Gene Cloning and Functional Analysis of PfSAD3 and PfSAD4 in Perilla frutescens
YIN Miao1,2, WANG Yao1,2, WANG Zhuang-Lin1,2, HU Ting1,2, CHEN Shu-Wei1,2, ZHOU Ya-Li1,2, XING Zhi1,2, WANG Ji-Ping1,2,*, LI Run-Zhi1,2
1 College of Agronomy, Shanxi Agricultural University, Taigu 030801, China; 2 Shanxi Engineering Research Center for Genetics and Metabolism of Specific Crops, Taigu 030801, China
Abstract：Δ9-stearoyl-ACP dehydrogenase (SAD) is a key enzyme involved in the synthesis and accumulation of unsaturated fatty acids in plants. In this study, to explore the role of the SAD enzyme in the fatty acid biosynthesis of Perilla frutescens, PfSAD3 (GENE_023515) and PfSAD4 (GENE_042129) genes were cloned and bioinformatic analysis and functional verification on them were conducted. Using the seeds of 'Jinzisu 1' as the experiment material, two full-length sequences of PfSAD3 and PfSAD4 were obtained, with the ORF lengths of 1 173 and 1 116 bp, respectively. MEGA 11.0 was used to construct evolutionary trees for the protein sequences of PfSAD3 and PfSAD4, as well as the SAD sequences of Arabidopsis thaliana and other species, it was found that PfSAD3 was closely related to LcSAD of Leucas cephalotes, and PfSAD4 was closely related to RcSAD of Ricinus communis. After constructing the yeast (Saccharomyces cerevisiae) expression vectors pYES2.0-PfSAD3 and pYES2.0-PfSAD4, PfSAD3 and PfSAD4 were transformed into the wild-type yeast strain INVSc1 and the defective yeast strain BY4389, respectively. The results showed that overexpression of PfSAD3 and PfSAD4 significantly increased the total fatty acid content and unsaturated fatty acid content in yeast (P<0.05), and restored the ability of the defective yeast to synthesize palmitoleic acid (C16:1) and oleic acid (C18:1). The substrate specificity of the PfSAD3 and PfSAD4 proteins was analyzed by adding exogenous fatty acids. The result showed that compared to PfSAD3, PfSAD4 had a more substantial substrate preference ratio for C18:0, and exhibited stronger enzyme activity. The functions of PfSAD3 and PfSAD4 were verified by overexpressing them in wild tobacco (Nicotiana tabacum). The result showed that the total fatty acid content and unsaturated fatty acid content in transgenic tobacco leaves were significantly increased (P<0.05), which was consistent with the results of yeast experiments. This study provides scientific bases and molecular targets for further elucidating the molecular regulatory mechanism of unsaturated fatty acid synthesis in oil crops such as P. frutescens, and for cultivating new oil crop varieties rich in oils through genetic engineering methods.
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