Isolation And Characterization Of MYB, Basic Helix-loop-helix And WD40Transcription Factors Genes Involved In Persimmon Proanthocyanidin Metabolism

Persimmon(Diospyros kaki Thunb.) originates in China, and China is the largest cultivator and producer worldwide. But an overwhelming amount of persimmons cultivated in China are astringent which take a lot of money and labors to de-astringency and process before eating every year. Persimmon fruit is astringent because it accumulates abundant proanthocyanidins (PAs, also called condensed tannin) in its fruit cells. Tannin, as one of the final products of flavonoid biosynthesis pathway, its biosynthesis pathway was controlled by a series of structural genes, and expressions of these structural genes were controlled by transcription factors (TFs). As it was reported in many researches, regulation of the structural gene expression in the flavonoid pathway was orchestrated by a ternary complex MYB-bHLH-WD40(MBW), composing of transcription factors from R2R3-MYB, MYC Like basic helix-loop-helix (bHLH), and WD40classes. In this research, we have isolated MYB, bHLH and WD40transcription factor genes from sweet persimmon'Luotiantianshi'which originated in China and done some tannin biosynthesis regulation studies.1. Tannin content analysis in developing persimmon fruits. The materials used in the research was'Luotiantianshi'(pollination-constant and nonastringent originated in China, C-PCNA),'Maekawa-Jiro'(PCNA originated in Japan, J-PCNA),'Nishimura-wase'(pollination-variant and nonastringent originated in Japan, J-PVNA) and'Mopanshi'(pollination-constant and astringent originated in China, C-PCA). In the four materials included, soluble tannin contents were at the highest level at5weeks after full bloom (WAB), and reduced with fruits development. At13WAB'Maekawa-Jiro'and 'Nishimura-wase'almost lost their astringency.'Luotiantianshi'lost its astringency after21WAB, and'Mopanshi'could not lose astringency naturally on the tree. The tannin stopped accumulation in'Maekawa-Jiro'and 'Nishimura-wase'at early stage of fruits development, but in'Luotiantianshi'and'Mopanshi'tannin accumulation lasted to the late stage of fruits development. 2. Tannin content analysis in fruits of'Luotiantianshi'treated with ethanol.'Luotiantianshi'fruits were treated with35%ethanol in vitro at17WAB. On the second day after treatment, the fruits lost astringency and the tannin cell color darkened. Soluble tannin content decreased dramatically; meanwhile insoluble tannin content increased correspondingly. The soluble tannin changed to insoluble tannin which was a sign that the fruits lost astringency by "coagulation effect"3. Molecular cloning and analysis of persimmon MYB gene. The sequences of Arabidopsis and grape MYB transcription factors were used as query sequences against D. kaki ESTs database using BLAST analysis. One significant match DC587440was found. The sequence was subsequently amplified from'Luotiantianshi'. After3'RACE and5'Hi-TAIL PCR amplification, the full length1152bp was obtained which had an ORF of843bp, encoding280amino acid residues. It was named as DkPA1and the sequence was submitted to GenBank. DkPA1had two conserved MYB domains and belonged to R2R3MYB transcription factors. Phylogenetic analysis showed DkPA1shared high similarity with MYB transcription factors from other species which were reported to be involved in tannin biosynthesis regulation. In quantitative real-time PCR analysis (qRT-PCR), expression pattern of DkPAl was correlated with tannin accumulation in'Luotiantianshi' and'Mopanshi'; also coincided with expression of flavonoid biosynthesis structural genes DkDHD, DkSCPL, DkF3'H, DkF3'5'Hand DkANR.4. Molecular cloning and analysis of persimmon bHLH gene. The sequence of Arabidopsis bHLH transcription factor AtTT8was used as a query sequence to probe the D. kaki ESTs database. One significant match DC587220was found. The sequence was subsequently amplified from'Luotiantianshi'. After3'RACE and5'Hi-TAIL PCR amplification, the full length2687bp was obtained which had an ORF of2160bp, encoding719amino acid residues. It was named as DkMYC1and the sequence was submitted to GenBank. DkMYC1had the typical basic Helix-Loop-Helix domain and belonged to the bHLH subgroup â…¢f. Phylogenetic analysis showed DkMYCl shared high similarity with bHLH transcription factors from Arabidopsis and grape which were reported to be involved in tannin biosynthesis regulation. In qRT-PCR analysis, expression pattern of DkMYC1correlated with tannin accumulation in'Luotiantianshi','Maekawa-Jiro'and'Mopanshi', also coincided with expression of flavonoid biosynthesis structural genes DkANR and DkF3'5'H. Promoters of DkLAR and DkANR were isolated from'Luotiantianshi', and several bHLH-binding cis-motifs (MYCATERD1and MYCCONSENSUSAT) were found in the promoter region of DkANR, which further supported that it was regulated by DkMYC1.5. Molecular cloning and analysis of persimmon WD40gene. Based on homology cloning method, we isolated a WD40fragments from'Luotiantianshi'. After3'RACE and5'Hi-TAIL PCR amplification, the full length1640bp was obtained which had an ORF of1014bp, encoding337amino acid residues. It was named as DkWD40and the sequence was submitted to GenBank. DkWD40had four conserved WD40repeat domains. Phylogenetic analysis showed DkWD40shared high similarity with WD40transcription factors from grape which was reported to be involved in tannin biosynthesis regulation. DkWD40showed coincident expression pattern with genes involved in tannin coagulation, but it didnot coincide very well with structural genes (DkDHD, DkSCPL, DkF3'H, DkF3'5'H and DkANR) in tannin biosynthesis during fruits development. In ethanol treatment, DkWD40also showed similar expression pattern with DkADHl which involved in tannin coagulation. When DkWD40overexpressed in Arabidopsis, seed coat where tannin concentrated was black brown.6. Tissue specific expression analysis. Expressions of DkPAl,DkMYCl and DkWD40were analysed in stem, leaf, flower, calyx, fruit peel, flesh and seed of four materials included. The results showed the three transcription factor genes expressed in almost all the organs and tissues included, rather than fruits specific expressed.7. Identification of'Huashi1'.'Huashi1'has been cultivated as a sweet persimmon. However its origin and astringent type was not clear. In this research, the tannin content, tannin cell size and fruits characters were measured. Combined with molecular markers amplification results, it was confirmed that'Huashi1'was a PVA type persimmon originated in Japan and it had big potential in persimmon breeding. In this research, MYB transcription factor gene DkPAl, bHLH gene DkMYC1and WD40gene DkWD40were isolated from'Luotiantianshi'(pollination-constant and nonastringent originated in China, C-PCNA). Expressions of the three genes were analyzed in developing persimmon fruits with different astringent type, and in ethanol treated persimmon fruits. Combined with tannin content analysis and expression of structural genes, it is inferred that DkPA1and DkMYC1were directly involved in tannin metabolism of developing persimmon fruits. However, DkWD40were involved in soluble tannin converting to insoluble tannin.