OverviewOperating ProcessResearch ProcessPractical ProblemExpected Research ResultsProtocols

Overview

The goal of this project is to investigate how the MS2 VLP delivery mechanism is expressed in tobacco plastids (Wang et al., 2016) in order to preserve the stability of artificial microRNA (amiRNA) and enhance the efficacy of PM-RNAi in C. rata. Prior to plastid transformation, we will test the target gene's validity in E. coli by expressing the matching vector. In the chitatin synthase gene 1 (CHS 1; Yu et al., 2020 Insect) experimental design, for instance, the amiRNA skeleton is derived from the bantam miRNA of the bantam miRNA (tc-ba; Ballyet al. 2020).

Research meaning

Spodoptera litura is an extremely omnivorous pests that feeds on cruciferous vegetables, cotton, soybeans and tobacco. Currently, chemical pesticides are the main tool used to prevent and control moths. However, repeated use of pesticides over an extended period of time has made moths resistant to many pesticides and less sensitive to transgenic Bt cotton, which has resulted in frequent outbreaks of the species and significant financial losses. Additionally, using traditional chemical pesticides results in adverse effects on human health and the environment, as well as drug resistance in pests. Thus, it is necessary to investigate ecologically friendly pest management techniques. Plant protection experts have been closely examining the RNA interference (RNAi) anti-insect technology that has been created recently because to its high efficiency, precision, environmental friendliness, and lack of pollution.Plant plastids, or chloroplasts, are utilized in pest control and have the ability to express external genes efficiently because of their large genome copy number (Lietal., 2023). The goal of this study is to regulate S. litura through the application of plastid-mediated RNAi (PM-RNAi) technology.
           Previous findings indicate that small interfering RNA (siRNA) rather than long double-stranded RNA (dsRNA; Fu et al., 2022; Li et al., 2023) is a more efficient molecule for plants to mediate RNAi against lepidopteran pests, in contrast to the RNAi trigger in Coleoptera. Consequently, it is preferable to use PM-RNAi to regulate lepidoptera in order to express siRNA or artificial microRNA (amiRNA) in plastids. Furthermore, RNAi's insecticidal efficiency is impacted by the fact that lepidopteran pests have more active dsRNA-degrading nuclease than Coleopteran pests (Shukla et al., 2016). In order to enhance PM-RNAi's effectiveness against Spodoptera litura, amiRNA coated with polypeptides or proteins (L et al., 2023).Is it possible to produce proteins in the tobacco plastid, such virus-like particles (VLPs) of MS2 phages (Wang et al., 2016), to encase the amiRNA and safeguard its stability, thereby enhancing PM-RNAi's potential to control pests?

Purpose of research

To verify  plastid-mediated RNAi technology for effective control of Spodoptera litura.

To verify whether MS2 VLP can wrap and protect amiRNA and enhance the efficacy of plastid-mediated RNAi against Spodoptera litura.

Operating Process

1. Bacterial feeding experiment

1-1. Bacterial expression vector construction and expression

On the basis of the pET28a vector, we constructed the bacterial expression vector p1-p4 (Figure 1).

Figure 1. Bacterial expression vector profiles. A.  p1, expressing only the MS2 VLP delivery system (PT 7:2 CP). B .  p2, expressing only amiRNA (T7: Tc-ba-miR-CHS 1). C . p3, expressing both the amiRNA and MS2 VLP delivery systems (PT 7: Tc-ba-miR-CHS 1 / PT 7:2 CP). D . p4, expressing both amiRNA and GFP (PT 7: Tc-ba-miR-CHS 1 / PT 7: GFP). CP, coat protein; GFP, green fluorescent protein; TAT, perforating membrane peptide; pac, packaging site.

Following construction, the vector was inserted into the B2 strain of E. coli. This strain was created by deleting the rnc gene encoding the endoribonuclease RNaseIII from the BL21 (DE 3) strain (Ma et al., 2020). This allowed the strain to express RNA efficiently and produce protein using the MS2 VLP delivery system.

1-2. Biological test of Spodoptera litura

The cultured transformed bacteria were smeared on the surface of wild tobacco leaves or stirred in artificial feed to feed Spodoptera litura larvae for insect resistance test, and the weight change, pupation rate, mortality rate and the level of inhibition of corresponding target factors by RNAi were recorded.

2. The plant feeding experiment

2-1. Construction of Tobacco Plastid Transformation Vector

Tobacco plastid transformation vector 5-8 was constructed (Figure 2). Foreign genes were added in the plastid genome by tr nfM and Among the trnG genes, the screening marker genes were aadA with spectacular omycin resistance (Wu et al.2017）.

