Cloning And Analysis Of Glycosyltransferase Gene Related To Anthocyanin Synthesis In ’Meitan Taicha’ Purple Bud Mutant

Leaf color is one of the important genetic characteristics of tea trees.When anthocyanins and their glycosides largely accumulate in the leaves of tea leaves,the leaves of tea trees will appear reddish purple.Anthocyanins belongs to flavonoids and gives red or purple color to the plants,which are catalyzed by glycosyltransferases to form more stable and more water-soluble anthocyanins with glycosyl groups.Glycosyltransferases responsing to catalyze anthocyanins and glycosyl groups to form anthocynidins are UDP-glycosyltransferases,which rely on Uridine Diphosphate to perform catalytic reaction.Although the mechanism of the purple color of tea bud leaves has been studied in depth in recent years,the glycosyltransferase involved in the synthesis of anthocyanins in purple tea tree has not been systematically reported.Therefore,it is of great significance for the mechanism of purple shoot formation in tea trees to screen the glycosyltransferase genes involved in the anthocyanin synthesis of purple tea tree and analysis its related functions.Based on our previous research of the leaves of ‘Meitan taicha’ purple bud mutant were employed as materials,using the leaves of green bud tea plant as the control.The total content of the main pigments namely chlorophyll,carotenoids and anthocyanins were assayed and the main types of anthocyanins were determined by UHPLC-MS firstly.Then based on NCBI-blastp and local BLAST software analysis,eight glycosyltransferase genes sequences in the tea tree transcriptome database and the tea tree genome were screened and were cloned.Bioinformation analysis of the resultanted genes were performed using conserved domain analysis and phylogenetic tree analysis.Their expression profiles in purple mutants and control green tea plants were detected by real-time PCR.Finally,seven glycosyltransferases genes were analyzed by prokaryotic expression.The results were shown as follows.1.The total amount of chlorophyll,carotenoids and anthocyanins in the shoots of the two-leaf shoots of purple bud tea was 1.51-,1.21-and 6.44-fold higher than in green bud tea tree respectively.The total amount of anthocyanins was significantly higher than that of green shoots,which was 6.44 times that of new shoots of green tea.Ultra-high performance liquid chromatography-mass spectrometry.It was found that there are three types of anthocyanins in the leaves of the tea shoots,namely cyaniding 3-O-galactoside,cyanidin-3-O-(6-coumaroyl)-galactoside and delphinis-3-O-(6-coumaroyl)-galactoside.In addition,the content of these three anthocynins in the new shoots of purple tea tree were significantly higher than that of green shoot tea tree.These results indicated that the high concentration accumulation of anthocyanins is the main reason that the leaves of the purple bud tea tree leaves used in this experiment presented reddish purple.2.Eight genes encoding glycosyltransfers were isolated from Camellia sinensis designated as CsUGT78A14,CsUGT78A15,CsUGT79B28,CsUGT79B29,TEA014249.1,TEA022583.1,TEA008907.1 and TEA008918.1,which have an open reading frame(ORF)of 1 383 bp,1 365 bp,1 383 bp,1 374 bp,1 482 bp,825 bp,1 440 bp,and 1 116 bp respectively.Conserved domain analysis indicated that the proteins encoded by genes TEA022583.1,TEA008907.1,CsUGT78A14,CsUGT79B29,and CsUGT79B28 may be responsible for the synthesis of anthocyanin 3-O-glucoside and the proteins encoded by genes TEA014249.1 and CsUGT78A15 may be responsible for the synthesis of anthocyanin3-O-galactoside.Phylogenetic tree analysis indicated that the proteins encoded by the genes CsUGT78A14,CsUGT78A15 and TEA022583.1 belonged to the 3GT class,the proteins encoded by the gene TEA014249.1 belonged to the 7GT class or 3’GT class and the proteins encoded by the genes CsUGT79B28,CsUGT79B29 and TEA008907.1belonged to the GGT class.(q)RT-PCR quantitative analysis showed that the transcriptional levels of CsUGT78A14,TEA022583.1,and TEA014249.1 in the shoots of purple buds were higher than those of green buds,while other genes presented opposite expression trends.3.The prokaryotic expression vectors of CsUGT78A14,CsUGT78A15,CsUGT79B28,CsUGT79B29,TEA014249.1,TEA008907.1 and TEA022583.1genes were constructed and transformed into E.coli HI-Control BL21(DE3),respectively.After inducing by IPTG,seven fusion proteins of about 65 k Da,65 k Da,66 k Da,66 k Da,69 k Da,67 k Da,43 k Da were epressed,which were consistent with the predicted molecular weight respectively.Then the resultant proteins were purified using a His-TrapHP(GE Healthcare)purification column to obtain a relatively single target protein band.