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| − | <h1>
| + | </body> |
| − | Experimental summary
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| − | </h1> | + | |
| − | <p id="para"> 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. <br><br>
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| − | 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. </p>
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| − | | + | |
| − | <h1>
| + | |
| − | Objectives </h1>
| + | |
| − | <p id="para">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 1. Target mRNA selection
| + | |
| − | </h2>
| + | |
| − | <p id="para">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 2. siRNA and shRNA design
| + | |
| − | </h2>
| + | |
| − | <p id="para">
| + | |
| − | 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.
| + | |
| − | <br><br>
| + | |
| − | These criteria include:<br><br>
| + | |
| − | Target site criteria: <br>
| + | |
| − | Not being in the first 75 bases from the start codon <br>
| + | |
| − | Not being in the intron.<br><br>
| + | |
| − | Nucleotide content of siRNA: <br>
| + | |
| − | GC content of ~50% GC content. <br>
| + | |
| − | UU overhangs in 3′-end (increase siRNA stability)<br>
| + | |
| − | Weak base pairing at 5′-end of the antisense strand (presence of A/U) <br>
| + | |
| − | Strong base pairing at 5′-end of the sense strand (presence of G/C) <br>
| + | |
| − | 5′-end of the antisense strand start with C (Insect agoraute2 prefers 5’ C)<br>
| + | |
| − | Based on these criteria, siRNAs that may target the Phyllotreta striolata genes were designed (Table 2-1).<br>
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 3. siRNA synthesis
| + | |
| − | </h2>
| + | |
| − | <p id="para">
| + | |
| − | 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).
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 4. In vitro transcription of shRNA
| + | |
| − | </h2>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 4.1 DNA Oligo Template Design
| + | |
| − | </h2>
| + | |
| − | | + | |
| − | <p id="para">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 4.2 Fill-in reaction to generate transcription templates
| + | |
| − | </h2>
| + | |
| − | | + | |
| − | <p id="para">
| + | |
| − | Each fill-in Reaction was set up with two Oligos<br>
| + | |
| − | 1.0 µl ——P1 Oligo (100 pmoles)<br>
| + | |
| − | 1.0 µl ——P2 Oligo (100 pmoles)<br>
| + | |
| − | 2.0 µl ——10 x buffer 2 (NEB)<br>
| + | |
| − | 0.5 µl ——50 X dNTPs (10 mM)<br>
| + | |
| − | 0.5 µl ——Klenow Fragment exo– DNA Polymerase (5 U/ ml)<br>
| + | |
| − | 15 µl ——RNase-Free Water<br>
| + | |
| − | 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. <br><br>
| + | |
| − | | + | |
| − | The integrity of shRNA templates was identified through 3% agarose gel eletrophoresis (Fig. 4-1). </p>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 4.3 In vitro transcription
| + | |
| − | </h2>
| + | |
| − | | + | |
| − | <p id="para">
| + | |
| − | 1. in vitro transcription reaction was set up using the prepared template. <br>
| + | |
| − | 10.7 µl ——RNase-Free Water<br>
| + | |
| − | 2.0 µl ——Fill-In Reaction product<br>
| + | |
| − | 2.0 µl ——10 x T7 RNA Polymerase Buffer (NEB)<br>
| + | |
| − | 2.8 µl ——100mM MgSO4 (NEB)<br>
| + | |
| − | 1.0 µl ——NTP Mix (80 mM each NTP)<br>
| + | |
| − | 1.5 µl ——T7 RNA Polymerase (50 U/ µl)<br>
| + | |
| − | 20 ul Total reaction volume<br><br>
| + | |
| − | 2. Incubate the reaction mixtures for 2-3 hours at 37ºC.<br><br>
| + | |
| − | 3. Add 1 µl RNase-Free DNase I (1 Unit/ml) to remove the DNA template, 37ºC 15 min.<br><br>
| + | |
| − | 4. Heat the reaction mixtures for 15 minutes at 70ºC to inactivate the enzyme.<br><br>
| + | |
| − | 5. Extract with Phenol/Chloroform.<br>
| + | |
| − | a. Add 100 µl RNase-Free Water to dilute the reaction.<br>
| + | |
| − | b. Add 120 µl phenol/chloroform and vortex briefly to mix.<br>