Figure 2. Atlas of the tobacco plastid expression vector. A. And p5, expressing only the MS2 VLP delivery system (P accD: 2 CP). B . And p6, expressing only amiRNA (Prrn: Tc-ba-miR-CHS 1). C . And p7, expressing both the amiRNA and MS2 VLP delivery system (Prrn: Tc-ba-miR-CHS 1 / P accD: 2 CP). D . And p8, expressing both amiRNA and GFP (Prrn: Tc-ba-miR-CHS 1 / P accD: 2 GFP).amiRNA Expression driven by the tobacco 16 S rRNA promoter (P rrn), 2 CP of MS2 driven by the tobacco (P accD) gene, screening marker gene aadA, driven by the promoter (Cr P psbA) of the Chlamydomyonas reinhardtias psbA gene. CP, coat protein; GFP, green fluorescent protein; TAT, perforated membrane peptide;

2-2. Acquisition of homogenized plastid-transformed tobacco

The plastid vector transformed tobacco by gene gun (Wu et al., 2017）.

2-3. Biological test of Spodoptera litura

While insect resistance was tested by feeding Spodoptera litura on isolated leaves, fresh leaves were changed daily to record larval weight changes, pupation rate, lethality, and levels of corresponding target genes inhibited by RNAi.

3. Experimental scheme

3-1. The vector p1-p8 was constructed by restriction-ligation

3-2. obtained plastid transgenic tobacco using the gene gun method.

The plasmid p5-p8 was bombarded into the tobacco plastid using a gene gun (PDS 1000/HHe, Biorad, USA). After the gene gun bombardment, the leaves were cut into small pieces and transferred to the healing induction medium containing tacamycin for screening. After obtaining the resistance, the buds continued to be induced on the regenerated medium containing monamycin until the resistant buds were obtained. Finally, the resistant buds were transferred to the rooting medium to induce rooting. The homogenization level of plastid transgenic seedlings was identified by Southern blot, and fully homogenized resistant seedlings were selected and transplanted into the soil, domesticated, and continued to grow. The accumulation of amiRNA in plastid-transformed plants was determined by semiquantitative Northern blot hybridization.

4.Technology roadmap

What skeleton is chosen

We chose the skeleton of the red insects(Pupation failure rate is more well documented in the literature)

We used to use the plant skeleton, but later we checked the literature that the effect of the animal skeleton was better than the plant skeleton, so we replaced the animal skeleton(Eventually working in insects)

Why to express the amiRNA

The long RNA is not effective, and the short is effective because there are highly active dsRNA-degrading enzymes in the intestinal fluid of Lepidoptera.

How to select the effective fragment

Skeleton more than 100 bp, that 20 bp is how to predict hotspots with software.

Why do you choose the MS2 VLPs

MS2 can effectively wrap the amiRNA and protect it from degradation.

MS2 phage virus-like particles (MS2 VLP) containing specific RNA and fragments have favorable features of stability, biocompatibility and biodegradability, making them suitable miRNA carriers.

This indicates that the TAT peptide was successfully displayed on the surface of VLPs, which could cause VLPs to penetrate the cell membrane of various cells.

Why did you choose the delivery system

Others have done it in animals, and the technology is more mature.

Formation of multicomplex nanoparticles and liposomes is widely used to protect dsRNA from ribonuclease degradation, and it is unlikely that these encapsulation systems will be incorporated into plastids to stabilize plastid-expressed dsRNA.

How the vector is constructed

Target genes

Bacteria vector

Mass transfer vector

Why do you choose B2 strain

Because it can express both nucleic acids and proteins.

B2 (BL21ARNC)strain: E. coli BL21(DE3) strain with the rnc gene missing from RNaselll(RNasell is knocked out to increase expression)

How the amiRNA is cleaved

We have a total of 138 bp, of which the effective fragment is only 21 bp, but after transcription, the skeleton is cut off at the fixed point, releasing the effective amiRNA.

How do you verify that the protein wraps the RNA

We look at the electron microscope, but we only see the virus particles, we can’t see whether the protein is encased in RNA. Although, we don’t have direct evidence that the protein wraps up the target gene, but we’ve done indirect experiments where the protein enters the cell and it’s definitely digested, and then the gene is still detected in the cell, so we suspect that the protein wraps up RNA, but whether the RNA is coupled to the protein surface or wraps up the protein we don’t know.

Research Process

1. The bacterial expression vector p1-p4 has been constructed and transferred to the B2 strain for expression.

2. To complete the expression level detection of proteins and amiRNA in bacteria.

2-1. Protein expression in the B 2 strain

The expression of B2 strains expressing different plasmids was induced by IPTG (isopropyl- β -D-thiogalactoside). In the B2 strain, 2 CP was detected, 2 CP in B2 in plasmid p1 and p3 and GFP in plasmid p4 (Figure 4A).

2-2. The amiRNA expression in the B 2 strain

B2 strains expressing different plasmids were induced by IPTG (isopropyl- β -D-thiogalactoside).2 CP could not be detected in B2 strains when expressing empty plasmids pET28a and p2; 2 CP could be detected in B2 strains expressing plasmids p1 and p3, and GFP in plasmid p4 (Figure 4B).

Figure 4 protein and amiRNA expression in B 2 strain. A . The expression of proteins in bacteria was determined by SDS-PAGE. B . Northern blot The expression of miRNA in the bacteria was examined.

