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Transgenic Research

, Volume 27, Issue 2, pp 155–166 | Cite as

Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B

  • Jing Yang
  • Guojie Xing
  • Lu Niu
  • Hongli He
  • Dongquan Guo
  • Qian Du
  • Xueyan Qian
  • Yao Yao
  • Haiyun Li
  • Xiaofang Zhong
  • Xiangdong Yang
Original Paper

Abstract

Soybean oil contains approximately 20% oleic acid and 63% polyunsaturated fatty acids, which limits its uses in food products and industrial applications because of its poor oxidative stability. Increasing the oleic acid content in soybean seeds provides improved oxidative stability and is also beneficial to human health. Endoplasmic reticulum-associated delta-12 fatty acid desaturase 2 (FAD2) is the key enzyme responsible for converting oleic acid (18:1) precursors to linoleic acid (18:2) in the lipid biosynthetic pathway. In this study, a 390-bp conserved sequence of GmFAD2-1B was used to trigger a fragment of RNAi-mediated gene knockdown, and a seed-specific promoter of the β-conglycinin alpha subunit gene was employed to downregulate the expression of this gene in soybean seeds to increase the oleic acid content. PCR and Southern blot analysis showed that the T-DNA had inserted into the soybean genome and was stably inherited by the progeny. In addition, the expression analysis indicated that GmFAD2-1B was significantly downregulated in the seeds by RNAi-mediated post-transcription gene knockdown driven by the seed-specific promoter. The oleic acid content significantly increased from 20 to ~ 80% in the transgenic seeds, and the linoleic and linolenic acid content decreased concomitantly in the transgenic lines compared with that in the wild types. The fatty acid profiles also exhibited steady changes in three consecutive generations. However, the total protein and oil contents and agronomic traits of the transgenic lines did not show a significant difference compared with the wild types.

Keywords

Soybean RNAi-mediated gene knockdown Fatty acid profile Oleic acid 

Introduction

Soybean [Glycine max (L.) Merr] is the most important leguminous crop in the world for feed and food products because of its high quality and contents of protein (~ 40%) and oil (~ 20%) in the seeds (Clemente and Cahoon 2009). Soybean oil accounts for 80% of vegetable oil worldwide and is composed of approximately 17% saturated fatty acids (palmitic acid and stearic acid), 20% monounsaturated fatty acids (oleic acid) and 63% polyunsaturated fatty acids (linoleic acid and linolenic acid) (Pham et al. 2010; Demorest et al. 2016). The oleic acid content is significantly less than that in competing oils, such as canola (~ 61%) and olive (~ 75%) (Teres et al. 2008). This fatty acid profile limits the use of soybean oil in food products and industrial applications because of poor oxidative stability, which results from the relatively high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid. These types of fatty acids are usually associated with auto-oxidation and undesirable flavor changes as well as the generation of a preponderance of trans-fatty acids during hydrogenation (Ascherio et al. 1999; Demorest et al. 2016). Trans-fatty acids are also related to coronary heart disease and plaque buildup plaque in the arteries (Ascherio et al. 1999). Oleic acid, a monounsaturated fatty acid, has been linked to considerable health and cooking benefits such as greater shelf stability and potential nutritional benefits compared with saturated and polyunsaturated fatty acids (Warner et al. 1994; Kinney et al. 2002). Increasing the seed oil quantity and oleic acid content would be an important step towards addressing these market needs; moreover, these traits are key targets for plant breeding.

The oleic acid content can be significantly increased by downregulating the expression or mutagenesis of FAD2, reticulum-associated delta-12 fatty acid desaturase 2 enzyme, in many plants (Mroczka et al. 2010; Stoutjesdijk et al. 2000, 2002; Jiang et al. 2017). FAD2 is the key enzyme responsible for converting oleic to linoleic acid by inserting a double bond at the 12th carbon in the fatty acid hydrocarbon chain (Okuley et al. 1994). Three FAD2s have been identified in soybean: FAD2-1A, FAD2-1B and FAD2-2 (Miquel 1994). Expression studies show that FAD2-1A and FAD2-1B express primarily during embryogenesis, whereas FAD2-2 expresses constitutively (Heppard et al. 1996; Tang et al. 2005). Further investigation shows that FAD2-1A and FAD2-1B are closely related with a shared genomic organization containing a single intron and 99% identity in the amino acid sequence; the only difference is at the 24th amino acid residue (Schlueter et al. 2007; Tang et al. 2005). A previous study showed that FAD2-1A and FAD2-1B enzyme activities were likely modulated by post-translational regulatory mechanisms. The enhanced degradation of FAD2-1A at high growth temperature is partially rescued by treating the cultures with MG132, a 26S proteasome-specific inhibitor (Tang et al. 2005).

