Clone And Function Analysis Of OsSIZ1 And OsAPX2 In Rice

This thesis includes two parts: 1) analysis of the T-DNA insertion mutant siz1 of rice and the study of the function of OsSIZ1; 2) clone and functional analysis of OsAPX2.Rice is an important food crop and also regarded as one of the model plant of the grass family and monocotyledon. The genome of rice had been sequenced,then it is urgent to annotate the functions of gene in rice genome. Mutant, particularly the tagging mutant, is the best material to clone gene .Up to date, scientists have created a pool that owns many mutants, and there are many researchers are devoted to study these mutants and the mutant gene's functions. Our Lab also has created a considerable mutant pool, and found some interesting mutants which referring to agricultural traits in rice. Here two mutants was reported, they are enhance tapping mutants .One gene is the homolog of AtSIZ1 gene, which was predicted to code a SUMO E3 ligase in Arabidopsis, the decrease of gene expression caused dense lateral roots in rice mutant ;the other gene codes an acrobat peroxidase and whose null mutant exhibit drawf and sterile .Firstly, we briefly show a T-DNA mutant siz1,which come out more lateral roots and the lateral roots is denser than wide type .Analysis of the T-DNA flanking sequence indicates that the T-DNA tagger was inserted in the third intron of the candidate gene OsSIZ1(OSJNBb0079L11.3)on Chr.5 in rice. The mutant phenotype cosegregated with T-DNA tagger and the candidate gene is the homolog of SUMO ligase E3(AtSIZ1)and the similarity of nucleic acid is 51.63%. In the mutant, the expression level of OsSIZ1 has severely decreased compared with Wild type and there is not new Tos17 insertion site in the mutant. The genetic analysis indicates that the candidate site is a single gene and meets to the Mendel's law. The promoter of the gene drives GUS gene expressed in root, leave, shoot, flower and pollen. The fusion protein of OsSIZ1 and GFP driven by 35S promoter was located in nuclear and cell peripheral .Otherwise, the IAA level was higher in root, but lower in shoot of young seedling than that of wild type, external use IAA can depress the length and numbers of lateral root, but induce that of wild type. The OsSIZ1 from wild type plant can restore the mutant phenotype to wild type in rice root. At the same time, we introduced OsSIZ1 to Arabidopsis mutant siz1-2,the homologous gene of OsSIZ1, to study the function of AtSIZ1.The transgenic plant of siz1-2 carrying with the can grow higher and stronger than mutant. The result suggests that OsSIZ1 has similar functions to AtSIZ1,and OsSIZ1 influenced rice growth and development.Secondly, in Mutant apx2, the T-DNA tagger was inserted in the fourth intron of OsAPX2. The mutant apx2 was a null mutant so that the apx2 mutant grew drawf and the homozygote was sterile and can't produce fruits. The anther developing abnormally leads to the vitality of pollen decrease severely. There is no new Tos17 insertion site in genome and the drawf and sterile phenotypes cosegregated with T-DNA tagging. That means the mutant gene maybe be responsible for the phenotype. So we introduced the full length cDNA of OsAPX2 to homozygotic mutant by A.G infection to do a complementation experiment. The transgenic plant T0 grows higher than mutant near to the height of wild type plant,and the flower is partly fertile and can fruit. The promoter of OsAPX2 drive GUS gene expressed in root, leave, node, flower and pollen. The fusion protein of OsAPX2 and GFP was located in cytoplasm and cell membrane in the root of transgenic tobacco. Histochemistry stain for mutant, wild type and over expression transgenic plant show that, in mutant leaves, there are more ROS and H2O2 than in WT. Transformed gene mutant leaves by DAB and NBT, but in the restore plants, the contents of ROS and H2O2 is same as that of wild type. It suggests that mutation of OsAPX2 results in that ROS and H2O2 accumulated in leaves of mutant, particularly accumulated much H2O2. Moreover, we find that excessive expression of OsAPX2 in rice can't enhance the ability to sweep off ROS, instead cause oxidative stress to rice. These results reveal that there is a precise mechanism of ROS elimination to keep the balance of ROS in plant and OsAPX2 play an important role in rice growth and flower development. Clone of OsAPX2 gives more evidences to explain the relationship between ROS and APX.