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[目的]克隆高羊茅FaGF14-B和FaGF14-C基因并进行基因的差异表达分析,为后续基因功能研究提供依据。[方法]以高羊茅转录组学测序获得的FaGF14-B和FaGF14-C基因序列片段为模板,利用3’RACE和5’RACE方法从高羊茅中扩增出FaGF14-B和FaGF14-C基因的全长cDNA序列,命名为FaGF14-B和Fa GF14-C,并进行核酸序列分析、基因编码蛋白分析、蛋白保守结构域分析、系统进化树分析与差异表达分析。[结果]FaGF14-B基因序列cDNA全长1 548 bp,具有完整的开放阅读框(ORF,449~1 228 bp),编码蛋白为260个氨基酸;FaGF14-C基因序列c DNA全长1 250 bp,具有完整的开放阅读框(ORF,66~848 bp),编码蛋白含有261个氨基酸;GF14-B和GF14-C蛋白都具有典型的14-3-3蛋白结构域,二级结构包含9个保守的α-螺旋及不保守的N-末端和C-末端。系统进化树分析表明,高羊茅Fa GF14-B和FaGF14-C与禾本科植物GF14蛋白具有较高的相似性,位于在同一个进化支上,亲缘关系较近;荧光定量PCR分析表明:FaGF14-B和FaGF14-C基因应答氮胁迫处理。[结论]该研究可为进一步筛选抗低氮胁迫相关基因、创制耐低氮的牧草新种质奠定理论基础。
[Objective] The aim of this study was to clone the genes of FaGF14-B and FaGF14-C of tall fescue and to analyze the differentially expressed genes. [Method] The sequences of FaGF14-B and FaGF14-C gene sequences obtained from transcriptomic analysis of Tall Fescue were used as templates. FaGF14-B and FaGF14-C were amplified from tall fescue by 3’RACE and 5’RACE methods The full-length cDNA sequences of the genes were named FaGF14-B and Fa GF14-C. Nucleotide sequence analysis, gene encoding protein analysis, protein conserved domain analysis, phylogenetic tree analysis and differential expression analysis were performed. [Result] The full-length cDNA of FaGF14-B gene was 1 548 bp in length and contained a complete open reading frame (ORF, 449-1 228 bp) encoding a protein of 260 amino acids. The FaGF14-C gene cDNA sequence was 1 250 bp in length , With a complete open reading frame (ORF, 66-848 bp) encoding a protein containing 261 amino acids; the GF14-B and GF14-C proteins all have a typical 14-3-3 protein domain and the secondary structure contains 9 Conserved alpha-helices and non-conserved N- and C-termini. Phylogenetic tree analysis showed that FaGF14-B and FaGF14-C of tall fescue had higher similarity with gramineous plant GF14 protein, located in the same clade, and their genetic relationship was closer. Fluorescent quantitative PCR analysis showed that FaGF14 B and FaGF14-C genes in response to nitrogen stress. [Conclusion] This study could lay the theoretical foundation for further screening of the genes related to low-nitrogen-tolerance and establishing new germplasm of low-nitrogen-tolerant forage grass.