Research On Salt Tolerant Peroxidases In Wheat And Arsenic Resistant Transgenic Plant

Soil salinity is a major abiotic stress limiting crop production worldwide. Under the pressure of food deficiency, population growth and environment deterioration, it is very important to enhance the salt tolerance of cereals. Peroxidase (POD) is one of the key enzymes in the defense system of plants under stress, so it is necessary to study the functions of POD in order to cultivate wheat with improved salt tolerance.In the previous study, TaPOD gene was cloned from the wheat Shanrong No.3 (SR3) via RACE technology and analyzed its function. In this paper, we transformed the Atpod deficient Arabidopsis with the 35S1301/TaPOD vector and carried through functional analysis. We construct the pUN-GFP/TaPOD vector used to transient expression analysis in Arabidopsis/onion, which showed that the fusion protein was targeted to cytoplasm or the membrane of the cytoplasm. Meanwhile, we analyzed the T₁ generation of transgenic SR3 wheat with salt treatment, PCR assay, and POD activity assay. With TaPOD gene specific primer, we cloned the different POD genes from the genome DNA of SR3, JN177 and A.elongatum respectively. The sequences analysis indicated that TaPOD gene came from parent JN177. With Dnaman and NCBI Spidey analysis we found the differences of POD between SR3 and A.elongatum mainly lied in the introns, their exons had the same size with high similarity.Earlier Southern blotting result showed that TaPOD had multiply copies in SR3 genome. Used the Chinese Spring DNA of nullisomic lines as template, the PCR analysis with the TaPOD specific primer showed that the TaPOD was located in the chromosome 7B, which indicated that the TaPOD might exist in the same chromosome in the form of multiply copies.According to the earlier two-dimensional electrophoresis results, we cloned designed two segments- TaPOX9 and TaSOD, which had 99% similarity to peroxidase 9 in Triticum monococcum L. and 99% similarity to SOD3.1 in Triticum aestivum respectively. RT-PCR revealed that the transcription of TaPOX and TaSOD were different in different tissues. A possible stress-responding mechanism of this gene has been discussed.In our study, we cloned the arsBC gene, which was related to As tolerance, from E.coli by PCR. We constructed the vector of pROK2/arsBC and transformed it into A.thaliana. Kan selection and subsequent PCR assay indicated the arsBC gene inserted into the A.thaliana genomes. In this paper, we made statistic analysis about the separation ratio in the transgenic Arabidopsis T₃ generation of arsBC, found 2 strains to be homozygous. In the RT-PCR followed, one of the lines had high level of arsBC expression. When treated with 100mM arsenate in 1/2 MS medium for two weeks, the transgenic plants displayed enhanced tolerance than the control (Col-0) .