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GLS-shRNA-2 template with T7 promoter

This DNA oligo This DNA oligo consists of T7 promoter (TAATACGACTCACTATA) and GLS-shRNA-2 transcription template (GGCGTACATCGATCAACGTCCTTGCTTCGACGTTGATCGATGTACGCCTT), We use this template for GLS-shRNA-2 generation through in vitro transcription system. With the DNA oligo of GLS-shRNA-2 template with T7 promoter, we add T7 RNA polymerase, NTP and in vitro transcription buffer, ARK-shRNA-1 can be generated within 30 minutes. GLS-shRNA-2 is designed to silence Phyllotreta striolata Glutathione S-transferase gene, the target site on the Glutathione S-transferase mRNA is 576-596 after AUG.

1. Usage

In our project, GLS-shRNA-2 was used to silence Glutathione S-transferase gene of phylotreta striolata. GLS-shRNA-2 solution (10ng/ml) can be sprayed on leaves of cruciferous plants, ingestion of the GLS-shRNA-2 by P. striolata will induce the RNAi mechanism in the insect and lead to its death.

2. Biology

Eukaryotic organisms including insects possess this RNAi mechanism for sequence-specific gene silencing that is triggered by the introduction of double-stranded RNA (dsRNA). Once introduced into the cell, the dsRNA is cleaved into small interfering RNA (siRNA) by an enzyme called Dicer, producing multiple siRNAs. One strand of each siRNA is loaded into Argonaute, an endonuclease, to form an RNA-induced Silencing Complex (RISC), and guiding the RISC to the target mRNA, resulting in the effective cleavage and subsequent degradation of the target mRNA. RNAi mechanism can be triggered by introducing either dsRNA, siRNA or shRNA.

The target site for GLS-shRNA-2 on the Glutathione S-transferase mRNA is 576-595 after AUG. Glutathione S-transferase catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification. When gene for Glutathione S-transferase is silenced, phylotreta striolata is killed.

3. Characterization

3.1 Design of GLS-shRNA-2

GLS-shRNA-2 was designed based on the mRNA sequences of Glutathione S-transferase. Factors that affect in vitro transcription efficiency, such as the requirement of a ‘GG’ 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, and the presence of asymmetry and energy valley within the shRNA; were considered.
These criteria include:

Target site:
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, GLS-shRNA-2 that may target the Phyllotreta striolata Glutathione S-transferase gene was designed (Table 1).

Table 1. Characteristics of ALR-shRNA-2

shRNA	GLS-shRNA-2
Sequence
GC% of antisense strand	48
GC% at 5′-end of antisense strand	48
GC% at 3′-end of antisense strand	48
Target site	576-595 after AUG

3.2 In vitro transcription of GLS-shRNA-2

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 an 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. DNA Oligos (Table 2) were ordered.

2) Fill-in reaction to generate ALR-shRNA-2 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 GLS-shRNA-2 templates was identified through 3% agarose gel eletrophoresis (Fig. 1). Agarose gel electrophoresis showed that the GLS-shRNA-2 template was successfully generated. Sharp bands can be seen on the gel, and the size of the bands is around 67 bases, which is the correct size.

Fig 1. Detection of GLS-shRNA-2 templates by agarose gel (3%). The integrity of shRNA templates was identified through 3% agarose gel eletrophoresis (200 V, 30 min)

3) In vitro transcription

1. in vitro transcription reaction was set up using the prepared template.
10.7 µl RNase-Free Water
2.0 µl Fill-In Reaction product
2.0 µl 10 x T7 RNA Polymerase Buffer (NEB)
2.8 µl 100mM MgSO4 (NEB)
1.0 µl NTP Mix (80 mM each NTP)
1.5 µl T7 RNA Polymerase (50 U/ µl)
20 ul Total reaction volume

2. Incubate the reaction mixtures for 2-3 hours at 37ºC.
3. Add 1 µl RNase-Free DNase I (1 Unit/ml) to remove the DNA template, 37ºC 15 min.

