| In this work, PCNA type'Xiaoguo-tianshi'was the main material which origined in China. The PCNA origined in Japan and the non-PCNA were used as comparisons. The laccase fragments from Diospyros kaki Thunb. in tannin biosynthesis pathway were isolated by homology-based cloning method. The 3'RACE (Rapid amplification cDNA End) and Genomic Walking PCR methods were used to obtain the full length cDNA of DkLAC. The bioinformatics analyses were performed in the full length cDNA sequences to predict the genie probable function. In addition, qRT-PCR was performed to analyze the genes spatiotemporal expression patterns which are responsible to the tannin biosynthesis in different type of fruit during the fruit development, and the different tissues. Moreover, to further confirm the function of target gene DkLAC1, the ectopic expression of DkLAC1 in Arabidopsis thaliana tt10 mutation was performed to induce PA polymerizes. The transient expression of the DkLAC1 in protoplast indicated that the gene probably located in the ectoplasm or vacuole. The promoter of target gene was cloned by Genomic Walking PCR method. Our data indicated there were many transcript factors binding cis-motifs, such as MYBCORE, MYC-BOX, W-BOX, in the promoter region of the tannin pathway genes in persimmon. These studies would contribute to the mechanism of the Chinese PCNA nature loss of astringency and genetic improvement in non-PCNA or J-PCNA persimmon. The main results of this work are as follows:1. Developmental pattern of tannin cell:the size of C-PCNA tannin cell is less than 30×103μm2 during fruit development, the accumulation of tannin continues until 10-15WAF. However, the C-PCNA can naturally loss the astringent in the late fruit developmental period. It indicates that the polymerization process runs during the C-PCNA fruit development, especially in the late fruit developmental period. The tannin cell developmental pattern in C-PCNA is different from J-PCNA and non-PCNA. During the C-PCNA tannin cell development period, the soluble tannin is polymerizing into insoluble tannin.2. DkLACl gene cloning and analyses:the full length cDNA sequence of DkLAC1 gene was isolated from Diospyros kaki Thunb.'Xiaoguo-tianshi'. The 1794bp cDNA sequence contains the ORF of the laccase, which encodes 569 amino acids. There are His-rich motifs conserved in LAC protein and the Cu2+-binding domains in the DkLAC1 protein sequence. Expression pattern of DkLACl gene is coincident with the tannin cell development pattern of 'Xiaoguo-Tianshi'.3. Promoter cloning and analyses:the DkLACl promoter sequence was obtained. Our data revealed that not only the normal promoter cis-motifs, such as the TATA-BOX and CAAT-BOX, but also the binding cis-motifs of the PA pathway genes, such as MYBCORE, MYC-BOX, and W-BOX were existed in the promoter region. It provides evidence that the DkLAC1 gene is regulated by the transcript factors, such as MYB, MYC et al., in the PA pathway.4. The transporter of tannin, DkGST, cloning and analyses:Based on the DkGST sequence information in the NCBI Diospyros EST database, we further designed our primers, and two transcripts were cloned. Notably, different transcripts of DkGST genes differentially expressed in different type of persimmons during fruit development. The same exhibition takes place in the expression of DkLAR and DkMYB genes. It may be caused by differentially expressional factor genes, or the expression abundance of the transcripts is significantly diverse in the different types of persimmons.5. Genetic transformation and mesophyll protoplast transient transformation:the ectopic expression of DkLAC1 in Arabidopsis thaliana transparent testa10 mutation induces PA polymerize. This indicates that the expression of the DkLAC1 gene complement the Arabidopsis tt10 mutant. Our present results of protoplast transient transformation was coincided with the bioinformatics analyses played which were performed using the PSORT database and suggessted that this gene probably located in the vacuole or ectoplasm. |
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