Significant progress has been made in downregulating the expression of FAD2 to enhance the oleic acid content in soybean seeds (Demorest et al. 2016; Hoshino et al. 2010; Murad et al. 2014; Pham et al. 2010; Stoutjesdijk et al. 2002; Wang and Xu 2008; Wanger et al. 2011; Chen et al. 2011; Zhang et al. 2014; Anai et al. 2008; Dierking and Bilyeu 2009). RNAi-mediated gene knockdown is a good method to downregulate GmFAD2. The oleic acid content increases from 20 to 51.71–57% in transgenic soybean seeds by antisense-mediated GmFAD2 knockdown (Stoutjesdijk et al. 2002; Zhang et al. 2014). Similarly, RNAi-mediated knockdown of the FAD2-1A and FAD2-1B genes triggered by inverted repeat fragments increases the oleic acid content from 20 to more than 80% (Wang and Xu 2008; Wanger et al. 2011). The RNA interference fragments of soybean genes FAD2-1 and FatB are introduced into soybean embryonic axes by particle bombardment, which leads to a significant increase in oleic acid content (up to 94.58%) and a decrease in palmitic acid content (to < 3%) (Murad et al. 2014). In addition, some other approaches have been developed to enhance the oleic acid content in soybean, for example, a combination of mutant alleles of FAD2-1A and FAD2-1B or stacking targeted mutations within FAD2-1A, FAD2-1B and FAD3, which can produce soybean lines with high oleic acid (~ 80%) and low palmitic acid (< 3%) contents (Demorest et al. 2016; Pham et al. 2010). These results demonstrate that members of the GmFAD2 gene family play an important role in metabolically engineering oil seed plants (Pham et al. 2010), and RNAi is an effective measure for improving the oil quality of soybean seeds.

In this study, our objective was to create transgenic soybean lines with high oleic acid content by downregulating GmFAD2-1B with dsRNA interference. Transgenic lines were obtained by Agrobacterium-mediated genetic transformation, and the expression pattern, fatty acid profiles, protein and oil contents were analyzed. The results confirmed that GmFAD2-1B was effectively suppressed by a single RNAi construct, and the fatty acid profile of the transgenic soybean was improved significantly.

Materials and methods

Nucleic acid isolation for gene cloning and vector construction

Fresh enlarged soybean leaves of Williams 82 were collected for RNA extraction with TRIzol reagent (Invitrogen). The RNA was reverse-transcribed to cDNA according to the manufacturer’s instructions (SuperScript III Reverse Transcriptase and DNase I, TansGen Biotech, Beijing, China). According to the CDS sequence of GmFAD2-1B (GenBank accession no. EU908061), a 390-bp conserved fragment (primers listed in Table S1: GmFad2-F/R) was amplified from the cDNA, and the PCR reaction was performed at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s with a final extension at 72 °C for 7 min with a 0.4-μM final concentration of primers. The RNAi fragment was then subcloned into the vector plasmid pTF101.1 with sense and antisense directions.

Genomic DNA of Williams 82 was extracted using the high-salt CTAB method for promoter cloning (Tel-zur et al. 1999). For specific expression of the GmFAD2-1B RNAi fragments in soybean seeds, a 1413-bp seed-specific promoter of the soybean β-conglycinin alpha subunit gene (PBCS) was isolated from Williams 82 according to the method described by Imoto et al. (2008) (primers listed in Table S1: PBCS-F/R). The PBCS was then subcloned into the pTF101.1 binary vector with the phosphinothricin acetyl transferase (bar) resistance gene (encoding phosphinothricin N-acetyltransferase, PAT) as the plant selection marker, which was driven by a modified CaMV 35S promoter (Hardegger et al. 1999; GenBank: GI3319906). The constructed plasmid pTF101.1-PBCS-GmFAD2-1B was transformed into the competent Agrobacterium tumefaciens strain EHA101 by the freeze-thaw method (Holsters et al. 1978; Nishiguchi et al. 1987).

