Effects Of Jasmonic Acid And TTG1(TESTA GLABRA1) On Proanthocyanidin Synthesis In Hairy Roots Of Brassica Juncea

Brassica juncea(L.)belongs to the family of Cruciferae Brassica plants,which has a variety of seed colors including yellow and black.The endosperm color of yellow seed and black seed is yellow,however the seed coat color of yellow seed and black seed is transparent and black respectively.The main reason for the color difference of seed coat between yellow seed and black seed is the presence of proanthocyanidins in the black seed coat.Although the key enzyme genes involved in the flavonoid metabolism have been well studied,the functional analysis of the flavonoid metabolic genes in the hairy root system has not been reported yet.Hairy roots have the characteristics of rapid growth,simple induction and single structure.It is rapid and efficient to analyze the secondary metabolism and to identify the related genes function in the hairy root system.Therefore,the establishment of B.juncea hairy root for the regulatory analysis of proanthocyanidins synthesis provides promising applications.In this study,the hairy root system of B.juncea induced by Agrobacterium rhizogenes A4 strain was established.The studies of the effects of bacterial concentration and different sources of explants on hairy root induction showed that the average rooting rate was 71.5% when the OD value of A.rhizogenes A4 reached to 0.8 and that the rooting rate of hypocotyls was significantly higher than the cotyledons and up to 87.3% after the infection.Subsequently,the GUS gene was overexpressed in the hairy roots of two cultivated materials PM(Purple-leaf Mustard)and SY(Sichuan Yellow)respectively.The staining results showed that the GUS gene could be successfully transferred into the hairy roots of Brassica and expressed stably.TTG1(TESTA GLABRA1)is a WD40 type protein,which is involved in the regulation of proanthocyanidins biosynthesis in Arabidopsis.In this research,two genes,TTG1 and GUS,were introduced into the wild-type A.rhizogenes A4,which were used to generate transgenic PM and SY hairy roots.Three out of six basta resistant hairy roots overexpressing TTG1 or GUS genes from PM and SY were tested by PCR to identify the successful integration of this two genes into the genome.Meanwhile,qRT-PCR was performed to test the expression of PA biosynthetic genes in transgenic hairy roots.It was found that overexpression of TTG1 gene in Sichuan yellow seeds inhibited the expression of DFR(Dihydroflavonol4-reductase),ANS(Anthocyanidin synthase)and BAN(Anthocyanidin reductase)enzyme genes.Overexpression of TTG1 in purple mustard promoted the expression of DFR,BAN and ANS genes.This study showed that TTG1 transcription factor has an inhibitory effect on the enzyme genes in SY hairy roots,and it has a promoting effect on enzyme genes in PM hairy roots.Moreover,the jasmonic acid treatment inhibited the expression of CHS(Chalcone synthase),CHI(Chalcone isomerase)and ANS,induced the expression of DFR in the wild-type PM hairy roots.Overexpression of TTG1 in PM materials increased the expression of other key enzyme genes in the proanthocyanidins pathway.