The primers used for gene cloning are listed in Supplementary Data Table S1. Full-length coding sequences or fragments were amplified from cDNA. The amplified sequences were cloned into the pDONR/Zeo plasmid by BP cloning (Invitrogen, Waltham, MA, USA). After verification by sequencing, the resultant plasmids were cloned into the destination vectors (Supplementary Data Table S2) by LR cloning (Invitrogen). Tomato transgenic plants were generated as previously described [6 (link)].
Bp cloning
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BP cloning is a laboratory technique used for the insertion of DNA fragments into plasmid vectors. It enables the efficient and accurate transfer of genetic material for various applications in molecular biology and genomics.
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7 protocols using bp cloning
1
Cloning and Validation of Tomato Genes
2
Gateway Cloning of Protein Constructs
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Full-length complementary coding regions (cDNAs) of KAT2, AKT2, AtCPK6, and AtCPK6 promoter (2 kbp upstream of start codon) were introduced into a Gateway pDONR207 vector by recombination, according to the manufacturer’s instructions (BP cloning; Invitrogen). Primers used for BP cloning are listed in Supplementary Table S3 . Gateway LR recombinations were then performed between entry clones and several destination vectors: pGEMGWC for heterologous expression in Xenopus laevis oocytes [35 (link)], pDEST15 for recombinant protein production in Rosetta strain [35 (link)]; and pEarleyGate101 and 102 for CFP and YFP fusion, respectively [45 (link)].
3
High-Throughput Mutagenesis Protocol
4
Engineered mfap4 Promoter Driven Turquoise2
5
Gene Cloning and Overexpression in Nicotiana
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The primers used for gene cloning are listed in Supplementary Table 1 . Full length coding sequences for ectopic overexpression and fragments for VIGS experiments were amplified from cDNA. The amplified sequences were cloned into the pDONR/Zeo plasmid by BP cloning (Invitrogen, Waltham, MA, United States). After verification by sequencing, resultant plasmids were used for LR cloning into the destination plasmids pTRV2 for VIGS and pMDC83 for overexpression in N. benthamiana. All plasmids were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation.
6
Engineered mfap4 Promoter Driven Turquoise2
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The mfap4 promoter was PCR amplified using the primers 5′-CATGTTCTCGAGGCGTTTCTTGGTACAGCTGG-3′ and 5′-CATGTTGGATCCCACGATctaaagtcatgaagaaaga-3′. The product was subsequently cloned into p5E MCS using XhoI and BamHI sites. The native start codon was mutated using the primer 5′-CTGAGCTGTTGAGGAGAGAGTGAGAAG[ATT]GCAGTAAGTTCTGTGGCTGTTTTATTCC-3′ by inverse PCR with the backbone primers 5′-GTAAGTTCTGTGGCTGTTTTATTC-3′ and 5′-CTTCTCACTCTCTCCTCAACAG-3′. The final p5E mfap4 was then assembled by Gibson assembly using this single stranded oligo and the backbone.
To generate pME Turquoise2, we used the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTggaccatggtgagcaagggcgaggag-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTttacttgtacagctcgtccat-3′ to amplify off pmTurquoise2 H2A (Addgene plasmid #3620235 (link)). The PCR product was subsequently cloned into pDONR221 by BP cloning (Invitrogen) to generate pME Turquoise2.
The mfap4:turquoise transgene construct was subsequently constructed by recombining p5E mfap4, pME Turquoise and p3E polyA into pDestTol2pA2 to generate pDestTol2; mfap4:turquoise.
To generate pME Turquoise2, we used the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTggaccatggtgagcaagggcgaggag-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTttacttgtacagctcgtccat-3′ to amplify off pmTurquoise2 H2A (Addgene plasmid #3620235 (link)). The PCR product was subsequently cloned into pDONR221 by BP cloning (Invitrogen) to generate pME Turquoise2.
The mfap4:turquoise transgene construct was subsequently constructed by recombining p5E mfap4, pME Turquoise and p3E polyA into pDestTol2pA2 to generate pDestTol2; mfap4:turquoise.
7
Genetic Manipulation of T. reesei
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To delete ste20, two approximately 2.0-kb DNA fragments of ste20 upstream and downstream non-coding regions were amplified from TU-6 genomic DNA and ligated into pDONOR-pyr4 via BP-cloning (Invitrogen) to yield the disruption vector pDONOR-ste20-pyr4, which was used to transform T. reesei after linearization with I-SceI. The pDONOR-pyr4 plasmid was obtained as described previously [52 (link)]. To construct the sho1 promoter replacement vector, the 2.0-kb upstream flanking sequence and 2.0-kb fragment starting from the initiation code ATG of the sho1 were amplified from the genomic DNA of TU-6, digested with HindIII/AscI and NotI/SpeI, respectively, and ligated into the corresponding sites of pMD-Ptcu1-pry4 sequentially to obtain pMD-Ptcu1-sho1. Similarly, the 2.2-kb upstream region of the sln1/ypd1 encoding sequence digested by HindIII/AscI and the 2.0-kb fragment containing the sln1/ypd1 ORF region digested by NotI/SpeI were ligated into pMD-Ptcu1-pyr4 to obtain pMD-Ptcu1-sln1 and pMD-Ptcu1-ypd1, respectively. The pMD-Ptcu1-pyr4 plasmid was obtained as described previously [31 (link)].
Fungal transformation was performed as described by Penttila et al. [53 (link)]. Transformants were selected on the minimal medium for uridine prototroph. Anchored PCR was performed to verify the correct integration events.
Fungal transformation was performed as described by Penttila et al. [53 (link)]. Transformants were selected on the minimal medium for uridine prototroph. Anchored PCR was performed to verify the correct integration events.
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