Agrobacterium-mediated transformation

Two soybean cultivars, Williams 82 and Shennong 9, were used as the recipient for Agrobacterium-mediated genetic transformation as described by Zhang et al. (2014) with slight modification. In brief, mature soybean seeds were placed in plates and surface-sterilized using chlorine gas (2 ml concentrated HCl added into 50 ml 5.25% NaClO) for 10–16 h. After blowing 2 h on the benchtop to remove Cl2, the sterilized seeds were placed on germination media at 23 °C for 24 h [B5 inorganic salt, B5 organic salt, 2% sucrose, 1 mg ml−1 cytokinin 6-benzylaminopurine (6-BA), 0.2% agar, pH 5.8]. After germination, the seed coats and primary shoots were removed from the imbibed seeds, and then cotyledonary nodes were cut 3–4 times with a scalpel dipped in A. tumefaciens. The explants were infected in A. tumefaciens suspension for 30 min and placed on co-cultivation media [10% B5 inorganic salt and B5 organic salt, 3% sucrose, 20 mmol l−1 2-[N-morpholino] ethanesulfonic acid (MES), 0.25 mg l−1 gibberellic acid (GA3), 1.67 mg l−1 N-6-benzylaminopurine (BAP), 200 μm l−1 acetosyringone (As), 400 mg l−1 l-cysteine, 1 mmol l−1 dithiothreitol (DTT) and 0.5% agar, pH 5.2] at 25 °C in the dark for 5 days. After co-cultivation, the explants were induced for shoots on induced media (10% B5 inorganic salt and B5 organic salt, 3% sucrose, 3 mol l−1 MES, 0.2% agar, 1.67 mg l−1 BAP, 250 mg l−1 cefotaxime sodium, 100 mg l−1 amoxicillin, pH 5.7) at 28 °C and 16-h light/8-h dark periods for 2 weeks. Then, the hypocotyls of the explants were cut again and transferred to induced media containing 5 mg l−1 glufosinate for another 2 weeks. The induced multiple shoots were cultured on elongation media [Murashige Skoog (MS) inorganic salts, B5 organic salt, 3 mol l−1 MES, 3% sucrose, 50 mg l−1 aspartic acid, 100 mg l−1 pyroglutamic acid, 0.1 mg l−1 indole acetic acid (IAA), 0.5 mg l−1 GA3, 1 mg l−1 zeatin riboside, 250 mg l−1 cefotaxime sodium, 100 mg l−1 amoxicillin, 0.2% agar, pH 5.8] at 25 °C in 16-h light/8-h dark periods. The remaining protocols were as per the methods published by Zhang et al. (2014).

Detection of transgenic soybean plants

LibertyLink strip analysis, Basta painting, PCR and Southern blot analysis were used to detect the transgenic soybean plants. T0 transgenic soybean plants were tested by LibertyLink strips to determine the PAT protein content following the manufacturer’s instructions (Envirologix Inc., Portland, ME, USA). T3 and T4 trangenic soybean lines and wild types were painted with Basta (135 g l−1) with 1:1000 dilution when the second trifoliolate leaves fully enlarged (Envirologix Inc., Portland, ME, USA).

For the PCR test, leaves of the T3 and T4 transgenic soybean and wild types were used to extract DNA with a simple homogenization and ethanol precipitation method (Edwards et al. 1991). Primers (Table S1: BAR-F/R and GmFad2-F1/R1) used in PCR amplification were designed according to bar and the GmFad2-1B sense fragment and its upstream promoter sequence, respectively. The PCR reaction was performed at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 45 s with a final extension at 72 °C for 10 min with 0.2-μM final concentrations of primers. The plasmid with the GmFAD2-1B interference cassette and wild-type soybean were used as the positive and negative controls, respectively.

