CN117448321A - Double-stranded RNA for biological pest control and application thereof - Google Patents
Double-stranded RNA for biological pest control and application thereof Download PDFInfo
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- CN117448321A CN117448321A CN202310253703.4A CN202310253703A CN117448321A CN 117448321 A CN117448321 A CN 117448321A CN 202310253703 A CN202310253703 A CN 202310253703A CN 117448321 A CN117448321 A CN 117448321A
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- stranded rna
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Abstract
The application relates to the field of biopesticides, in particular to double-stranded RNA for biological pest control and application thereof. The application also provides RNA for causing pest AChE gene silencing, which is a cotton bollworm primer pair or a combination of the cotton bollworm primer pair and one or two of a plutella xylostella primer pair and a beet armyworm primer pair. The application also provides engineering bacteria for expressing the RNA and target RNA produced by the engineering bacteria, wherein the target RNA is double-stranded RNA for biological pest control. In addition, the application also provides wettable powder of double-stranded RNA. The prepared wettable powder is subjected to pesticide effect test, and the result shows that the RNA preparation is effective for preventing and controlling various pests and has up to 95% protection on vegetables.
Description
Technical Field
The invention relates to the field of biological pesticides, and relates to double-stranded RNA for biological pest control and application thereof.
Background
RNA Interference (RNAi) technology is a new technology for pest control, and by utilizing the self RNA regulation mechanism in the pest body, a target gene is sent into the body in the form of double-stranded RNA, so that gene silencing is caused, and the pest death is caused. The technology is environment-friendly, simple and convenient, and becomes an excellent substitute in the large environment where the chemical pesticide environmental pollution is serious, the drug resistance is increased and the residual problem can not be solved.
With the gradual enhancement of the detoxification capability of insects on chemical pesticides and the gradual reduction of the sensitivity on target sites of pesticides, the mechanism research on RNA interference technology is more thorough, the application of transgenic plant culture, disease control and the like is more mature, and people begin to apply the RNA interference technology to pest control. Researchers are continually searching for efficient target genes, better double-stranded RNA introduction modes, proper double-stranded RNA use frequencies and the like to improve the silencing effect of RNA interference, and RNA interference technology is successfully applied to various pests such as diptera, coleoptera, lepidoptera, hymenoptera, hemiptera, orthoptera and the like under laboratory conditions, so that RNA interference has shown great potential in pest control.
Screening for genes of interest for RNA interference is generally initiated from several aspects including direct lethal genes, genes related to oviposition or mating of pests affecting genes like vitellogenin receptor (Vitellogenin recepotor, vgr), genes related to growth and development of pests such as juvenile hormone and ecdysone, genes related to pest resistance and immunity, and functional genes such as genes related to olfaction to interfere with pest identification of crops, etc. by reducing resistance of pests to long-term pesticide use.
In this paper by Yojana et al (Yojana R.Chikane, vishal V.Dawkar, ranjit S.Barbale, priyadarshini V.Tilak, vidya S.Gupta, ashak P.Giri.RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera [ J ]. Pesticide Biochemistry and Physiology,2016,133), tens of candidate enzymes/proteins of trypsin, chymotrypsin, glutathione S transferase, cathepsin L, fatty acid binding protein, esterase, catalase, superoxide dismutase, chitin deacetylase were compared and it was found that feeding the cotton bollworm Helicoverpa armigera after in vitro synthesis of double stranded RNA resulted in good RNA interference effects, resulting in the highest mortality, and thus the selection of the acetylcholinesterase gene for the engineering bacteria induced expression double stranded RNA experiments.
The most important and fundamental role of the essential esterase-acetylcholinesterase (AChE) in organisms is to hydrolyze acetylcholines (ACh) into acetic acid and choline, so as to ensure normal transmission of nerve signals and prevent nerve convulsions and spasm caused by excessive accumulation of the acetylcholines in the synaptic cleft. In other words, the acetylcholinesterase can play a role in disturbing the nervous system of the insect as long as the acetylcholinesterase is inactivated or the activity is inhibited, so that the nervous system of the insect is in an excited state for a long time, and other physiological activities and reproductive survival of the insect are affected. Thus, the acetylcholinesterase gene belongs to a direct lethal gene.
Since 1914 theory of acetylcholine was isolated and identified in 1932, studies on acetylcholinesterase have been continued, and it has been found that the function of acetylcholinesterase is not limited to basic catalysis, but it affects cell adhesion, such as in bone cells, with bone cell matrix; there is also a relationship with cell adhesion in nerve cells, such as promotion of axon growth, induction of synapse formation, participation in migration activity of nerve cells, and the like. In addition, acetylcholinesterase can promote the generation of blood platelet to solve the problem of thrombocytopenia after stem cell transplantation, can be used as a marker of apoptosis to promote the apoptosis of cells, can influence the metabolism and aggregation of amyloid, can regulate the activity of dopamine-cholinergic nerves, can be used for the learning and memory functions of rats, and the like. In short, acetylcholinesterase and acetylcholinesterase inhibitors have been widely used in various fields such as medicine, agriculture, and biological research, gradually departing from the theory.
In invertebrates, choline is only hydrolyzed by acetylcholinesterase, and thus the action of acetylcholinesterase is more important in invertebrates, and its species may be more complex and diverse than in vertebrates. The genes encoding acetylcholinesterase are called Ace genes, and there are mostly only 2, ace 1 and Ace 2, respectively.
The lepidoptera pests which harm crops are various, and common insects which harm vegetables are cotton bollworms Helicoverpa armigera, plutella xylostella Plutella xylostella, asparagus caterpillar Spodoptera exigua, prodenia litura Spodoptera litura Fabricius, spodoptera frugiperda Spodoptera frugiperda and the like, wherein:
the cotton bollworms are mainly crops such as cabbage, corn, tomatoes and the like, the egg laying amount of the cotton bollworms is greatly different at different temperatures, the maximum egg laying amount of the cotton bollworms can be 900 or more, the cotton bollworms are hatched into larvae in 2 to 4 days, the larvae are 4mm long, the adults are 15 to 18mm long, the wings are 32 to 35mm wide, the front wings are provided with irregular black spots and light gray wavy lines, and the rear wings are white in color. At a proper temperature, 31-36 days are needed for one complete generation, 3 generations occur rarely in one season, 8 generations are more, and leaves can be eaten after 2 years, so that vegetable yield reduction is caused;
the plutella xylostella is named as a little blue worm, most of larvae are dark brown, the body length is 9-12 mm, the heads are yellow brown, patterns are visible on the chest backboard, the pupas are yellow green or gray brown, the body length is 7-9 mm, and the larvae are wrapped by silk cocoons. The insect is most serious under drought and warm conditions, the generation of the insect is less than 5 generations in one year, the generation of the insect is more than 18 generations, the diamond back moth has stronger adaptability and drug resistance, is one of 20 most resistant insect species reported at present, has higher resistance to common organophosphorus and pyrethroid pesticides, and has small selectable range;
The spodoptera exigua, also called corn exidoptera, belongs to the family of lepidoptera exidoptera, is a lepidoptera pest which is difficult to control, and is harmful to various vegetables, such as soybean, cabbage, red bean, eggplant, potato and the like. The egg period of the beet armyworm is 2-5 days, the adult body is 11-14 mm long, the wing width is 23-28 mm, the front wing is provided with triangle black spots, the insect is generally gray brown, a large amount of crops are taken when the larvae grow to 3-4 years old, the leaves can be completely eaten when the larvae are serious, and the beet armyworm can take 5-8 generations in one year under the growth condition;
cotton bollworms Helicoverpa armigera, plutella xylostella Plutella xylostella and asparagus caterpillar Spodoptera exigua are lepidoptera pests, and have great influence and harm to the growth of agricultural crops in China, so that the quality and yield of agricultural products are very influenced, and direct harm is generated to the economy, environment and grain requirements of China. Therefore, the three pests are taken as main research objects, the RNA interference technology is used for only identifying the corresponding mRNA sequence, the characteristics of no harm to non-target organisms are achieved, and biological pesticides which are harmless to human bodies and the environment and can prevent and treat the pests are researched, so that a plurality of laboratory theoretical basis is provided for developing novel RNA interference medicaments as a reference.
