Cloning, Sequencing Analysis And Expression Of Chitinase Class Ⅰ Gene From Medicago Sativa L.

Chitinase (EC3.2.2.14, Chitinase) is referred to as Chi, which is widely distributed, chitin is its natural substrate. It is a major component of cell wall of pathogenic fungi, chitinase inhibits fungal growth by degrading chitin in the cell wall of pathogenic fungi, and cell wall debris degraded can enhance disease-resistance of host plant itself. In addition, some chitinases have activity of lysozyme, can lead to pathogen death by damaging the internal structure of the pathogen. These features of chitinase make it one of the focuses of the current disease-resistance gene breeding.Alfalfa (Medicago sativa L.) is a perennial legume, famous with high yield and good palatability. However, in recent years, alfalfa disease is one of the constraint factors to its development. Basing on this, the study is by transferring exogenous chitinase gene into to the plants to enhance the plants disease-resistance to reach the goal of preventing plant disease, it lays the theorical foundation for further transferring exogenous chitinase gene into the alfalfa.The main results are as follows:1. The full-length cDNA had been cloned by RACE, with primers designed according to an EST sequence segment of Medicago truncatula that had been registered in NCBI (GenBank accession number: AF167323) and template with RNA of"Zhongmu No.6 alfalfa", amplified 400bp fragment (3'-RACE)and 800bp fragment(5'-RACE)respectively;2. Bioinformatics analysis indicated that the full-length sequence of the gene was 1132 bp, containing an ORF (open reading frame) of 930bp to encode 309 amino acids. The gene was named as MsChiâ… (GenBank accession number: HM224449). The clustering analysis indicated the high amino acids alignment homologies of MsChiâ… with chitinase Classâ… gene of other plants; Function analysis showed that: the protein was hydrophilic and transmembrane protein, belongsing to the chitinase 19th family;3. Constructing the plant expression vector MsChi-pCAMBIA1302 by the method of restriction enzymes (Ncoâ… and Speâ… ) and introducing into agrobacterium LBA4404 by the method of freeze-thaw is for the transformation of tobacco;4. Transgenic tobacco plants were obtained by co-culture, selection and regeneration, the PCR, RT-PCR molecular detection indicated that the gene had been successfully integrated into the tobacco genome;5. Prokaryotic expression vector MsChi-pEASY-E1 was constructed and induced protein expression with 1mmol/L IPTG. SDS-PAGE analysis showed that: the molecular weight of induced protein was between 30KD and 40KD.