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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (4): 978-987.doi: 10.3724/SP.J.1006.2023.24071

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

Study on VIGE system mediated by different plant viruses in cotton

LEI Jian-Feng1(), LI Yue2, DAI Pei-Hong2, ZHAO Yi2, YOU Yang-Zi2, JIA Jian-Guo1, ZHAO Shuai1, QU Yan-Ying1,*(), LIU Xiao-Dong2,*()   

  1. 1College of Agronomy, Xinjiang Agricultural University/Research Center of Cotton Engineering, Ministry of Education, Urumqi 830052, Xinjiang, China
    2College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
  • Received:2022-03-30 Accepted:2022-07-21 Online:2023-04-12 Published:2022-08-17
  • Contact: *E-mail: xiaodongliu75@aliyun.com;E-mail: xjyyq5322@126.com
  • Supported by:
    Natural Science Foundation of Xinjiang Uygur Autonomous Region(2022D01B23);Basic Research Funds for Universities in the Autonomous Region(XJEDU2022J007)

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Abstract:

Plant virus-mediated sgRNA delivery and expression has tremendous advantage over traditional transformation of intact editing vectors for gene editing, because sgRNA expression can be rapidly amplified and be accumulated along with virus replication and movement, resulting in efficient gene editing efficiency. In this study, to develop and apply more plant virus-mediated gene editing (VIGE) systems in cotton, the overexpressing Cas9 (Cas9-OE) cotton was used as VIGE receptor and the cotton leaf crumple virus (CLCrV) and tobacco rattle virus (TRV) mediated VIGE systems were established in cotton. First of all, both CLCrV and TRV-mediated VIGE could target and knock out the GhBsrk1 and GhMAPKKK2 in subgroup A and subgroup D genomic sequences, which verified the feasibility and effectiveness of these two systems. Further quantificaiton of the editing efficiency of GhBsrk1 and GhMAPKKK2 genes by these two systems showed that both CLCrV and TRV-mediated VIGE could produce high-efficiency gene editing efficiency, and there was no significant difference in gene editing efficiency between the two systems. This study also explored the effect of sgRNAs driven by cotton endogenous U6 promoter and Arabidopsis U6 promoter on VIGE gene editing efficiency. The results showed that both promoter-mediated VIGE were able to produce high-efficiency gene editing efficiency, and there was no significant difference in gene editing efficiency based on these two promoter-mediated VIGE systems. The above results indicate that more plant virus vectors can be developed for the application of cotton gene editing research, thus obtaining an efficient sgRNA screening system.

Key words: cotton, CLCrV, TRV, VIGE, gene editing

Table 1

Primer sequences used in this study"

引物 Primer name 序列 Sequence (5'-3') 目的 Destination
GhBsrk1-sgRNA1F: GATTGTTGCACTTAGCTCCATGGC 构建AtU6-26::GhBsrk1-sgRNA和GhU6-5P::GhBsrk1-sgRNA: 5'-GTTGCACTTAGCTCCATGGC-3'
Construction of AtU6-26::GhBsrk1-sgRNA and GhU6-5P::GhBsrk1-sgRNA: 5'-GTTGCACTTAGCTCCATGGC-3'
GhBsrk1-sgRNA2F: AAGTGTTGCACTTAGCTCCATGGC
GhBsrk1-sgRNA1R: AAACGCCATGGAGCTAAGTGCAAC
GhMAPKKK2-sgRNA1F: GATTGAGGGTTCCCAGCTGACATA 构建AtU6-26::GhMAPKKK2-sgRNA和GhU6-5P::GhMAPKKK2-sgRNA: 5'-GAGGGTTCCCAGCTGACATA-3'
Construction of AtU6-26::GhMAPKKK2-sgRNA and GhU6-5P::GhMAPKKK2-sgRNA: 5'-GAGGGTTCCCAGCTGACATA-3'
GhMAPKKK2-sgRNA2F: AAGTGAGGGTTCCCAGCTGACATA
GhMAPKKK2-sgRNA1R: AAACTATGTCAGCTGGGAACCCTC
M-GhBsrk1F: GTTTAGAATACAATGCAGAAACTTTC PCR扩增涵盖GhBsrk1基因A亚组和D亚组靶位点区域
PCR amplification contains the target site regions of GhBsrk1 gene subgroup A and subgroup D
M-GhBsrk1R: GACAAGTTTAAGCACACACTTC
M-MAPPPK2F: CCATGTCGTAGCTTATAAAGG PCR扩增涵盖GhMAPKKK2基因A亚组和D亚组靶位点区域
PCR amplification contains the target site regions of GhMAPKKK2 gene subgroup A and subgroup D
M-MAPPPK2R: CGATTCATTCACGAACTCATG
GhUBQ7F: GAAGGCATTCCACCTGACCAAC PCR扩增GhUBQ7基因部分片段
PCR amplification of partial fragments of GhUBQ7 gene
GhUBQ7R: CTTGACCTTCTTCTTCTTGTGCTTG
Cas9F: GTCATTACGGACGAGTACAAG qRT-PCR分析Cas9 mRNA表达量
The mRNA relative expression level of Cas9 by qRT-PCR
Cas9R: AGGTAGCAGATCCGATTCTTT