Genomic DNA was extracted from enlarged soybean leaves using a modified high-salt CTAB method (Tel-zur et al. 1999) for Southern hybridization. Approximately 20 μg genomic DNA of each sample was digested with Hind III (New England Biolabs Inc., Beverly, MA), separated on 1% agarose gel and transferred onto a Hybond N+ nylon membrane using the alkaline transfer buffer as recommended by the supplier (GE Amersham, RPN303B). The bar probe was amplified as described above (Table S1: Bar-F/R) and purified with the EasyPure Quick Gel Extraction Kit (TansGen Biotech, Beijing, China). About 1 μg purified probe was labeled with digoxigenin-(DIG-)11-dUTP with DIG High Prime DNA labeling reagents (Labeling and Detection Starter Kit I, 11745832910; Roche Applied Science, Indianapolis, IN, USA) for 20 h and then inactivated at 65 °C for 10 min. Hybridization was performed at 42 °C for 14–16 h. The washing conditions were at 25 °C for 5 min with 2 × SSC and 0.1% SDS and at 66 °C for 15 min with 0.5 × SSC and 0.1% SDS. Chemical staining was carried out at room temperature with BCIP/NBT as substrate until the signal was clearly detected (Labeling and Detection Starter Kit I, 11745832910; Roche Applied Science, Indianapolis, IN, USA).

Expression analysis of different tissues

Different tissues of the transgenic plants and wild types were collected for expression analysis. When the fourth trifoliolate leaves had fully enlarged, RNA was extracted from the roots, stems and fourth trifoliolates of the transgenic and wild-type plants. Buds and ~ 1 cm soybean seeds of these plants were also used for RNA extraction. Total RNA was isolated from ~ 100 mg of ground tissues per sample using the EasyPure® Plant RNA Kit (TansGen Biotech, Beijing, China), according to the manufacturer’s instructions. Two micrograms of total RNA from each sample was reverse-transcribed using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TansGen Biotech, Beijing, China) systems with oligo (dT) 18 as primer. Quantitative PCR was performed on the real-time PCR system (Applied Biosystems, 7900HT, USA) with TransStart® Top Green qPCR SuperMix (TansGen Biotech, Beijing, China). The PCR conditions were 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. GmActin (GeneBank ID no. NM 001289231) was used as the positive internal control, and the gene primers for the qRT-PCR were designed using the conserved sequence of GmFAD2-1B (Fig. 1A, long arrow) and GmActin (Table S1: GmFad2-F2/R2 and GmAct-F/R; final concentration was 0.4 μM). The relative expression level was determined using the comparative 2−ΔΔCt method (Livak and Schmittgen 2001), and the data were processed by SigmaPlot, version 13.0.
Fig. 1

Schematic maps of the GmFAD2-1B and RNAi vector; Agrobacterium-mediated transformation of soybean and LibertyLink strip test. A The CDS sequence of GmFAD2-1B. Short arrows: primer-binding sites for RNAi fragment amplification; long arrows: primer-binding sites for qRT-PCR. B Schematic representation of the recombinant plasmid pTF101.1-PBCS-GmFAD2-1B. RB and LB represent the right and left borders of the T-DNA, respectively. GmFAD2-1B and bar inserts were driven by the PBCS promoter and the modified CaMV 35S promoter, respectively. Arrows: binding sites of GmFad2-F1/R1. C Transformation and regeneration of soybean. a Germinated soybean seeds; b infection with A. tumeficiens; c co-cultivation; d co-cultivation after 5 days; e induced green shoots; f regenerated plantlets. D LibertyLink strip test of part of the T0 transgenic soybean. WT, Williams 82; numbers, transgenic soybean

