RCH1:HDT1,HDT2-RNAi
(Plasmid #108446)

• Purpose: Arabidopsis transformation, RNAi vector targeting HDT1/HDT2 with RCH1 promoter
• Depositing Lab: Ton Bisseling
• Publication: Li et al Plant Cell. 2017 Sep;29(9):2183-2196. doi: 10.1105/tpc.17.00366. Epub 2017 Aug 30.

Backbone

• Vector backbone: pK7GWIWG2
• Vector type: Plant Expression, RNAi

Growth in Bacteria

• Bacterial Resistance(s): Spectinomycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert 1

• Gene/Insert name: HDT1
• Species: A. thaliana (mustard weed)
• Entrez Gene: HDA3 (a.k.a. AT3G44750, ATHD2A, HD2A, HDT1, HISTONE DEACETYLASE 2A, histone deacetylase 3)
• Promoter: RCH1

Cloning Information for Gene/Insert 1

• Cloning method: Gateway Cloning
• 5′ sequencing primer: unknown

Gene/Insert 2

• Gene/Insert name: HDT2
• Species: A. thaliana (mustard weed)
• Entrez Gene: HD2B (a.k.a. AT5G22650, ARABIDOPSIS HISTONE DEACETYLASE 2, ATHD2, ATHD2B, HD2, HDA4, HDT02, HDT2, HISTONE DEACETYLASE, HISTONE DEACETYLASE 2, MDJ22.7, MDJ22_7, histone deacetylase 2B)
• Promoter: RCH1

Cloning Information for Gene/Insert 2

• Cloning method: Gateway Cloning
• 5′ sequencing primer: unknown

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Full sequence is not available for this plasmid. Please see the diagnostic digest and partial sequencing results as references for verification

To build the pRCH1:HDT1HDT2 RNAi construct (hdt1,2i), the CaMV 35S promoter in pK7GWIWG2 vector (Limpens et al., 2005) was replaced with RCH1 promoter [pK7GWIWG2(II)]. The RCH1 promoter was first amplified on genomic DNA and cloned into pENTR-D-TOPO. Then it was cut out from the vector by HindIII (partial digestion) and XbaI and recombined with two fragments of the pK7GWIWG2(II) vector in a three-point ligation. The two fragments of pK7GWIWG2(II) vector were obtained by digestion either with HindII and NcoI, or with SpeI (compatible with XbaI) and HindIII. The whole RNAi cassette including the RCH1 promoter was cut out from the vector using ApaI and HindIII and ligated into the pBnRGW binary vector digested with the same enzymes and in such way creating pBnRRGWIWG vector. The RNAi target sequences of HDT1 and HDT2 coding sequences (0.6 kb for each) were combined in one amplicon using a two-step PCR, following the procedure described by Franssen et al. (2015). In brief, the first step PCRs introduced short overlaps (15 bp) in PCR fragments of HDT1 and HDT2 coding sequences with HDT1rnai-F and HDT1rnai-R primers, or with HDT2rnai-F and HDT2rnai-R primers. These two fragments were used as templates in the second-step PCR with HDT1rnai-F and HDT2rnai-R primers. The final PCR fragment was introduced into pENTR-D-TOPO vector and recombined in inverse-repeat orientation into the pBnRRGWIWG binary vector by a LR Gateway reaction (Invitrogen).