CRISPR/Cas9 genome editing construct for CsMS editing was generated using the pHEE401E vector tagged with GFP. Two sgRNAs were driven by the U6 promoter, and the Cas9 protein was driven by the egg cell-specific promoter [34 (link)]. The empty pHEE401E vector was used as a control. Two sgRNAs were targeted against cucumber malate synthase gene (CsaV3_1G009520) designed using CRISPR-GE tool (http://skl.scau.edu.cn/ ) [35 (link)]. For analysis the activity of CsLBD16(LATERAL ORGAN BOUNDARIESDOMAIN, Csa3G398920) which homolog of the Arabidopsis LATERAL ORGAN BOUNDARIES-DOMAIN 16 (AtLBD16, At2g42430) under nematode parasitism, the vector pCAMBIA1391 carrying GUS gene was driven by the 2000 bp promoter region of the CsLBD16 gene, generating a pCsLBD16::GUS recombinant construct. The primer's sequences for construction of two vectors are listed in Table 1 .
List of primers used in this study
| Primer name | Application | Primer (5'–3') |
|---|---|---|
| qCsMS-F | Relative expression | GCCTTGTTGTTTGTCGCTGA |
| qCsMS-R | TTAGTCGCCGGATCAAACCC | |
| qCsLBD16-F | CAGAAACCCTAATGGATTCAGGAAG | |
| qCsLBD16-R | GTGGGCTTGGGTTGTTCGTAATTTG | |
| qCsTUB-F | CATTCTCTCTTGGAACACACTGA | |
| qCsTUB-R | TCAAACTGGCAGTTAAAGATGAAA | |
| pCsLBD16-F | pCAMBIA-1391 | GCGCGCCAAGCTTGGCTGCAGACCTAAGTCCGAAGCCATAAGTGAC |
| pCsLBD16-R | TCTTAGAATTCCCGGGGATCCGGGAAAATAGAAGAAATGGCCGTGC | |
| CsMSDT1-BsF | CsMS-pHEE401E | ATATATGGTCTCGATTGAGAGGCTACGACGTTCCAGGTT |
| CsMSDT1-F0 | TGAGAGGCTACGACGTTCCAGGTTTTAGAGCTAGAAATAGC | |
| CsMSDT2-R0 | AACTGCTAATTTTCGACGCTCTCAATCTCTTAGTCGACTCTAC | |
| CsMSDT2-BsR | ATTATTGGTCTCGAAACTGCTAATTTTCGACGCTCTCAA | |
| GFP-F | GFP characterization | CAAGGGCGAGGAGCTGTTCACCG |
| GFP-R | CAGCTCGTCCATGCCGTGAGTGA | |
| CsMSCsa9-F | Mutant characterization | GCTTGGGATGTATTCCGAATCA |
| CsMSCsa9-R | GGATGAAGATTTACCTGGAGTG |
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