Fatty acid analysis of the transgenic lines

Twenty seeds of the T2–T4 transgenic plants and wild types were harvested for fatty acid composition analysis using gas chromatography (GC, Agilent 7890, Wilmington, DE, USA) (Wang and Xu 2008; Zhang et al. 2014; Murad et al. 2014). Individual fatty acid content was reported as the relative percentage of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acids in the extracted fatty acids. The procedure for the fatty acid extraction was as follows: soybean seeds were ground to powder using a mortar and pestle, and then the powder was dried in a glass flask overnight in an oven at 90 °C. Two milliliters of sodium methoxide (0.5 M l−1) was added to each flask, and then the samples were placed in a water bath at 30 °C for 30 min. Lipids were extracted with n-hexane after being transesterfied with sodium methoxide. Then, the samples were separated into phases for 45 min, and 1 ml supernatant was separated using Hewlett-Packard 6890 GC supplied with a hydrogen flame ionization detector and a capillary column (HP INNOWax: 30 M × 0.25 mM × 0.25 μM) with an N2 carrier at 2 ml min−1. The chromatographic conditions were set—initial temperature, 100 °C; final temperature, 250 °C; injection temperature, 250 °C; rate, 15 °C min−1 up to 250 °C—and remained at 250 °C for 7 min. The retention times of the fatty acid methyl esters (FAMEs) were compared with those of the standards (37-component FAME mix: Supelco 47885-U), and relative fatty acid compositions were then calculated as the percentage that each fatty acid represented of the total measured fatty acid (Wang and Xu 2008; Zhang et al. 2014; Murad et al. 2014).

Protein and oil analyses and investigation of agronomic traits

Field tests were conducted using a randomized complete block design with two replicates of hillplots at Gongzhuling in 2014–2016 (Martin et al. 1990). For an analysis of total seed nitrogen, the seeds were ground in a coffee grinder after drying at 60 °C. Then, the powder was weighed to approximately 0.2 g in small tin capsules (LECO, St. Joseph, MI). The percentage of total nitrogen in the grain powder was determined using a LECO CHN 2000 analyzer (LECO, St. Joseph, MI) (Hwang et al.2014). The seed protein percentage was calculated as follows: total nitrogen percentage × 6.25 (Jung et al. 2003; Hwang et al. 2014). The seed oil percentage was determined with approximately 10 g of seeds by using a Maran Pulsed NMR (Resonance Instruments, Witney, Oxfordshire, UK), followed by the field induction decay-spin echo procedure (Rubel 1994). Protein and oil concentrations were expressed on a percentage dry weight basis. The agronomic traits were investigated including plant height, stem diameter, branch number, node number, pod number, seed number, seed yield and 100-seed weights.

Results

Agrobacterium-mediated transformation and generation of the transgenic plants

The conserved sequence of GmFAD2-1B was cloned from Williams 82 as an RNAi trigger fragment (Fig. 1A, short arrows) and subcloned into the pTF101.1 vector to downregulate this gene. To express GmFAD2-1B sense and anti-sense sequences in soybean seed, the PBCS promoter, which was expressed especially in the soybean cotyledon and embryonic axis, was used to drive the RNAi fragment (Fig. 1B). The bar resistance gene, a screening marker, was activated by the modified CaMV 35S promoter (Hardegger et al. 1999; GenBank: GI3319906). These two ORFs were subcloned into the expression vector pTF101.1 in the opposite orientations (Fig. 1B).

The cotyledonary nodes of mature soybean seed were used as explants for Agrobacterium-mediated transformation (Fig. 1C). The transformation method had a relatively steady efficiency (Zhang et al. 2014). Two soybean cultivars, Williams 82 and Shennong 9, were used as the wild types, because Williams 82 is a model plant with the whole genome sequence available and Shennong 9 is a native variety with good agricultural traits in Northeastern China. The regenerated plants were tested using the LibertyLink strip, and the positive ones that showed bands in test lines were transplanted into soil for breeding seeds (Fig. 1D).

PCR and Southern blot analysis

Southern blot analysis was conducted to confirm integration of insertion in the T2 generation (Fig. 2a). Southern blot was performed with approximately 20 μg of genomic DNA digested by Hind III from the T2 transgenic lines and their wild types with the bar fragment as the probe (Fig. 2a). Selected samples for Southern blot analysis were based on the fatty acid analysis of the T2 transgenic lines (Table 1). The results showed that bar was integrated into the soybean genomes with 1–2 copies. The transgenic lines with a single copy (Fig. 2b), 8-3 and 9-1, were selected for the next analysis because multi-copies of the inserted sequences could recombine during mitosis and be lost.
Fig. 2

