG). The soluble analysis of this fusion protein revealed that it exists in the form of inclusion bodies.3. The construction of plant expression vectorThe target gene CmLGT was linked with plant expression vector pCAMBIA2301g by restriction enzyme digestion, ligation, transformation, and then transformed into Agrobacterium tumefaciens LBA4404. It showed that the above plant Expression vector with the target gene was successfully transformed in Agrobacterium tumefaciens LBA4404 by using BamH I and Sac I double restriction enzyme digestion and PCR confirmation.4. Genetic transformation of tobaccoThe plant expression vector containing limonoids glycosyltransferase gene CmLGT was transformed into wild tobacco plants through employing leaf disc method by Agrobacterium tumefaciens-mediated, nine strains of transgenic tobacco were obtained by means of resistance screening, GUS staining and PCR amplification test.