特早熟春性甘蓝型油菜sBnFLD基因的克隆及表达

Cloning and expression of sBnFLD gene from spring rapeseed(Brassica napus L.)

【目的】克隆特早熟春性甘蓝型油菜A基因组上的sBnFLD基因,并对其进行表达研究,为该基因的功能及其在成花转变中的作用研究奠定基础。【方法】根据GenBank中已报道的拟南芥和白菜型油菜FLD同源基因的保守序列设计引物,采用PCR和RT-PCR扩增特早熟春性甘蓝型油菜86号品系(光周期不敏感)的FLD同源基因,用qRT-PCR技术检测sBnFLD基因在86号品系不同发育时期茎、叶和茎尖中的表达情况。【结果】克隆出了sBnFLD基因,命名为sBnFLD,在GenBank中的登录号为KR003079.1。sBnFLD基因cDNA全长2 376bp,有3个内含子,4个外显子,编码791个氨基酸残基,分子量86.5ku,等电点8.5;sBnFLD为非分泌蛋白和非膜蛋白;sBnFLD蛋白N端有2个保守的结构域α螺旋结构域(SWIRM)和NAD(P)-binding-8结构域,该蛋白有多个α螺旋和β折叠。生物信息学分析显示,sBnFLD蛋白与已报道的甘蓝型油菜未知蛋白(CDX73929.1)和电子克隆的白菜型油菜FLD(XP_009135110.1)氨基酸序列相似性达99%,与拟南芥FLD氨基酸序列相似性达87%。qRT-PCR分析结果显示,sBnFLD基因在油菜苗期和现蕾初期茎、叶、茎尖中均有表达,但在蕾期茎尖中表达量最高。【结论】克隆出的sBnFLD基因为甘蓝型油菜的FLD同源基因,该基因在春性特早熟甘蓝型油菜开花调控中可能起着重要的调节作用。

【Objective】The research cloned sBnFLD gene and analyzed its expression.【Method】The primers were designed according to conversed sequence of published FLDin Arabidopsis and B.rapa.Homologues cloning and reverse transcription PCR（RT-PCR）were applied to clone the gene and analyze its expression.【Result】The FLDhomologue gene was cloned in B.napus and named sBnFLD（accession No.KR003079.1）.The CDS sequence of sBnFLD was 2 376 bp,containing 3introns and 4exons,and encoding aprotein of 791 amino acid residues with a predicted molecular weight of 86.5ku and a theoretical pI of 8.5.sBnFLD was nerther secreted protein nor non-membrane protein.N-terminal of sBnFLD had two conserved domains,SWIRM and NAD（P）-binding-8.sBnFLD is a instability protein composed with alpha helix and beta folding.Bioinformatics analysis showed that the sBnFLD had 99% similarity with unknown protein CDX73929.1in B.napus and prediction protein XP_009135110.1in B.rapa,and had 87%similari-ty with FLD in A.thaliana.The expression analysis of sBnFLDgene using qRT-PCR showed that the sBnFLDgene was expression in stem,seeding and shoot tips at seeding and bud stage.However,the relative expression level of the gene was the highest in shoot tips at bud stage.【Conclusion】As homologous gene of FLD,sBnFLD might play a role in flowering regulation.