Identification Of N-glycosidase Activity For Ribosome Inactivating Proteins From Camellia Sinensis

All the time, pests and diseases have caused a serious impact on plant growth, especially cash crops, such as tea and tobacco. Many kinds of plant diseases and pests spread fast and are not easy to control, so comprehensive prevention and protection are urgently needed, more effective method is to foster resistant varieties. With the molecular biology and genetic technology fast development, it provide a good platform to foster resistant varieties, we can reveal the mechanism of plant resistance for genetic breeding. The study will lay the foundation for high yield cash crops and cultivating high quality varieties.We isolated a full-length cDNA of CsRIP gene from a green leafhopper-resistant tea genotype-Fuzao2's leaves when tea responsed to green leafhopper, then we cloned the genome sequence by3'RACE and5'RACE. After eight separate genomes sequenced, we found they had a high homology and could be divided into three categories, named respectively CsRIPⅠ, CsRIPⅡ and CsRIPⅢ. Through the PCR technology testing their mRNA expression, we found that CsRIPⅠ highly expressed in tea leaves when tea trees were feeded by little green leafhoppers, it may play an important role in plant insect resistance mechanism, CsRIPⅡ specifically expressed in the cotyledon tissue, we conjectured it could be a storage protein gene.CsRIP gene encode a protein, the protein is a kind of toxins inactivating ribosome and restraining protein synthesis. The protein has various functions such as antiviral, antifungal, antitumor and it widely exist in plants, it has increasingly become one of most important topics of the research on plant resistance. We make use of the TAIL-PCR method for cloning tea tree seed specific promoter and insect induced the promoter. Through the analysis of their sequences, we find the two promoters have multiple DNA components, CsRIP genes may be induced and regulated by abscisic acid, jasmonic acid methyl ester, mechanical damage, heat and salicylic acid. Through CsRIPI gene prokaryotic expressing, SDS-PAGE electrophoresis analyzing and soluble analysising, we find that the target protein express the largest amount by IPTG induced after4h. CsRIPⅠ gene successfully transformed into tobacco by agrobacterium will lay the foundation for researching CsRIPⅠ gene function and cultivating resistance plants.