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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (8): 1481-1490.doi: 10.3724/SP.J.1006.2021.04214

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

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 Online:2021-08-12 Published:2021-02-25
  • Contact: YU Wei-Chang E-mail:zahngwang@foxmail.com;wyu@szu.edu.cn
  • 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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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

Fig. 1

Construction of genome editing vector Primer 1 and primer 2 containing gRNA1 and gRNA2 sequences, are used to synthesize sgRNA by PCR with pCBC-DT1T2 as template. The synthesized sgRNA was cloned into pSKE401 plasmid by Bsa I restriction enzyme sites to obtain FAD2T1T1 genome editing construct. LB, RB denote T-DNA left and right border sequences; U6-26p, U6-29p are promoters while U6-29T and U6-26T are terminators of Arabidopsis U6 gene; 35Sp-Cas9-NosT, 35Sp-NptII-polyA are gene expression cassettes of Cas9 and NptII genes."

Fig. 2

FAD2A and FAD2B gene alignment and the selection of CRISPR target sites in peanut Black dots denote identical nucleotides; T1 and T2 indicate the target sequences of genome editing; PAM sequences are underlined with red lines."

Fig. 3

Gene transformation and analysis of transgenic lines in peanut A: gene transformation vector. T1 and T2 are gRNA1 and gRNA2 target sites; B: transgenic calli (left) and plantlets (right); C: PCR identification of transgenic plants. The amplified band is Cas9 gene fragment, and the arrow denotes 400 bp DNA marker; D: sequence analysis of genome editing mutants."

Table 1

Mutation sites of FAD2A gene"

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

Table 2

Mutation sites of FAD2B gene"

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
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