Revision as of 19:41, 23 September 2018

Experimental summary

Short interfering RNAs (siRNAs) and their corresponding shRNAs were designed based on the mRNA sequences of their target genes. siRNAs were synthesized by a bio-company, shRNAs were produced by in vitro transcription. The efficiency of both siRNAs and shRNAs in mediating RNAi in Phyllotreta striolata were examined. Experimental results show that both siRNAs and shRNA could successfully silence their target genes, which was demonstrated by the survival rate decrease after siRNA or shRNA treatment. Results also show that GC percentage of the total siRNA/shRNA, the 5’ end of the siRNA/shRNA, the 3’ end of the siRNA/shRNA affect the RNAi efficiency.

In addition, we tested the attraction effect of sucrose and lemon yellow on P. S. Results show that sucrose plus lemon yellow has the best attraction effect.

Objectives

The objective of our project is to trigger the RNAi mechanism in Phyllotreta striolata, which could lead to the death of the beetle, by topical application of exogenous shRNA/siRNAs.

1. Target mRNA selection

Based on the Phyllotreta striolata transcriptome sequence data provided by Professor Weichang Yu, we selected our target mRNAs, which are the mRNAs of Arginine Kinase, Glutathione S-Transferase and Aldose Reductase. These three genes encode important enzymes that are involved in metabolic pathways. The mRNA sequences for arginine kinase (Fig. 1-1), Glutathione S-Transferase(Fig. 1-2), and Aldose Reductase(Fig. 1-3) were obtained.

2. siRNA and shRNA design

Based on the Phyllotreta striolata transcriptome sequence data provided by Professor Weichang Yu, we selected our target mRNAs, which are the mRNAs of Arginine Kinase, Glutathione S-Transferase and Aldose Reductase. These three genes encode important enzymes that are involved in metabolic pathways. The mRNA sequences for arginine kinase (Fig. 1-1), Glutathione S-Transferase(Fig. 1-2), and Aldose Reductase(Fig. 1-3) were obtained. Based on the mRNA sequences, we designed 7 double strand siRNAs and 7 corresponding single strand shRNAs. Factors that affect in vitro transcription efficiency, such as the requirement of a ‘GG’ or ‘GA’ dinucleotide at the start of the transcript; and factors that affect RNAi efficiency, such as distance of target region to transcription start site, nucleotide composition, absence of secondary structures in the target site, and siRNA and the presence of asymmetry and energy valley within the siRNA; were considered during siRNA/shRNA designing.

These criteria include:

Target site criteria:
Not being in the first 75 bases from the start codon
Not being in the intron.

Nucleotide content of siRNA:
GC content of ~50% GC content.
UU overhangs in 3′-end (increase siRNA stability)
Weak base pairing at 5′-end of the antisense strand (presence of A/U)
Strong base pairing at 5′-end of the sense strand (presence of G/C)
5′-end of the antisense strand start with C (Insect agoraute2 prefers 5’ C)
Based on these criteria, siRNAs that may target the Phyllotreta striolata genes were designed (Table 2-1).

3. siRNA synthesis

We select one double strand siRNA for each target mRNA (ARK, GLS, ALR) and sent out the siRNA sequences for direct synthesis. The integrity of siRNA was identified through 15% denaturing polyacrylamide gel eletrophoresis (Fig. 3).

4.In vitro transcription of shRNA

4.1 DNA Oligo Template Design

For primer 1, convert the sense strand of the siRNA sequence to the corresponding DNA sequence, add a 17 base T7 promoter sequence (TAATACGACTCACTATA) to the 5’end of the DNA sequence, add a 8 base loop sequence to the 3’-end of the DNA sequence. For primer 2, add the antisense sequence complementary to the loop sequence to the 3’-end of the DNA sequence. add 2 AA’s to the 5’-end of the Primer 2 oligo. 5 pairs of DNA Oligo (Table 4-1) were ordered.

4.2 Fill-in reaction to generate transcription templates

Each fill-in Reaction was set up with two Oligos
1.0 µl ——P1 Oligo (100 pmoles)
1.0 µl ——P2 Oligo (100 pmoles)
2.0 µl ——10 x buffer 2 (NEB)
0.5 µl ——50 X dNTPs (10 mM)
0.5 µl ——Klenow Fragment exo– DNA Polymerase (5 U/ ml)
15 µl ——RNase-Free Water
20 µl Total reaction volume, incubate the reaction mixtures for 2 hours at 37ºC, then 25 min at 75 ºC, cool at room temperature for 2 minutes.

The integrity of shRNA templates was identified through 3% agarose gel eletrophoresis (Fig. 4-1).