XU Yu-chao et al.Journal of Integrative Agriculture 2016,15(3):528-536 529 ERF superfamily can be grouped into five main subfamilies. 2.Results AP2,CBF/DREB,ERF,RAV and one soloist,At4g13040 (Sakuma et al.2002;Nakano et al.2006).They played a 2.1.Cloning the BrcERF-B3 cDNAs from cabbage different role in the regulation of the plant.and there was a mutual regulation relation among them (Zhao et al.2006). The BrcERF-B3 genes were cloned from two different cab- Ethylene-responsive transcription factors (ERFs)were bage lines (Fig.1),and both of their open reading frame firstly identified from tobacco as GCC box-binding proteins (ORF)fragments were 807 bp in length.The sequencing and could either induce or repress the expression of genes results showed that nucleotide sequences of two BrcERF-B3 containing the GCC box and related elements in their pro genes were identical. moters.Sequence analysis showed that ERFs contained a Based on the similarity of the amino acid sequences highly conserved,plant specific DNA-binding domain(DBD) of their DNA-binding domains(DBD),a phylogenetic tree consisting of 58-59 amino acids (Ohme-Takagi and Shinshi was created from the deduced amino acid sequences of 1995).At present,the percentage of total ERFgenes in all BrcERF-B3 and other ERF proteins from Arabidopsis.The the AP2/ERF family has been reported in several plants, result revealed that BrcERF-B3 belonged to the B3 group such as Arabidopsis(44.2%),populus trichocarpa(45.5%). of the ERF subfamily(Figs.2 and 3).The alignment of the Chinese cabbage (45.7%).Vitis vinifera(55%)and rice ERF-B3 showed that DBD of BrcERF-B3 shared a high (48.2%)(Sakuma et al.2002:Nakano et al.2006;Zhuang degree of sequence homology with ERF-B3 from other et al.2008.2009:Li et al.2013).Based on the sequence species.The DBD of BrcERF-B3 consisted of 3 anti-par- identities of their DBD,the ERF subfamily members can be allel B-sheet and 1 a-helix,besides,the F(phenylalanine) classified into six small subgroups(B1 through B6)(Sakuma in BrERF-B3 was replaced by T(threonine)in BrcERF-B3. etal.2002). N(asparagine)in BnERF-B3 was replaced by D(aspartic ERF family genes were reported mostly in responses to acid)in BrcERF-B3,respectively,these residues appeared biotic and abiotic stresses (Gutterson and Reuber 2004: to be responsible for binding specificity(Fig.4).BrcERF-B3 Kizis et al.2001).The group IX ERF genes in cotton may exhibited much greater similarity(95%)to those of BrERF- be involved in jasmonate (JA),ethylene (ET)responses B3(Fig.5). (Champion et al.2009).Arabidopsis ERF family mem- bers B3 subgroup AtERF98 regulated ABA synthesis and 2.2.Analysis of the expression of BrcERF-B3 gene involved in salt stress(Zhang et al.2012).It is reported that AtERF13 and AtERF15 regulate ABA response pos- BrcERF-B3 was completed at different leaf development itively (McGrath et al.2005),however,few researches stages by qRT-PCR.The gene expression profile presented associated with flower development have been found. in Fig.6 showed BrcERF-B3 notably increased during Ro- Non-heading Chinese cabbage(Brassica rapa ssp.chin- sette stage,then significantly decreased in maintainer line, ensis)is a cross-pollinated crop that is widely cultivated in East Asia.We created a stamen-petalody mutant line through chemical mutagenesis,in which stamens convert- ed into petaloid and flower turning into unisexual female flower.We previously obtained an ERF gene fragment by cDNA-AFLP technique,and it was named as BrcERF-B3 bp (not logged).However,the transcriptional regulatory 2000 function of BrcERF-B3 and its expression levels remains 1000 BrcERF-B3 unclear.In this study,we cloned the BrcERF-B3 gene 500 from the leaves and analyzed the expression levels of BrcERF-B3 in different organs in two lines.In addition, 250 we also analyzed the effects of BrcERF-B3 responses to 100 biotic and abiotic stresses.These results indicated that the expression level of BrcERF-B3 expressed only in mutant stamen at the early bud stage.The BrcERF-B3 existed different expression profiles in response to plant hormone, cold and NaCl treatments.These works will provide theoretical base for further studying stamen-petalody in Fig.1 The open reading frame of BrcERF-B3 amplification non-heading Chinese cabbage. products.M.DL2000 marker;1,mutant;2,maintainer.XU Yu-chao et al. Journal of Integrative Agriculture 