| + | |
| − | c. Spin in a microfuge for 1 minute at full speed.<br>
| + | |
| − | d. Carefully pipette off the top aqueous phase and transfer to a clean tube.<br><br>
| + | |
| − | 6. Precipitate the shRNA.<br>
| + | |
| − | a. To the recovered aqueous phase, add 1/10 vol. of 3 M Sodium Acetate (pH 5.2).<br>
| + | |
| − | b. Add 2.5 volumes of 95-100% ethanol.<br>
| + | |
| − | c. Incubate for 15 minutes on ice.<br>
| + | |
| − | d. Pellet the shRNA in a microfuge by spinning at full speed for 15 minutes.<br>
| + | |
| − | e. Remove the supernatant.<br>
| + | |
| − | f. Carefully wash the pellet once with 70% ethanol.<br>
| + | |
| − | g. Air dry the pellet for only 2-5 minutes.<br><br>
| + | |
| − | 7. Add 100 µl of the 1 X Annealing Buffer to the shRNA pellet and resuspend the shRNA.<br><br>
| + | |
| − | 8. The integrity of shRNA was identified through 3% agarose gel eletrophoresis (Fig. 4-2)<br>
| + | |
| − | The procedure of the shRNA in vitro transcription system is illustrated in Fig. 4-3.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h2>
| + | |
| − | 5. RNAi efficiency test
| + | |
| − | </h2>
| + | |
| − | | + | |
| − | <p id="para">
| + | |
| − | Adult P. striolata were obtained from Shenzhen University field station, and kept in glass bottles. The tissue culture seedlings of Chinese cabbage, Brassica chinensis leaves were placed into the above bottles (Fig.5-1). <br><br>
| + | |
| − | The solutions of siRNA/shRNA (10 ng/mL) were separately sprayed onto the leaves of Chinese cabbage every third day, each solution has two repeats. Around twenty adult beetles of P. striolata were tested per siRNA/shRNA sample. The survival rates of adult beetles, were recorded at different days after siRNA/shRNA treatment (Table 5-1, Fig. 5-1). RNAi efficiencies, between siRNA and shRNA (Fig. 5-2), and between different shRNAs (Fig. 5-3), which was demonstrated by the survival rate decrease after siRNA or shRNA treatment, were compared. <br><br>
| + | |
| − | Results show that all the samples tested, except ALR-siRNA-1, and AlR-shRNA-1, could trigger RNAi mechanism, which was demonstrated by the survival rate decrease after treatment (Table 5-1, Fig. 5-1). After 11 days of treatment, there was a slight decrease of survival rate in water treatment (100% to 94%), in ALR-siRNA-1 (100%-81%) and in ALR-shRNA-1(100%-100%). The survival rate decrease of other treatments are significant (between 34.5%-85%). The differences of RNAi efficiency between siRNA and its corresponding shRNA, which have the same target site, are not significant (Fig. 5-2). But the nucleotide content of siRNA/shRNA seems play a role in RNAi efficiency (Fig. 5-3). When the antisense strand of the siRNA/shRNA has a weak base pairing at 3′-end (presence of A/U), but a strong base pairing at 5′-end (presence of G/C), such as ALR-siRNA-1 and ALR-shRNA-1 (Table 5-3), the RNAi efficiency of this siRNA/shRNA is very low. This result may be caused by the failure of the antisense strand to be loaded into RNA-induced silencing complex (RISC), due to the lower free energy level at the 5’ end comparing to that at the 3’-end.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h1>
| + | |
| − | Conclusions
| + | |
| − | </h1>
| + | |
| − | <p id="para"> Our project shows that the topical spray application of sequence specific siRNA/shRNA to a target insect is an effective, simple, safe and a relatively inexpensive technology for insect control. Our project also shows that the nucleotide content of shRNA seems play a role in RNAi efficiency, thus this study not only provides an environmentally friendly approach for pest control, our results are also important for the design of efficient siRNA in order to silence genes in P. striolata and provide a basis for similar studies in other organisms. </p>
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