530 XU Yu-chao et al.Journal of Integrative Agriculture 2016,15(3):528-536 AtCBF1 91 AtCBF2 99 AtCBF3 AtDREB-A1 92 DREB AtDREB-A4 AtDREB-A5 AtDREB-A3 52 AtDREB-A2 AtDREB-A6 BrcERF-B3 99 AtERF1 AtERF2 99 AtERF5 AtERF6 ERF 99 AtERF3 68 AtERF7 AtERF9 84 99 AtERF4 AtERF8 AtAP2 AP2 100 AtRAV1 RAV AtRAV2 Fig.2 The phylogenetic analysis between BrcERF-B3 and the Arabidopsis thaliana AP2/ERF superfamily transcription factors. The phylogenetic tree was produced with the neighbor-joining method by the MEGA 5 software.The numbers above the branches indicated the reliability percent of bootstrap values from 1 000 replicates.BrcERF-B3 was boxed.The same as below. while the expression of BrcERF-B3 in mutant firstly subtly 2.3.Investigating the responses of BrcERF-B3 on decreased at rosette stage,then significantly increased,and different stresses keep a higher levels,suggested that BrcERF-B3 enhanced reproductive growth development.The expression profile of After ABA and MeJA treatments,similar trend were found BrcERF-B3 gene showed unobvious expression difference in expression of BrcERF-B3 in mutant and BrcERF-B3 was during flower development stages in maintainer line,but up-regulated and then down-regulated as time goes on.In the gene expressed significantly higher in mutant than in maintainer,the expression of BrcERF-B3 was decreased maintainer at the flower development(bud diameter <2 mm) compared to 0 h excepting at 1 h MeJA treatment.The stages indicated that BrcERF-B3 might enhance abnormal expression of BrcERF-B3 existed significant difference flower development in mutant(Fig.7). between maintainer line and mutant under ABA treatment RT-PCR was carried out on cDNA derived from floral and at 1,2,and 12 h MeJA treatment.During the cold organs to learn more about the BrcERF-B3 expression treatment,BrcERF-B3 gene initially increased at 1 h and profile.The results showed that BrcERF-B3 expressed subsequently slowly reached peaks at the transcription strongly in petal,stamen and carpal in mutant and slightly level at 4 and 2 h in mutant and maintainer,respectively. expressed in sepal,petal and carpel in maintainer.Partic- then decreased slowly.The expression of the gene in ularly.BrcERF-B3 expressed obviously higher in mutant mutant was significantly higher than in maintainer at 2-12 h. stamen than in maintainer stamen,which meant that The transcription levels of BrcERF-B3 in mutant and BrcERF-B3 gene acted a positive role in the formation of maintainer was obviously down-regulated in the NaCl mutant stamen (Fig.8). treatment (Fig.9).530 XU Yu-chao et al. Journal of Integrative Agriculture 2016, 15(3): 528–536 while the expression of BrcERF-B3 in mutant firstly subtly decreased at rosette stage, then significantly increased, and keep a higher levels, suggested that BrcERF-B3 enhanced reproductive growth development. The expression profile of BrcERF-B3 gene showed unobvious expression difference during flower development stages in maintainer line, but the gene expressed significantly higher in mutant than in maintainer at the flower development (bud diameter <2 mm) stages indicated that BrcERF-B3 might enhance abnormal flower development in mutant (Fig. 7). RT-PCR was carried out on cDNA derived from floral organs to learn more about the BrcERF-B3 expression profile. The results showed that BrcERF-B3 expressed strongly in petal, stamen and carpal in mutant and slightly expressed in sepal, petal and carpel in maintainer. Partic￾ularly, BrcERF-B3 expressed obviously higher in mutant stamen than in maintainer stamen, which meant that BrcERF-B3 gene acted a positive role in the formation of mutant stamen (Fig. 8). 2.3. Investigating the responses of BrcERF-B3 on different stresses After ABA and MeJA treatments, similar trend were found in expression of BrcERF-B3 in mutant and BrcERF-B3 was up-regulated and then down-regulated as time goes on. In maintainer, the expression of BrcERF-B3 was decreased compared to 0 h excepting at 1 h MeJA treatment. The expression of BrcERF-B3 existed significant difference between maintainer line and mutant under ABA treatment and at 1, 2, and 12 h MeJA treatment. During the cold treatment, BrcERF-B3 gene initially increased at 1 h and subsequently slowly reached peaks at the transcription level at 4 and 2 h in mutant and maintainer, respectively, then decreased slowly. The expression of the gene in mutant was significantly higher than in maintainer at 2–12 h. The transcription levels of BrcERF-B3 in mutant and maintainer was obviously down-regulated in the NaCl treatment (Fig. 9). Fig. 2 The phylogenetic analysis between BrcERF-B3 and the Arabidopsis thaliana AP2/ERF superfamily transcription factors. The phylogenetic tree was produced with the neighbor-joining method by the MEGA 5 software. The numbers above the branches indicated the reliability percent of bootstrap values from 1 000 replicates. BrcERF-B3 was boxed. The same as below