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作物学报 ›› 2019, Vol. 45 ›› Issue (6): 839-847.doi: 10.3724/SP.J.1006.2019.84157

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用CRISPR/Cas9技术创制大豆高油酸突变系

侯智红,吴艳,程群,董利东,芦思佳,南海洋,甘卓然,刘宝辉()   

  1. 广州大学生命科学学院, 广东广州 510006
  • 收稿日期:2018-11-21 接受日期:2019-01-19 出版日期:2019-06-12 网络出版日期:2019-06-12
  • 通讯作者: 刘宝辉
  • 作者简介:E-mail: 739425367@qq.com
  • 基金资助:
    本研究由国家自然科学基金项目(31771815, 31701445, 31801384)

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 Published:2019-06-12 Published online:2019-06-12
  • Contact: Bao-Hui LIU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China.(31771815, 31701445, 31801384)

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摘要：

大豆是重要的油料作物, 其种子脂肪酸中油酸含量是评价大豆油脂品质的重要指标之一。本研究设计了分别由AtU3d、AtU3b和AtU6-1启动子驱动、长20 bp的guide RNA (gRNA)靶点以靶向编辑GmFAD2-1A基因的外显子区, 首先将这3个靶点一起组装到pYLCRISPR/Cas9-DB载体上, 然后利用农杆菌介导的方法转化大豆材料华夏3号。通过PCR技术及测序分析对T1代转基因大豆植株靶点编辑情况进行检测, 获得纯合GmFAD2-1A大豆突变体。GmFAD2-1A突变大豆植株在株高、主茎节数、单枝分枝数、叶形、花色、种皮色、种脐色、生育期等方面与对照大豆植株没有显著差异; 而GmFAD2-1A突变体大豆种子油酸含量显著高于对照大豆品种华夏3号, 说明GmFAD2-1A是油酸代谢过程中的关键基因。本研究利用CRISPR/Cas9技术成功对控制大豆油酸基因GmFAD2-1A进行编辑, 获得稳定的纯合GmFAD2-1A大豆突变体材料, 为高油酸育种提供了新的种质资源并创建了方法。

关键词: 大豆, 脂肪酸, 油酸, GmFAD2-1A, CRISPR/Cas9

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

图1

pYLgRNA及pYLCRISPR/Cas9-DB质粒图"

图2

3个靶点分别在GmFAD2-1A基因内的位置 左边和右边白色方框代表5' 和3' 非编码区; 黑色方框代表CDS; 实线代表内含子。"

表1

引物序列"

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

图3

pYLCRISPR/Cas9-FAD2-1A-gRNA载体构建示意图"

表2

大豆遗传转化中使用的培养基及成分"

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

图4

重组质粒鉴定 A: 菌落PCR检测电泳; M: DNA marker (DM2000); 1: H2O空白对照; 2: pYLCRISPR/Cas9-DB质粒; 3~7: FAD2-1A基因敲除阳性单菌落。B: Asc I酶切鉴定pYLCRISPR/Cas9-FAD2-1A-gRNA载体; M: 1 kb DNA ladder marker; 1: pYLCRISPR/Cas9-FAD2-1A- gRNA。"

图5

转化过程及叶片涂抹草铵膦筛选结果 A: 农杆菌介导的子叶节法转化大豆; 1: 无菌苗的萌发; 2: 共培养; 3: 丛生芽的诱导; 4: 丛生芽的伸长; 5: 诱导生根; 6: 移栽土里。 B: T0转基因大豆植株草铵膦涂抹鉴定; a: 阳性转基因植株; b: 阴性转基因植株。"

图6

GmFAD2-1A突变体鉴定 A: T1代转基因大豆植株的PCR检测; M: DNA marker (DM2000); 1: H2O 空白对照; 2: H3野生型; 3~7: 转基因大豆草铵膦抗性阳性苗。B: GmFAD2-1A突变体与野生型序列比对分析; C: GmFAD2-1A突变体与野生型蛋白序列比对结果。"

表3

转基因大豆表型性状"

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

图7

GmFAD2-1A突变体表型及其脂肪酸组分含量 OA: 油酸; LA: 亚油酸; ALA: 亚麻酸; PA: 棕榈酸; SA: 硬脂酸。"

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