Functional Study Of Calcium/Calmodulin-regulated Receptor-like Kinase Gene MeCRLK1 From Cassava (Manihot Esculenta)in Response To Low Temperature

As a tropical and subtropical plants, cassava is sensitive to low temperature. For studying the molecular mechanism of cassava low temperature sensitivity,we began to research from the signal perception in low temperature signal pathways. As the signal receptor, plant receptor like kinase play important roles in signal transduction pathway.Thus in this study, we cloned a putative calcium/calmodulin-regulated receptor-like kinase gene by RT-PCR from cassava cultivar SC8 and named it as MeCRLKI (GenBank Accession No.KP256022). Then we did the bioinformatic analysis of MeCRLKI,prokaryotic expression and subcellular localization, gene expression profile under low temperature and transfer MeCRLK1 into Arabidopsis to reveal its effect on cold resistance of transgenic Arabidopsis. The results show as below:(1) The MeCRLK1 gene CDS sequence is 1296 bp in length and encoded a protein of 431 amino acids. The protein molecular formula is C2118H3391N605O636S18, molecular weight is about 48.08 kD, and theoretical pI is 8.11. The protein has multiple different phosphorylation sites, 3 predicted CaM binding sites, 4 disorder, and 1 potential globular domains. The structure analysis indicated that the protein sequence has one transmembrane domain, a S_TKc domain, and without signal peptide in N terminal. The protein subcellular localized on membrane. The protein sequence alignment between MeCRLKl and AtCRLKl indicated that the similarity is 79.8%, the identify is 77.6%. The sequence differences between MeCRLK1 and AtCRLK1 is most in the C terminal.(2) At room temperature, the expression of MeCRLKl gene is highest in cassavaroots, followed by the expression in leaves. Low temperature treated for 0.5?2 h, decreased the expression of MeCRLKl in roots and leaves. However, the expression changes in shoots was not obviously. Low temperature treated for 3 h later, the gene expression in roots, shoots and leaves gradually increases. Thus it speculate that MeCRLKl has no resistance to low temperature.(3) Subcellular localization showed that MeCRLK1 mainly on the plasma membranes of cassava embryo. It predicted that the function of MeCRLKl may be associated with stomatal movement involves abiotic stress response.(4) Phenotype changes occurred on MeCRLKl transgenic Arabidopsis thaliana seedling root and potted seedlings leaves, but no changes on the resistance to low temperature. activity of the cold related enzymes SOD and POD slightly higher than(5) control, but the CAT enzyme activity difference was not significant in transgenic Arabidopsis.It predicted that MeCRLKl genes did not play roles to improve the cold resistance ability, but response to the abiotic or biotic stresses which closely related to the root, through regulating root growth.(6) MeCRLK1 protein can be induced to expression in E. Coli BL21, however the quantity is low. Preliminary optimization results showed that under the induced condition in bacterial liquid concentration of OD600 = 1.0, the IPTG 0.4 mmol/L, 20? , and cultivating for 6 h,the protein expression quantity is relatively high,but still exist in the form of inclusion body protein. Thus it is still need further fumble for its protein expression conditions, for the separation and purification of recombinant proteins, as well as lay the foundation for subsequent experiment research.The results lay the foundations for the further validate MeCRLKl gene function.