Isolation And Identification Of Protein Kinase MeMAPK1 And MeMAPK8 From Cassava

Cassava (Manihot esculenta Crantz), belong to Euphorbiaceae plants, is one of the three major crops in the world. The loss of cassava production is about one-third due to the infection ofXanthomonas axonopodis pv.manihotis, which can cause important infulence on the agricultural production. MAPK (Mitogen activated protein kinase), a protein kinase in the MAPK cascade pathway, play a vital role in disease resistance pathway. Although a large number of MAPKs have been found in plants, there are few studies on MAPK signaling pathways in Cassava. In Arabidopsis, it has been shown that AtMAPK3 and AtMAPK6 play a key role in plant disease resistance. In this study, according to Cassava genome information and similarity blast, MeMAPK1 and MeMAPK8, the homologue of AtMAPK3 and AtMAPK6 was identified. Furthermore, the function of MeMAPKl and MeMAPK8 was studied by biochemical function prediction, in vitro complementary experiments and genetic transformation in the PTI pathway. The main findings are as follows:(1) Based on the MAPKs member of Cassava and the genetic relationship among Cassava.Arabidopsis and rice, we found two genes possibly involved in disease resistance and named MeMAPKl and MeMAPK8. The full length cDNA of the MeMAPK1 and MeMAPK8 gene was cloned by RT-PCR. The full length of MeMAPK1 is 1215 bp,encoding amino acid 404aa.The full length of MeMAPK8 is 1098bp, encoding amino acid is 365aa. The bioinformatics analysis of the gene suggested that MeMAPKl and MeMAPK8 possibly function as protein kinase.(2) The expression of MeMAPKl and MeMAPK8 was analyzed by qRT-PCR under SA, JA,Eth, flg22, drought, cold and Xam04 treatment. The results showed that the expression of MeMAPK1 was the highest under the drought and cold stress.MeMAPK8 was induced by the hormone and pathogen treatment, which indicated that the gene could be have the function in hormone response and disease resistance. The expression of MeMAPK8 showed that the gene was increased and subsequently decreased. These results suggested that MeMAPKl and MeMAPK8 were not only regulated the plant disease resistance pathways also regulated the plant growth and development.(3) To detect the subcellar location, MeMAPK1-GFP and MeMAPK8-GFP vectors were constructed, transformed to Arabidopsis protoplast and observed under microscope. The results showed that MeMAPK1 located in the cytoplasm, MeMAPK8 located in the cell membrane and nucleus of Arabidopsis protoplasts.(4) The overexpression vectors pJG053-MeMAPK1-GFP and pJG053-MeMAPK8-GFP were introduced into Arabidopsis transgenic plants by Agrobacterium-mediated transformation.The five transgenic lines were detected and the results showed that MeMAPKl and MeMAPK8 were inserted the genome and can effectively expressed in Arabidopsis.(5) After Xam04 and PstDC3000 inoculation, the transgenic plants showed higher resistance compare to Col-0 and atmpk3, atmpk6 mutant, indicating MeMAPKl and MeMAPK8 involved in Xam04 resistance. However, atmpk3 showed higher resistance compare to Col-0 and atmpk6. And we detected MeMAPK8 is less resistant to PstDC3000 than MeMAPK1.(6) To confirm which protein interacts with MeMAPKl and MeMAPK8, yeast two-hybrid cDNA library of Cassava was screened. Seven possibly interacting protein were found and the interaction will be further confirmed.(7) The prokaryotic expression vector was constructed and the expression of MeMAPKI and MeMAPK8 were in E.coli (DE3).Westem blotting showed that the fusion protein could be expressed in E.coli (DE3).