Isolation And Identification Of Receptor Like Cytoplasm Gene MeBIK1from Cassava

Cassava,an important starchy food for the people in the tropical areas of the world, is a novel energy crop. One of the most limiting problems of this crop is cassava bacterial blight (CBB) disease, caused by Xanthomonas axonopodis pv manihotis(Xam) Plant innate immunity which recognises conserved microbe specific molecules is the first line of defence to protect cassava from this devastating disease. The plant innate immune system provides broad spectrum everlasting protection and has been a new choice for effective plant breeding for disease resistance. Therefore, isolation and identification of cassava innate immunity-related genes will be helpful to illustrate the molecular mechanism of cassava and pathogen interaction andthe novel resistant genes will provide new genetic resources for disease-resistant breeding.In this study, based on sequence similarity to the amino acid sequence of AtBIK1 (LOC_ At2g39660) in Arabidopsis PTI pathway a,gene which was predicted to code receptor-like protein kinase were identified in the cassava genome database. The function were characterized with expression profile, genetic complementation of loss-of-function mutant, transgenic research. The main results were as following:1. Based on the Arabidopsis known knowledge and the released cassava genome databases, we search of the release of cassava homologous genome database, the candidate of the cassava PTI pathway receptor protein kinase gene, the result showed that the length of the cDNA is 1230 bp. It encodes 409 amino acids containing a conserved kinase domain and named MeBIK1 as the gene highly homologous with the gene of Arabidopsis AtBIKl.2. Arabidopsis protoplasts transient expression system showed that MeBIK1 located on the cell membrane.3. qRT-PCR showed that MeBIKl gene expression was hihgly induced by the flagellin stimulation and this induction indicates. MeBIK1 was involved in plant innate immune system. At 1h after induction the expression is highest and subsequently the expression is gradually decreased.4. Genetic complementation experiments to Arabidopsis thaliana atbikl mutant showed that MeBIK1 can restore PTI pathway of Arabidopsis mutants atbikl.5. Different concentrations of 2,4-D and picloram were applied to induce somatic embryogenesis. When 12 mg/L of picloram was supplemented to MS medium, production of somatic embryos reach to 83.3% and much higher than induction by 2,4-D and other concentrations of picloram., We also induced TMS60444, SC6, SC8, SC124, Arg7's somatic embryos by different concentrations picloram. The results showed that TMS60444's embryogenesis rate can be up to 89.2%, while SC8 is 83.3%.L6. To explore transformation into cassava FEC by Agrobacterium-mediated genetic transformation, different Agrobacterium bacterium concentration and time were setted.The results showed that the FEC survival rate was highest when bacterial concentration was OD600=0.4 and infection time was 30 min.8. Histochemical staining showed the temporary expression rate of gus gene reached more than 84.21%.9. MeBIK-GFP fragment was amplied by PCR after DNA extraction and 2 of 4 regenerate plants after transformation indicating a purpose strip,the positive rate is 50%.