Cloning And Sequence Analysis Of A Chitinase Gene From Vigna Sesquipedalis And Construction Of The Plant Expression Vector

The aim of the study was to lay foundations for transfer of the gene encoding the chitinase ~nd for further acquisition of the Eansgemc plants of Citrullus lanalus resistant to infection by Fusarium oxy.~7porwn f. rnveum, with cloning.and sequence analysis of a chitinasc gene and cobstuction of the plant expression vector. At first, the quality of the DNA extracted from difi~rent oz~mns of the 慫aoshu 2?(V sesquzpedalis) was compared, and the effect of the [INA as template on PCR products was studied, but no difference observed between them, respectively. PCR conditions for amplification of 慫aoshu 2?cbitinase mature protein gene were optimized, and the optimal concentrations arq: Mg~ 1.5ninml/L,dNTP 0.2mmol/L and primer 0.2 ~?mol/L. A pair of primers :P1 (5?gaattcgagcagtgt ggaagc caagcgggtggtgc3? andP2 (5?ggatcGtcgacgatggggtgtagatt3? were designed according to ten N-terminal amino acids ~f chitlnase mature protein from J?gna sesquipedalis, which was induced, purified and sequenced by our lab, and to the C-terminal amino acids of the same protein gene of flgna w2gu1cu?ata from NCBI (ienebank. The particular class I chitinase~ mature protein gene from Vigna sesquz~~edaIis and class I chitinase signal peptide gm~e from Phaseo!us vulgaris were amplified by PCR. The two genes were. cloned into T-vector. The complete sequence of the particular chitinase mature protehi gene from J~?gna sesquz~edalis was determined and its amino acids sequence was deduced. Chitinase mature protein gene sequence and their amino acids sequence from Vigna sesquzpedali.s, ~?gi~a unguiczdata.and Phaseolus vulgarls were compared and analysed. The results showed that 44 Restriction Enzyme sites ~re found in chitinase mature protein gene from Vigna sesquzpedalis and J攊gna ungidculata respectively. Only 41 Restriction Enzyme sites were detetmined in chitinase mature protein gene from Phaseolus vulgaris, and the types and sites of enzymes were different with those from J?gna. The homology between the cbitinase mature protein genes from ~gna sesquipedalis and Vigna unguiculata is 98.3%, tMt from 2 I?gna sesquzpedalis and Phaseolus vulgaris is 86.1%; while the homology between two amino acids sequence of the chitinase mature protein from Vigna sesquipedalis and 1?gna unguiculata is 97.3%, that from Vigna sesquipedalis and Phaseolus vidgaris is 86.2%. Moreover, seven class I chitinase mature protein genes and amino acids sequences from Vigna sesquipedalis, J4gna unguiczdata., Phaseolus vulgaris, Pisum sativum, Oryza sativa,Nicotiana tabacum and Triticum aestivum were compared. The results showed that the gene and amino acid sequence homology between seven species are over 44.3% and 47.90/o, respectively. In addition, conservation of three sequences: EQCGSQAGGALCP, CCSQFOWCGST and CQSQC in the 40 amino acids sequence at the beginning of the N-terminal chitinase mature protein is very high, The conservation of eight Cys at the sites of 3,12,17,18,24,31,36,40 is 100% Finally, the chitinase signal peptide gene from Phaseolus vu?garis and particular chitinase mature protein gene from Vigna sesquipedolis were constructed into one chimera gene. The chimera gene recombinated into pBIl2l binary vector, and the plant expression vector of complete class I chitinase gene was obtained.