In recent years, the global extreme climate is frequently generated, drought and waterlogging are frequently generated, human activities are aggravated and the like, so that serious ecological environment damage is caused, the population quantity of pests is greatly increased, the occurrence frequency of pests is also increased, huge losses are caused to global agricultural economy, and therefore, pest control becomes the center of gravity of agricultural development. The existing common pest control means mainly comprise agricultural control, physical control, chemical control and biological control.
The phenomenon of RNA interference has been discovered since 1998 and has been discovered in various organisms to date and proved to be highly conserved. However, at present, related researches on lepidoptera RNA interference reaction are relatively few in related documents, and in most cases, double-stranded RNA is directly synthesized in vitro, but the double-stranded RNA synthesized in vitro is unstable in environment (light irradiation and temperature can inactivate the RNA synthesized in vitro), is easy to decompose in vivo after being eaten by pests, is difficult to reach a pest action target, has small yield, has high cost and the like, and the selling price of an in vitro synthesis kit is 6556 yuan/20 times. In addition, multiple pest damages may exist in farmland at the same time, however, one RNAi agent can only specifically kill specific pests, has no broad spectrum and is difficult to be accepted by farmers.
Therefore, in order to develop double-stranded RNA preparation and apply RNA interference commercially, the inventor mainly explores the possibility of culturing double-stranded RNA in engineering bacteria, and tries to construct engineering bacteria corresponding to various double-stranded RNA aiming at the gene of key enzyme acetylcholinesterase in various agricultural pests as a target gene. And a plurality of engineering bacteria for synthesizing double-stranded RNA are mixed to prepare wettable powder for controlling field pests.
Specifically, the double-stranded RNA is introduced by feeding, so that the research result can be used in a field experiment in a future mode. The method for generating double-stranded RNA is improved, an expensive in-vitro synthesis kit is changed into engineering bacteria which are continuously generated in energy, namely, more stable double-stranded RNA is cultured by using escherichia coli or bacillus subtilis, and a series of problems that the double-stranded RNA synthesized in vitro is high in price, difficult to absorb, unstable and the like are solved. The wettable powder of double-stranded RNA is prepared by adding a carrier, a surfactant and an RNase inhibitor into double-stranded RNA, and is used for controlling field pests. And attempts to re-etch the technology into other genes of other lepidopterans (such as plutella xylostella Plutella xylostella and asparagus caterpillar Spodoptera exigua) so as to provide a new experience for the RNA interference technology-mediated treatment mode.
Because double-stranded RNA is very easy to degrade, the inventor develops a set of preparation formula, and the preparation formula is placed at room temperature to test the RNA content and the biological activity change of the double-stranded RNA in one year, so that a foundation is laid for the commercial application of subsequent double-stranded RNA preparations.
Disclosure of Invention
In order to provide a double-stranded RNA preparation method capable of enhancing pest gene silencing effect and improving pest killing effect, the application provides a double-stranded RNA preparation method for causing pest AChE gene silencing.
In a first aspect, the present application provides an RNA that causes pest AChE gene silencing, which is a cotton bollworm primer pair, or a combination of a cotton bollworm primer pair with one or both of a plutella xylostella primer pair, a asparagus caterpillar primer pair, wherein:
cotton bollworm a: an upstream primer F GTGGAGACTCAACGAAGATC, seq_1;
a downstream primer R CTCTTAGACCACATAATGAACTC, seq_2;
plutella xylostella P1: an upstream primer F ATTGTTTGGAGAATCGTCCG, seq_3;
a downstream primer R GGATTCAGTTCTCTGACCGC, seq_4; or (b)
Plutella xylostella P2: an upstream primer F GGCAAGAATTACTCACCCGA, seq_5;
a downstream primer R CGGAAAAGCGAGATTCAAAC, seq_6;
beet armyworm S1: an upstream primer F GGCTGTGTCGGTTTCATTG, seq_7;
a downstream primer R GATTCAACTCTCTAACTGCCTGC, seq_8; or (b)
Beet armyworm S2: an upstream primer F GGGAGAAGAAATGTGGAATCC, seq_9;
downstream primer R GCCGACTCACCAAATAATGTTA, seq_10.
In a second aspect, the present application provides an engineered bacterium expressing RNA, the engineered bacterium comprising: RNA, vectors and competent cells that cause pest AChE gene silencing.
The carrier isT Easy vector.
The competent cells are E.coli, preferably E.coli HT115 (DE 3).
The preparation method of the engineering bacteria comprises the following steps:
1) Preparation of total RNA of pests: extracting intestinal and brain tissues of corresponding pests by using an RNA prepure animal tissue total RNA extraction kit to obtain total RNA of the pests;
2) Preparation of template DNA:
the total RNA solution of the pest prepared in the step 1) is synthesized into single-stranded cDNA by using an M-MuLV first-strand cDNA synthesis kit, amplified into double strands by polymerase chain reaction (Polymerase chain reaction, PCR) technology, and simultaneously can be utilized to locate template DNA corresponding to target double-stranded RNA;
3) Preparation of plasmids: 1. Mu.L of vector, 2. Mu.L of template DNA prepared in step 2), 1. Mu.L of T4 DNA Ligase, 2X Rapid Ligation Buffer. Mu.L, ddH 2 O1. Mu.L; standing overnight at 4deg.C to obtain plasmid;
4) Transformation of plasmids: e, preparing competent cells from escherichia coli, adding the competent cells into the plasmid prepared in the step 1), carrying out heat shock reaction after ice bath, resuscitating in an LB liquid medium, concentrating, discarding supernatant, culturing the precipitate on a plate containing ampicillin, picking single bacterial colonies, placing the single bacterial colonies in the LB liquid medium containing ampicillin again, and culturing to obtain the engineering bacteria expressing RNA.
The method comprises the following steps:
in the step 2):
ampicillin-containing resistant plates with ampicillin concentration of 50. Mu.g/mL;
ampicillin-containing LB liquid medium, ampicillin concentration of 50. Mu.g/mL;
LB liquid medium composition: 1.0g of sodium chloride, 1.0g of tryptone, 0.5g of yeast extract powder and 100mL of deionized water, preparing a culture solution according to the proportion, adjusting the pH to 7+/-0.2 (25 ℃), and sterilizing at high temperature and high pressure to obtain a sterile culture medium.
The gene segments of three different kinds of insects, namely cotton bollworm Helicoverpa armigera, plutella xylostella Plutella xylostella and asparagus caterpillar Spodoptera exigua, are respectively transferred into escherichia coli, and the maturity of the method in genetic engineering ensures that engineering bacteria can be successfully prepared. The engineering bacteria are inspected and found: the sequence has been successfully linked to a vector, and it is known by comparing the known acetylcholinesterase gene sequence that the target gene sequence has been contained in the plasmid, and that the mutation has occurred in a very individual gene, but the mutation does not affect the subsequent induced expression and the effect of double-stranded RNA. Thus, it was confirmed that the plasmid had been successfully transferred into E.coli.
