Construction Of CDNA Library Of Chinese Wild Vitis Amurensis With Resistance To Cold-stress And Analysis Of Expressed Sequence Tags

Temperature is an important non-biological factor of plant growth and development. Plants are often damaged by chilling. Vitis vinifera, the main varieties currently cultivated in grape production are weak in cold-resistance, thus the study of cold hardiness of grape and cloning cold tolerance genes is good for cold for the new grape hardy varieties cultivation. In China, the wild grape resources are rich, and according to previous researches, Chinese wild Vitis amurensis is the highest resistant to cold stress in Vitis L. The test materials used in this study is Heilongjiang Seedling, which is a clone of Chinese wild Vitis amurensis, and performs highly resistant to cold weather. Construct the cDNA library through utilizing SMART^TM cDNA Library Construction Kit. Expression and characteristics analysis of EST sequence based on Bioinformatics method provide theory basis for molecular mechanism studies at the cloning of cold tolerance genes and functional expression analysis. The results are as follows:Total RNA was extracted from the leaves of Heilongjiang Seedling at 0h, 2h, 6h ,12h, 24h, 48h at 4℃respectively with SDS/phenol method modified partially, and pooled equal amounts of RNA. The titer of primary library was 2.67×10⁶ pfu/mL, and the recombination rate was 98.5% . The titer of amplified library was 1.53×10⁹ pfu/mL, and the recombination rate was 98.2% .Randomly chosing clones, 227 sequences with high quality were obtained, and all of them have submitted to GenBank database, The GenBank accession number are as follows, JG294234-JG294243,JG392010-JG392092,JK006548-JK006681. Using Blastx program of NCBI for homologue search, there are 46 EST sequences matched to the genes whose protein function were unclassified, 20.3%; 181 EST sequences showed high homologue to 12 clusters of gene with known function, 79.7%. 38 EST sequences was related to stress resistance. These genes included that Defence related-protein, such as endochitinase, pathogenesis-related protein 10, immunophilin, abscisic stress ripening protein, salt-tolerance protein, metallothionein-like protein; relating to Signal transduction, such as ethylene response factor 5, calmodulin, GTP binding protein; Stress induced protein, such as early responsive to dehydration, heat stable protein, polyubiquitin, SUMO protein, F-box family protein, DnaJ protein, hypersensitive-induced reaction protein, ozone-responsive stress-related protein-like; Transcriptional regulaters, such as MYB49, AP2 domain class transcription factor, BZIP domain class transcription factor, RD22-like protein. These substances may play a role in cold-resistance responsein grapes.