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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (6): 839-847.doi: 10.3724/SP.J.1006.2019.84157

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

Creation of high oleic acid soybean mutation plants by CRISPR/Cas9

Zhi-Hong HOU,Yan WU,Qun CHENG,Li-Dong DONG,Si-Jia LU,Hai-Yang NAN,Zhuo-Ran GAN,Bao-Hui LIU()   

  1. School of Life Sciences, Guangzhou University, Guangzhou 510006, Guangdong, China
  • Received:2018-11-21 Accepted:2019-01-19 Online:2019-06-12 Published:2019-06-12
  • Contact: Bao-Hui LIU E-mail:Liubh@iga.ac.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China.(31771815, 31701445, 31801384)

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Abstract:

Oleic acid content is one of the essential indicators to evaluate quality of oil in soybean. Three sites of 20 nt guide RNA (gRNA) targeted to the exon of GmFAD2-1A were designed and transcribed from the AtU3d, AtU3b, and AtU6-1 promoters, respectively. The three target sites of gRNA were ligated to the vector pYLCRISPR/Cas9-DB, and then the recombinant plasmid was transformed into a soybean cultivar Huaxia 3 by Agrobacterium-mediated transformation. The sequences near the editing site were analyzed by the PCR method and sequencing from T1 transgenic soybean plants, homozygous GmFAD2-1A mutants were obtained using CRISPR/Cas9 technology. The agronomic traits such as plant height, main stem number, branching number per plant, leaf shape, flower color, seed coat color, hilum color and growth period were no significant difference between the transgenic soybeans and non-transformed controls. However, the content of oleic acid in the transgenic soybean seed was significantly higher than that of the control cultivar Huaxia 3, indicating that GmFAD2-1A was a key gene during synthesis of oleic acid. We succeeded in editing the GmFAD2-1A by CRISPR/Cas9 technology in soybean and obtained homozygous mutant materials, which provides new germplasm resources and method for the breeding of high oleic acid.

Key words: soybean, fatty acid, oleic acid, GmFAD2-1A, CRISPR/Cas9

Fig. 1

Maps of pYLgRNA and pYLCRISPR/Cas9-DB vectors"

Fig. 2

Positions of three targets in the GmFAD2-1A gene locus The white boxes on the left and the right represent 5' and 3' noncoding regions; the black box stands for CDS; the solid line represents the intron."

Table 1

Primer sequence"

引物名称
Primer name		 寡核苷酸序列
Oligonucleotide sequence (5'-3')
FAD-AtU3d-T1-F1 gtcACAAAGCCACCATTCACTGT
FAD-AtU3d-T1-R1 aaacACAGTGAATGGTGGCTTTG
FAD-AtU3b-T2-F2 gtcAACCAAAATCCAAAGTTGCA
FAD-AtU3b-T2-R2 aaacTGCAACTTTGGATTTTGGT
FAD-AtU6-1-T3-F3 attGTTTGGCTGCTATGTGTTTA
FAD-AtU6-1-T3-R3 aaacTAAACACATAGCAGCCAAA
FAD2-1A-CasF-Test GAGGGATTGTAGTTCTGTTG
FAD2-1A-CasR-Test CTATGGCCCATTGGTTGCTC
U-F CTCCGTTTTACCTGTGGAATCG
gRNA-R CGGAGGAAAATTCCATCCAC
B1’ TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG
B2 AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC
B2’ TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG
B3 AGCGTGggtctcGtcttGGTCCATCCACTCCAAGCTC
B3’ TTCAGAggtctcTaagaCACTGGAATCGGCAGCAAAGG
BL AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC
Bar-F TGCCAGTTCCCGTGCTTGAA
Bar-R CTGCACCATCGTCAACCACTA

Fig. 3

Schematic diagram of the pYLCRISPR/Cas9-FAD2-1A-gRNA vector construction"

Table 2

Mediums and compositions in soybean transformation"