Southern blot, Basta painting and PCR analysis of transgenic plants. a Southern blot analysis of the T2 transgenic lines with bar as the probe. bar, phosphinothricin acetyl transferase resistance gene; M, 15-kb DNA marker; P, positive plasmid; W82 and SN9, Williams 82 and Shennong 9, respectively; numbers, transgenic lines. Arrows: single copy in transgenic lines 8-3 and 9-1. b Basta painting in the fields using Basta (135 g l−1) 1:1000 diluted solution; c, d PCR analysis of the T3–T4 transgenic lines with PBCS-GmFAD2-1B and bar primers. M, DL2000 marker; P, positive plasmid; W82 and SN9, Williams 82 and Shennong 9, respectively; 8-3 and 9-1, transgenic soybean lines

Table 1

Fatty acid profiles in the T2 transgenic lines

Soybean lines

Fatty acids (mol%)

ODP

16:0

18:0

18:1

18:2

18:3

W82

10.19

5.00

22.00

56.05

6.76

0.74

8-1

7.19

3.80

71.57

11.52

5.93

0.20

8-2

7.63

4.09

63.10

18.67

6.51

0.29

8-3

6.70

3.58

78.83

5.78

5.11

0.12

8-4

7.01

3.74

75.96

7.51

5.77

0.15

8-5

7.23

3.79

71.05

12.22

5.71

0.20

SN9

11.00

3.97

19.74

57.23

8.06

0.77

9-1

7.17

4.53

78.28

5.12

4.90

0.11

9-2

8.25

4.09

67.59

13.13

6.94

0.23

9-3

6.68

3.69

78.72

5.76

5.15

0.12

ODP = (18:1 + 18:2) mol%/(18:1 + 18:2 + 18:3) mol%. 16:0, 18:0, 18:1, 18:2 and 18:3 represented palmitic, stearic, oleic, linoleic and linolenic acids, respectively

The Basta painting and PCR analysis were performed to confirm transgenic positivity of the T3 and T4 generations. The T3 and T4 generations of transgenic lines 8-3 and 9-1 noticeably resisted the herbicide compared with their wild types (Fig. 2b). The PCR was performed using primers designed from the fragment between the PBCS and sense of GmFAD2-1B (Table S1: GmFad2-F1/R1; showed in Fig. 1b). The transgenic lines were homozygous in the T3 generations (Fig. 2c, d) using this pair of primers and bar-specific primers, and these results also suggested that RNAi fragments and bar were transformed into soybean and stably inherited by the progeny.

Expression level of GmFAD2-1B decreased remarkably in transgenic seeds

Different tissues from the T4 generation of transgenic lines, 8-3 and 9-1, and wild types were collected for GmFAD2-1B expression analysis (Fig. 3). In the same developmental stage, expression levels of GmFAD2-1B in the roots of all the samples were similar, consistent with the results of a previous study (Zhang et al. 2014). In the flowers, expression levels of GmFAD2-1B increased moderately and reached the peak in the seeds of Williams 82 and Shennong 9. However, in transgenic lines, the expression levels of GmFAD2-1B reached their peak in flowers. The dramatic change was that the knockdown of GmFAD2-1B caused a significant decrease in the expression of GmFAD2-1B in seed tissue in the transgenic lines versus their wild types. The reason for these results could be the seed-specific PBCS promoter, which especially activated the RNAi fragment in the seed and downregulated GmFAD2-1B expression. The increased expression in the stem and flower in transgenic plants could also be caused by downregulation of this gene in the seeds. These results indicated that the PBCS promoter had played a key role in changing the expression of GmFAD2-1B in the transgenic seeds, like in the results of previous studies (Zhang et al. 2014; Imoto et al. 2008).
Fig. 3

Real-time PCR analysis of GmFAD2-1B in the different tissues from T4 generations of transgenic lines

Oleic acid content increased by RNAi-mediated knockdown of GmFAD2-1B

Fatty acid compositions of the T2–T4 seeds of transgenic plants were analyzed using gas chromatography. An oleic desaturation proportion (ODP) value was used to assess the cumulative effect of GmFAD2-1B activity (Wang and Xu 2008; Liu et al. 2002). The ODP values of the wild types were 0.74 and 0.77, and 0.11 to 0.29 of the T2 transgenic lines, indicating that 71–89% of GmFAD2-1B activities were reduced in these lines by RNAi-mediated gene knockdown (Table 1). In addition, the oleic acid contents in the T2 transgenic lines increased from 22.00 to 78.83% and from 19.74 to 78.28% compared with Williams 82 and Shennong 9, respectively. Concomitantly, the contents of linoleic acid decreased from 56.05 to 5.78% in 8-3 and 57.23 to 5.12% in 9-1 compared with Williams 82 and Shennong 9 (Table 1). The linolenic acid contents were also decreased in the transgenic lines (from 6.76 to 5.11 and 8.06 to 4.90% in 8-3 and 9-1, respectively) (Table 1).

