Preliminary Study On Function Of The Transcription Factor JERFs

【Backgrounds】ERFs(Ethylene Responsive Factors) are specific family of the transcription factors in plants,which play the key regulatory roles during the plants growth, development and stresses response.Five new members of the ERF proteins were isolated from the tomato expression cDNA library by the yeast one hybrid method with a four times repeated GCC box present in the promoter of tomato NP24 as bait.They could bind both ethylene responsive element and jasmonate responsive element,and named as JERF1/JERF3/TERF1/TERF2/TSRF1,which are unified to called JERFs(Jasmonate and ethylene responsive element binding factors).The reported experiments showed that the JERFs participated in the pathways of biotic and abiotic stresses responsive in the tobacco. However,it is to be further studied that whether the JERFs have the same function in other plants and what the mechanism of JERFs in regulating the interaction between different plant stress response signaling pathways is.【Objects】To elucidate the effect and the basis of TERF2 in enhancing rice seedling tolerance to the chilling stress;To obtain the transgenic Arabidopsis thaliana overexpressing Tag-JERFs fused proteins,which provided the significant materials for studying the physiological functions of JERFs;To screen and identify the important proteins interacting with JERFs in Arabidopsis.【Methods】Biochemical methods,RT-PCR and Real-time PCR techniques were used to research the effects of TERF2 on the physiological and biochemical parameters and the genes expression related to the resistance of rice in seedling stage.Furthermore,in order to investigate the molecular basis of TERF2 in regulating the key genes expression which are related to the chilling stress response in seedling stage rice the cis-elements in the promoters of the target genes were analyzed.At the same time,the Tag-JERFs plant overexpression vectors were constructed by molecular clone techniques and the constructs were electropotated into the Agrobacterium tumefaciens LBA4404.Arabidopsis plants were transformed by the floral dip method and the transgenic Arabidopsis plants overexpression Tag-JERFs fused protein were screened and identified.Therefore,the functions of JERFs in the Arabidopsis different signal pathways were studied with physiological experiments.【Results】(1)ERFs transcription factor TERF2 did not only affect the growth and development of the rice,but also improve the chilling tolerance of the seedling stage rice.(2) TERF2 could improve the osmoregulatory substances contents such as the soluble sugars and proline.Through stabilizing the chloroplast biogenesis-related genes(OsHAP3A,OsHAP3B and OsHAP3C) and transmembrane proteins genes(OsLti6αand OsLti6b) expression,antioxidant enzymes genes(SODB,APX1 and APXb) expression of the leaves during cold stress in seedling rice plant were activated,then the chlorophyll content was maintained,the MDA accumulation was reduced,the stability and integrity of the plasma membranes was maintained.So that it can enhance the seedling rice tolerance to the chilling by decreasing the losses of the photosynthetic rate and membrane permeability.(3) TERF2 could activate the regulatory proteins genes(OsMYB,OsICE1 and OsCDPK7) and cold responsive functional genes(OsSPDS2 and OsEnol) expression of the rice under normal conditions,thereby improving the seedling rice tolerance to the chilling.(4) TERF2 activated the target genes expression at the transcriptional level,mainly by binding with the GCC-box and/or DRE/CRT cis-elements in the promoters of the downstream target genes.(5) The His-TERF2 fused gene plant overexpression vector was constructed and the construct was transformed to the Arabidopsis plants by the floral dip method.Six lines of the transgenic Arabidopsis with one copy were obtained by screening and identifying.(6)The HA-JERFs fused gene plant overexpression vectors were constructed and the constructs were transformed to the Arabidopsis plants by the floral dip method.Nine lines of the transgenic HA-TSRF1 Arabidopsis with one copy,six lines of the transgenic HA-JERF1 Arabidopsis with one copy,two lines of the transgenic HA-JERF3 Arabidopsis with one copy,three lines of the transgenic HA-TERF1 Arabidopsis with one copy,three lines of the transgenic HA-TERF2 Arabidopsis with one copy,were obtained by screening and identifying,separately.(7) JERF1,JERF3 and TERF1 all could decrease the sensitivity of seeds germination to the salt in Arabidopsis,but TERF2 could improve the sensitivity of seeds germination to the salt;JERF1 and JERF3 could reduce the sensitivity of seeds germination and seedling growth to the mannitol in Arabidopsis,while TERF1 and TERF2 could enhance the sensitivity of seeds germination and seedling growth to the mannitol and ABA;JERF1 and JERF3 could lessen the sensitivity of seeds germination to the glucose in Arabidopsis,while TERF1 and TERF2 could raise the sensitivity of seeds germination to the glucose;Moreover,JERF1,JERF3,TERF1 and TERF2 all were involved in the 'triple response' cauese by the ethylene in Arabidopsis.The results showed that the JERFs transcription factors participated in many siganls transduction pathways.【Conclusions】(1)TERF2 could active the regulatory and cold responsive target genes expression at the transcriptional level mainly through binding with the GCC-box and/or DRE/CRT cis-elements in the promoters of the downstream target genes.So as to improve the seedling rice tolerance to the chilling,then by promoting the physiological and biochemical mechanism responsive to the cold in rice.Furthermore,it does not affect the growth and development for the rice in full growth period.(2) Six types of the transgenic Arabidopsis plants were obtained,which could overexpression His-TERF2,HA-TSRF1,HA-JERF1,HA-JERF3,HA-TERF1 and HA-TERF2 fuse protein respectively.(3) The transcription factors JERFs have the important regulatory roles in the osmotic stress response,ABA,sugar and ethylene signals pathways of the Arabidopsis.