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作物学报 ›› 2021, Vol. 47 ›› Issue (8): 1481-1490.doi: 10.3724/SP.J.1006.2021.04214

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

CRISPR/Cas9编辑花生FAD2基因研究

张旺(), 冼俊霖, 孙超, 王春明, 石丽, 于为常*()   

  1. 深圳大学生命与海洋学院/广东省植物表观遗传学重点实验室, 广东深圳 518071
  • 收稿日期:2020-09-19 接受日期:2021-01-13 出版日期:2021-08-12 网络出版日期:2021-02-25
  • 通讯作者: 于为常
  • 作者简介:E-mail: zahngwang@foxmail.com
  • 基金资助:
    国家自然科学基金项目(31671766);深圳市科创委基础研究项目(JCYJ20190808143207457);深圳市科创委基础研究项目(JCYJ20180305124101630);深圳市科创委基础研究项目(JCYJ20170818094958663)

Preliminary study of genome editing of peanut FAD2 genes by CRISPR/Cas9

ZHANG Wang(), XIAN Jun-Lin, SUN Chao, WANG Chun-Ming, SHI Li, YU Wei-Chang*()   

  1. College of Life Sciences and Oceanography, Shenzhen University/Guangdong Provincial Key Laboratory for Plant Epigenetics, Shenzhen 518071, Guangdong, China
  • Received:2020-09-19 Accepted:2021-01-13 Published:2021-08-12 Published online:2021-02-25
  • Contact: YU Wei-Chang
  • Supported by:
    National Natural Science Foundation of China(31671766);Shenzhen Commission of Science and Technology Innovation Projects(JCYJ20190808143207457);Shenzhen Commission of Science and Technology Innovation Projects(JCYJ20180305124101630);Shenzhen Commission of Science and Technology Innovation Projects(JCYJ20170818094958663)

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摘要：

油酸脱氢酶(Δ12FAD或FAD2)是催化油酸(OA)的C12位上脱氢生成双不饱和亚油酸(LA)的关键酶, 它控制油酸、亚油酸的含量及其比值(O/L)。研究表明, AhFAD2是油酸生成亚油酸的关键基因, 决定花生种子中油酸和亚油酸的相对含量。本研究根据AhFAD2基因序列, 设计了相应sgRNA序列, 并构建了旨在敲除FAD2A和FAD2B两个花生油酸脱氢酶的CRISPR/Cas9基因编辑载体。经花生基因转化后, 通过对转基因花生突变位点基因组序列分析找出基因突变体。对靶基因分析发现, 16株转基因花生含有29个FAD2A基因突变, 其中16个突变引起蛋白质序列变化; 11株转基因花生含有30个FAD2B基因突变, 其中17个突变引起蛋白质序列变化。FAD2A和FAD2B蛋白质序列的变化可影响花生油酸脱氢酶的活性, 改变油酸催化脱氢, 使亚油酸合成受阻, 油酸含量增加。本研究为研究花生脂肪酸合成及高油酸花生育种提供了宝贵的基因突变体材料。

关键词: 花生, 油酸, 亚油酸, FAD2, 基因组编辑

Abstract:

Oleate dehydrogenase (Δ12FAD or FAD2) is the key enzyme catalyzing the dehydrogenation of oleic acid (OA) at the C12 position to produce diunsaturated linoleic acid (LA). It controls the contents and ratios (O/L) of oleic acid and linoleic acid in plants. Increasing evidences in molecular biology research indicate that AhFAD2 is the key gene for the conversion of oleic acid to linoleic acid, and determines the relative content of oleic acid and linoleic acid in peanut seeds. In this study, the corresponding sgRNA sequences were designed based on AhFAD2 gene sequences, and a CRISPR/Cas9 gene editing vector was constructed to mutate the peanut FAD2A and FAD2B genes. After peanut gene transformation, gene mutations were identified by genomic sequence analysis of transgenic peanut flanking the sgRNA target sites. Target gene analysis indicated that 29 mutations of FAD2A gene in 16 transgenic peanut plants were obtained, among which 16 mutations caused protein sequence changes; 30 mutations in 11 transgenic peanut plants contained mutations in FAD2B gene, among which 17 mutations caused changes in protein sequence. Changes in the protein sequences of the FAD2A and FAD2B genes might affect the enzyme activity, change the catalytic dehydrogenation of oleic acid, hinder the synthesis of linoleic acid, and thus increase the content of peanut oleic acid. These FAD2 gene mutants are valuable in the study of fatty acid metabolism and the breeding of high oleic peanuts.

