Genome-Wide Analysis And Expression Involved In Flavonoids Synthesis Of The Mulberry MYB Transcription Factor

The transcription factor, also called as the trans-acting factor, is a protein molecule with special structure to regulate the gene expression. The transcription factor can be widely found in the eucaryon, of which the MYB transcription factor is one of the biggest families among eucaryon with a segment of highly conservative MYB sequence combined with DNA. The MYB transcription factor participates in the initiation of transcription under the RNA polymerase to regulate the gene expression. Klempnauer identified the oncogene v-myb to cause the acute myeloblastic leukemia in 1982. Hence, the MYB was named. The later research found that there exist corresponding protooncogene c-myb in the zooblast. The Paz-Ares firstly separated and identified ZmMYB gene from the corn in 1987 with the following MYB genes identified in a large number of plants. At present, we have conducted systematic research on the MYB gene in various plants including rice, Arabidopsis, grape, corn, poplar and apple etc. The research indicated that MYB family play an important regulation role in the plants’ growth and development, metabolic regulation, photosynthesis, cell differentiation, cell cycle, hormone and environmental factor response and leaf organ formation etc.Mulberry is a perennial woody plant with high economic value. Except for the main food source of bombyx mori, its leaf, peel and fruit can be taken as medicine. For example, the modern pharmacology found that the morus is composed by abundant bioactive substance including polyphenol, flavone, polysaccharide and multiple vitamins etc with the efficacy of decreasing the blood sugar, delaying the aging and inhibiting bacterial as well as antioxidant etc. The peel is full of resveratrol that can prevent the oxidation of low density lipoprotein, potential cancer, cardiovascular disease and virus with immune regulation. Due to the sour and sweet taste as well as rich nutrition, the morus has been widely favored by the people. According to the report, the large amount of flavonoid, especially the rich anthocyanin in the mature morus can resist inflammation, oxidant and tumor as well as regulating the blood fat with high biological activity. The key enzyme expression in the flavonoid biosynthesis pathway was mainly regulated by the upper stream transcription factors, of which the MYB transcription factor is one of the main regulatory genes of flavonoid biosynthesis pathway. For now, the reports about the research on the MYB transcription factor in the morus to regulate the flavonoid biosynthesis pathway are quite rare. With the completion of morus notabili’s whole-genome sequencing, it would be possible to conduct the gene analysis on the MYB family, which also provides theoretical basis to analyze the key regulatory factor of morus’s anthocyanin biosynthesis pathway.This paper identified the morus notabili protein sequence with MYB structural domain in the whole genome database through bioinformatics method, then selected the possible transcription factors to participate in the flavonoid biosynthesis and then carried out the bioinformatics analysis. Meanwhile, we also extracted the total flavone and cDNA at different development stages of medium maturing morus 5801, applied qRT-PCR to study the transcriptional levels of flavonoid synthetic gene and the previously identified MYB gene at different stages of morus and then conducted correlation analysis on the total amount of flavonoid, flavonoid synthetic gene and MYB gene’s transcriptional level changes and finally studied the relation between the flavonoid structural gene and MYB transcription factor. The results of this research are as follows:1、Bioinformatic analysis of MYB genes in the mulberry genomeThe bioinformatics method was adopted to identify and obtain 128 transcription factors with MYB structural domain from the morus notabili’s genome including 35 MYB-related,90 R2R3-MYB and 3 3RMYB. Next, this paper analyzed the identified 90 R2R3-MYB genetic information, amino acid conservative sequence and phylogenetic relationships. According to the N-terminal conservation and C-terminal mutability of R2R3-MYB, we divided the morus notabili R2R3-MYB into 33 subgroups including 25 subgroups (S1-S25) commonly owned by the arabidopsis as well as 8 subgroups (S26-S33) only owned by morus.In order to study the MYB gene relevant to the morus flavonoid biosynthesis pathway, we attempted to find out the fourth, fifth, sixth and seventh subgroups relevant to the flavonoid biosynthesis pathway via the evolutionary relationship between the morus and Arabidopsis MYB. It was discovered that the sixth subgroup did not have the gene of marus notabili. The research showed that the gene of this subgroup (such as PAP1) has played important role in the anthocyanin synthesis. This experiment did not detect the expression of anthocyanin synthesis genes ANS previously, which may be related to its gene deletion; besides, we found the genes MnMYB4-1,MnMYB4-2, MnMYB5-1, MnMYB5-2 and MnMYB7-1 of the fourth, fifth and seventh subgroups of morus notabili as well as the genes MnMYB4 and MnMYB27-1 relevant to flavonoid biosynthesis selected by the same branch (G1) of those subgroups to conduct the further analysis.The regulatory sites predication was conducted on the upper stream promoter sequence of MnMYB4-1, MnMYB4-2, MnMYB5-1, MnMYB5-2, MnMYB7-1,MnMYB4 and MnMYB27-1 in the plantCARE database. The results showed that those genes have a large number of regulatory components relevant to the photoresponse at the upper stream, which indicated that the regulation of gene was closely related to the light. Besides, we also found the action site MBSI of MYB transcription factor relevant to the flavonoid biosynthesis at the upper stream of MnMYB4 and MnMYB27-1. It was assumed that those two genes may participate in the flavonoid biosynthesis as the upstream regulatory genes.2、Flavonoids synthesis related gene expression analysisIn order to study further the relationship between the relevant MYB genes and the flavonoid biosynthetic pathway, we’ve collected the mulberries of 5801, which is a kind of mulberry species in different growth stages including S1 (green), S2 (changing from green to red), S3 (changing from red to purple) and S4 (purple) to extract RNA and the general flavone in these four periods. Through detection of the expression of the relevant flavonoid biosynthetic structural genes in Mulberry and the related MYB genes in different growth periods of Mulberry by qRT-PCR, it turns out that:The total flavonoid content of this Mulberry variety in the periods of S2 and S4 remains at a high level, which separately is 0.82% and 1.04%. However when it’s in the periods of S1 and S3, the total flavonoid content is at a low level, which is 0.51% and 0.62% respectively.Both of MaMYB5-1 and MaMYB7-1 show comparatively low expression quantity in the above four growth stages of Mulberry 5801 with negative correlation having been established between both of them and the relevant flavonoid biosynthetic genes in Mulberry, such as CHS1, CHS2, CHI1, F3H1, F3’H1 and ANS. However Both of MaMYB5-1 and MaMYB7-1 are positively correlated with ARN, the original related anthocyanin biosynthetic genes with the correlation values separately up to 0.906 and 0.902, indicating that MaMYB7-1 and MaMYB5-1 might play a role in regulating the synthesis of the original anthocyanin in Mulberry 5801.The expression analysis on MaMYB4-1, MaMYB4-2, MaMYB4 and MaMYB27-1 reveals that they’re positively correlated with the regulation of the structural genes.There’s a significant positive correlation between MaMYB4 and FLS, the flavonol biosynthetic genes with the correlation value up to 0.952. However MaMYB4 plays a significant role in the negative regulation of ANR, the original related anthocyanin biosynthetic genes, indicating that this transcription factor might activate or inhibit the synthesis of FLS and ANR.