Construction Of Grapevine Cdna Library And Expression Analysis Of Genes Related To Fruit Development Of Table And Wine Grape

Grape is an important fruit tree in the word and is also one of the most important fruit crop in China. The planting area and output of the grape in China list top position in the world, especially for the table grape, the cultivate area and yield have been in the first place in the world for many years. But the discovery of novel genes and the study of genes related to the fruit quality of grapevine just start the first step. In this research, EST-SSR markers were developed and microarray was also used to analysis the different expression of genes related to the development of table and wine grape on the basis of a constructed cDNA library from flower and fruit of grapevine. The main results are as follows:1. Construction of the cDNA library of grape flower and berry is the importantly fundamental work for the study on the molecular mechanism of the development of grape flower and berry, in which it can facilitate the cloning, characterization and utilization of the important genes involved. In this research, a cDNA library of flower and berry of grapevine’Summer Black’, a well known grape cultivar in its high popularity in cultivation and great berry quality, was constructed with Creator SMART cDNA Construction Kit. This library has a titer of1.2×106pfu/mL, and a capacity of6.0x106pfu. The recombination efficiency was about99%, and the insert sizes were about1.0-3.0kb based on PCR analysis of30randomly chosen clones, suggesting that the cDNA library of grape flower and fruit has been successfully constructed for the use in further study.2. A total of6,230EST sequences were produced from7,561clones in a cDNA library generated from grapevine’Summer Black’(Vitis vinifera x V. labrusca.) flower and fruit tissues in this study. After cluster and assembly analysis of the datasets,3,582unigenes (GenBank accession numbers GW836604to GW840185) were established. The BLASTx result showed that all of the3,582unigenes including913unigenes with known function, mainly involved to material transport and metabolism, translation and modification category, general function prediction, energy production and conversion and so on,12unigenes with unknown function, and381new genes. The mapping result showed that289 new grapevine EST sequences could be mapped on the19grapevine chromosomes.3. A total of3,582grapevine EST sequences that were from the cDNA library were screened by the use of MISA software to search for SSR motifs and540SSR loci were identified from the459grape EST sequences. The frequency of EST-SSRs identified was15.08%. All the540SSR loci included135(3.77%) dinucleotides,270(7.54%) trinucleotides,59(1.65%) tetranucleotides,29(0.81%) pentanucleotides, and47(1.31%) hexanucleotides. Fourty-seven pairs of primers were designed against the some EST sequences by the software Primer3.0Plus, and the PCR products of these primers were detected by PAGE. Among the47pairs of EST-SSR primers,20pairs could amplify distinct PCR bands that were the anticipated products. Eighteen pairs of the EST-SSR primers could amplify polymorphic bands.4. A microarray system comprising15,403ESTs was used to obtain an overall view on the different expression profiles of quality formation-related genes involved in fruit development of table and wine grapes. The result showed that2,493genes exhibited at least2.0-fold differences in expression levels, with1,244genes being up-regulated and1,249being down-regulated during the entire fruit development stage. The expression patterns from the microarray analysis were validated with quantitative real-time polymerase chain reaction analysis of19selected genes of interest, and only18genes show patterns similar to the changes measured by microarray analysis. Following BLAST2GO analysis, only172differentially expressed genes (including52up-regulated and120down-regulated) got GO annotation, which belong to the different leves of molecular function, cellular component and biology process. According to annotation, the differently expressed genes were mainly involved in the metabolism process of metabolic, cellular and so on, related to binding, catalytic activity and so on, distributed in the whole cell or organelle. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the differently expressed genes between table and wine grape were involved in several pathways primarily focused on Carbohydrates metabolism, Biosynthesis of secondary metabolism, Lipid metabolism and Energy metabolism.