1.College of Life Science and Agro-Forestry, Qiqihar University;2.Branch of Animal Husbandry and Veterinary of Heilongjiang Academy of Agricultural Sciences
Training Plan of Young Creative Talents of Average Four-year College of Heilongjiang (UNPYSCT-2017153)， Innovative Research Project for Graduate Students of Qiqihar University (YJSCX2020041)
棉子糖系寡糖（Raffinose oligosaccharides, RFOs）是植物体内一种重要的渗透调节物质，肌醇半乳糖苷合成酶（Galactinol synthase, GolS）是RFOs合成过程中的关键酶。本研究从大豆中克隆了GmGolS1基因。序列分析结果显示，GmGolS1基因位于大豆3号染色体，其序列内部有3个内含子，ORF全长1020 bp，编码的多肽含有339个氨基酸，分子量38.82 kDa，等电点5.47。GmGolS1氨基酸序列具有植物GolS蛋白的共同特征：一个保守的丝氨酸磷酸化位点、一个锰离子结合相关的DXD元件和一个羧基末端疏水性五肽。蛋白系统进化分析结果显示，GmGolS1与沙东青AmGolS和蒺藜苜蓿MtGolS的亲缘关系较近。实时荧光定量PCR结果显示GmGolS1在大豆幼苗中可以不同程度的应答高温、低温、干旱及高盐胁迫，且高温胁迫下GmGolS1的表达量升高最明显。构建GmGolS1植物表达载体并转化烟草，获得4株转基因烟草株系。GolS酶活性检测结果显示，转基因烟草中GmGolS1转录后的产物发挥了GolS的催化活性。高温胁迫下GmGolS1转基因烟草的植株表现及电解质渗透率、可溶性糖含量和丙二醛含量的检测结果表明，GmGolS1提高了转基因烟草的耐高温胁迫能力。
Raffinose oligosaccharides (RFOs) are one of important osmoregulatory substance in plants. Galactinol synthase (GolS) is a key enzyme in the synthesis of RFOs. In this study, GmGolS1 was cloned from soybean based on sequence homology. GmGolS1 was found on chromosome 3 with three introns. The full length open reading frame (ORF) of GmGolS1 was 1020 bp, which encodes 339 amino acids with the molecular weight of 38.82 kDa, and the isoelectric point of 5.47. The amino acid sequence GmGolS1 has characteristics that are commonly detected in plant GolS proteins: a conserved serine phosphorylation site, a manganese ion binding associated DXD element, and a carboxy-terminal hydrophobic pentapeptide. The phylogenetic analysis showed that GmGolS1 was closely related to AmGolS and MtGolS. Real-time fluorescence quantitative PCR analysis showed that GmGolS1 was inducible upon heat, low temperature, drought and salt stresses in different degrees in seedlings, and the most abundant expression was observed under heat stress treatment. By transforming GmGolS1 was into tobacco, four transgenic lines were identified using genomic DNA PCR and real-time fluorescence quantitative PCR. Test for GolS enzyme activity showed that the transcribed product of GmGolS1 in transgenic tobacco played the catalytic activity of GolS. Gained from plant performance, electrolyte leakage, soluble carbohydrate content and malondialdehyde content of GmGolS1 transgenic tobacco under heat stress, GmGolS1 was found correlating with improved heat resistance in transgenic tobacco.