Selection Of Mapk2 Mutant By Silencing The MAPK2 Gene Using RNA Interference In Arabidopsis Thaliana

Plants are exposed to a number of different conditions that can adversely affect growth, development and homeostasis. As part of their response to environmental changes, plants modulate the expression of specific genes by transducing extracellular stimuli into intracellular message by the signal transductiong cascades, thereby perceiving the diverse stimuli and the changes of environment factors. Among these, Mitogen-activated protein kinase (MAPK) and the MAP kinase cascade play a very important role in this signal transduction pathway. It has been established that MAPK cascade involves in stress responses, such as wounding, drought, cold, high salt treatments, and pathogen infection, and plant hormone stimuli. A database survey has recently found that there are at least 20 MAPK family members in the whole genome of Arabidopsis Plant MAPKs can be classified into four different subfamilies based on the analysis of their amino acid sequences. Most known members of the family belong to subgroups A and B. They have been most frequently found to be involved in environmental responses, hormonal responses and cell division. But nothing is yet known about members of subfamily C. RNAi (RNA interference) is a post-transcriptional gene silencing phenomena that founded in plants, animals and fungi. In nature, RNAi may play an important biological role in protecting the genome against instabilities caused by transposons and repetitive sequences, and be an ancient antiviral response/protection mechanism, or have integral functions in the regulation of endogenous genes in development. RNAi has become an important technique to reveal gene functions through makes gene inactivation without change the genomes. It may be also act as a powerful tool to characterize the genes that involved in the complex signal transduction pathway.In our experiment, a 650bp fragment of the Arabidopsis MAPK2 gene was prepared by the Polymerase Chain Reations (PCR) using genome DNA as template. The fragment was inserted in the two multi-clone sites of binary vector pFGC5941, as anti-sense and sense orientations, respectively. The construct pFGCmpk2ds was transformed into Agrobacterium tumefaciensLBA4404 strain. Arabidopsis thaliana was transform by floral dip method. The T2 population wad screened fro mutants by glufosinate-ammonnium, and 25 transformed lines were obtained. Total RNAs from wild type and putative mutant lines were isolated, and the MAPK2 mRNA levels of the transformed lines were measured by PCR. The result indicated that the mRNA leavel of the mapk2 gene in transformed lines is obviously lower than that in WT, it is proved that the transformed lines of mapk2 are the RNAi silencing mutants.After primary analysis, some distinguishing phonetypes of the RNAi silencing mutants of mapk2 can be observed. mapk2 has more rosette leaves (average 27.7) than WT (average 11.5), some of the mutants have more rosette-liked structures on the blots. It is also can be observed that the apical dominance is absent from the mutant when and after flowering. The phenotype of the mapk2 may indicate that the MAPK2 involves in the developmental regulation of the Arabiopsis, suggesting that there is a link between MAPK2 and auxin signal transduction, but it needs more evidences to prove it.