2016, 15(3): 528–536 529 ERF superfamily can be grouped into five main subfamilies, AP2, CBF/DREB, ERF, RAV and one soloist, At4g13040 (Sakuma et al. 2002; Nakano et al. 2006). They played a different role in the regulation of the plant, and there was a mutual regulation relation among them (Zhao et al. 2006). Ethylene-responsive transcription factors (ERFs) were firstly identified from tobacco as GCC box-binding proteins and could either induce or repress the expression of genes containing the GCC box and related elements in their pro￾moters. Sequence analysis showed that ERFs contained a highly conserved, plant specific DNA-binding domain (DBD) consisting of 58–59 amino acids (Ohme-Takagi and Shinshi 1995). At present, the percentage of total ERF genes in all the AP2/ERF family has been reported in several plants, such as Arabidopsis (44.2%), populus trichocarpa (45.5%), Chinese cabbage (45.7%), Vitis vinifera (55%) and rice (48.2%) (Sakuma et al. 2002; Nakano et al. 2006; Zhuang et al. 2008, 2009; Li et al. 2013). Based on the sequence identities of their DBD, the ERF subfamily members can be classified into six small subgroups (B1 through B6) (Sakuma et al. 2002). ERF family genes were reported mostly in responses to biotic and abiotic stresses (Gutterson and Reuber 2004; Kizis et al. 2001). The group IX ERF genes in cotton may be involved in jasmonate (JA), ethylene (ET) responses (Champion et al. 2009). Arabidopsis ERF family mem￾bers B3 subgroup AtERF98 regulated ABA synthesis and involved in salt stress (Zhang et al. 2012). It is reported that AtERF13 and AtERF15 regulate ABA response pos￾itively (McGrath et al. 2005), however, few researches associated with flower development have been found. Non-heading Chinese cabbage (Brassica rapa ssp. chin￾ensis) is a cross-pollinated crop that is widely cultivated in East Asia. We created a stamen-petalody mutant line through chemical mutagenesis, in which stamens convert￾ed into petaloid and flower turning into unisexual female flower. We previously obtained an ERF gene fragment by cDNA-AFLP technique, and it was named as BrcERF-B3 (not logged). However, the transcriptional regulatory function of BrcERF-B3 and its expression levels remains unclear. In this study, we cloned the BrcERF-B3 gene from the leaves and analyzed the expression levels of BrcERF-B3 in different organs in two lines. In addition, we also analyzed the effects of BrcERF-B3 responses to biotic and abiotic stresses. These results indicated that the expression level of BrcERF-B3 expressed only in mutant stamen at the early bud stage. The BrcERF-B3 existed different expression profiles in response to plant hormone, cold and NaCl treatments. These works will provide theoretical base for further studying stamen-petalody in non-heading Chinese cabbage. 2. Results 2.1. Cloning the BrcERF-B3 cDNAs from cabbage The BrcERF-B3 genes were cloned from two different cab￾bage lines (Fig. 1), and both of their open reading frame (ORF) fragments were 807 bp in length. The sequencing results showed that nucleotide sequences of two BrcERF-B3 genes were identical. Based on the similarity of the amino acid sequences of their DNA-binding domains (DBD), a phylogenetic tree was created from the deduced amino acid sequences of BrcERF-B3 and other ERF proteins from Arabidopsis. The result revealed that BrcERF-B3 belonged to the B3 group of the ERF subfamily (Figs. 2 and 3). The alignment of the ERF-B3 showed that DBD of BrcERF-B3 shared a high degree of sequence homology with ERF-B3 from other species. The DBD of BrcERF-B3 consisted of 3 anti-par￾allel β-sheet and 1 α-helix, besides, the F (phenylalanine) in BrERF-B3 was replaced by T (threonine) in BrcERF-B3, N (asparagine) in BnERF-B3 was replaced by D (aspartic acid) in BrcERF-B3, respectively, these residues appeared to be responsible for binding specificity (Fig. 4). BrcERF-B3 exhibited much greater similarity (95%) to those of BrERF￾B3 (Fig. 5). 2.2. Analysis of the expression of BrcERF-B3 gene BrcERF-B3 was completed at different leaf development stages by qRT-PCR. The gene expression profile presented in Fig. 6 showed BrcERF-B3 notably increased during Ro￾sette stage, then significantly decreased in maintainer line, M bp 2 000 1000 750 500 250 100 1 2 BrcERF-B3 Fig. 1 The open reading frame of BrcERF-B3 amplification products. M, DL2000 marker; 1, mutant; 2, maintainer