In a third aspect, the present application provides a double stranded RNA produced by an engineering bacterium, which is expressed by induction by the following method: under the induction of IPTG, the engineering bacteria generate double-stranded RNA, and the double-stranded RNA is centrifugated and freeze-dried.
The inducer is Isopropyl-beta-D-Thiogalactoside;
the induction expression method provided by the application comprises the following steps: picking single colony, placing the single colony into ampicillin-containing LB liquid culture medium solution for culturing overnight, taking overnight culture bacterial liquid into ampicillin-containing 2 XYT culture medium solution, carrying out shake culture until OD600 is more than 0.8, adding IPTG solution, continuing shake culture to obtain double-stranded RNA at 4 ℃ and 4,000rpm, centrifuging, discarding supernatant to obtain bacterial mud, and drying to obtain the ampicillin-containing strain.
The LB liquid medium comprises the following main components: 1.0g of sodium chloride, 1.0g of tryptone, 0.5g of yeast extract powder and 100mL of deionized water; preparing culture solution according to the above proportion, regulating pH to 7+ -0.2 (25deg.C), and sterilizing at high temperature under high pressure to obtain sterile culture medium.
The 2 XYT culture medium comprises the following main components: sodium chloride 0.5g, tryptone 1.6g, yeast extract 1.0g and deionized water 100mL; preparing culture solution according to the above proportion, regulating pH to 7+ -0.2 (25deg.C), and sterilizing at high temperature under high pressure to obtain sterile culture medium.
Ampicillin was present in the various ampicillin-containing media at a concentration of 50. Mu.g/mL.
IPTG (Isopropyl-. Beta. -D-thiogalactoside, IPTG) solution 238mg of IPTG was dissolved in 10mL of deionized water and filtered through a 0.22 μm filter.
The centrifugation conditions are as follows: centrifuge at 4℃for 10min at 4,000 rpm.
The drying is freeze drying or spray drying, and is carried out by adopting a conventional method or adopting the following method to prepare the product: pre-freezing for 4-5 h at-80 ℃, opening a vacuum freeze dryer for pre-freezing in advance for half an hour, removing a cover of a 50mL centrifuge tube filled with pre-frozen bacterial mud, covering a layer of film on a tube mouth, reserving a plurality of air holes on the film (the operation avoids impurities falling into the freeze-drying process and ensures the purity of freeze-dried products), placing the centrifuge tube on a centrifuge tube rack, putting the centrifuge tube into the vacuum freeze dryer, and performing vacuum freezing for 18h at the parameters of the cold trap temperature of-50 to-53 ℃ and the pressure of 6-6.50 Pa to obtain the freeze-dried products.
In a fourth aspect, the present application also provides a wettable powder comprising double stranded RNA, comprising: double stranded RNA, a vector, a surfactant, and an RNase inhibitor.
Specifically, the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 30-150 parts of a carrier, 2-9 parts of a surfactant and 2-9 parts of an RNase inhibitor.
Preferably, the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 60-120 parts of a carrier, 2-5 parts of a surfactant and 2-6 parts of an RNase inhibitor.
Further preferably, the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 60-90 parts of a carrier, 3.5-5 parts of a surfactant and 3.5-5 parts of an RNase inhibitor.
Optimally, the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 60 parts of a carrier, 4.2 parts of a surfactant and 4.9 parts of an RNase inhibitor.
In the wettable powder, the following components are contained in the mixture:
the double-stranded RNA is freeze-dried powder.
The surfactant is Sodium Dodecyl Sulfate (SDS), nekal (BX), sodium lignin Sulfonate (SL), calcium lignin sulfonate (CL), preferably calcium lignin sulfonate; the dosage of the surfactant is 3-7% of the sum of the masses of the carrier and the double-stranded RNA, and is specifically 3%, 5% and 7%;
the carrier is kaolin, bentonite, diatomite or white carbon black, preferably white carbon black; the mass of the vector and the double-stranded RNA is 3-15:1, specifically 15:1, 12:1, 9:1, 6:1, 3:1.
The rnase inhibitor is urea or Sodium Dodecyl Sulfate (SDS), preferably urea; the amount of RNase inhibitor is 3-7%, specifically 3%, 5%, 7% of the sum of the vector and double-stranded RNA.
The application also provides a preparation method of the wettable powder, which is to uniformly mix any double-stranded RNA with a carrier, a surfactant and an RNase inhibitor, and freeze-dry or spray-dry the mixture to prepare the wettable powder.
The application also provides application of the wettable powder in preparing pesticides for preventing and controlling lepidopteran pests.
The application also provides a using method of the wettable powder: diluting the wettable powder with water for 200-400 times, and then spraying on the leaf surfaces of green vegetables.
In summary, the present application has the following beneficial effects:
1. the pest RNA extracted from the midgut of the larvae of cotton bollworms, plutella xylostellas and asparagus caterpillars is taken as an original gene sequence, cDNA is generated through reverse transcription, and a target acetylcholinesterase gene is amplified and positioned through PCR, and is connected with the target geneT Easy vector, then transformed into competent cells of escherichia coli to obtain double-stranded RNA engineering bacteria.
2. After the engineering bacteria provided by the application generate double-stranded RNA under the induction of IPTG, feeding the four-instar larvae, and finding that the target genes are silenced; up to 60% mortality was observed in the second instar larvae fed on the insect, the dead bodies blackened, while the other non-dead bodies grew slowly, with individuals significantly less than the negative control.
3. In the RNA interference experiment, the RNA including double-stranded RNA is easy to degrade by RNase, so that the preservation of the double-stranded RNA becomes a difficult problem. Thus, four commonly used rnase inhibitors, urea, sodium dodecyl sulfate, vanadyl ribonucleoside complex and protein inhibitor RNasin, were tried to extend the storage time of double stranded RNA. The results indicate that urea and RNasin have a high degree of inhibition of rnase and have the potential to act as highly potent inhibitors, but the low cost of urea has made it remarkable in view of the cost of commercialization.
4. The wettable powder provided by the application can prolong the effective period of being capable of being maintained for at least six months at normal temperature.
5. The wettable powder provided by the application is used for carrying out indoor potted plant leaf surface spraying experiments on cotton bollworms, plutella xylostellas and asparagus caterpillars, three insects all show RNA interference effects on the third day after application, the growth of the insects is limited, the insects die to different degrees within seven days after application, and the death rate is 85%. The preparation provided by the application also has up to 95% protection on green vegetables, and compared with green vegetables without application, the RNA interference preparation has good effect on crop protection.
Drawings
Fig. 1: preparing an experimental flow chart by single-gene engineering bacteria;
fig. 2: PCR electrophoresis diagram of the acetylcholinesterase gene of cotton bollworm;
fig. 3: PCR electrophoresis patterns of beet armyworm and plutella xylostella acetylcholinesterase genes, wherein S1 and S2 represent 2 segments of target genes of beet armyworm, P1 and P2 represent 2 segments of target genes of plutella xylostella, and 3 samples are one gene;
fig. 4: sequencing result diagram of engineering bacteria of cotton bollworm, asterisks indicate that the sequencing result is consistent with the original gene.
