Bioinformatics and Functional Analysis of High Oleic Acid-Related Gene GmSAM22 in Soybean [Glycine max (L.) Merr.] |
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| Authors: | Shuo Qu Qi Cai Huimin Cui Lamboro Abraham Yaolei Jiao Guilong Ma Piwu Wang |
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| Affiliation: | 1 College of Agronomy, Jilin Agricultural University, Changchun, 1300118, China2 College of Plant Protection, Agricultural University, Changchun, 1300118, China |
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| Abstract: | High yield, high quality, stable yield, adaptability to growth period, and modern mechanization are the basic requirements for crops in the 21st century. Soybean oleic acid is a natural unsaturated fatty acid with strong antioxidant properties and stability. Known as a safe fatty acid, it has the ability to successfully prevent cardiovascular and cerebrovascular disorders. Improving the fatty acid composition of soybean seeds, can not only speed up the breeding process of high-quality high-oil and high-oleic soybeans, but also have important significance in human health, and provide the possibility for the development of soybean oil as a new energy source. Hence, the aim of this study was to analyze the high oleic acid elated gene GmSAM22 in soybean. In this research the soybean oleic acid-related gene GmSAM22 was screened out by Genome-wide association analysis, a 662 bp fragment was acquired by specific PCR amplification, and the pMD18T cloning vector was linked by the use of a seamless cloning technique. Bioinformatics analysis of the signal peptide prediction, subcellular localization, protein hydrophobicity, transmembrane region analysis, a phosphorylation site, protein secondary and tertiary structure and protein interaction analysis of the protein encoded by the SAM22 gene was carried out. The plasmid of the gene editing vector is pBK041. The overexpression vector was transformed from pCAMBIA3301 as the base vector, and overexpression vector were designed. Positive plants were obtained by genetic transformation by the pollen tube channel method. Fluorescence quantitative PCR was performed on the T2 generation plants to detect the relative expression levels in different tissues. Southern Blot was used to detect the presence of hybridization signal. Screening genes BAR, 35S, and NOS in plants were identified by conventional PCR. 10 seeds with high and low oleic acid content were chosen for quantitative PCR identification, and finally, the concentration and morphology of soybean fatty acids were identified by near-far infrared spectroscopy. On 10 seeds with an upper and lower oleic acid content, a quantitative fluorescence analysis was done. In Southern blot hybridization, the SAM22 gene was integrated into the recipient soybean plant in hands of a sole copy. Fluorescence quantitative PCR appeared that the average relative expression of the SAM22 gene in roots, stems, leaves, and seeds was 1.70, 1.67, 3.83, and 4.41, respectively. Positive expression seeds had a 4.77% increase in oleic acid content. The level of oleic acid in the altered seeds was reduced by 4.13% when compared to CK, and it was discovered that the GmSAM22 gene could be a regulatory and secondary gene that promotes the conversion of stearic acid to oleic acid in soybean. There has not been a discussion of gene cloning or functional verification. The cloning and genetic transformation of the soybean SAM22 gene can effectively increase the content of oleic acid, which lays a foundation for the study of soybean with high oleic acid. |
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| Keywords: | Soybean SAM22 gene" target=" _blank" >SAM22 gene oleic acid cloning CRISPR/Cas9 expression vector |
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