Tea Beta-1, 3 - Glucanase Cdna Fragment Cloning And Sequence Analysis

Tea(Camellia sinensis (L) O.kuntze) is one of the most important economic plants in china. Fungal is known to be the major tea disease, which influences the quality and quantity of tea. It is necessary to control fungal disease and cultivate new variety for fungal resistance by genetic engineering techniques. In the paper, we studied on cloning a 1396bp fragment of β-1,3-glucanase cDNA of tea at the first time. The results are reported as follows: Total RNA from young leaves of tea was isolated by chomczynsk's single step method of RNA isolation ,which was partially modified on the basis of the features of biochemistry composition in fresh tea leaves. Two degenerate primers were designed and synthesized according to the highly conservative sequences among the known β-1,3-glucanase genes . A cDNA fragment of 366bp(RTPCR.1) was amplified by RT- PCR,which was subsequently cloned to PGEM-T Easy Vector for sequencing analysis. The result of sequencing analysis showed that the nucleotide and deduced amino acids sequences had high identity with the corresponding part of β-1,3-glucanase cDNAs of other plants published. The results indicate that the special PCR product is a cDNA fragment of β-1,3-glucanase of tea plant. Two special primers were designed based on the above sequence of the RT-PCR product. A 1252bp fragment(3RACE.l) was amplified by 3'race reaction. The analysis of the product of 3'race shows that there is a 249bp overlapping region between the 3'end of the RTPCR.1 fragment and the 5'end of the 3 RACE. 1 fragment ,and the nucleotide and deduced amino acid sequences share 50%-90% homologous identical to the corresponding part of β-1,3-glucanase gene family of Arabidopsis thaliana. A 1369bp successive sequence of the 3'end of the β-1,3-glucanase gene cDNA was obtained by spliced the overlapping region.