萌发培养基
Germination
medium		 共培养培养基
Co-cultivation
medium		 诱导培养基
Shoot induction medium		 伸长培养基
Shoot elongation medium		 生根培养基
Rooting
medium
MS合成盐 MS salt mixture — — — 1× 1×
B5合成盐 B5 salt mixture 1× 1/10× 1× — —
2-(4-吗啉)乙磺酸 MES (g L-1) — 4.2 0.6 0.6 —
6-苄基腺嘌呤 6-BAP (mg L-1) — 3.2 1.6 — —
赤霉素 GA3 (mg L-1) — — — 0.5 —
羧苄青霉素Car (mg L-1) — — 50 50 —
二硫苏糖醇 DTT (mg L-1) — 150 — — —
替卡西林 Tic (mg L-1) — — 100 100 25
头孢霉素 Cef (mg L-1) — — 75 75 25
L-天冬酰胺 L-Asp (mg L-1) — — — 50 —
谷氨酰胺 Glu (mg L-1) — — — 50 —
草铵膦 Glufosinate (mg L-1) — — 250 — —
萌发培养基
Germination
medium		 共培养培养基
Co-cultivation
medium		 诱导培养基
Shoot induction
medium		 伸长培养基
Shoot elongation medium		 生根培养基
Rooting
medium
吲哚乙酸 IAA (mg L-1) — — — 0.1 —
玉米素 ZR (mg L-1) — — — 1 —
吲哚丁酸 IBA (mg L-1) — — — — 1
蔗糖 Sucrose (g L-1) 20 30 30 30 20
琼脂 Agar (g L-1) 12 6.5 8.5 9 12
pH 5.8 5.4 5.7 5.6 5.8

Fig. 4

Identification of recombinant plasmids A: Electrophoresis of PCR detection of clony; M: DNA marker (DM2000); 1: H2O blank control; 2: pYLCRISPR/Cas9-DB plasmid; 3-7: FAD2-1A gene knockout positive single clones. B: Identification of the pYLCRISPR/Cas9-FAD2-1A-gRNA plasmid digested with Asc I; M: 1 kb DNA ladder marker; 1: pYLCRISPR/Cas9- FAD2-1A-gRNA."

Fig. 5

Conversion process and the results of leaf painting A: transformation soybean cotyledonine by agrobacterium; 1: germination of sterile seedlings; 2: co-cultivation; 3: shoot induction; 4: shoot elongation; 5: rooting of the resistant shoot; 6: seedling acclimatization. B: identification of the herbicide resistance of the leaves of T0 transgenic soybean plants using glufosinate; a: positive transgenic plant; b: negative transgenic plant."

Fig. 6

Identification of GmFAD2-1A mutant A: PCR detection of T1 transgenic soybean; M: DNA marker (DM2000); 1: H2O blank control; 2: WT H3; 3-7: positive seedling of glufosinate tolerence of transgenic soybean. B: sequence alignment of GmFAD2-1A mutant compared to the WT line; C: sequence alignment of GmFAD2-1A mutant and wild-type protein."

Table 3

Agronomic traits of the transgenic soybean"

农艺性状
Agronomic trait		 华夏3号
Huaxia 3		 GmFAD2-1A突变体
GmFAD2-1A mutant
株高 Plant height (cm) 58.53±1.47 57.60±0.54
主茎节数 Main stem number 11±0 11±0
单株分枝数 Branching number per plant 4±0 4±0
叶形 Leaf shape 椭圆形 Oval 椭圆形 Oval
花色 Flower color 白色 White 白色 White
种皮色 Seed coat color 黄色 Yellow 黄色 Yellow
种脐色 Hilum color 浅褐色 Pale brown 浅褐色 Pale brown
生育期 Growth period (d) 107±0 107±0
蛋白 Protein (%) 40.47±0.54 40.61±0.12
油脂 Oil (%) 20.06±0.12 20.5±0.16

Fig. 7

GmFAD2-1A mutants phenotype and the fatty acids content OA: oleic acid; LA: linoleic acid; ALA: linolenic acid; PA: palmitic acid; SA: stearic acid."

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