Chapter 2 Materials and methods
2.2 Gateway cloning
2.2.1 Generation of 35S-MYB and 35S-mMYB constructs
MYB gene sequences were amplified with gene-specific primers (Appendix Primer Table 1, that had a NetPrimer program score higher than 80) that contained attB1 and attB2 recombination sequences to allow integration into a Gateway donor vector. PCR amplification was performed using high fidelity KOD Hot Start DNA Polymerase (Novagen) with the following cycling conditions: 1 cycle of 95oC/2 min, 35 cycles of 95oC/20 sec, 55oC/10 sec, 70oC for 20 sec/kb extension time according to amplicon size, and 1 cycle of 70oC for 10 min. PCR products were analysed by agarose gel electrophoresis and products of the desired sizes were excised from the gel and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). The BP reactions were
48 performed with Gateway BP ClonaseTM II enzyme mix (Invitrogen), to integrate the purified PCR products into the pDONOR/ZEO vector (Invitrogen) to generate pENTRY-
MYB vectors. The BP enzyme reaction mixture was transformed into E. coli Alpha-Select Gold Efficiency competent cells (Bioline) by heat shock. After recovery at 37°C for one hour, the bacteria were grown overnight at 37°C on low salt Luria Broth (LB) plates containing Zeocin (Invitrogen) at a concentration of 50 μg/mL. Positive clones were subcultured in liquid low-salt Luria broth (LSLB) overnight at 37°C, and plasmids were extracted from the culture using an AxyPrepTM Plasmid Miniprep Kit (Axygen). Plasmids were then screened by restriction enzyme digestion and were then subject to DNA sequencing to confirm the correct MYB constructs, using ABI PRISM BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) under recommended cycling conditions. The sequencing products were precipitated and analysed at John Curtin School of Medical Research, Australian National University, Canberra.
The pENTRY-mMYB vectors, which served as miR159-resistant versions, were obtained by performing site-directed mutagenesis of the miR159 binding site on the sequence confirmed pENTRY-MYB vectors (Appendix Primer Table 2, Liu and Naismith, 2008). The mutated miR159 binding site in the pENTRY-mMYB vectors was confirmed by restriction enzyme digestion and DNA sequencing.
All entry clones were integrated into the pDESTINATION vector pGWB602Ω (Nakamura et al., 2010) to generate the corresponding binary vectors expressing 35S-
MYB via LR reactions using Gateway LR ClonaseTM II enzyme mix (Invitrogen). The LR enzyme reaction mixture was transformed into E. coli via electroporation. Transformed bacteria were grown overnight at 37°C on LB plates containing Spectinomycin at a concentration of 50 μg/mL, and positive clones were subcultured in
49 liquid LB overnight. After plasmid extraction, the expression vectors of 35S-MYB were screened and confirmed by restriction enzyme digestion.
2.2.2 Generation of MYB81-33 and mMYB81-33 constructs
The MYB81-33 construct was generated by mutating the miR159 binding site of MYB81
to make it identical to the miR159 binding site of MYB33. The pENTRY-MYB81-33 and pENTRY-mMYB81-33 (miR159-resistant version) vectors were obtained by performing site-directed mutagenesis of the miR159 binding site on the sequencing confirmed pENTRY-MYB81 vectors (Appendix Primer Table 4). The mutations in the pENTRY- MYB81-33 and mMYB81-33 vectors were confirmed by restriction enzyme digestion and DNA sequencing. The entry clones were recombined into the destination vector pGWB602Ω to generate the corresponding binary vectors expressing 35S-MYB81-33 and
35S-mMYB81-33 via Gateway LR reactions (Invitrogen). Expression vectors were screened and confirmed by restriction enzyme digestion.
2.2.3 Generation of MYB33-mSL constructs
The MYB33-mSL1, MYB33-mSL2 and MYB33-mSL2-2 constructs were obtained by performing site-directed mutagenesis of the SL1 or SL2 region of a MYB33 genomic fragment in the pDONR/Zeo vector (Appendix Primer Table 5). This 4356 bp MYB33
genomic fragment includes the 1991 bp of genomic sequences upstream of the MYB33
start codon, the whole MYB33 coding region and 585 bp of sequences downstream of the
MYB33 stop codon (Li et al., 2014b). Following this, the mMYB33-mSL2-2, which served as a miR159-resistant version, was achieved by site-directed mutagenesis of the miR159 binding site on the sequencing confirmed pENTRY-MYB33-mSL2-2 vector. The mutations in the pENTRY-MYB33-mSL vectors were confirmed by restriction enzyme digestion and DNA sequencing. The entry clones were recombined into the destination
50 vector pMDC123 (Curtis and Grossniklaus, 2003) via Gateway LR reactions (Invitrogen). Expression vectors were screened and confirmed by restriction enzyme digestion.
