Origin And Preliminary Study On The Function Of The OsAMI10 Gene In Rice

Synthesis of amide can not only store nitrogen but also eliminate toxicity caused by high concentration of ammonia.High concentration of ammonia will make photophosphorylation uncoupling and influence the formation of ATP. Thus, It’s important to study the function of the genes related to amide metabolism. Horizontal gene transfer (HGT) is the movement of genetic material between different organisms with reproductive isolation other than via vertical transmission. HGT has been shown to be an important factor in the evolution of many organisms, such as increasing genetic variation, enhancing function and improving adaption to environment.Previous studies indicated that the amidase family in land plants originated from horizontal gene transfer events and took bacteria as the donor. For the further research, a member of the amidase family of the terrestrial plant, OsAMI10 gene of rice, was studied in this research by using the bioinformatics and reverse-genetics methods. The main results were as follows:1. The sequence of OsAMI10 gene in Oryza sativa was used as a query to search in the NCBI nr protein database and Phytozome database. The results indicated that apart from Picea abies, there was at least one homologous gene in land plants genome with completed sequence. The evolutionary analysis of representative species genome indicated homologous genes of AMI10 0 in Physcomitrella patens, Solanum lycopersicum and Gossypium raimondii were produced by replication after other species differentiation. Two homologous genes of Brachypodium distachyon were produced by tandem repeat. Comparing intron phase and position of these genes showed that although some evolutionary mechanism, such deletion and gain, were found in intron, intron position of AMI10 gene was conserved in most of plant.2. Phylogenetic tree of AMI10 homologous genes which were found in nr database was structured, and results showed AMI 10 gene was single-origin in plant. Phylogenetic tree classified AMI 10 homologous genes into two group, A and B. The origin of the plant AMI 10 gene was the result of an ancient horizontal gene transfer event from bacteria that were classified into A group. In this study, results indicated that AMI10 gene originated from horizontal gene transfer that took bacteria as donor before the differentiation of Klebsormidium flaccidum and Coccomyxa subellipsoidea.3. A pair of primers, LP and RP, and a T-DNA boundary primer, BP, were used to identify homozygous mutants by PCR. Then, the gene expression of osami 10 of homozygous mutant plants identified by PCR were analyzed. The results showed that there were not significant difference between osami 10 homozygous mutants,and wildtype in term of the gene expression of OsAMI10, for T-DNA was inserted into intron.4. Both the phenotype of overexpressing plants and RNAi plants are significantly different with wild-type plants.Some important traits of widetype,overexpression and RNA interference plants were measured within growth stage of rice.The results indicated that the plant height of overexpressing plants is higher than widetype,the hundred-grain weight is lighter than widetype significantly. Tillers, effective panicles and hundred-grain weight differed significantly between RNAi plants and widetype plants.5. Rice OsAMI10 gene may be associated with chlorophyll synthesis.Chlorophyll content of widetype and RNAi plants were measured in four-leaf stage of rice. The consequence revealed chlorophyll a,chlorophyll b and chlorophyll content of RNAi plants were significantly lower than widetype, which indicated that OsAMI10 gene interference affected the synthesis of chlorophyll. Therefore, we estimate that OsAMI10 gene may be associated with chlorophyll synthesis.6. Rice OsAMI10 gene was regulated by nitrogen.To identify the effect of nitrogen on gene expression of OsAMI10, four-leaf stage seedlings were studied under nitrogen-difficient stress. The results showed gene expression of OsAMI10 was induced by N-difficient stress treatment. Gene expression reached its lowest level when seedlings were incubated under low nitrogen after 6h. Then, gene expression rose slightly and remained stable. After recovering nitrogen 1h, gene expression level reached normal condition.7. The protein coded by OsAMI10 gene had amidase activity. Prediction of OsAMI10 protein structure domain indicated that the protein belonged to amidase family and might have amidase activity. To further test this hypothesis, glutaminase activity of leaf of widetype, overexpression and RNAi plants were measured in four-leaf stage of rice.There was very significant difference within widetype, overexpression and RNAi plants by t-test. The results indicated that the protein coded by OsAMI10 gene had amidase activity, which was consistent with previous prediction.