Fig. 5: the asterisk represents that the sequencing result is consistent with the original gene;
fig. 6: the asterisk represents that the sequencing result is consistent with the original gene;
Fig. 7: beet armyworm Ace 1 engineering bacteria sequencing result diagram, asterisks represent that the sequencing result is consistent with the original gene;
fig. 8: beet armyworm Ace 2 engineering bacteria sequencing result diagram, asterisks represent that the sequencing result is consistent with the original gene;
fig. 9: the electrophoresis pattern of double-stranded RNA induction of cotton bollworm shows that 4 samples are respectively induced at 37 ℃ for 4 hours, 5 hours and 6 hours and induced at 32 ℃ for 6 hours;
fig. 10: the double-stranded RNA induction electrophoresis patterns of plutella xylostella and asparagus caterpillar are used for experiments by adopting various induction times (4 h, 5h and 6 h) and induction temperatures (37 ℃ and 32 ℃), and double-stranded RNA with brighter bands is selected for subsequent stomach toxicity experiments. S1 and S2 represent double-stranded RNA corresponding to 2 segments of target genes of asparagus caterpillar, and P1 and P2 represent double-stranded RNA corresponding to 2 segments of target genes of diamond back moth;
fig. 11: gene silencing levels of cotton bollworms, data in the figures refer to mean ± standard deviation (SEM). Asterisks indicate significant differences (< 0.005, <0.001, <0.0001,);
fig. 12: the gene silencing level of plutella xylostella is shown as mean ± SEM. Asterisks indicate significant differences (< 0.005, <0.001, <0.0001,);
fig. 13: gene silencing levels of asparagus caterpillar, data in the figures refer to mean ± SEM. Asterisks indicate significant differences (< 0.005, <0.001, <0.0001,);
Fig. 14: four vectors were electrophoretically verified. The steps are as follows from left to right: kaolin, bentonite, diatomite and white carbon black;
fig. 15: RNA concentrations of the four vectors corresponding to the master powder were compared. Data in the figures refer to mean ± SEM. Asterisks represent significant differences (< 0.005, <0.001, < 0.0001);
fig. 16: different mass ratio electrophoresis patterns of white carbon black and raw materials;
fig. 17: the data in the figures refer to mean ± SEM. Asterisks represent significant differences (< 0.005, <0.001, < 0.0001);
fig. 18: electrophoretogram of different rnase inhibitors;
fig. 19: electrophoresis patterns of urea with different addition amounts;
fig. 20: RNA concentration at different addition levels of different inhibitors. Data in the figures refer to mean ± SEM. Asterisks represent significant differences (< 0.005, <0.001, < 0.0001);
fig. 21: electropherograms of different surfactants at the same mass ratio;
fig. 22: RNA content of different surfactant addition amounts. Data in the figures refer to mean ± SEM. Asterisks represent significant differences (< 0.005, <0.001, < 0.0001);
fig. 23: destination strips at different storage times;
fig. 24: green vegetable leaf survival rate.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
1. Experimental equipment (equipment name, model and manufacturer respectively):
sterile super clean bench (model SW-CJ-IFD), constant temperature incubator (model DHG-9070A), shanghai-constant technology instruments Co., ltd;
a high pressure steam sterilizing pot, LDZX-50KBS, shanghai Shen An medical equipment factory;
constant temperature shaker, COS-200B, shanghai Bilang instruments Co., ltd;
vortex mixer, XW-80A, shanghai precision industries, ltd;
nucleic acid electrophoresis tank (model HE-200), gel imager (model Tanon 2500), shanghai Tencenters Co., ltd;
a constant temperature heater, GL-150B, manufactured by Linbell instruments, inc.;
an ultraviolet spectrophotometer, UV1902PC, available from phoenix optics group limited;
an illumination incubator, blue Pard, available from Shanghai-constant technology instruments Co., ltd;
reverse transcription-fluorescence quantitative PCR instrument, light Cycle 96, available from Shanghai Luo pharmaceutical Co.
2. RNA store Reagent reagent, RNA prep pure animal tissue total RNA extraction kit, agarose in electrophoresis experiment, 6× DNA loading buffer, DNA marker II (molecular weight standard) and DNA marker IV (molecular weight standard), centrifugal column type large-scale Agarose gel DNA recovery kit, 10×PCR buffer, ddH 2 O, etc., all purchased from tiangen biochemical technology (beijing) limited. Wherein:
the total RNA extraction kit for the RNA prep pure animal tissue comprises the following components: 30ml of lysate RL, 1 ml of deproteinized solution RW, 12ml of rinsing solution RW, and RNase Free ddH 2 O15 ml, RNase Free adsorption column CR3 (comprising 2ml collecting tube) 50 sets (hereinafter abbreviated as adsorption column), RNase Free filtration column CS (comprising 2ml collecting tube) 50 sets, RDD buffer 4ml, RNase-Free DNase I1 set, DNase I dry powder, and Proteinase K.
3. DEPC Water (diethyl pyr)The primer and dNTP Mixture (with Mg) are used for PCR, and the primer and dNTP Mixture are used for PCR 2+ 25 mM), 50 XTAE buffer for electrophoresis, 4S red plus nucleic acid stain (10,000 Xaqueous solution), sterile CaCl 2 Solution (1M), T4 DNA Ligase1, 2X Rapid Ligation Buffer, ddH 2 O, etc., all from the division of bioengineering (Shanghai). Wherein:
the M-MuLV first strand cDNA synthesis kit comprises: 5 Xreaction Buffer 10Mm dNTP Mix,Oligo-dT Primer (0.5. Mu.g/. Mu.l), random Primer p (dN) 6 (0.2μg/μl)，RNase Inhibitor(20U/μl)，M-MuLV Reverse Transcriptase(200U/μl)，RNase-free ddH 2 O. 4. Preparation of culture Medium
LB liquid medium, its main ingredients: 1.0g of sodium chloride, 1.0g of tryptone, 0.5g of yeast extract powder and 100mL of deionized water;
2 XYT medium, its main ingredients: sodium chloride 0.5g, tryptone 1.6g, yeast extract 1.0g and deionized water 100mL.
The preparation method of each culture medium comprises the following steps: preparing culture solution according to the above proportion, regulating pH to 7+ -0.2 (25deg.C), and sterilizing at high temperature under high pressure to obtain sterile culture medium.
5. IPTG (Isopropyl-. Beta. -D-thiogalactoside, isopropyl-. Beta. -D-thiogalactoside, IPTG), trizol reagent, were all purchased from Biotechnology (Shanghai) Inc.
6. Isopropyl alcohol and ethanol, available from Shanghai Taitan technologies Co., ltd;
7. trans Script one step gDNA removal and cDNA synthesis super mix, perfect start green qPCR super mix, all available from Beijing all gold biotechnology Co.
8. 1×TAE solution, available from Shanghai Biyun biotechnology Co.
Example 1: design of primer pairs
The primers for PCR were synthesized based on the acetylcholinesterase genes corresponding to double-stranded RNAs causing RNA interference reaction in the documents of Yojana et al [ document 1], gaddelapati et al [ document 2] and Zhao jin et al [ document 3], and the primer sequences are shown in Table 1.
TABLE 1 primer sequences for cotton bollworms, plutella xylostella and asparagus caterpillar
F represents the upstream primer, R represents the downstream primer, bp represents the primer length, and Accession No. is the sequence number found on NCBI.
