Construction Of EST Library From The Fruit Of Chinese Bayberry (Myrica Rubra Sieb. Et. Zucc.)

Chinese bayberry is one of Chinese characteristic fruits with unique flavor, bright color, high juiciness, and abundant nutrition and medicine values. Furthermore, Chinese bayberry has great benefits to human health. To make a comprehensive understanding of mechanisms involved in ripening and quality formation of fruit, we carried out the studies on fruit EST. The main results were summarized as follow:(1) Construction of the four fruit cDNA libraries of Chinese bayberryTotal RNA were extracted from the green immature, pink ripe, red ripe and dark red ripe fruits of Biqi Cultivars with improved Bugos protocol, mRNA were purified with polyAT Isolation Systems, double-strands cDNAs were synthesised with LD-PCR method, then digested with Sfi I enzyme and ligated with pDNR-LIB vector, after transformation, four cDNA libraries were constructed, which consisted of 1.18×10~6 cfu/ml, 3.92×10~5 cfu/ml, 1.49×10~5 cfu/ml and 2.01×10~5 cfu/ml, with recombination rates 94.74%, 94.74%, 100%, 94.74%, respectively. The inserts size mostly ranged from 300 bp to 1,000 bp. The results suggested that the library can be suitable for screening low abundant mRNA for cDNA clones.(2) Construction of EST library included 2000 ESTsTwo thousand ESTs were obtained with 500 clones randomly selected from each cDNA library. Three hundred and ninety five Unigenes obtained were analyzed using BLAST tools. Three hundred and fifteen functional genes (79.75%) have homology sequence from reported plant genes. According to the function, the Unigenes were classified with different catalogues, and 14.18% of unigenes were related to disease resistance and stress response, which indicated that stress defense system was strengthened during fruit ripening. Fruit ripening related genes, such as ACC oxidase and polygalacturonase, and fruit quality related genes, such as Myb , Thaumatin-like protein, were identified.(3) Construction of an MPCR system to screen high frequency genesAnalysis of ESTs results indicated that 10 Unigenes, with frequencies in range of 1.55% to 20.4%, appeared quite often in cDNA libraries. The total frequencies of these 10 Unigenes reached 57.00%. According to the sequences of these 10 high frequency genes, MPCR primers were designed and a system was successfully established to screen these high frequency genes. The system was validated with sequenced clones.