Construction of the transformation vector and genetic transformation

To construct the overexpression vector, we amplified the CmWRKY15-1 full-length cDNA sequence via PCR after the addition of the enzymatic sites for XbaI and SacI using gene-specific primers (Supplemental Table 1). We purified the PCR product for ligation into a pTOPO vector to generate a p-TOPO-CmWRKY15-1 construct, which was confirmed by sequencing, and digested it with XbaI and SacI to release the PCR product for subcloning into a pBI121 vector containing the cauliflower mosaic virus (CaMV) 35S promoter53. To generate an RNAi vector, pHANNIBAL was used as an intermediate vector. By using PCR, we amplified a 200-bp sense and a 200-bp antisense fragment from CmWRKY15-1 containing the XhoI/KpnI and ClaI/HindIII restriction sites, respectively. The two fragments were digested with enzymes and then inserted into both sides of a PDK intron to yield a RNA hairpin construct. We then connected the hairpin RNA construct of pHANNIBAL to pBI121 to generate a CmWRKY15-1 gene silencing vector. We subsequently introduced the pBI121-CmWRKY15-1 overexpression construct and RNAi pRNAi-CmWRKY15-1 construct into Agrobacterium (Agrobacterium tumefaciens) strain EHA105 and then separately transformed Jinba and C029 with pRNAi-CmWRKY15-154.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.