Wherein:
1,Yojana R.Chikate,Vishal V.Dawkar,Ranjit S.Barbole,Priyadarshini V.Tilak,Vidya S.Gupta,Ashok P.Giri.RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera[J ]. Pesticide Biochemistry and Physiology,2016,133;
document 2,Gaddelapati Sharath Chandra,Ramaswamy Asokan,Maligeppagol Manamohan,Nallur Krishna Kumar.Enhancing RNAi by using concatemerized double-structured RNA [ J ]. Pest Management Science,2019,75 (2);
document 3,Zhao Jing,Hao Dejun,Xiao Liubin,Tan Yongan,Jiang Yiping,Bai Lixin,Wang Kai.Molecular and functional properties of two Spodoptera exigua acetylcholinesterase genes[J ]. Archives of Insect Biochemistry and Physiology,2019,101 (3);
example 2: extraction of total RNA from pests
1. Experimental materials:
1.1 test insects cotton bollworm Helicoverpa armigera, plutella xylostella Plutella xylostella and Spod-optera exigua and foods thereof were purchased from Henan province family cloud biopesticide Co.
The experimental object is 2-year larva, and the culture condition of the illumination incubator is as follows: the illumination is carried out for 16 hours, the temperature is 26-28 ℃, and the humidity is about 40%.
1.2 arrangement of liquids:
the total RNA extraction kit for extracting the total RNA of the RNA prep pure animal tissue is used, and before the total RNA is used, the total RNA extraction kit comprises the following components:
the preparation method of the 1% beta-mercaptoethanol lysate RL comprises the following steps: beta-mercaptoethanol was added to the lysate RL to a final concentration of 1%, e.g., 10. Mu.l beta-mercaptoethanol was added to 1ml lysate RL.
Preparing DNase I mixed solution: DNase I dry powder (1500U) was dissolved in 550. Mu.l RNase Free ddH 2 And (3) in the step O, mixing gently, sub-packaging, and storing at-30 to-15 ℃ for 9 months to obtain DNase I storage solution.
2. Extraction of Total RNA (for flow chart see FIG. 1)
The total RNA is extracted by using a kit for extracting total RNA from animal tissues of RNA prep pure from intestinal and brain tissues, and the extraction of cotton bollworms, plutella xylostella and asparagus caterpillar is the same, and the following is taken as an example:
1) Homogenizing:
dissecting the bollworm Helicoverpa armigera, taking out the intestines and brain, and soaking in RNA store Reagent reagent for later use.
20mg of cotton bollworm midgut or brain tissue was weighed, 300. Mu.L of a lysate RL of 1% beta-mercaptoethanol was added, and the tissue was thoroughly ground with a grinding pestle. ( And (3) injection: because the brain is difficult to grind, the brain can be crushed and then added into the lysate )
2) After homogenization, 590. Mu.L of RNase Free ddH was added 2 O and 10 mu L of protease K are mixed uniformly, water bath is carried out for 20min at 56 ℃, and centrifugation is carried out for 5min at 12,000rpm, and the supernatant is taken out and placed in another clean collecting tube for standby.
3) Adding absolute ethyl alcohol with the volume which is 0.5 times of the volume of the supernatant liquid into the collecting pipe containing the supernatant liquid in the step 2), uniformly mixing, transferring into an adsorption column, centrifuging for 1min at 12,000rpm, pouring out waste liquid, and placing the adsorption column into the collecting pipe.
4) 350. Mu.L deproteinized liquid RW1 was added to the collection tube of step 3), centrifuged at 12,000rpm for 1min, the waste liquid was discarded, and the adsorption column was placed in the collection tube.
5) 70. Mu.L of RDD buffer and 10. Mu.L of DNase I were prepared, added to the collection tube of step 4), left to stand at room temperature for 15min, then 350. Mu.L of deproteinized liquid RW1 was added, centrifuged at 12,000rpm for 1min, the waste liquid was discarded, and the adsorption column was placed in the collection tube.
Preparing DNase I mixed solution: adding 10 μl DNase I stock solution into a new RNase Free centrifuge tube, adding 70 μl RDD buffer solution, and gently mixing.
6) Add 500. Mu.L of rinse RW (ethanol before use) to the collection tube of step 5), centrifuge for 1min at 12,000rpm at room temperature, discard the waste liquid, and place the column into the collection tube.
7) 500. Mu.L of the rinse liquid RW was again added to the collection tube of step 6), the mixture was left to stand at room temperature for 3 minutes at 12,000rpm, the waste liquid was discarded, and the adsorption column was placed in the collection tube.
8) After air-drying at room temperature for several minutes, the residual rinse solution was thoroughly removed. Finally, placing the adsorption column into a new centrifuge tube, suspending and dripping 50 mu L of RNase Free ddH into the middle of the adsorption film 2 O, standing for 2min, centrifuging at 12,000rpm for 2min, and collecting RNA solution, namely total RNA solution of the pests.
Example 3: preparation of template DNA
The pest total RNA solution was synthesized into single-stranded cDNA using M-MuLV first strand cDNA synthesis kit, and then amplified into double-stranded DNA by polymerase chain reaction (Polymerase chain reaction, PCR) technique. The preparation methods of the cotton bollworms, the plutella xylostella and the asparagus caterpillar are the same, and the cotton bollworms are still taken as examples below:
1. reverse transcription of RNA
The total RNA solution of pest prepared in example 2 was used to synthesize single-stranded cDNA using M-MuLV first strand cDNA synthesis kit, and the whole experiment was performed on ice.
1) The system solution of the total RNA reverse transcription system 1 was added to the ice-bathed test tube, shaken well and centrifuged for 3sec, and the supernatant was removed.
The system solution of the total RNA reverse transcription system 1 is as follows: 5. Mu.L of pest total RNA solution, 1. Mu. L, RNase-free ddH each for the upstream and downstream primers in the corresponding pair of primers in Table 1 2 O 6μL。
2) Placing the test tube into a 65 ℃ water bath kettle for 5min, transferring into an ice bath, placing for 30sec, centrifuging for 3sec, and removing supernatant;
3) The test tube was put back in ice bath, the system solution of the total RNA reverse transcription system 2 was added, and after gentle mixing, the mixture was centrifuged for 3sec to remove the supernatant.
System solution of total RNA reverse transcription system 2: 12. Mu.L of total RNA solution of pests, 4. Mu.L of 5 Xreaction Buffer, 1. Mu.L of RNase Inhibitor, 2. Mu. L, M-MuLV Reverse Transcriptase. Mu.L of dNTP Mix.
If the total RNA concentration is lower, the amount of total RNA can be increased and the amount of enzyme-free water can be reduced in an appropriate amount.
4) Reverse transcription reaction was performed on a PCR instrument under the following reaction conditions: pre-reacting at 25 ℃ for 10min; cDNA synthesis at 45℃for 60min; the reaction was terminated at 70℃for 10min to obtain a template cDNA solution.
2. Amplification of cDNA
The single-stranded cDNA prepared above is extremely unstable and amplified into double-stranded DNA by the polymerase chain reaction (Polymerase chain reaction, PCR) technique.
The PCR system is as follows: taq plus DNA polymerase 1. Mu.L, template cDNA solution 2. Mu.L, upstream primer (10. Mu.M) 2. Mu.L, downstream primer (10. Mu.M) 2. Mu.L, dNTP mix (with Mg) 2+ )(25mM)1μL、10×PCR buffer 5μL、ddH 2 O37. Mu.L. (the primers are the cotton bollworm upstream primer and downstream primer in Table 1).
