玉米Gln1-3 gDNA序列分离、基因结构、保守功能域与等位变异分析

谷氨酰胺合成酶家族是高等植物体内氨态氮同化酶, 是植物体内氮利用与循环的核心构件。玉米Gln1-3基因编码叶肉细胞中的细胞质同工酶, 与全株氮向籽粒蛋白质同化与氮利用效率及产量紧密相关。本研究以玉米自交系辽白371为材料, 采用PCR与接头步移(walking)方法分离得到Gln1-3基因区域基因组DNA(gDNA)全长4 571 bp, 起始密码子至终止密码子序列长3 062 bp。将该基因在GenBank注册, 登录号为EU338535, 并进行了详细注释。该基因由10个外显子、9个内含子组成, 剪接方式主要为5′供位保守的GU与3′受位AG模式。编码的GS1蛋白由356个氨基酸组成, 分子量39.2 kD, 等电点 (pI) 为5.202。保守功能域分析表明, 氨基末端外显子2到外显子6为氨离子结合结构域, 羧基末端外显子8与外显子9构成ATP酶结构域, 在胞内行使催化功能。对50个玉米自交系Gln1-3基因重要分析区域进行了等位变异分析, 鉴定出364个等位变异, 其中313个为SNP变异, 占86%。

Genomic DNA Sequence, Gene Structure, Conserved Domains, and Natural Alleles of Gln1-3 Gene in Maize

Glutamine synthetase gene family, the core elements of in vivo nitrogen use efficiency (NUE) and nitrogen recycle, encodes enzymes catalyzing the nitrogen assimilation from ammonium ion into protein in higher plant. Gln1-3 is a member of GS family that encodes the cytosolic isozyme expressed in mesophyll cells of maize (Zea mays L.). The knowledge about the genomic se-quence of the gene is the premise for the natural alleles, functional studies, and molecular marker development. In the present study, 4 571 bp of genomic region of Gln1-3 was sequenced using a maize inbred line Liaobai 371 with PCR walking strategy. The 3 062 bp coding region of the gene was comprised of ten exons that were separated by nine introns. Totally, six out of nine splice sites followed splicing mechanism with conserved GU sequences at 5′ donor sites and AG at 3′ acceptor sites. The se- quence has been submitted to GenBank (Accession No.: EU338535) and annotated in details. Encoded GS1 protein, molecular weight of 39.2 kD, is comprised of 356 amino acids. Its isoelectric point (pI) is 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 and exon 9 at carboxyl terminal consisted of an ATPase domain to fulfill the synthesis activity. A total of 364 sites of natural variation at impor-tant and target region of Gln1-3 gene were identified among 50 maize inbred lines, and 313 (86%) sites of the total (364) were SNP variations. The results provide a good basis for analyzing the key candidate genes associated with NUE and grain yield under different nitrogen supplies levels.