Cloning And Function Analysis Of Rice(Oryza Sativa L.) Mitochondria Serine Hydroxymethyl Transferase Gene From Photorespiration Mutant

The sequencing of rice genome, a important crop, and constructing of T-DNA insertion mutant pools have provided good conditions for rice functional genomics. Many important agricultural character mutants have been isolated so far, in which some genes causing mutant phenotypes have also been cloned. These works are beneficial for learning the genetics of agricultural characters. For closely related to photosynthesis, the studying of chlorophyll deficient mutations is significance. In this study, after obtaining lots of chlorophyll deficient mutants, a gene relation to photorespiration was cloned and the functions were studied. The most studying contents are listed as follows: 1 Identification of mutant phenotypeWhen planted in culture room, the mutant phenotype was difficult to discern from wild at first, but after 15 days since sowing, the mutant phenotype appeared gradually. Before dying, the mutants grew faster than wild, but the seedlings were so slender that it couldn't sustain the weight of leafs and began to be curved. 2 Cloning of the mutated geneIt was supposed that the mutant phenotype was relation to T-DNA by GUS staining. Using PCR-walking technique, the flanking sequence of T-DNA left border was isolated easily. After BLAST, the T-DNA was proven to be inserted into a gene, predicted coding rice mitochondria serine hydroxymethyltransferase (SHMT). It was certain that the tagged gene co-segregated with mutant phenotype by PCR method. The normal gene in wild was amplified and transformed to mutant, and after regeneration, the mutant phenotype disappeared. The complementary experiment proven the cloned gene is responsible for the chlorophyll deficient mutant phenotype.3 The expression patterns of OsmSHMT Using enhancer trap vector which can indicate expression patterns of tagged gene, the different parts were examined with GUS assay. The leaf blade and root were stained strongly, but the flower was barely. 4 The mutant phenotype was restored in high CO2 concentrationFrom bioinformatic analysis, the OsmSHMT gene takes part in photorespiration process. For proving the result, the OsmSHMT mutant was cultured in high CO2 concentration, after 2 weeks, no any observable mutant phenotype could be discerned from wild.5 OsmSHMT gene prokaryotic expressionAfter transformation of OsmSHMT-pET28a expression vector into E.coli strain BL21(DE3), high yield of recombinant protein was achieved.6 The changes of photosynthesis in mutantFor determining the changes of photosynthesis, the chlorophyll fluorescence curve was examined, and it observed that the chlorophyll fluorescence quenching was influenced greatly in mutant, especially in high light intensity. In low light intensity, comparing qP with qN, it was evident that the qN was declined more seriously. It might be caused by the enhanced membrane permeability. In high light intensity, the qP was also declined serious, it might be caused by damaged Calvin cycle.7 The changes of chloroplast ultrastructure and protein complexes on thylakoidCompared with wild type, the quantity and volume of chloroplasts in mutant mesophyll were decrease. In chloroplast of wild, the granum thylakoids were distributed more regular than mutant. It might be caused by the stroma thylakoid damage. As for the changes of protein complexes on thylakoid, from BN-gel, it was observed that most strips of mutant moved slower than wild. It might be caused by the loose structure of injured protein complexes.In conclusion, the mutation of OsmSHMT damages photorespiration, and because the Calvin cycle needs photorespiration to sustain, the injured Calvin cycle leads to declined light energy converting and the excess light energy promotes reactive oxygen species(ROS) producing, then the membrane, the protein complexes and the chlorophyll were all injured by ROS, at last, the chlorophyll deficient mutant phenotype appears.