羽衣甘蓝SEPALLATA-like基因的系统发育与表达分析

Phylogenetic and expression analysis of SEPALLATA-like gene in Brassica oleracea L.var.acephala

E类MADS-box基因SEPALLATA(SEP)-like在被子植物生殖生长特别是花器官发育方面具有重要作用。为分析羽衣甘蓝E功能MADS-box基因SEP-like基因的序列特征及其在花发育过程中的时空表达模式,以羽衣甘蓝品系‘14 line’为试材,利用cDNA末端快速扩增(Rapid amplification of cDNA ends,RACE)技术克隆了SEP直系同源基因BroaSEP1/2/3(GenBank登录号:KC967957、KC967958、KC967960)。序列和系统进化树分析表明,这3个基因分别与野甘蓝(Brassica oleracea var.oleracea)、芜菁Brassica rapa、萝卜Raphanus sativus、甘蓝型油菜Brassica napus的SEP1、SEP2、SEP3基因具有很高的同源性。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每一基因都有其亚家族特异的C-末端功能域SEPⅠ和SEPⅡ基序。BroaSEP1、BroaSEP2、BroaSEP3基因的开放阅读框长度分别为801 bp、759 bp、753 bp,分别编码266、252、250个氨基酸残基。半定量RT-PCR和实时荧光定量PCR研究结果表明,BroaSEP1、BroaSEP2、BroaSEP3在发育的花芽中特异性表达,但是表达水平在不同发育时期以及野生型、多瓣型和少瓣型花芽中存在明显差异。

The E class MADS-box genes SEPALLATA(SEP)-like play critical roles in angiosperm reproductive growth,especially in floral organ differentiation.To analyze the sequence characteristics and spatio-temporal expression patterns of E-function MADS-box SEP-like genes during kale(Brassica oleracea L.var.acephala)flower development,BroaSEP1/2/3(GenBank No.KC967957,KC967958,KC967960)homologues,three kale SEP MADS-box gene,were isolated from the kale variety‘Fourteen Line’using Rapid amplification of cDNA ends(RACE).Sequence and phylogenetic analysis indicated that these three SEP genes had a high degree of identity with SEP1,SEP2,SEP3 from Brassica oleracea var.oleracea,Brassica rapa,Raphanus sativus and Brassica napus,respectively.Alignment of the predicted amino acid sequences from these genes,along with previously published subfamily members,demonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins:the MADS domain,intervening(I)domain and keratin-like(K)domain,and the C-terminal domain SEPⅠand SEPⅡmotif.The longest open reading frame deduced from the cDNA sequences of BroaSEP1,BroaSEP2,and BroaSEP3 appeared to be 801 bp,759 bp,753 bp in length,respectively,which encoded proteins of 266,252,and 250 amino acids respectively.Expression analyses using semi-quantitative RT-PCR and quantitative real-time PCR indicate that BroaSEP1/2/3 are specifically expressed in floral buds of kale during flower development process.The expression levels of the three genes are very different at different developmental stages,also in wild type,mutant flower with increased petals,and mutant flower with decreased petals.These different patterns of gene expression maybe cause the flowers to increase or decrease the petal number.