In the T3 and T4 generations, two transgenic lines, 8-3 and 9-1, were selected to continue the analysis because of the relatively lower ODP values in the T2 generation and single-copy T-DNA insertion in Southern blot analysis (Fig. 4a). Fatty acid profiles in these generations were similar to those in the T2 transgenic plants with ~ 75 to 80% 18:1 fatty acid contents, which was significantly decreased compared with the wild-type soybean. The content of linoleic acid also decreased significantly (from ~ 57 to < 6%) in the transgenic lines compared with that in the wild types (Fig. 4a). Activity of GmFAD2-1B in the three successive generations was almost completely silenced by introducing intron-containing sense and antisense fragments (Figs. 3, 4a), indicating that RNAi-mediated gene knockdown was effective: Stable inheritance was confirmed by PCR and Southern blot analyses.
Fig. 4

Fatty acid profiles and the contents of protein and oil in soybean seeds. a Fatty acid profiles of the seeds from T3 and T4 transgenic lines and wild types. 16:0, 18:0, 18:1, 18:2 and 18:3 represented palmitic, stearic, oleic, linoleic and linolenic acids, respectively. **Significant difference, p < 0.05. b Protein and fat contents in the seeds from T4 transgenic lines and wild types. W82, Williams 82; SN9, Shennong 9. 8-3 and 9-1 were two transgenic lines

No effects on protein and oil contents and agronomic traits by RNAi-mediated knockdown of GmFAD2-1B

To evaluate the effect of downregulation of GmFAD2-1B on the total protein and oil contents, seeds of the T4 transgenic lines were analyzed for changes in the protein and oil contents. The results showed there were no obvious differences in the protein and oil contents between the transgenic lines and wild types (Fig. 4b). No significant differences were observed between the wild type and transgenic plants regarding the agronomic traits investigated, including plant height, stem diameter, branch number, node number, pod number, seed number, seed yield and 100-seed weights in the fields (data not shown). These results suggested that RNAi-mediated gene knockdown had no effect on the total protein and oil contents or agronomic traits of the transgenic plants despite the different expression patterns of GmFAD2-1B in the stem, leaf and flower compared with wild types.

Discussions

The fatty acid profile of soybean oil limits its application in food products: approximately 20% oleic acid and 60% linolenic acid contents affect the oxidative stability of the oil. Many attempts have been made to improve soybean oil quality, including RNAi, induced mutations and site-directed mutagenesis using sequence-specific nucleases. For RNAi, sense (Mroczka et al. 2010), antisense (Zhang et al. 2014) and both strands (Wang and Xu 2008; Wanger et al. 2011) mediating downregulation of FAD2 expression are all effective ways to enhance the oleic acid content in soybean seeds. A previous study confirmed that oleic acid was elevated from 20 to ~ 85% using a full-length FAD2 cDNA sense suppression construct (Knowlton 1999), and a moderate polyunsaturated fatty acid was reduced to ~ 20% by using a FAD2 intron sequence in a sense suppression construct (Fillatti and Joanne 2009). However, the oleic acid content was 51.71–57% in transgenic soybean seeds using an antisense sequence of FAD2 in a suppression construct (Stoutjesdijk et al. 2002; Zhang et al. 2014). Inverted-repeat sequence-mediated gene knockdown was more effective than antisense-mediated gene knockdown (Wang and Xu 2008; Wanger et al. 2011; Murad et al. 2014). The RNAi trigger fragment of GmFAD2-1 activated by soybean lectin promoter was transformed into soybean genome and the phosphotransferase (NPT II) as selectable markers. The oleic acid contents in T1 transgenic lines ranged from 71.5 to 81.9% (Wang and Xu 2008). Two other studies also obtained high oleic acid soybean contents (80 and 94.8%, respectively) by knocked down GmFAD2-1 and FatB under the control of soybean seed 7Sα’ or CaMV 35S constitutive promoter (Wanger et al. 2011; Murad et al. 2014). In this study, the inverted repeat fragment of GmFAD2-1B (GenBank accession no. EU908061) was selected as the RNAi trigger sequence driven by seed-specific promoter PBCS and the bar rsistance gene driven by CaMV 35S as the plant-selectable marker instead of the NPT II gene. These sequences are constructed into a single vector and transformed into the soybean genome by Agrobacterium-mediated transformation. We tracked the T1–T4 transgenic soybean lines and confirmed that the RNAi fragment of GmFAD2-1B and the bar resistance gene are inherited stably. The fatty acid profiles also exhibit steady changes in three consecutive generations. The content of oleic acid in the T4 transgenic soybean seed is ~ 75 to 80% and the linoleic acid content decreased from ~ 57 to < 6% compared with wild types.

