Biological Function Analysis Of Rice Embryo Specific Gene OsESG1

RLKs (Receptor-like Kinases) is a member of plant protein kinases family. Asbeing receptors of signal moleculars, RLKs play important roles in plant growth anddevelopment, down stream genes transcription and expression regulation, andparticipated in stress responses by phosphorylation and dephosphorylation ofintracellular target genes. RLKs are known to be divided into four subfamiliesaccording to the difference of extracellular domains. SRLKs(S-domain RLKs)，whichare characterized by S-domain in the extracellular part, is one of the more importantsubfamily with about147members in rice. Up to now, partial of SRLK genes havebeen isolated and identified, and were further verified to participated inself-incompatibility and developmental process, but few focused on abiotic sressresistance process.In the former research, a rice SRLK gene, OsESG1(HE584611), was identified.The antisense expression vector of OsESG1driven by35S promoter was constructed,and the anti-sense (AOsESG1,AS) transgenic rice was harvested.In this research, the prokaryotic expression vector of OsESG1was furtherconstructed, and the preliminary study on protein expression was carried out.Combined with the OsESG1expression patterns analysis under different treatmentconditions, the phenotype of AS transgenic plants, the related physical indexs and thebiological functions under drought, salinity and IAA treatments were analysed.Combined with the results of high throughput sequencing, the function of OsESG1could be further clarified, and the data could provide clues and evidence for theconstruction of gene regulatory network. The results were listed as follows:1, Prokaryotic expression of OsESG1: the prokaryotic expression vector ofOsESG1-pET-28a(+) was constructed and transformed into competent cells of E.colistrain BL21for expression. After induction by IPTG, a protein product of about 80kDa could be seen by SDS-PAGE analysis. The expression of OsESG1attained tothe maximum level when induced by IPTG for7h, and the protein expression levelstayed this level after that. The0.1mmol IPTG was also be vertified to be the bestinduction concentration for OsESG1expression.2, The expression patterns analysis of OsESG1: the expression patterns ofOsESG1were carried out by semi-quantitive RT-PCR. The tissue expression patternananlysis showed that OsESG1is highly expressed in embryo of rice seeds. Theinduced expression pattern analysis showed that, the expression of OsESG1could beinduced by ABA, GA3and Me-Ja in the leave of rice, and expression level ofOsESG1decreased by IAAtreatment in the root of rice. The expression of OsESG1inrice root could be influenced by drought, cold and salt stresses, and there were anobvious increase of OsESG1when treated by drought.3, The biological function analysis of OsESG1: function analysis were carried outusing AS transgenic rice as meterials. The phenotype observation showed that thenumber of adventitious root of AS rice decreased, and the growth of primary root wasinbibited compared with those of wild type plants. The results indicated that OsESG1may participate in the formation of adventitious roots, and play an important role inroot development process. Determination of physical indexs showed that, the MDAcontent, ion leakage increased and the activity of CAT, APX decreased in transgenicrice after drought treatment, indicating the drought tolerant ability in transgenic ticedecreased. OsESG1participates in the formation of adventitious roots and plays animportant role in drought resistance. We also found that leaf senescence induced bydark in AS is more serious than those of control, indicated that OsESG1may take partin the dark-induced leaf senescence in rice.4, High throuthput sequencing and digital gene expression profiling: differentexpression genes were screened by RNA-Seq using AS and WT of “zhonghua11” asmaterials. The expression of two Aux/IAA genes increased in AS but seven SAURsgenes decreased, indicating that the function of IAA was influence in rice, which isconsist with phenotype of root growth inhibition in transgenic rice. Also, the theexpression of isopentenyl transferase genes downregulated and the expression of cytokinin dehydrogenase upregulated in AS, leading the level of cytokinin in ricedecreased, which is in accordence with more serious leaf senescence that induced bydark in AS. The expression of peroxidase and the WRKY were all downregulated inAS, which leading to the drought tolerance decrease inAS rice.In summary, OsESG1could be expressed80kDa protein product using prokaryoticexpression system. Biological function analysis of OsESG1found that, OsESG1takepart in development of rice root system through regulation of IAApathway and it playroles in leaf senescence induced by dark in rice. More importantly, OsESG1participates in rice drought tolerance by regulating the expression of preoxidase andWRKY transcription factors, which is confirmed by RNA-Seq. The results of highthroughput sequncing using wild type and transgenic rice as materials showed that,the expression of partical genes in IAA metabolism and stress response pathways arechanged and verified, which shed some light for further OsESG1function analysisand construction of OsESG1regulation network.