Part:BBa_K4286130

shRNA (RPMK1-1)-1

RPMK1-1, which is one of the two PATHOGENICITY MAP KINASE 1 (PMK1) homologues, RPMK1-1 and RPMK1-2, is encoded by Rhizoctonia solani isolate R5 mitogen activated protein kinase gene. RPMK1 genes were strongly induced during the initial infection process. PMK1 homologues in other fungal pathogens are essential for the formation of appressorium, the fungal infection structures required for penetration of the plant cuticle, as well as invasive growth once inside the plant tissues and overall viability of the pathogen within the plant. Thus, we interfered with the expression of RPMK1-1 though this shRNA in order to prevent R.solani infection, suppress the viability of R.solani in rice, and ultimately cure rice sheath blight.

Next, the RPMK1-1 sequences found by this method were analyzed, queried or predicted on the National Center for Biotechnology Information (NCBI) website whether there were multiple spliced versions of mRNA, and if there were, the homologous region was taken as the target region of RNAi interference target. However, it may be due to the lack of relevant research and literature support, and the corresponding mRNA has no variant. Also, the total nucleic acid database blast was carried out on the target CDS to query the similarity of homologous genes in adjacent species, and shRNA was designed in non-conserved regions to improve the species specificity of our shRNA and ensure biological safety.

And then these gene fragments were analyzed by siRNA Design websites (http://rnaidesigner.thermofisher.com/rnaiexpress/setOption.do?designOption=shrna&pid=8294778592286769918). The program scans the DNA sequence of a gene fragment and calculates the binding energy of sense and antisense siRNAs based on the sequence pattern. The higher the score, the stronger the binding ability of the siRNA to the target sequence. Based on the principle of shRNA design, we selected the fragments with high potential siRNA activity from a series of sequences.

For biosafety, the candidate RNAi fragments were submitted to the total mRNA database for blast, and the sequence similarity was compared. Focus on species with more than 90% similarity and their nucleic acid fragments to ensure that there is no matching of common species (human, rice, dog, wheat, etc.) to ensure the specificity of the sequence.

Finally, the chosen RNAi fragment is assembled in the order of sense RNAi fragment — loop — antisense RNAi fragment.

[1]Tiwari, I.M., Jesuraj, A., Kamboj, R. et al. Host Delivered RNAi, an efficient approach to increase rice resistance to sheath blight pathogen (Rhizoctonia solani). Sci Rep 7, 7521 (2017). https://doi.org/10.1038/s41598-017-07749-w

Sequencing

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]