Construction Of Yeast Hybrid CDNA Library And Library Sreening By DkPDC2 Promoter In Luotian Tianshi Persimmon(Diospyros Kaki Thunb.)

The mechanism of natural deastringency in Japanese pollination-constant and non-astringent(JPCNA) persimmon caused by the declined percentage of tannins cell relative to other tanning cells along with the fruit development(called “dilution effect”). Chinese pollination-constant and non-astringent(CPCNA) persimmon involves not only with the tannins cells “dilution effect”, but also directly related to the “coagulation effect”. Previous studies indicated that the “coagulation effect” in CPCNA tightly related with the key genes in the metabolism of acetaldehyde, alcohol dehydrogenase gene(ADH) and pyruvate decarboxylase gene(PDC). In this study, the CPCNA types ‘Luotian Tianshi’ and ‘Eshi 1’ were the main materials, compared with JPCNA type ‘Youhou’ and non-PCNA type ‘Mopanshi’. Firstly, all members of ADH and PDC family and their promoter sequences were isolated from all materials and compared their sequences similarities and differences respectively. Then fruis of ‘Luotian Tianshi’(CPCNA) at different development stages(5, 10, 15, 20 and 25 weeks after flowering) and different fruit tissues(flower, leaf, calyx, peel, pulp, seed and core) were mixed to construct yeast hybrid cDNA library. Finally, ADH and PDC promoter sequence were used to construct bait vectors for cDNA library screening in order to find out the transcription factors invovled in “coagulation effect” in CPCNA. These results will further provide scientific basis to explain the molecular mechanism of natural loss of astringency in CPCNA.The main results of this research are as follows:1. The sequence of three ADH gene DkADH1, DkADH2, DkADH3 and four PDC gene DkPDC1, DkPDC2, DkPDC3, DkPDC5 in ‘Mopanshi’(non-PCNA) were obtained from NCBI Nucleotide database. Since DkPDC5 was a partial cDNA sequence and had a high sequence identity with DkPDC2, in later research DkPDC2 was main studied rather than DkPDC5. Six genes were isolated from different astringent types(CPCNA, JPCNA and non-PCNA) and translated. Gene and amino acid sequence alignments showed the same results that the same gene in different astringent types has a high sequence identity except several bases or amino acid differences.2. Based on cDNA full length, several ADH and PDC promoter sequence(except DkADH3 and DkPDC1, not full length) were obtained through genome walking. Different astringent types promoter sequence alignments showed that DkPDC2 promoter was same in two CPCNA types ‘Luotian-tianshi’ and ‘Eshi 1’ but differed from JPCNA type ‘Youhou’ and non-PCNA type ‘Mopanshi’ in the core promoter region of ATG upstream which has sequence indels.3. ‘Luotian Tianshi’(CPCNA) that from different development stages(5, 10, 15, 20 and 25 weeks after flowering) and different fruit tissues(flower, leaf, calyx, peel, pulp, seed and core) were mixed to construct yeast hybrid cDNA library. Evaluating the quality of the constructed cDNA library through the processing steps. RNA and mRNA quality were up to standard. The transfomation efficiency of primary-, secondary-, and tranformed Y187 yeast library were 100 %, and the average insert sequence length were above 1.3 kb, total clone numbers were above 1×106.4. ‘Luotian Tianshi’(CPCNA) DkPDC2 promoter combined with homologous recombination was used to construct pPDC2-Ab Ai prey vector, which was transformed into Y1 HGold strain to construct Y1HGold[pPDC2-AbAi] strain. Transcription activation experiment showed that the minimal concentration of AbA of Y1HGold[pPDC2-AbAi] strain was 100 ng/m L. Using Y1HGold[pPDC2-AbAi] strain to screen yeast hybrid cDNA library obtained massive positive clones, select several of which for PCR amplification, the length of most interacted fragments were 1-2 kb.In summary, different astringent types ADH and PDC has little differences at cDNA level, but DkPDC2 has significant differences at promoter level, which implied that the promoter differences may influence DkPDC2 gene transcriptional level change. The construction of ‘Luotian Tianshi’ yeast hybrid library provide a new direction to discover new gene involved with natural de-astringency in CPCNA at transcriptional level.