Study On Cloning The Gene Of β-1, 3-glucanase In Tea Plant And Its Expression In Escherichia Coli

Tea plant [Camellia Sinensis (L.) O. Kuntzes] is one of the important industrial crops and export agricultural products in China. Since a long time , fungal diseases have been one of the principal causes of tea plant growth and tea yield , which results the national tea great economic losses , so how to control fungal diseases has been one of the concerned questions by the breeders . Since human started to cultivate tea plants, a lot of measures have been applied to control the fungal diseases, such as breeding the fungus-resistant cultivars by conventional breeding ways , the application of pesticides , and colligated ways to reduce dosage of agrochemicals and rudimental agrochemicals . But due to the limited inheritance resources, the high variability of fungal and the pollution of the pesticides etc. , which leads to applying the technology of gene engineering to breed the anti-pathogens cultivars becomes one of the recent research focuses of agricultural biological technology . In this paper , the full-length cDNA of β-1,3-glucanase of tea plant had been cloned and studied at the first time . This gene was expressed in E.coli and its protein activeness was determined, which was not only contribute to the gene engineering research on anti-fungal pathogens of tea plant , but make a base for applying the biological technology to breed the tea cultivars. In this work, the experiment results show that : 1. According to the partial known 3'-terminal sequence of β-1,3-glucanase of tea plant , we designed and synthesized the GSP premier for 5'-RACE reaction. A 1005bp fragment was amplified by 5'-RACE which was subsequently analyzed in GenBank date-base after sequencing , the results showed that the sequence of the nucleotide and deduced amino acids had high identity with the corresponding part of β-1,3-glucanase cDNAs of other plants published . The results indicated that this specific product may be the partial fragment of β-1,3-glucanase of tea plant . 2. According to the partial known 3'- terminal sequence and cloned 5'-terminal sequence, we designed and synthesized two terminal GSP premiers . The full-length fragment of β-1,3-glucanase cDNA was amplified PCR by using high-fidelity DNA polymerase , and the sequencing analysis showed that the full-length of β-1,3-glucanase cDNA was composed of 2002bp, and the ORF was 1488bp, coding for 495 amino acids . The full-length fragment was subsequently analyzed in GenBank and Swiss-Port date-bases ,and the results showed that the deduced amino acids had 30~70% identity with the corresponding part of β-1,3-glucanase cDNAs of other plants published . The results indicated that this specific product may be the full-length fragment of β-1,3-glucanase of tea plant . 3. The ORF sequence of β-1,3-glucanase was cloned into expression vector pET32a and expressed in BL21trxB(DE3) . The molecular weight of the fusion and target protein induced by IPTG was about 73kDa , and 53.5kDa respectively accorded with the estimated molecular weight 52.9kDa . Its enzyme activity was analyzed by thin-layer chromatography, but no activity was determined.