Cloning Of Nonspecific Lipid Transfer Protein Genes In Chimonanthus Praecox And Function Analysis Of Genes

Nonspecific lipid transfer protein(nsLTP) is a kind of pathogenesis-related (PR) proteins that have been located in cell membrane. These proteins were so named because of their ability to transfer phosphatidylcholine, phosphatidylinositol and phosphatidylserine in vitro. The assay for antibacterial and antifungal show that they have ability to inhibit bacterial and fungal,they also have potential to inhibit pests. Many assays also indicate that these proteins might be involved in formation of cutin or wax. So it is very significant.Nonspecific lipid transfer protein have been isolated from many plants and cloned many genes of nonspecific lipid transfer proteins(nsLTP). Two nsLTP genes have been isolated from Calycanthus praecox's cDNA library though EST tags and has been carried on the sequence analysis and the function to it forecast. In order to confirm the function two nsLTP's expression vectors have been costructed, as well as have transfered Nicotiana tobacum.Transgene plants have been got and analysised. The main research as fellow:1.Get the lipid transfer protein genes(nsLTP genes)Get the lipid transfer protein genes in library stochastic choice clone, distill DNA plasmid,take the library clone's plasmid as the template,using EST sequence DW222703 special primer we can get estimate size (388bp) special fragment ,using EST sequence DW223106 special primer we can get estimate size (446bp) special fragment.Meanwhile, using library clone vector pTriplEx2 spceial primer to expand for DW222703 approximately the 613bp special fragment and for DW223106 approximately the 653bp special fragment.Though analysis of biomformation and alignment of internet, CH0092 and CH1477 clones from library respectively stand for genes of full-long cDNA of DW222703 and DW223106. 2.Sequence analysisAccording to the sequence analysis of the goal gene nsLTP92 and gene nsLTP1477 , it discovers that these genes are respectively composed by 613 and 653 nucleotides, respectively including 360 and 351 nucleotides ORE, and is respectively translated to 119 and 116 amino acids. This protein theory molecular weight is respectively 12.1 and 11.7kD, the isoelectric point is respectively 8.57 and 9.05. 3.construct the expression vectorsThe purpose gene is in the clone vector pTripEx2, cuts DNA plasmid from the vaccine activated with X-balI and SmalI enzyme, and finally be translated into pc2301 vector, construct successfully express vector verified by enzyme digest. 4.Transferring foreign genes to tobacco genomeNsLTP92 and nsLTP1477 genes were respectively transfer into tobacco genome by agrobacterium mediated transformation, Leaf dish as host. More than ten transfergene plants are obtained for each gene by using different transformation. 5. Identification of transgenic plantTransformed plants were firstly stained by GUS solution, showing that foreign gene had been introduced and regenerated into tobacco genomes. They were further confirmed by PCR assay. GUS positive plants could be amplified a stable band with molecular weight conforming to positive control; however GUS negative plant could not. 6.aphid-resistant bioassay in transgenic plantsAnalysis and stastics to transgenic plants of aphid-resistant bioassay, the result of analysis and stastics explains transgene plants of the tag nsLTP92-1,nsLTP92-3,nsLTP92-4,nsLTP92-5 have the aphid-resistance when P=0.05, but others haven't. The result also show nsLTP1477 gene haven't the aphid-resistance. 7. Observation of transgenic plants in greenhouse.Growth of transgenic plants nsLTP92 and nsLTP1477 were observated in greenhouse. In the same condition, comparisons were infected by Phytophtora parasitica var. nicotianae Tucker. so survey about disease index were carried on all transgenic plants in the same time. Difference were found for transgenic plants nsLTP92 and nsLTP1477 to resist Phytophtora parasitica.