Shaking the above system, centrifuging, and performing PCR reaction on a PCR instrument under the following reaction conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 45sec; annealing at 51-58 ℃ for 15sec; extending at 72℃for 45sec, wherein denaturation, annealing, and extension were performed for 30 cycles, followed by final extension at 72℃for 10min, to obtain a template DNA.
(explanation:
1. reasons for setting the reaction conditions: the temperature can be more suitable by using the step annealing temperature, and finally, DNA with the deepest band is selected for recovery, so that high-quality DNA can be obtained.
2. The template DNA products are divided into 5 kinds, 1 kind of cotton bollworms, 2 kinds of plutella xylostella and asparagus caterpillar respectively
Experimental example 1: verification of DNA agarose gel electrophoresis
To verify that the template DNA prepared in example 3 was the target DNA, agarose gel electrophoresis was used for examination and verification.
1. The detection mode is as follows:
a1% agarose solution was prepared from 0.4g agarose and 40mL 1 XTAE solution, and the agarose solution was heated to dissolve completely. After cooling to about 40 ℃, add the following 1:10,000. Mu.L of 4S red plus nucleic acid stain was added, thoroughly shaken and poured into a glass plate, and a comb was inserted, and allowed to solidify after cooling.
After complete solidification, the comb was removed, the glass plate was taken out and placed in an electrophoresis tank, and 5. Mu.L of the template DNA solution was mixed with 1. Mu.L of 6X DNA loading buffer and then applied. The electrophoresis conditions were 120V, run for about 30min until the sample was run to two thirds of the gel, take out the electrophoresis gel, and observe the electrophoresis result in an imaging recorder.
The template DNA solutions were template DNAs of cotton bollworms, plutella xylostella and asparagus caterpillar, respectively, prepared in accordance with the method of example 3.
2. Detection standard:
the target gene of the cotton bollworm Helicoverpa armigera is 308bp, and if the target gene is compared with a marker, a clear band exists at about 300bp, the target gene is considered to be successful in verification;
similarly, if the other four fragments had a band at 500bp, the PCR experiment was considered to be successful in amplification. After verification is successful, the DNA is obtained again by gel recovery experiments, and the method can remove nonspecifically bound impurity DNA, only select the required DNA band, ensure the purity of plasmid connection and reduce the possibility of false positive plasmids.
3. Detection result: see FIGS. 2 and 3
The length of the target cDNA of the cotton bollworm Helicoverpa armigera is 308bp, and the 300bp part in FIG. 2 has obvious bands, so that the obtained target gene can be proved.
The cDNA lengths of the plutella xylostella Plutella xylostella and the spodoptera exigua Spodoptera exigua are 400-500 bp, and as shown in fig. 3, obvious bands are arranged at the positions of less than 500bp, which indicates that the next ligation transformation experiment can be carried out. In the figure, the beet armyworm fragment 1 (S1), the beet armyworm fragment 2 (S2), the plutella xylostella fragment 1 (P1) and the plutella xylostella fragment 2 (P2) are sequentially arranged from left to right, and the gene lengths are respectively 470 bp,417bp,498bp and 487bp. S1 and S2 represent 2-segment target genes of asparagus caterpillar, and P1 and P2 represent 2-segment target genes of plutella xylostella. 3 samples were one gene.
Example 4: preparation of engineering bacteria for expressing RNA
1. Experimental materials
Coli HT115 (DE 3) strain, available from Wohan vast, biotechnology Inc.
T Easy vector, available from Promega (Beijing) Biotechnology Co.
Glycerin, ampicillin, sodium chloride used in preparing the culture medium, tryptone, yeast extract, agar, etc. were purchased from Shanghai Taitan technologies Co.
The final ampicillin media concentration was typically 50. Mu.g/mL, and the usual storage concentration of ampicillin was 100mg/mL, i.e.1 g of ampicillin was dissolved in 10mL of deionized water. After dissolution, the cells were sterilized by filtration through a 0.22 μm filter, and frozen at-20 ℃. When used, 25. Mu.L of ampicillin was added to each 50mL of medium after dissolution at room temperature.
2. Attachment of carriers
2.1 preparation of plasmid:
the reaction system is as follows:t Easy vector 1. Mu.L, template DNA (prepared in example 3, one of them) or control DNA (without any DNA added) 2. Mu.L, T4 DNA Ligase 1. Mu.L, 2X Rapid Ligation Buffer. Mu.L, ddH 2 O 1μL。
The plasmid was obtained by standing overnight at 4 ℃.
2.2 transformation of plasmid
1) Coli HT115 (DE 3) strain was first made into competent cells prior to transformation:
in the first step, a small amount of bacterial liquid of HT115 (DE 3) strain in glycerol pipe is taken to be streaked on a non-resistance plate, and the culture is carried out at 37 ℃ for overnight.
And secondly, picking single colonies into LB liquid medium solution, and culturing at 37 ℃ for 14 hours until the later logarithmic growth phase.
Next, at 1:50, inoculating the bacterial liquid into LB liquid culture medium, oscillating at 37deg.C to OD 600 Approximately 0.5 (note: OD) 600 Is the absorbance of the solution at a wavelength of 600 nm). Transferring the bacterial liquid into two new centrifuge tubes, placing the two new centrifuge tubes on crushed ice for 10min, centrifuging the bacterial liquid at 4,000rpm and 4 ℃ for 10min after the bacterial liquid is cooled, and discarding the supernatant. 10mL of pre-chilled 0.1mol/L sterile CaCl was added 2 The cells were suspended in the solution, centrifuged at 4,000rpm for 10min at 4℃in crushed ice for 30min, and the supernatant was discarded. 4mL of pre-chilled 0.1mol/L sterile CaCl containing 15% glycerol was added 2 And (5) fully and uniformly mixing the solutions, and placing the solutions in crushed ice for ice bath for 10min to obtain competent cells. Split charging 100 uL/branch, storing and-80 ℃.
Competent cells are extremely easy to inactivate, the thawing time is not too long, and the transformation experiment needs to be carried out on ice in the early stage.
2) mu.L of competent cells was taken, 5. Mu.L of the plasmid prepared in step 1) was added thereto, and the mixture was placed in crushed ice for 30min in an ice bath. After heat shock at 42℃for 60sec and ice bath for 2min, 900. Mu.L of LB liquid medium was added and resuscitated in a shaking table at 37℃for 1 hour. After centrifugation at 4℃and 4,000rpm for 5min to concentrate the bacterial solution and discard 800. Mu.L of supernatant, 100. Mu.L was plated on ampicillin (50. Mu.g/mL) resistant plates and incubated overnight at 37℃until single colonies grew. Single colonies were picked and placed in ampicillin (50. Mu.g/mL) resistant LB liquid medium, and cultured overnight at 37℃to obtain an engineering bacterium expressing RNA.
( Explanation: the final products are divided into 5 kinds, 1 kind of cotton bollworms, 2 kinds of plutella xylostella and asparagus caterpillar respectively )
Experimental example 2: verification of engineering bacteria-agarose gel electrophoresis
And preliminarily judging whether the transformation is successful or not according to whether the size of the band is consistent with the size of the target gene. The main experimental operation principle is that the DNA in the bacterial liquid is amplified again by the same primer, then agarose gel electrophoresis is carried out, if the 300bp part has obvious band, the bacteria contains the target gene, namely the transformation is successful.
