| Pinus massoniana, a kind of important tree species in Southern China, is widely used in construction, furniture and paper production。Pine wilt disease casued by Bursaphelenchus xylophilus has already caused serious damage to chinese forestry. Being of the complexity of pathogenic mechanism of the disease, there is no effective way to prevent and cure it so far. Therefore, studying on the interaction between p. massoniana and B.xylophilus can reveal the resistant mechanism of the host plant and the pathogenic mechanism, which provide further information for selection and reasonable use of resistant, In the paper, to elucidate the resistant mechanism at the molecular level.suppression subtractive hybridization(SSH)was adapted to construct a P. massoniana leaf cDNA library induced by B. xylophilus. Illustrating the resistant mechanism of the plant and cloning relative genes will be beneficial to improving the resistance of P.massoniana.After inoculating with Bursaphelenchus xylophilus, the leaves from Pinus massoniana were harvested at 24,48 and 72hours respectively. A suppression subtractive hybridization library of Pinus massoniana was constructed using above leaves based on SMART method and PCR-Select cDNA Subtraction Kit. Fifty nine effective sequences were obtained from seventy randomly picked positive colonies. BlastX alignment results revealed that nine fragments showed similarity to function-unkonwn sequences and 6 didn't find any similar genes. Resistance related genes obtained in the SSH library were analysed that signal transduction,stress response,defence protein synthesis,transcriptional and other vital process were involved in disease defence process. Zinc-finger protein, phenylalanine ammonia-lyase,4-coumarate:CoA ligase, SPI1B,wound responsive protein, ABA-responsive and embryogenesis-associated gene;LEA-like protein, Rac-like GTP binding protein, calcium binding protein, cytochrome oxidase, NBS-LRR protein had played an important role in main resistance process.Analysis results of signal transduction related gene may also take part in triggering resistance response. There still need to study some of unknown gene sequence in the cDNA SSH library.In this paper the gene encoding phenylalanine ammonia-lyase(PAL),Cyclophilin and Chlorophyll a/b-Binding Protein(Cab) were amplified by RT-PCR and 5' rapid amplification of cDNA end(RACE) respectively. The cDNA of pal is 2700bp long,in which including 2157 open reading frame(ORF), encoding a protein of 718 amino acids residues.The predicted molecular mass is 78.20kD and theoretical isoelectric is 5.81. It shows as hydrophilic protein. The GenBank acession Number is GQ142010. And further analysis showed that it possess the PAL domain sequence GTITASGDLVPLSYIAG and no transmembrane regions. The cDNA of Cyclophilin is 1002bp, in which including 519bp ORF, encoding a protein of 172 amino acids residues. The predicted molecular mass is 18.212 kD and theoretical isoelectric is 8.82, It is a hydrophilic protein. The GenBank acession Number is GQ497815. And further analysis showed that it possess the Peptidyl-prolyl cis-trans isomerase domain FKGSSFHRVIPGFMCQGG,signal sonsensus sequence and transmembrane regions. The prokaryotic expression vector of cyp gene encoding protein was constructed by subcloning the fragment into pET-24a(+) and was expressed in Escherichia coli induced by IPTG. The cDNA of Cab is 1062bp, in which including 825bp ORF, encoding a protein of 274 amino acids residues. The predicted molecular mass is 28.98 kD and theoretical isoelectric is 5.24, It is a hydrophilic protein. The GenBank acession Number is GQ073386. And further analysis showed that it possess the chlorophyll a/b binding domain and no transmembrane regions. The prokaryotic expression vector of cab gene encoding protein was constructed by subcloning the fragment into pET-24a(+) and was expressed in Escherichia coli induced by IPTG. |
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