Functional Characterization of OsMAPK3 Gene from Rice (Oryza sativa L.) Required for Abiotic Stress and Herbicide Tolerance

Mitogen-activated protein kinase (MAPK) is one of the important components that know to participate in regulation of plant abiotic-and boitic- stress responses and in controlling of plant growth and development. MAPK genes belong to a multiple gene family. However, the physiological function of each individual MAPK in rice and how MAPK engages in various abiotic stresses tolerance have not yet completely determined. In this study, first we addressed the putative function of OsMAPK3 by making alignment of the amino acid and DNA sequences and conducted the phylogenetic relationship analysis of OsMAPK3 with other rice MAPK gene. Then to understand gene expression pattern of OsMAPK3, the OsMAPK3 promoter sequence was further analysis for identification of different cis-acting DNA elements. The result showed that OsMAPK3 and OsMAPK4 that already demonstrated to involve in plant abiotic stress tolerance are in the same group C. Their nucleotide sequences shared 69% similarity; while OsMAPK3 only share 19% to 51% of sequence similarities with other OsMAPK genes. Besides, several stress-, hormone- responsive and development-control related cis-acting DNA elements exist in the promoter of OsMAPK3, including DRE, HRE, CRE, ABRE, ARE, and Myb factor recognition site. When TNG67 rice seedlings were treated with different abiotic stresses and OsMAPK3 gene expression was determined, OsMAPK3 can be induced by ABA, NaCl, drought, H2O2 and SA. On the other hand, there is no effect of paraquat, CuSO4, CdCl2 and methyl jasmonate treatments
To reveal the effect of OsMAPK3 on abiotic stress tolerance of rice, we took Agrobacterium-mediated gene transfer method to produce both over-expressing and knockout OsMAPK3 transgenic rice plants and its effect on the tolerance capability of rice seedlings under drought and salt stresses was examined. The result indicated that over-expression of OsMAPK3 in rice lead to increase of higher survival ratio, chlorophyll content, Fv/Fm value, proline content and antioxidant activity under stress. This result provided conceivable functional evidence that over- expression of OsMAPK3 gene could improve the abiotic stress and herbicide tolerance. On the contrast, the OsMAPK3 knockout transgenic rice displayed intolerant phenotype against stresses. Moreover, the over-expression OsMAPK3 transgenic rice can promote new tiller formation under cold treatment and ends with new tiller at higher internodes position when reaching maturity. We concluded that these phenomena of new tiller formation may be related to the escape of cold damage and enhance survival of rice seedlings or delay the senescence to death of rice. In this thesis, we successfully transduced OsMAPK3 gene into rice and efficiently rise up the abiotic stress and herbicide tolerance. It will be worthy to note that compared to improve plant stress tolerance through transferring the signal transduction downstream gene, our approach seems better and benefit field management than traditional study.
In future, we hope to apply microarray and 2-D gel analysis techniques in characterization the gene and protein expression profiles between wild type and OsMAPK3 transgenic rice plants to understand the regulation mechanism of OsMAPK3 gene under stresses. In addition, we are dedicated to elucidate the effect of OsMAPK3 on branching (tiller)-formation related genes in rice as well as change the endogenous content of ethylene and auxin that lead to alteration of rice growth and development. Taken together, by transgenic approach this study demonstrated that OsMAPK3 gene can play an important role in regulation of abiotic stress and herbicide tolerance, also affect the tiller formation in rice.