Key words: peanut, oleic acid, linoleic acid, FAD2, genome editing

图1

基因组编辑载体的构建 合成含有gRNA1和gRNA2序列的引物1和引物2, 以pCBC-DT1T2载体为模版合成sgRNA片段, 并通过引物1和引物2上的Bsa I酶切位点, 克隆到pSKE401载体, 获得含2个靶点的基因组编辑载体FAD2T1T2。LB、RB分别为农杆菌T-DNA的左右边界; U6-26p、U6-29p为拟南芥U6基因启动子; U6-29T、U6-26T为转录终止子; 35Sp-Cas9-NosT、35Sp-NptII-polyA分别为Cas9和NptII基因表达框。"

图2

花生FAD2A, FAD2B基因序列分析及基因组编辑载体靶点的选择 黑点表示相同序列, T1和T2标记基因组编辑靶点位置, 红色下画线标记PAM (NGG)序列。"

图3

花生基因转化及转基因植株的分析 A: 转基因载体, T1、T2分别为gRNA1和gRNA2靶点序列; B: 转基因愈伤及再生植株, 左侧为抗性愈伤组织, 右侧为转基因植株; C: 转基因植株鉴定, 扩增条带为Cas9基因片段, 箭头指示400 bp PCR扩增产物; D: 突变体序列分析, 转基因植株通过测序检测基因编辑突变体。"

表1

FAD2A基因突变"

突变位点
Mutation site		 核酸Nucleic acid 密码子Codon 氨基酸Amino acid
突变前Before mutation 突变后
After mutation		 突变前Before
mutation		 突变后
After mutation		 突变前Before
mutation		 突变后
After mutation
40 A G AAG GAG K E
150 C A TCC TCA
172 G A GTG ATG V M
214 A C AAG CAG K Q
282 T C ACT ACC
333 C T TAC TAT
362 C T ACC ATC T I
384 T C GTT GTC
416 C A CGC CAC R H
419 A T CAC CTC H L
432 C A ACC ACA
448 G A GAC AAC D N
451 G T GAA TAA E 终止密码Stop codon
464, 465 CA TG CCA CTG P L
487 T C TGG CGG W R
515 C T CCA CTA P L
518 G A GGG GAG G E
528 C T ATC ATT
534 C T CTC CTT
668 插入序列7个碱基7 bp insertion (CTCAGGA) 引起移码突变Frame shift mutation
721 C T CTG TTG
800 A G TAT TGT Y C
807 G A CAG CAA
930 G A ACG ACA
963 T A CCT CCA
1017 C T TAC TAT
1039 T G TTT GTT F V
1047 A G AAA AAG
1105 A C AAG CAG K Q

表2

FAD2B基因突变"