4. Heat the reaction mixtures for 15 minutes at 70ºC to inactivate the enzyme.

5. Extract with Phenol/Chloroform.
a. Add 100 µl RNase-Free Water to dilute the reaction.
b. Add 120 µl phenol/chloroform and vortex briefly to mix.
c. Spin in a microfuge for 1 minute at full speed.
d. Carefully pipette off the top aqueous phase and transfer to a clean tube.

6. Precipitate the shRNA.
a. To the recovered aqueous phase, add 1/10 vol. of 3 M Sodium Acetate (pH 5.2).
b. Add 2.5 volumes of 95-100% ethanol.
c. Incubate for 15 minutes on ice.
d. Pellet the shRNA in a microfuge by spinning at full speed for 15 minutes.
e. Remove the supernatant.
f. Carefully wash the pellet once with 70% ethanol.
g. Air dry the pellet for only 2-5 minutes.

7. Add 100 µl of the 1 X Annealing Buffer to the shRNA pellet and resuspend the shRNA.
The procedure of the shRNA in vitro transcription system is illustrated in Fig. 2.

Fig. 2. Diagram illustrating the procedure of ALR-shRNA-2 in vitro transcription

The integrity of GLS-shRNA-2 was identified through 3% agarose gel eletrophoresis (Fig. 3) Agarose gel electrophoresis showed that the GLS-shRNA-2 was successfully transcribed. Sharp bands of around 50 bases in length were detected on the gel, the size of the bands is correct.

Fig 3. Detection of in vitro transcribed GLS-shRNA-2 by agarose gel (3%). The integrity of shRNA was identified through 3% agarose gel eletrophoresis (200 V, 30 min).

3.4 RNAi efficiency test

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. 4).

Fig.4. Adult P. striolata and Brassica chinensis leaves were placed into the glass bottles for RNAi efficiency test. ALR-shRNA-2 sample has two repeats.

The solutions of ALR-shRNA-2 (10 ng/mL) was 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 ALR-shRNA-2 treatment.

Results show that, ALR-shRNA-2 could trigger RNAi mechanism, which was demonstrated by the significant survival rate decrease after treatment, while the negative control sample showed slight survival rate decrease.

Fig. 5 The survival rate of Phyllotreta striolata at different days after ALR-shRNA-2 treatment.

4. References

[1] Baum J.A., Bogaert T., Clinton W., Heck G.R., Feldmann P., Ilagen O., Johnson S., Plaetinck G., Munyikwa T., Pleau M., Vaughn T., Roberts J. 2007: Control of coleopteran insect pests through RNA interference. Nat. Biotech. 25: 1322–1326.
[2] Gorden K.H. & Waterhouse P.M. 2007: RNAi for insect-proof plants. Nat. Biotech.25: 1231–1232.
[3] Macrae I.J., Zhou K., Li F., Repic A., Brooks A.N., Cande W.Z., Adams P.D., Doudna J.A. 2006: Structural basis for double-stranded RNA processing by Dicer. Science. 311 (5758): 195–8.
[4] Mao Y.B., CAI W.J., WANG J.W., HONG G.J., TAO X.Y., WANG L.J., HUANG Y.P., CHEN X.Y. 2007: Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat. Biotech. 25: 1307–1313.
[5] Turner C.T., Davy M.W., Macdiarmid R.M., Plummer K.M., Birch N.P., Newcomb R.D. 2006: RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding. Insect Mol. Biol. 15: 383–391.
[6] Wang M, Weiberg A, Lin F M, et al. Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection[J]. Nature Plants, 2016, 2(10):16151-16151.
[7] Zhao Y.Y., Yang, G., Pruski W., You M.S. 2008: Phyllotreta striolata (Coleoptera: Chrysomelidae): arginine kinase cloning and RNAi-based pest control. Eur J Entomol 105: 815–822.
[8] Eaton D.L., Bammler T.K. 1999: Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol. Sci. 49 (2): 156–64.

Sequence and Features