Difference between revisions of "Part:BBa K4653001"

Revision as of 08:31, 4 October 2023

shRNA(CHSIIIa)-1

In order to kill B. cinerea, the pathogen of grey mold and control the disease in tomato, we designed two pieces of shRNAs targeting the CHSIIIa gene of the pathogen, which is essential for its cell wall formation, based on RNAi technology.

Sequencing

Sequence and Features

Biology

Chitin synthase (CHS) catalyzes the synthesis of chitin, a major structural component of fungal cell walls consisting of β-1, 4-linked N-acetylglucosamine polymers. CHS can be divided into seven classes, and ascomycetes usually have representatives of all seven classes, with each enzyme having a specific role. CHS (III, V, and VI) are specific to molds, suggesting that they may play an important role in hyphal growth. Like other ascomycetes, B. cinerea contains two CHSIII genes. It has been shown that the BcchsIIIa gene is most expressed in the CHSIII genes, whereas the BcchsIIIb gene is not expressed in any of the growth conditions used. By silencing the important chitin synthase gene BcCHSIIIa, the formation of the cell wall of B. cinerea could be affected, thus killing pathogenic fungi.

Design of shRNA

After confirming the selection of targets BcCHSIIIa, we searched the cDNA library of B. cinerea according to the sequences or primes provided in the literature, and found the homologous cDNA sequence of B. cinerea. Then, the sequence was input into the National Center for Biotechnology Information (NCBI) website for analysis and prediction, and the CDS sequence of the target gene was input into the total nucleic acid database BLAST to query the homologous similarity of neighboring species. siRNA sequences were designed in non-conserved regions to ensure species-specific and biosafety of our shRNAs.

Next, we used a professional siRNA design website to predict the siRNA sequences that would effectively target the mRNA, and then screened out siRNA fragments with high potential activity in a series of predictions based on shRNA design principles. By making structural predictions of the mRNA, we ensured that the selected siRNA sequences targeted relatively loose positions in the mRNA structure. For biosafety reasons, we BLAST the candidate siRNA fragments into the total mRNA database to ensure that it does not target any genes of common species (such as human, tomato, dog, rice, wheat, etc.), ensuring sequence specificity.

Finally, we assembled the selected siRNA sequence into our shRNA in the sequence of siRNA sense strand - loop - reversed siRNA antisense strand.

Plasmid construction

We have assembled our shRNA in the sequence of siRNA sense strand - loop - reversed siRNA antisense strand, then sequence was assembled in the pET28a (+) plasmid. The recombinant vector was transferred into RNase-deficient E. coli HT115(DE3), and the large-scale fermentation production of shRNA in E. coli could be achieved by induction of IPTG. In our experiment, the results of treatment of different shRNAs at both phenotypic and molecular levels were analyzed to screen out the effective shRNAs.

Usage