突变位点
Mutation site		 核酸Nucleic acid 密码子Codon 氨基酸Amino acid
突变前
Before mutation		 突变后
After mutation		 突变前
Before mutation		 突变后
After mutation		 突变前
Before mutation		 突变后
After mutation
40 A G AAG GAG K E
150 A C TCA TCC
172 A G ATG GTG M V
214 A C AAG CAG K Q
282 C T ACC ACT
333 C T TAC TAT
362 C T ACC ATC T I
384 T C GTT GTC
416 C A CGC CAC R H
419 A T CAC CTC H L
432 A C ACA ACC
451 G T GAA TAA E 终止密码Stop codon
464 C T CCG CTG P L
487 T C TGG CGG W R
515 C T CCA CTA P L
518 G A GGG GAG G E
528 T C ATT ATC
534 T C CTT CTC
668 插入序列7个碱基 7 bp insertion (CTCAGGA) 引起移码突变Frame shift mutation
721 T C TTG CTG
800 A G TAT TGT Y C
807 G A CAG CAA
907 G A GCA ACA A T
930 G A ACG ACA
963 T A CCT CCA
982 G T GCA TCA A S
1017 C T TAC TAT
1039 G T GTT TTT V F
1047 A G AAA AAG
1105 C A CAG AAG Q K
[1] 万书波, 封海胜, 张建成. 打造强势花生产业, 参与国际竞争. 花生学报, 2003,32(增刊1):5-10.
Wan S B, Feng H S, Zhang J C. Development of a strong competitive peanut production in China. J Peanut Sci, 2003,32(S1):5-10 (in Chinese with English abstract).
[2] 张智猛, 戴良香, 李美, 于遒功, 张玉凤, 万书波. 花生种子产业现状与发展对策. 中国农业科技导报, 2013,15(1):30-37.
Zhang Z M, Dai L X, Li M, Yu Q G, Zhang Y F, Wan S B. Present status and development countermeasures of peanut seed industry. J Agric Sci Technol, 2013,15(1):30-37 (in Chinese with English abstract).
[3] 张立伟, 王辽卫. 我国花生产业发展状况、存在问题及政策建议. 中国油脂, 2020,45(11):116-122.
Zhang L W, Wang L W. Development status, existing problems and policy recommendation for peanut industry in China. China Oils Fats, 2020,45(11):116-122 (in Chinese with English abstract).
[4] 徐同成, 王文亮, 程安玮, 刘丽娜, 杜方岭. 花生油的营养价值及掺伪检测技术. 粮油加工, 2010, (8):29-32.
Xu T C, Wang W L, Cheng A W, Liu L N, Du F L. Nutritional value of peanut oil and method for the detection of fake peanut oil. Cereals Oils Proc, 2010, (8):29-32 (in Chinese).
[5] 王传堂, 朱立贵. 高油酸花生. 上海: 上海科技出版社, 2017. pp 27-35.
Wang C T, Zhu L G. High Oleic Peanut. Shanghai: Shanghai Scientific and Technical Publishers, 2017. pp 27-35(in Chinese).
[6] 李丽, 崔顺立, 穆国俊, 杨鑫雷, 侯名语, 李文平, 刘富强, 刘立峰. 高油酸花生遗传改良研究进展. 中国油料作物学报, 2019,41:986-997.
Li L, Cui S L, Mu G J, Yang X L, Hou M Y, Li W P, Liu F Q, Liu L F. Research progress of peanut breeding with high oleic acid. Chin J Oil Crop Sci, 2019,41:986-997 (in Chinese with English abstract).
[7] Miller J F, Zimmerman D C, Vick B A. Genetic control of high oleic acid content in sunflower oil. Crop Sci, 1987,275:923.
[8] Braddoc J C, Sims C A, O’Keefe S F. Flavor and oxidative stability of roasted high oleic acid peanuts. J Food Sci, 1995,60:489-493.
[9] 曹福亮, 王欢利, 郁万文, 程华. 高等植物脂肪酸去饱和酶及编码基因研究进展. 南京林业大学学报(自然科学版), 2012,36(2):125-132.
Cao F L, Wang H L, Yu W W, Cheng H. Progress of research on fatty acid desaturase and their coding genes in higher plant. J Nanjing For Univ (Nat Sci Edn), 2012,36(2):125-132 (in Chinese with English abstract).
[10] Ray T K, Holly S P, Knauft D A, Abbott A G, Powell G L. The primary defect in developing seed from the high oleate variety of peanut (Arachis hypogaea L.) is the absence of Δ12 desaturase activity. Plant Sci, 1993,91:15-21.
doi: 10.1016/0168-9452(93)90184-2
[11] Jung S, Swift D, Sengoku E, Patel M, Teulé F, Powell G, Moore K, Abbott A. The high oleate trait in the cultivated peanut (Arachis hypogaea L.): I. Isolation and characterization of two genes encoding microsomal oleoyl-PC desaturases. Mol Gen Genet, 2000,263:796-805.
doi: 10.1007/s004380000244
[12] Norden A J, Gorbet D W, Knauft D A. Variability in oil quality among peanut genotypes in the Florida breeding program. Peanut Sci, 1987,14:7-11.
doi: 10.3146/i0095-3679-14-1-3
[13] 刘耀光, 李构思, 张雅玲, 陈乐天. CRISPR/Cas植物基因组编辑技术研究进展. 华南农业大学学报, 2019,40(5):38-49.
Liu Y G, Li G S, Zhang Y L, Chen L T. Current advances on CRISPR/Cas genome editing technologies in plants. J South China Agric Univ, 2019,40(5):38-49 (in Chinese with English abstract).