1. Whether the plasmid was successfully introduced into E.coli cells:
the inspection process is divided into two steps:
1) Bacterial liquid PCR
The PCR system is as follows: 1. Mu.L of Taq plus DNA polymerase, 2. Mu.L of bacterial liquid DNA (prepared in example 3), 2. Mu.L of upstream primer (10. Mu.M) of the corresponding pest, 2. Mu.L of downstream primer (10. Mu.M) of the corresponding pest, dNTP mix (with Mg) 2+ )(25mM)1μL，10×PCR buffer5μL，ddH 2 O17μL。
Reaction conditions: pre-denatured at 94℃for 3min, denatured at 94℃for 45sec, annealed at 55℃for 15sec, extended at 72℃for 45sec, denatured, annealed, extended for 30 cycles, and then finally extended at 72℃for 10min.
2) The PCR reaction solution was subjected to electrophoresis verification, and the electrophoresis verification procedure was the same as that described in Experimental example 1.
2. By sequencing experiments, the sequence is observed to be successfully connected with the vector, and the known acetylcholinesterase gene sequence is compared, so that the target gene sequence is already contained in the plasmid, and the mutation of a very individual gene is already carried out, but the mutation does not influence the subsequent induced expression and the effect of double-stranded RNA. Thus, it was confirmed that the plasmid had been successfully transferred into E.coli HT115 (DE 3). (see in particular FIGS. 4-8)
Example 5: double-stranded RNA produced by engineering bacteria
Under the induction of IPTG (Isopropyl-beta-D-Thiogalactoside), the engineering bacteria generate double-stranded RNA.
1. Picking a single colony of the engineering bacteria prepared in the example 4, and culturing the single colony in 50mL of LB liquid medium containing 50ug/mL ampicillin at 37 ℃ and 200rpm overnight;
2. 1mL of the overnight culture broth is taken into 50mL of 2 XYT culture medium solution containing 50ug/mL of ampicillin, and the culture shaking is carried out at 37 ℃ and 200rpm until the OD600 is more than 0.8;
3. adding IPTG inducer to the solution to make the final concentration of the inducer be 0.4mM, and continuing shaking culture at 37 ℃ and 200rpm for several hours, then obtaining double-stranded RNA;
the inducer is Isopropyl-beta-D-thiogalactoside (IPTG) stored at a concentration of 0.1M. Weighing 238mg IPTG inducer by a weighing balance, putting the inducer in a 10mL quantitative bottle, fixing the volume of deionized water to a scale mark, filtering, sterilizing and packaging the inducer in a 1.5mL sterile EP tube by a 0.22 mu m filter membrane after the inducer is thoroughly dissolved, and storing the inducer at the temperature of minus 20 ℃;
4. subpackaging 1 ml/branch, centrifuging at 4 deg.C and 4,000rpm for 10min, discarding supernatant, and freeze drying the rest bacterial mud to obtain double-stranded RNA expressed by engineering bacteria;
the freeze drying method comprises the following steps: pre-freezing for 4-5 h at-80 ℃, opening a vacuum freeze dryer for pre-freezing in advance for half an hour, removing a cover of a 50mL centrifuge tube filled with pre-frozen bacterial mud, covering a layer of film on a tube mouth, reserving a plurality of air holes on the film (the operation avoids impurities falling into the freeze-drying process and ensures the purity of freeze-dried products), placing the centrifuge tube on a centrifuge tube rack, putting the centrifuge tube into the vacuum freeze dryer, and performing vacuum freezing for 18h at the parameters of the cold trap temperature of-50 to-53 ℃ and the pressure of 6-6.50 Pa to obtain the freeze-dried products.
Experimental example 3: feeding experiment
Detecting gene silencing effect of double-stranded RNA expressed by engineering bacteria in different lepidopterans by feeding, exploring tolerance of different lepidopterans to RNA interference, and detecting the death degree caused by the RNA interference to determine whether good RNA interference effect can be caused.
1. Experimental materials
1) Insects: cotton bollworm Helicoverpa armigera, plutella xylostella Plutella xylostella and asparagus caterpillar Spodoptera exigua larvae and feeds are purchased from Henan province cloud biopesticide limited. The stomach toxicity experiment is carried out in an illumination incubator, white light is irradiated for 16 hours and dark for 8 hours every day, the temperature is 26-28 ℃, and the relative humidity is 40%.
2) Primer: see Table 2
In the gene silencing efficiency experiment for measuring RNA interference by reverse transcription-fluorescence quantitative PCR, the gene primers and the internal reference gene primers were synthesized according to the documents of Yojana et al [ document 1], gaddelapati et al [ document 2] and jin et al [ document 3], as shown in Table 2.
Table 2: primer sequences for reverse transcription-fluorescent quantitative PCR
F represents the upstream primer and R represents the downstream primer.
2. Experimental method
The double-stranded RNAs (5 kinds) expressed by the engineering bacteria prepared in the example 5 are respectively extracted by Trizol reagent, and the specific extraction method is as follows:
After centrifugation, 500. Mu.L of Trizol reagent was added to the bacteria, and the mixture was left at room temperature for 10 minutes after thoroughly mixing. 200. Mu.L of chloroform was added thereto, and the mixture was vigorously shaken for 30sec, left at room temperature for 3min, and obvious delamination was observed, followed by centrifugation at 12,000rpm at 4℃for 10min. At this time, the sample is divided into three layers, the upper water phase is taken into a new EP tube, equal volume of isopropanol is added, and the mixture is fully and uniformly mixed and then placed for 20 minutes at room temperature. Centrifuge at 12,000rpm for 10min at 4℃and discard the supernatant.
1mL of 75% ethanol in DEPC treated water was added, and the mixture was centrifuged at 12,000rpm at 4℃for 3min, and the supernatant was discarded. Air-dried at room temperature for several minutes to completely remove ethanol and residual organic solvent. 50. Mu.L of RNase Free ddH was added 2 O dissolves RNA to obtain the extracted double-stranded RNA bacterial liquid.
3. Detecting the index:
3.1 electrophoresis verification:
the extracted double-stranded RNA was verified by agarose gel electrophoresis. The electrophoresis verification step is shown in experimental example 1.
3.2 stomach toxicity test-four-instar larvae
3.2.1 experimental method: respectively feeding double-stranded RNA bacterial liquids (prepared in the previous step 2) extracted from three pests such as cotton bollworms, asparagus caterpillar, plutella xylostella and the like into corresponding three larvae, wherein the asparagus caterpillar and the plutella xylostella respectively take one of the two larvae for feeding;
And three larvae were additionally fed with a bacterial liquid containing only HT115 (DE 3) competent cells as a control group.
Four-instar larvae are selected for feeding, 10 mu L of bacterial liquid is added into the feed every day, and dissecting is carried out after 3 days of feeding.
3.2.2 detecting positions and indexes: the brains were removed and total RNA was extracted from the cells, and the procedure was as in example 2.
3.2.3 reverse transcription-fluorescent quantitative PCR
Reverse transcription-fluorescence quantitative PCR (reverse transcription fluorescence quantitative polymerase chain reaction (reverse transcription quantitative polymerase chain reaction, RT-qPCR) refers to an experimental mode of performing PCR after reverse transcription of RNA into cDNA and combining with fluorescence quantitative detection, using Syber Green I as a fluorescent dye, utilizing signals of a fluorescent group to perform the function of monitoring the whole PCR process in real time, and finally obtaining a Ct value.
