| Grapevine is one of the most essential fruit varieties, as well as one of the most cultivated species all over the world. Its fruit can be taken fresh and used into wine. Besides that, as the raw stuffs for raisin and grape juice, it has great economic value. Vitis amurensis is one of the most important wild fruit resources, with very strong cold resistance, which is the main raw materials to make wine in north-eastern district of China. Veraison is the peculiar stage of non-climacteric fruits, in which great changes take place with rigidity, colour, shape and physiological changes of the berry. In order to get more information of functional genes, this paper constructed cDNA library of Vitis amurensis pericarp at veraison and took more analysis with EST sequencing and bioinformatics acknowledge. Main results are as below:1. The total RNA was isolated from 50% coloured pericarps of V. amurensis with modified CTAB method, and the double strands cDNA was synthesized with LD-PCR. With the pDNR-LIB as the vector, the recombinant plasmid was transformed into E.coli DH5αby the electroporation. The cDNA library of V. amurensis pericarp at veraison could be constructed, in which inserts ranged from 0.5 kb to 2 kb with average of approximate 0.8 kb. The titer of the unamplified library was 1.12x 106 pfu·mL-1, and the titer was up to 2.82×109 pfu·mL-1 after amplification. The percentage of recombination was nearly 100%, indicating that cDNA library kept high quality with integrity2. Randomly picking and sequencing 1009 single clones from cDNA library of V. amurensis pericarp at veraison, deleting vector sequences and low quality sequences,974 high quality sequences were obtained in total, which covered 96.54% in total. With the submission into GenBank. the accession numbers were GW666520～GW667493. Taking advantage of phrap software to combine them,710 unigenes were obtained, containing 126 contigs and 584 singlets. The non redundant sequences covered 72.90% of total sequences. The effective length of consensus sequences ranged from 102 bp to 1024 bp. the average of which was 431.35 bp.3. Endowing 710 unigenes with functional annotation, the results showed that 528 unigenes (representing 722 ESTs) contain highly homological with genes known in database. Based on the standard classification of Arabidopsis thaliana.528 unigenes were divided into 10 categories according to the functional description of genes by comparing with BlastX, which are energy metabolism with 17.46%, protein synthesis with 7.46%, transporters/intracellular traffic with 5.07%, disease/defence with 6.62%, cell structure with 1.83%, signal transduction with 3.24%. protein destination and storage with 2.82%, cell growth and divison with 1.27%, transcription with 1.83% and unclassified genes with 26.76% in total.4. Based on cDNA library of V. amurensis pericarp at veraison, this paper took full-length cloning and bioinformatics analysis from regulator gene concerning with anthocyanins synthesis. Full-length sequence of transcription factor MYBA3 obtained from cloning in V. amurensis pericarps at veraison is 764 bp, with coding 250 amino acids. Multiple compared with amino acid sequences of relevant genes of other species, the similarity with Vitis vinifera L. genes reached to 97%. And the signal peptide structure, hydrophobicity and secondary structure was predicted. |
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