[14] Zhang Y, Ma X, Xie X, Liu Y. CRISPR/Cas9-based genome editing in plants. Prog Mol Biol Trans, 2017,149:133-150.
[15] Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol, 2014,14:327.
doi: 10.1186/s12870-014-0327-y
[16] Livingstone D M, Birch R G. Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds. Mol Breed, 1999,5:43-51.
doi: 10.1023/A:1009658313170
[17] Jung S, Powell G, Moore K, Abbott A. The high oleate trait in the cultivated peanut [Arachis hypogaea L.]: II. Molecular basis and genetics of the trait. Mol Gen Genet, 2000,263:806-811.
doi: 10.1007/s004380000243
[18] Barkley N A, Chamberlin K D C, Wang M L, Pittman R N. Development of a real-time PCR genotyping assay to identify high oleic acid peanuts (Arachis hypogaea L.). Mol Breed, 2010,253:541-548.
[19] Chen Z, Wang M L, Barkley N A, Pittman R N. A simple allele-specific PCR assay for detecting FAD2 alleles in both A and B genomes of the cultivated peanut for high oleate trait selection. Plant Mol Biol Rep, 2010,283:542-548.
[20] Moore K M. High Oleic Acid Peanut, 1999, US Patent, 5945578.
[21] Horn M E, Eikenberry E J, Lanuza J E R. High stability peanut. 1999, US Patent, 5684232.
[22] Yu S L, Pan L J, Yang Q L, Min P, Ren Z K, Zhang H S. Comparison of the Δ12 fatty acid desaturase gene between high oleic and normal oleic peanut genotypes. J Genet Genome, 2008,3511:679-685.
[23] Chu Y, Holbrook C C, Ozias-Akins P. Two alleles of control the high oleic acid trait in cultivated peanut. Crop Sci, 2009,496:2029.
[24] Patel M, Jung S, Moore K, Powell G, Ainsworth C, Abbott A. High oleate peanut mutants result from a MITE insertion into the FAD2 gene. Theor Appl Genet, 2004,108:1492-1502.
pmid: 14968307
[25] 李拴柱, 宋江春, 王建玉, 张秀阁, 乔建礼, 刘宁. 高油酸花生遗传育种研究进展. 作物杂志, 2017, (3):6-12.
Li S Z, Song J C, Wang J Y, Zhang X G, Qiao J L, Liu N. Advances in genetics and breeding of high oleic acid peanut. Crops, 2017, (3):6-12 (in Chinese with English abstract).
[26] Wang M L, Tonnis B, An Y Q C, Pinnow D, Tishchenko V, Pederson G A. Newly identified natural high oleate mutant from Arachis hypogaea L. subsp. hypogaea. Mol Breed, 2015,359:186.
[27] Nadaf H L, Biradar K, Murthy G S S, Krishnaraj P U, Bhat R S, Pasha M A, Yerimani A S. Novel mutations in oleoyl PC desaturase (AhFAD2B) identified from new high oleic mutants induced by gamma rays in peanut. Crop Sci, 2017,575:2538-2546.
[28] Fang C Q, Wang C T, Wang P W, Tang Y Y, Wang X Z, Cui F G, Yu S L. Identification of a novel mutation in FAD2B from a peanut EMS mutant with elevated oleate content. J Oleo Sci, 2012,613:143-148.
[29] 庄伟建. 一种花生高油酸隐性突变系Ahfad2a-1植株. 2016, 中国专利, CN105519432 A.
Zhuang W J. A recessive high oleic peanut mutant Ahfad2a-1. 2016, Chinese Patent, CN105519432A (in Chinese).
[30] 陈四龙. 一种高油酸花生突变基因AhFAD2B-814及应用. 2018, 中国专利, CN108753803 B.
Chen S L. A high oleic peanut gene AhFAD2B-814 and its application. 2018, Chinese Patent, CN108753803 B (in Chinese).
[31] 徐霞. 高油酸花生基因工程育种的研究. 山东大学硕士学位论文, 山东济南, 2006.
Xu X. Studies on the Breeding of High Oleic Peanut by Genetic Engineering. MS Thesis of Shandong University, Jinan, Shandong, China, 2006 (in Chinese with English abstract).
[32] Yin D M, Deng S Z, Zhan K H, Cui D Q. High oleic peanut oils produced by hpRNA mediated genes silencing of oleate desaturase. Plant Mol Biol Rep, 2007,25:154-163.
doi: 10.1007/s11105-007-0017-0
[33] 李桂民. 双链RNA基因沉默在高油酸花生育种中的应用. 东北师范大学硕士学位论文, 吉林长春, 2005.
Li G M. Applications of dsRNA Gene Silencing in the Breeding of High Oleic Peanut. MS Thesis of Northeast Normal University, Changchun, Jilin, China, 2005 (in Chinese with English abstract).
[34] Yuan M, Zhu J, Gong L, He L, Lee C, Han S, Chen C, He G. Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing. BMC Biotechnol, 2019,19:24.
doi: 10.1186/s12896-019-0516-8 pmid: 31035982
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