| Animal husbandry plays a vital role in our country's economical industry.Alfalfa (Medicago sativa L),As a kind of important forage grass ,always considered as the king of forage.But in recent years plant disease and insect pest problem has been becoming increasingly severe and has became one of the primary restrictive factors for the alfalfa industrialized development in the northern area of our country.fungal disease such as brown spot,frosty mildew, rust become serious day by day; the insect pest type are also quite many and is mainly oriental army worm,aphids and so on at present.The plant breeding scientists have devoted in sharpen the alfalfa disease-resistant insect pest ability these days.Along with genetic engineering starting,it has provided one new method for plant disease protection in the environment friendly foundation.Many experiments have already proved that:chitinas mainly can degenerates fungus cell wall's chitin,thus achieves to suppresses the fungus growth,and simultaneously has both the insecticidal activity.Cowpea trypsin inhibitor which has the direct action to cause the insect unnatural death in the insect digesting system,holding the broad spectrum insecticidal activity.Our research has separately cloned these two genes from the alfalfa and the cowpea,constructed bivalent plant expression vector,and then transferred it into alfalfa through mediation of agrobacterium -tumefaciens LBA4404.We Obtained primarily resistant callus.1.Genomic DNA in the leaves of the cowpea was extracted with CTAB.The CpTI gene was amplified by PCR with the conserved sequence acted as primers,then cloned to pMD-18T vector and detected the recombinant by restriction enzyme method.The sequence has been registered in GeneBank(NO. EU088405).It is one of serine protease inhibitor gene family .After digesting by BamHâ… and Sacâ… ,the gene was cloned to pCUbi1303 between the promoter 35S and the terminator NOS.After detected by restriction enzyme and PCR,it indicated that the plant expression vector pCUbiCpTI1303 was constructed successfully, which provides a basis for the following transgene.2.CpTI gene was cloned into pGEX4T-1 vector.After detected by restriction enzyme,The recombinant pGEX4T-CpTI was obtained.The recombinant 37kDa fusion protein was produced after being induced with 0.08mmol/L IPTG.This indicates the molecular weight of the expressed protein fit in theory value well.3.Induced alfalfa which has been cultured for 15 days by salicylic acid and mechanical stick-method,Cloned a 984bp ChiA fragment separately from genomic DNA by PCR and total RNA by RT-PCR using artificial synthetic special primers.Then combined it with plasmid pMD-18T easy vector,obtained recombinant pMD-ChiA.After detected by restriction enzyme indicated that the nucleotide sequence from genomic DNA and total RNA completely to be consistent,The sequence has been registered in GeneBank(NO.EU294179).Sequence analysis indicated that the homology between this fragment and the reported sequence from LanZhou may be 100%.The amino acid has the model structure area such as:N-terminal signal peptide,Hevein domains(chitin-binding domain),variable spacer region,Catalytic domains.Indicated that it is Classâ… C hitinas gene.4.Digested pMD-ChiA and pMD18-T-CpTI separately,cloned them to pC35S1303 successively.After detected by restriction enzyme and PCR, it indicated that the bivalent plant expression vector was constructed successfully.5.By introducing the bivalent plant expression vector into agrobacterium tumefaciens LBA4404,obtained project strain LBA4404.Processed the seed of alfalfa with 9% NaClO.Cultured the aseptic seedling.The leaves and hypocotyls was used as the explants.After infected with Agrobacterium,the explants was co-cultured without light for 3days,then transferred into the callus inuction culture medium,finally the selected culture medium. |
