玉米Gln1-4的gDNA序列、基因结构、保守功能域与等位变异

Genomic DNA Sequence,Gene Structure,Conserved Domains,and Natural Alleles of Gln1-4 Gene in Maize

本研究旨在分离玉米谷氨酰胺合成酶(GS)家族重要成员Gln1-4 gDNA序列全长,分析基因结构、保守功能域与自然等位变异,为氮利用效率功能位点关联性分析奠定基础。利用PCR步移(walking)方法分离Gln1-4基因区域基因组DNA序列,用生物信息学方法分析基因结构与保守功能域,测序与序列比对法分析重要区域自然等位变异。结果表明,分离得到自交系Mo17 Gln1-4区域gDNA 3 724 bp,起始密码子至终止密码子序列长2 858 bp,登录到GenBank(登录号为EU369651),并注释。Gln1-4基因含10个外显子与9个内含子,18个剪接位点均为保守的5'供位GU与3′受位AG模式。编码的GS蛋白由356个氨基酸组成,分子量39.2 kD,等电点(pI)为5.202。氨基末端外显子2到外显子6为氨离子结合结构保守功能域;羧基末端外显子8与外显子9构成ATP酶活性保守功能域。Gln1-4与Gln1-3基因相比,在DNA序列、氨基酸序列、基因结构、保守功能域均很保守,氨基酸序列一致性达98.31%。52个玉米自交系的Gln1-4等位变异分析中,共鉴定出318个等位变异位点,其中242个SNP,45个Indels,占90%。该基因氮利用效率功能关联性分析区间应位于氨离子结合功能域与ATPase活性保守功能域中重要的变异位点,18个剪接位点。

Maize （Zea mays L.） cultivars with improved nitrogen use efficiency would be beneficial for low-input production systems and for environment since it would reduce the surface water pollution and the nitrate leaching into underground water. Glutamine synthetase genes families are the core elements for nitrogen assimilation and metabolism in maize plants. The objectives of this study were to isolate the genomic DNA sequence of Gln1-4, which is one of the important members of glutamine synthetase gene family, to analyze the gene structure, conserved domains and natural allelic variations and so that was to found basis for association analysis of the functional sites related to nitrogen use efficiency in maize. PCR walking strategy was applied to isolate the gDNA sequence of Gln1-4 and its flanking sequence. The gene structure was analyzed by aligning gDNA sequence and its’ mature mRNA sequence. The conserved domains were obtained by searching Conserved Domain Database （CDD） at NCBI. The natural allelic variations were evaluated by resequencing and aligning DNA sequences among 52 inbreds. A total of 3 724 bp gDNA sequence of Gln1-4 of Mo17 was assembled. The full length of the coding region was 2 858 bp, which was comprised of ten exons separated by nine introns. All 18 splicing sites were the conserved sequence of GU at 5＇ donor sites and AG at 3＇ acceptor sites. The sequence has been submitted to GenBank （Accession No.： EU369651） and annotated in details. Gln1-4 encodes a GS protein with molecular weight of 39.2 kD, which was comprised of 356 amino acids. Its isoelectric point （pI） was 5.202. Conserved domain searching results showed that the region from exon 2 to exon 6 at amino-terminal was an ammonium ion binding domain, and exon 8 to exon 9 at carboxyl terminal consisted of an ATPase activity domain. As compared with Gln1-3, the DNA sequence, amino acid sequence, gene structure and conserved domains for Gln1-4 were highly conserved with a 98.31% identity of amino acid sequence. A total of 318 types of natural DNA variation at important and target region of Gln1-4 gene were identified among 52 maize inbred lines, of which 242 was SNPs and 45 was small indels, comprising 90% of the total allelic variations. The analysis of the functional sites gene associated with nitrogen use efficiency of Gln1-4 in maize should focus on binding and catalyzing domains and splicing sites.