Fig. 1

Construction of CLCrV-sgRNA and TRV-sgRNA vectors"

Fig. 2

qRT-PCR analysis of Cas9 expressions Different letters indicate significant difference among different treatments at the 0.05 probability level."

Fig. 3

Identification of different CLCrV-sgRNA and TRV- sgRNA expression vectors by restriction enzyme digestions M: 2K Plus II DNA marker; 1: CLCrV-AtU6-26::GhBsrk1-sgRNA; 2: CLCrV-AtU6-26::GhMAPKKK2-sgRNA; 3: CLCrV-GhU6-5P:: GhBsrk1-sgRNA; 4: CLCrV-GhU6-5P::GhMAPKKK2-sgRNA; 5: TRV-AtU6-26::GhBsrk1-sgRNA; 6: TRV-AtU6-26::GhMAPKKK2- sgRNA; 7: TRV-GhU6-5P::GhBsrk1-sgRNA; 8: TRV-GhU6-5P:: GhMAPKKK2-sgRNA."

Fig. 4

CLCrV and TRV-mediated VIGE targeted mutagenesis of GhBsrk1 and GhMAPKKK2 in cotton M: 2K Plus II DNA marker. A and B: the detection of GhBsrk1-sgRNA targeted mutations. A: wild type served as a control, 1 and 2 are plant numbers inoculated with CLCrV-AtU6-26::GhBsrk1-sgRNA and TRV-AtU6-26::GhBsrk1-sgRNA respectively. The gel image shows digested PCR products of GhBsrk1 gene with Nco I and undigested PCR products. B: the undigested PCR products lacking the Nco I site (due to the presence of a mutation) that were subsequently purified, cloned, and analyzed by sequencing. C: GhBsrk1 mutation sequencing peak map; D and E: the detection of GhMAPKKK2-sgRNA targeted mutations. D: wild type served as a control, 1 and 2 are plant numbers inoculated with CLCrV-AtU6-26::GhMAPKKK2-sgRNA and TRV-AtU6-26::GhMAPKKK2-sgRNA, respectively. The gel image shows digested PCR products of GhMAPKKK2 gene with Nde I and undigested PCR products. E: the undigested PCR products lacking the Nde I site (due to the presence of a mutation) that were subsequently purified, cloned, and analyzed by sequencing. F: GhMAPKKK2 mutation sequencing peak map. Green color indicates the PAM sequence, underline in blue indicates the restriction site on the target sequence. M indicates the mutation sequence, insertions are denoted with red uppercase letters, deletions are shown as red dashes."

Fig. 5

Comparison of gene editing efficiency by CLCrV and TRV-mediated VIGE M: 2K Plus II DNA marker. A: wild type, 1-12 are individual plant numbers inoculated with CLCrV-AtU6-26::GhMAPKKK2-sgRNA, 13-25 are individual plant numbers inoculated with TRV-AtU6-26::GhMAPKKK2-sgRNA. B: wild type, 1-8 are individual plant numbers inoculated with CLCrV-AtU6-26::GhBsrk1-sgRNA, 9-15 are individual plant numbers inoculated with TRV-AtU6-26::GhBsrk1-sgRNA."

Fig. 6

Comparison of gene editing efficiency by different U6 promoters-mediated VIGE M: 2K Plus II DNA marker. A: wild type, 1-12 are individual plant numbers inoculated with CLCrV-AtU6-26::GhMAPKKK2-sgRNA; B: wild type, 1-11 are individual plant numbers inoculated with CLCrV-GhU6-5P::GhMAPKKK2-sgRNA."

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