Part:BBa_K4286125

shRNA (PG)-2

Polygalacturonase (PG) is a major pectin degrading enzyme of Rhizoctonia solani which hydrolyzes the α-1, 4-glycosidic linkage of D-galacturonic acid in pectin. Pathogenic fungi synthesize PG enzyme for initiation and its establishment during host infection, that is, PG genes were strongly induced during the initial infection process. What is more, pectinases including PG are the most important factor for the pathogenesis of plant fungi which help in decomposition of pectin in the plant cell wall. The degradation of pectin is important not only to weaken the cell wall to facilitate penetration and colonization of the host cell but also is a source of carbon during pathogen proliferation. Thus, we designed this shRNA molecule for the silencing of R.solani-encoded PG gene and suppression of rice sheath blight development.

Next, the PG 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]Rao, T.B., Chopperla, R., Methre, R. et al. Pectin induced transcriptome of a Rhizoctonia solani strain causing sheath blight disease in rice reveals insights on key genes and RNAi machinery for development of pathogen derived resistance. Plant Mol Biol 100, 59–71 (2019). https://doi.org/10.1007/s11103-019-00843-9 [2]Chen, X., Lili, L., Zhang, Y. et al. Functional analysis of polygalacturonase gene RsPG2 from Rhizoctonia solani, the pathogen of rice sheath blight. Eur J Plant Pathol 149, 491–502 (2017). https://doi.org/10.1007/s10658-017-1198-5

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]