Cultivating Rice Mutants Of OsMAPK4 And Function Analysis

Accumulation of salt in the soil causes deletetious effects in crop growth and leads to production reduce including rice,which is affecting the agriculture yield of our country even the world.Therefore, lots of researchers are devoting themselves to developing the mechnism of salinity tolerance,finding the key genes and Cultivating the transgenic crops .Mitogen-activited protein kinases(MAPKs) is a universal Ser/Thr protein kinases in all eukaryotes. When the plants are suffering the environmental stress such as drought,low temprature and high salinity, MAPKs can be activated to respond. In rice, the expression of OsMAPK4 up-regulated rapidly when the plants suffered from high salinity and low temperature.In this study,plant expression RNAi vector pCMI was constructed.And the rice was transformed by Agrobacterium Tumefaciens.Transgenic plants were obtained,and the T1generation was inspected by resistance identification.The tolerance to salinity of pCME12 transgenic T1 generation plants was analyzed.The transcriptinon level of the targeted genes was primarily analyzed by Real-time PCR.The DNA chip results are analyzing.The main results was summarized as follows:1. Construction of plant expression vectors pCMIPlant expression vectors pCMI was designed and constructed,which contained three fragments: OsMAPK4 Fragment,Anti-OsMAPK4 Fragment and pCAMBIA-1301 Intron.It can form hpRNA after transcription.2. Transformation and PCR identification of resistant seedlingsThe vectors pCMI and pME12 were transferred into rice"wuyoudao 1"by Agrobacterium Tumefaciens. 38 resistant seedlings were obtained,which were transformed with LBA4404(pCMI);23 resistant seedlings were obtained,which were transformed with LBA4404(pME12). PCR positive plants were 10 and 11 separately.3. Obtained T1 generation plants resistant to Bialaphos and PCR positive Screening T1 generation seedings by M0 culture containing 6mg/L Bialaphos,5 and 7 resistant pants were obtained,thereinto PCR identification was detected.9 and 12 plants were positive.4. Analysis of tolerance to salinity of pCME12 transgenic T1 generation plants The Rate of Normal growth tolerance to salinity of 11 pCME12 transgenic T1 generation plants were detected.Among them,7 plants were above 10% and 3 were above 20%,which suggested the transformated OsMAPK4 gene enhanced the tolerance to salinity of rice.5. Detection of OsMAPK4 transcription in normal and induced plants(1) selection of Real-time PCR primer:β-actin gene special 1 primer and OsMAPK4 gene special 1 primer(2) Real-time PCR identification to normal rice leaves:2 lower expression of OsMAPKs were detected from 5 RNAi plants.The expression level was 1/7 and 1/9 of wild plants. 2higher expression of OsMAPKs were detected from 3 pCME12 Tansgenic plants.The expression level was3.6 and2.3 times as wild plants.(3) Real-time PCR identification to 150mM NaCl induced 1h leaves.The results were: the transcription level of 2 RNAi pants were 0.41 and 0.69 of untreated plants and 2 pCME12 Tansgenic plants were 1.23 and 1.69 times of untreated plants.6. Transcriptom Analysis of transgenic plants(1)The differences of OsMAPK4 expression profiling were analyzed,suggesting 1022 genes up-expression,among them,116 genes were onlogy annotation.While 1654 genes down-expression,among them,260 genes were onlogy annotation.37 genes were the same.(2)37genes'annotation and function were collected and analyzed,13 genes were obtained , relevant tightly with salt tolerance,including signal transduction, gene expression regulation and salt-tolerant genes.