2.2.4 Generation of MYB81-SL constructs
The MYB81-SL construct, including the MYB81 genomic sequence (1361 bp) plus 15 nt of the 5’ UTR and 9 nt of the 3’ UTR, was synthesized by IDT (USA) and sub-cloned into pDONR/Zeo vector. MYB81-SL-6nt was generated by performing site-directed mutagenesis of deleting the 6-nt between two SLs in the pENTRY-MYB81-SL vector.
mMYB81-SL and mMYB81-SL-6nt, which served as miR159-resistant versions, were achieved by site-directed mutagenesis of the miR159 binding site on the sequencing confirmed pENTRY-MYB81-SL and pENTRY-MYB81-SL-6nt vectors, respectively (Appendix Primer Table 6). The mutations in the pENTRY-(m)MYB81-SL/SL-6nt vectors were confirmed by restriction enzyme digestion and DNA sequencing. The entry clones were recombined into the destination vector pGWB602Ω via Gateway LR reactions (Invitrogen). Expression vectors were screened and confirmed by restriction enzyme digestion.
2.2.5 Generation of the MIM159 decoy constructs
The artificial target mimics MIM159 for Arabidopsis and tobacco transformation (Todesco et al., 2010) were obtained from the European Arabidopsis Stock Centre (NASC) and sub-cloned into pDONR/Zeo via Gateway BP reaction. Sequencing confirmed entry clones of MIM159 were then integrated into the Gateway destination vector pMDC32 (Curtis and Grossniklaus, 2003) for Arabidopsis transformation; or integrated into Gateway destination vector pGWB602Ω for tobacco transformation, via Gateway LR reactions (Invitrogen). Expression vectors were screened and confirmed by restriction enzyme digestion.
51 To generate rice MIM159 construct, firstly, a 661 bp rice IPS1 gene (AY568759) was synthesized by IDT (USA) and sub-cloned into pDONR/Zeo via Gateway BP reaction. Next, site-directed mutagenesis was performed on the pENTRY vector to generate the rice miR159 binding site with a three-nt bulge at the cleavage site (Appendix Primer Table 8), which confirmed by the following restriction enzyme digestion and DNA sequencing. The entry clones were recombined into the modified destination vector pMDC32-Ubi (in which a Ubiquitin promoter had replaced a 35S promoter) via Gateway LR reactions (Invitrogen). The expression vector was screened and confirmed by restriction enzyme digestion.
2.2.6 Generation of mNtGAMYB2, mOsGAMYB and mLaMYB33 constructs
Tobacco GAMYB2 (NtGAMYB2) and rice GAMYB (OsGAMYB) gene sequences were amplified with gene-specific primers, which contained attB1 and attB2 recombination sequences, on the tobacco and rice cDNA templates, respectively (Appendix Primer Table 7 and 8). PCR amplification was performed using high fidelity KOD Hot Start DNA Polymerase (Novagen) with the following cycling conditions: 1 cycle of 95oC/2 min, 35 cycles of 95oC/20 sec, 55oC/10 sec, 70oC for 20 sec/kb extension time according to amplicon size, and 1 cycle of 70oC for 10 min. PCR products were analysed by agarose gel electrophoresis and products of the desired sizes were excised from the gel and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). The BP reactions were performed with Gateway BP ClonaseTM II enzyme mix (Invitrogen), to integrate the purified PCR products into the pDONOR/ZEO vector (Invitrogen) to generate pENTRY-NtGAMYB2 and pENTRY-OsGAMYB vectors. The LaMYB33
construct was obtained from the Liwang Qi Lab (Laboratory of Cell Biology, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, People’s Republic of China)
52 and sub-cloned into the pDONR/Zeo vector. Plasmids were then screened by restriction enzyme digestion and were then subject to DNA sequencing to confirm the NtGAMYB2 and OsGAMYB sequences.
The pENTRY-mGAMYB vectors, which served as miR159-resistant versions, were obtained by performing site-directed mutagenesis of the miR159 binding site on the verified pENTRY-GAMYB vectors (Appendix Primer Table 7 and 8). The mutated binding site in the pENTRY-mGAMYB vectors was confirmed by restriction enzyme digestion and DNA sequencing.
All entry clones were integrated into the destination vector pGWB602Ω to generate the corresponding binary vectors expressing 35S-mGAMYB via Gateway LR reactions (Invitrogen). Expression vectors were screened and confirmed by restriction enzyme digestion.