摘要
【目的】植物源环氧化脂肪酸(epoxy fatty acids,EFAs)是生产高值化工产品的优异可再生原材料。EFAs仅在一些野生植物种子中高水平合成和积累,难以规模化利用。通过在普通油料作物大豆(Glycine max (L.)Merr.)发育种子中组装环氧化脂肪酸合成途径,以期实现这类珍稀脂肪酸(unusual fatty acids,UFAs)的商业化绿色生产。【方法】通过构建琉璃菊(Stokesia laevis)脂肪酸环氧化酶(epoxygenase,SlEPX)基因种子特异表达载体,基于体细胞胚发生的粒子轰击法对大豆(cv. Jack)进行遗传转化,经连续选择和鉴定,获得表型稳定的高代转基因大豆株系。分别运用PCR和实时荧光定量PCR检测外源基因SlEPX的整合及在大豆发育种子中的表达谱。统计分析SlEPX转基因大豆籽粒形态和大小、百粒重以及种子萌发率等表型,应用气相色谱和凯氏定氮法测试种子油脂和蛋白等相关生理生化特性。【结果】外源基因SlEPX稳定整合于大豆基因组,且能在高代转基因大豆发育种子中正确有效表达。SlEPX转基因大豆种子新合成积累了2.9%的EFAs,相应的亚油酸(18﹕2Δ9,12)含量减低8%。与对照相比,SlEPX转基因大豆种子变长,表皮多皱褶。种子大小测量显示,转基因大豆小粒种子(粒径<4 mm)占比明显增加。转基因与对照大豆的种子发芽率无明显差异,然而转基因植株生长缓慢。转基因大豆种子油脂含量、蛋白质含量和百粒重分别减少5%、6%和8.28%。进一步生化分析发现,转基因大豆新合成的EFAs,绝大部分结合于卵磷脂(phosphatidylcholine,PC,占12.6%)分子,仅少量结合于甘油三酯(triacylglycerol,TAG,占2.3%)。这些数据表明在转基因大豆种子中,外源SlEPX酶能正确催化亚油酸(18﹕2Δ9,12)生成环氧化脂肪酸即斑鸠菊酸(vernolic acid,Va)(12-epoxy-18﹕1Δ9)。但是,绝大多数斑鸠菊酸积累于构成细胞膜的主要成分PC分子中,而没有转移整合进入贮藏的TAG分子。大量新合成的斑鸠菊酸结合于PC分子可能损伤宿主细胞膜稳态和生理反应,导致转基因大豆产生不利表型。【结论】在大豆发育种子中单独表达外源脂肪酸环氧化酶,能催化合成少量EFAs,但同时产生一些不利表型。在Sl EPX转基因大豆种子中共表达DGAT或PDAT,既可实现环氧化脂肪酸在TAG中富集,同时还能消除环氧化脂肪酸在细胞膜中的积累及其所导致的负效应。
【Objective】Epoxy fatty acids(EFAs) derived from plants are renewable materials for production of high-valued chemical products. Such unusual fatty acids(UFAs) are only highly synthesized and accumulated in some wild plant seeds, with difficult in utilization on a large scale. This study was conducted to assembly epoxy fatty acid biosynthesis pathway in developing seeds of soybean(Glycine max(L.) Merr) for commercial production of these unusual fatty acids. 【Method】In this paper, Sl EPX, an epoxygenase gene from Stokesia laevis(a high accumulator of EFAs) was cloned into pCAMBIA1301 expression vector driven by a seed-specific promoter. Soybean(cv. Jack) was genetically transformed using the particle bombardment method based on somatic embryogenesis system. The high-generation lines of SlEPX-transgenic soybean with stable phenotypes were obtained by continuous selection and identification. The integration of heterologous SlEPX gene and its expression profiles were examined by PCR and Real-time quantitative PCR, respectively. Seed phenotypic examinations were statistically analyzed including seed morphology, size, 100-seed weight and germination rate. Seed oil and protein contents and other physiological properties were measured by gas chromatography and Kjeldahl method. 【Result】The results showed that Sl EPX gene was stably integrated into soybean genome, with its accurate and effective expression in developing seeds of high-generation soybeans. EFAs were newly synthesized but low content(2.9%) in SlEPX-transgenic soybean seeds, and linoleic acid(18﹕2Δ9,12)was accordingly reduced by 8%. Compared to the control, the transgenic soybean seeds were a little longer and wrinkled seed coat. The percentage of small seeds(diameter <4mm) was increased significantly in the transgenic soybean. Seed germination rate had no difference between transgenic and control whereas the transgenic plant exhibited slow growth. The oil and protein content as well as 100-seed weight of transgenic soybean seeds were reduced by 5%, 6% and 8.28%, respectively. Further biochemical analysis demonstrated that newly-synthesized vernolic acid(one kind of EFAs) in the transgenic seeds mostly bound to phosphatidylcholine(PC)(12.6% of total fatty acid) while only a small amount existed in triacylglycerol(TAG)(2.3%). These data indicated that heterologous SlEPX enzyme did correctly catalyze oleic acid(18﹕2Δ9,12) to vernolic acid(Va)(12-epoxy-18﹕1Δ9) in the transgenic soybean seeds. However, most of Va molecules were accumulated in PC(the major cell membrane lipid) but not in storage TAG. A large amount of Va bound to PC could damage cell membrane homeostasis, causing unfavorable phenotypes in transgenic soybeans. 【Conclusion】 The present study revealed that the overexpression of a heterologous epoxygenase alone in soybean developing seeds can catalyze biosynthesis of EFAs at small amount, but results in some undesirable agronomy traits. It is needed to further co-express the acyltransferase that can specifically transfer the Va-acyl group from PC into TAG molecules in SlEPX-transgenic soybean for enriching epoxy fatty acids in TAG and simultaneously, to eliminate negative effect caused by EFA accumulation in cell membrane.
引文
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