大豆GmAAP 基因的克隆、生物信息学分析及亚细胞定位

本研究以大豆Williams 82为实验材料,采用RT-PCR技术克隆到了GmAAP 基因,其开放阅读框长度为1440 bp,编码479个氨基酸。生物信息学分析表明,GmAAP蛋白分子量为53.286 kD,理论等电点为8.93,不稳定指数为34.04,属于稳定的蛋白结构;其二级结构中α-螺旋、β-折叠、β-转角、无规卷曲分别占据47.18%、15.66%、2.71%、34.45%;跨膜区与疏水性分析显示其含有10个跨膜区,且构成跨膜区的氨基酸多为疏水性氨基酸;GmAAP与野生大豆AAP6亲缘关系最为相近,且序列比对的一致性为100%,可能为大豆AAP6 基因。构建融合表达载体,利用PEG-Ca2+介导法转化拟南芥原生质体进行亚细胞定位分析,结果显示GmAAP定位在质膜上。AAP能提高豆科植物对外源氮的吸收与利用率,进而增加种子内贮藏蛋白含量,提升大豆品质。本研究的结果可为GmAAP 功能 的进一步研究奠定基础,同时也为氮素的高效利用提供候选基因。

Cloning,bioinformatics analysis and subcellular localization of GmAAP gene from Glycine max

GmAAP gene was cloned by reverse transcription PCR (RT-PCR) from soybean Williams 82 in this study. Sequence analysis showed that the fragment length was 1440 bp encoding a complete open reading frame (ORF) of 479 amino acid residues. Bioinformatics analysis showed that the molecular mass of GmAAP protein was 53.286 kD, the theoretical isoelectric point was 8.93, and the instability index was 34.04. It was a stable protein. The α-helix, extended strand, β-turn, and random coil in the secondary structure account for 47.18%, 15.66%, 2.71%, and 34.45% respectively. The transmembrane region and hydrophobicity analysis showed that it contained 10 transmembrane regions, and the amino acids constituting the transmembrane region were mostly hydrophobic amino acids. GmAAP is most closely related to wild soybean GsAAP6 , and the consistency of sequence alignment is 100%, which might be the soybean AAP6 gene. In order to investigate the subcellular localization of GmAAP, fu? sion expression vector was constructed and then transformed into Arabidopsis protoplasts by method of PEG-Ca2+ mediated transformation. The results showed that GmAAP was localized on plasma membrane. AAP could increase the absorption and utilization of exogenous nitrogen in legumes, which further increased the storage protein content in seeds and improved soybean quality. The results might lay a foundation for further research on the function of GmAAP. Meanwhile, it provided candidate genes for the efficient use of nitrogen.