3. Preliminary biological testing of Spodoptera litura

B2 strains expressing different plasmids were fed to the first hatched larvae of Spodoptera litura. Due to time reasons, the larvae have not yet waited to pupate. Larlarvae feeding on strains expressing p2, p3 and p4 and p3 only. It means that amiRNA, the strain expressing both 2 CP and resulted in the smallest weight gain in the larvae (Figure 5A). The target gene CHS 1 of the p3 and p4 strains expressing the expression plasmid p2 was also significantly downregulated (Figure 5B). The result is that amiRNA can downregulate the target of insect pest, mark genes, and 2 CP may strengthen the control effect of Spodoptera litura.

Although some of the experiments have not been completed, preliminary results show that amiRN A can be effective in the control of NA and use CHS 1 as a target gene.

Figure 5. amiRNA expression in the strain. Nine days after feeding bacteria expressing different plasmids body weight (A) and the downregulation of the target gene CHS 1 (B).

Practical Problem

How to grow transgenic tobacco on a large scale

Transgenic plant planting is a policy problem, safe release, etc., countries can commercial planting only cotton, papaya and small poplar, soybean and corn mass into the stage of safe release, based on RNAI genetically modified crops, only the United States began commercial planting, for corn root, the country has not allowed commercial planting genetically modified tobacco (tobacco is a national taxpayer, not ordinary crops, planting is controlled, tobacco is not literally grow) theory and technical reserves, if the future countries if open policy can be used directly.

Do Chitin Synthase gene affect close relatives of Spodoptera litura

We consider sequence conservation when selecting targets, in principle selecting specific targets, but do not exclude high homology with other bugs, for example, species not sequenced. What would be better to control multiple pests at the same time. We are more worried about targeting the natural enemies of moth.

Does gene expression affect tobacco's own growth

Generally, it does not affect, the dsRNA expressed in plastids generally does not exceed 1% of the RNA used, the expression is not enough to affect the plant phenotype, transgenic plants rarely have a different phenotype from the wild type, but because we do not have it.

Expected Research Results

1. Perfect the bacterial feeding experiments.

2. Completed the construction of the plastid expression vector.

3. To obtain homogenized plastid-transformed tobacco.

4. For protein and amiRNA expression in plastid transformed tobacco.

5. Complete the biological tests of the plastid transformed plants fed with the Spodoptera litura, including the pest mortality rate, the pupation rate, and the expression level of the target genes.

Protocols

PCR amplification fragments

1. PCR reaction system

1. Place in a PCR amplifier (Adjust the time according to the instructions).

Agarose gel preparation for electrophoresis

1. Prepare with 1% agarose gelatin.
2. Weigh 0.5 g of agarose into a Conical flask and add 50 ml of Tris-acetate EDTA(TAE).
3. Heat in the microwave until transparent.
4. Select the comb according to the fragment length and insert a comb at the top of the mold to form the sample well.
5. After the gel is polymerized, the comb can be removed.
6. Place gel in the electrophoresis chamber. Gels should be completely submerged in buffer to allow ion flow and prevent gel drying.

Loading buffer

1. Load the marker into the first well using a micropipette and allow all the samples to “fall” into each of the following wells
2. Fill the upper and lower chambers with running buffer and apply an electric potential on the gel at a constant voltage of 120 V for 30min.
3. Remove the gel from the gel tray and expose the gel to UV light. DNA bands show up as fluorescent bands.

Recycling of DNA from Agarose Gels

1. Excise a gel slice containing the DNA of interest.
2. Place the gel slice containing the DNA sample into an EP tube. Add 30μl of XP2 Binding Buffer.
3. Melt the gel slice at 70°C for 15 minutes. Make sure that the gel slice is melted.
4. Pour in the Spin column to collect Tube and centrifugal (12000rpm 1min).
5. Discard the buffer.
6. Add 700μl SPW Buffer to elute 2 times. Centrifuge the solution (12000rpm 2min).
7. Replace the lower part with an EP tube.
8. Add 30μl Elution buffer and heat for 2 min at 60°C. Centrifuge the solution (12000rpm 1min).
9. Nucleic acid quantification.

Culture monoclonal colonies

1. Add 900 μl of antibiotic-free LB liquid medium and incubate at 180 rpm shaking for 45 min.
2. Centrifuge at 6000 rpm for 5 min.
3. Inoculate onto Luria-Bertani (LB) medium containing Kanamycin. Incubate the culture overnight at 37°C.

Isolate and purify plasmids

1. Add 200 µl of Alkaline lysis solution II to each bacterial suspension.
2. Add 150 µl of ice-cold Alkaline lysis solution III.
3. Centrifuge the bacterial lysate at 12000 rmp for 5 min at 4°C in a microfuge. Transfer the supernatant to a fresh tube.
4. Add an equal volume of phenol: chloroform.
5. Adding ethanol into the supernatant. Mix the solution by vortexing.
6. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 min at 4°C in a microfuge.

Plasmid restriction digestion

1. Restriction digestion system

1. The centrifuge tube was placed in a 37 ℃ water bath overnight