Cloning And Expression Analysis Of Anthocyanin Biosynthesis Genes In Red-Leaf Mustard

Root mustard is a type of pickled mustard plant stem originating from Sichuan, China. In English, it is commonly known as Sichuan vegetable or Chinese pickled vegetable, including the several other types in the Sichuan province itself, the pickle is made from the fist-size root of Brassica juncea. Red-leaf mustard is a unique cultivar of the root mustard, which has a stunning purple leaf color caused by the accumulation of anthocyanins in leaves. In recent years, with the research of synthetic pigments and the health concerns, many synthetic pigment was no longer in use, and hence the anthocyanins that people gradually accepted as a safe natural pigments has formed a huge market. In plants, a well-known physiological funcion of the flavonoid pigments is the recruitment of polinators and seed dispersers, Besides providing beautiful pigmentation in flowers, fruits, seeds, and leaves, flavonoids also have key roles in signaling between plants and microbes, in male fertility of some secies, in defense as antimicrobial agents and feeding deterrents, and in UV protection. As one of the most important water-soluble flavonoid pigments, anthocyanins are the most conspicuous one, its biosynthesis pathway has been studied extensively in model organism. Most structural genes and regulatory genes in the pathway have been isolated. Howerver, there are no reports on this subject in red-leaf mustard. Therefore, the cloning and expression analysis of structral genes in red-leaf mustard will promote further studies on new cultivars breeding and the utilization of narural pigments.In this research, cDNA encoding PAL, CHS, CHI, F3' H, DFR and ANS were isolated by using specific or degenerate primers from the leaves of the red-leaf mustard, and their expression(expression under environment stresses and the expression in red/common leaf mustard) was investigated by real-time quantitative RT-PCR. The main results were showed as follows: According to the conserved motifs of cloned genes involved in anthocyanin synthesis, specific primers or degenerate primers were used to amplify the six structural genes from red-leaf mustard cultivar. The CHI gene and DFR gene were cloned by using homology-based cloning strategy, the complementary DNA of CHI gene(Genbank Accesion:GU230157) has an open reading frame of 759bp, and the DFR gene is 1157bp; the cDNA fragments of PAL(Genbank Accesion:GQ505065), CHS(Genbank Accesion:GU230158), F3'H, ANS(Genbank Accesion:GU230160) genes were obtained by using specific primers. Sequence analysis showed that these genes shared high similarity with those from other plants. The results of phylogenetic analysis revealed that the sysematic evolution of those six genes were up to their distinct evolutionary pathways.The impact of environmental stress upon red-leaf mustard anthocyanins production and the expression of structural genes involved in their biosynthesis such as chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR) and anthocyanindin synthase (ANS) was studied. The results of real-time RT-PCR analysis suggested that the amount of anthocyanin and the expression of those structural genes required a period of time to reach the peak. The expression of DFR gene and ANS gene induced by low-temperature and light-stress led us to the conclusion that the two genes are the important subject for characterizing the molecular responses to temperature and light in the leaf of red-leaf mustard. Furthermore, the low-temperature and light-stress might induce different regulation mechanisms in the anthocyanin biosynthesis.Genes encoding chalcone synthase (CHS), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR) and anthocyanindin synthase (ANS) were assayed by using quantitative real-time RT-PCR, in order to test the transcription levels in the leaf of red-leaf mustard and common root-mustard. The result indicated that the expression of structural genes(CHS, F3'H, DFR and ANS genes) in red-leaf mustard was much higher than the expression in common root-mustard, and the expressions reached the highest level in the fourth leaf. These results suggested that the absence of pigment may be caused by the structural genes down-regulated in the common root-mustard cultivars.