Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (2): 191-200.doi: 10.3864/j.issn.0578-1752.2019.02.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Phenotypic Analysis of Epoxygenase-Transgenic Soybeans

HAO QingTing,ZHANG Fei,JI XiaJie,XUE JinAi,LI RunZhi()   

  1. Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2018-07-13 Accepted:2018-10-11 Online:2019-01-16 Published:2019-01-21
  • Contact: RunZhi LI E-mail:rli2001@126.com

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Abstract:

【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, SlEPX, 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 SlEPX 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.

Key words: Glycine max L., epoxy fatty acids, epoxygenase, transgenic soybean, phenotype analysis

Table 1

Primer sequences"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 大小
Size (bp )
SlE-PCR F:AATCTTGCATCCTCACTGGTTTA 23
R:TGTGGTGCAGATAAGTGATTACG 23
Actin-qPCR F:CAGAAAGGATCTATATGGCAAC 22
R:ATTTTCTTTCTGGTGGAGCTAC 22
SlE-qPCR F:TCAGGCAAGAAGTACGATAGA 21
R:AACCCGATTCAGGAGACC 18

Fig. 1

Detection of the target gene SlEPX in the transgenic soybean genome by PCR analysis M: DNA marker; WT: Wild-type soybean (negative control); P: Plasmid DNA (positive control); 1-4: SlEPX-transgenic soybean lines"

Fig. 2

Expression analysis of SlEPX in the transgenic soybean seeds at different development stages"

Fig. 3

Fatty acid composition in seed oil of the SlEPX- transgenic soybeans at different seed development stages ** and * indicates statistically significant differences at P<0.01 and P<0.05, respectively. The same as below"

Fig. 4

Distribution of vernolic acid (Va) in TAG and PC lipids in the SlEPX-transgenic soybean seeds"

Fig. 5

Total fatty acid and protein percent of the SlEPX- transgenic soybeans"

Fig. 6

Comparison of seed traits between SlEPX-transgenic and wild-type soybean A: Seed germination rate; B: 100-seed weights; C: Seed morphology of transgenic (right) and wild-type (left) soybean; D: Seed size (grain diameter); E: Transgenic (left) and wild-type (right) soybean plants"

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