In addition to FAD2 knockdown by RNAi-mediated post-transcriptional gene silencing (PTGS), other approaches to elevating the oleic acid contents in different crops have been reported. For example, allele mutants of FAD2 or FAD3 reduced the linolenic acid content in soybean seed oil (Hoshino et al. 2014; Pham et al. 2010, 2012). This method was also applied in safflower (Liu et al. 2013) and sunflower (Schuppert et al. 2006) oils to enhance the oil quality. However, multiple FAD2 genes control the oleic acid content in soybean (Pham et al. 2010), cotton (Liu et al. 1999), safflower (Cao et al. 2013) and flax (Chen et al. 2015; Khadake et al. 2009), providing techniques that can make multiple paralogs of a gene more attractive. In our study, the conserved sequence of GmFAD2-1B as an RNAi trigger fragment was transformed into the soybean genomes, and the fatty acid profile was improved significantly with oleic acid content ~ 77% in three consecutive generations. These results confirmed that GmFAD2-1Bs are effectively suppressed with a single RNAi construct and inherited stably. Concurrently, the protein and oil contents of the transgenic plants were not affected by altering the fatty acid synthesis. The expression pattern of GmFAD2-1B in different soybean tissues and agronomic traits of the T4 transgenic lines were analyzed. Because of the RNAi-trigger fragments driven by PBCS, the expression level of GmFAD2-1B in the soybean seeds decreased remarkably compared with non-transformation controls. Different expression patterns of GmFAD2-1B in the stem, leaf and flower of transgenic and wild-type soybean lines had almost no effect on the agronomic traits of these lines. Compared with constitutive promoters (Bellucci et al. 1999), the endogenous seed-specific promoter will have fewer effects on the health of the plant because GmFAD2-1B expresses especially in seed and converts oleic acid to linoleic acid in the lipid biosynthetic pathway.

RNAi-mediated gene knockdown driven by the seed-specific promoter for improvement of soybean oil quality remains a competitive method compared with gene editing and allele mutation; it has no effects on the protein and oil contents and agronomic performance and can be used as a tool for breeders or researchers to study the effects of altered fatty acid content in other systems.

Notes

Acknowledgement

This work was supported by grants from the China National Novel Transgenic Organisms Breeding Project (2016ZX08004-003), National Natural Science Foundation of China (31671764), and Jilin Provincial Agricultural Science and Technology Innovation Project in China (CXGC2017JQ013).

Author contributions

JY performed the PCR, Southern blot and fatty acid profile analysis; GX performed the soybean transformation; LN and HH constructed the RNAi vector and analyzed the expression pattern and protein and fat contents; DG and QD designed the primers for PCR and analyzed the agronomic traits of the transgenic and control plants; XQ and YY performed the LibertyLink strip test; HL checked the manuscript. XZ and XY are co-corresponding authors. All authors have read and approved the final version of the manuscript.

Supplementary material

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Supplementary material 1 (DOCX 13 kb)

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Jilin Provincial Key Laboratory of Agricultural Biotechnology, Agro-Biotechnology InstituteJilin Academy of Agricultural SciencesChangchunChina

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