1) Preparation of template cDNA
Preparing a reverse transcription system: template RNA (prepared in example 5) 7. Mu.L, random Primer 1. Mu.L, 2 XS Reaction Mix 10. Mu.L, trans Script RT/RI Enzyme Mix 1. Mu.L, gDNA reverse 1. Mu.L.
The reverse transcription reaction conditions were as follows: pre-reaction, cDNA synthesis, heat inactivation, wherein the pre-reaction is 10min at 25 ℃, the cDNA synthesis is 15min at 42 ℃, and the heat inactivation is 5sec at 85 ℃. (the objective is to remove TransScript RT/RI enzymes and gDNA remote)
2) And (3) PCR reaction:
preparing a real-time quantitative PCR system solution, wherein the preparation system comprises the following steps: 3. Mu.L of template cDNA, 0.4. Mu.L of upstream primer (10. Mu.M), 0.4. Mu.L of downstream primer (10. Mu.M), 2X Perfect Start Green qPCR Super Mix. Mu.L, ddH 2 O6.2μL。
The prepared sample is added into an eight-joint tube seed feeding machine, and the reaction conditions are as follows: 94℃for 30sec,94℃for 5sec,53℃for 15sec,72℃for 10sec, the latter three 45 cycles.
( And (3) injection: the upstream primer and the downstream primer corresponding to the cDNA of the cotton bollworm A template are the upstream primer and the downstream primer in the table 2; the upstream primer and the downstream primer corresponding to the cDNA of the plutella xylostella P1 template are the upstream primer and the downstream primer of the plutella xylostella P1 shown in Table 2, and the like )
3.2.4 investigation of the index-investigation of the degree of silencing:
1) The investigation method comprises the following steps: the qPCR is carried out by the three-step method, and the three-step method can improve the amplification efficiency. Finally, the data is checked in the computer and is processed.
2) By detecting the expression level of acetylcholinesterase gene in pest, it is determined whether the gene in pest is silenced after feeding double-stranded RNA, the silencing degree and the introduced double-stranded RNA is actually functioning.
In the measurement, a concept of an internal gene is introduced for correcting a sample error in an experiment, which means that a relatively constant gene is expressed in an organism, and the expression amount of the measured gene in the organism is obtained by subtracting the number of the measured samples from the reference. More realistic data can be obtained by the reference gene. The reference gene is generally selected from the beta-actin gene.
After the Ct value of each sample is obtained, 2-DeltaDeltaCt is plotted on the ordinate and each double-stranded RNA gene is plotted on the abscissa. Wherein,
ΔΔct= Δct (positive sample) - Δct (negative sample) (formula 1)
Delta Ct (positive sample) =ct (positive sample, using the gene primer of interest) -Ct (positive sample, using the internal reference gene primer)
(2)
Delta Ct (negative sample) =ct (negative sample, using the gene primer of interest) -Ct (negative sample, using the internal reference gene primer)
(3)
In the above formula:
ct value, C represents Cycle, t represents threshold, i.e. the number of PCR cycles each reaction undergoes to reach the set threshold, so that the more template copy number, the faster it reaches the threshold Cycle number, the smaller the Ct value.
The reference gene is generally selected from the beta-actin gene;
3.3 stomach toxicity test-second instar larvae
The second-instar larvae are divided into a plurality of groups, such as a dosing group, a negative control group and the like, wherein 10 pieces of the second-instar larvae are respectively added into the feed every day, and 10 mu L of the drug is added into the feed every day.
The prepared target RNA (prepared in example 5) is added into the drug adding group, and the double-stranded RNA after centrifugation is added with enzyme-free water with equal concentration again to feed insects. The negative control is colibacillus liquid without adding plasmid, namely pure HT115 (DE 3) competent cells are cultured overnight in 50mL LB liquid culture medium under the same condition, after the bacterial liquid is turbid, 1mL bacterial liquid with multiple tubes is subpackaged, the bacterial liquid is centrifugated for 10min at 4 ℃ and 4,000rpm, the supernatant is discarded, the sediment is stored at-80 ℃, and equal concentration non-enzyme water is added when the bacterial liquid is used, so that the bacterial liquid can be fed.
4. Experimental results
4.1 verification of double-stranded RNA: see FIGS. 9 and 10
To determine the optimal induction temperature and time, the target gene length of cotton bollworm Helicoverpa armigera was 308bp:
as can be seen from FIG. 9, when the induction times are the same, there is a distinct band at 37℃corresponding to 300bp, whereas no band appears at 32 ℃; when the induction temperature is the same, a band appears in the induction for 5 hours, but does not appear in the induction for 4 hours, so that the generation of double-stranded RNA can not be ensured under the condition of too low temperature or short induction time, and therefore, the induction for 5 hours at 37 ℃ is selected as the optimal induction condition for carrying out subsequent stomach toxicity experiments, and besides, the bright band at 100bp in the figure is other RNA in bacterial liquid, such as 5S RNA, 16S RNA, 23S RNA and other impurities.
In the induction experiments of plutella xylostella Plutella xylostella and spodoptera exigua Spodoptera exigua, experiments were also performed using various induction times (4 h, 5h and 6 h) and induction temperatures (37 ℃ and 32 ℃), as can be seen from fig. 10, most of the bands were evident at 500bp, several samples were most evident, the optimal time was recorded, and the subsequent stomach toxicity experiments were performed. The optimal induction temperature is 37 ℃, the optimal induction time of the plutella xylostella P1 and the spodoptera exigua S1 is 6h, and the optimal induction time of the plutella xylostella P2 and the spodoptera exigua S2 is 5h.
4.2 reverse transcription-fluorescent quantitative PCR: see FIGS. 11, 12, 13
The reverse transcription-fluorescence quantitative PCR is utilized to obtain the expression quantity of the acetylcholinesterase gene in the pest body, and compared with the pest fed with the escherichia coli only, the expression quantity of the gene in the pest body is reduced, namely the gene silencing efficiency and the RNA interference effect can be seen. After the measured expression levels of the respective genes, the degree of inhibition of each sample RNA, i.e., the value of 2-DeltaCt, can be calculated, and FIGS. 11 to 13 can be made on the ordinate thereof.
As can be seen from FIGS. 11, 12 and 13, the gene silencing effect of P2, namely, the 2 nd segment target gene of plutella xylostella Plutella xylostella is the best, while the inhibition effect of S1, namely, the 1 st segment gene of asparagus caterpillar Spodoptera exigua is the least. The silencing effect of the cotton bollworm Helicoverpa armigera gene is also excellent, and is inferior to that of P2.
Overall, the silencing effect was within 20% of P1, and the next step was to conduct a gastric toxicity experiment to observe the mortality of these samples for smaller larvae.
4.3RNA interference
Different degrees of death were observed for two-instar larvae feeding the artificially synthesized double stranded RNAs to cotton bollworm Helicoverpa armigera, plutella xylostella Plutella xylostella and asparagus caterpillar Spodoptera exigua.
For cotton bollworm Helicoverpa armigera, to determine the RNA interference effect of the acetylcholinesterase gene at different induction times, double-stranded RNA after induction for 5h and 6h was fed, respectively, and compared with a negative control group, i.e., bacterial liquid containing only HT115 (DE 3), 10 pieces each. After feeding, the larvae have death phenomena, the death rates are 40% and 60% respectively, the bodies of the larvae are blackened, the growth degree of other non-dead insects is slow, and the individual is obviously smaller than that of a negative control, and is blocked in the development process.
2) The RNA interference gastric toxicity experiments of plutella xylostella Plutella xylostella and asparagus caterpillar Spodoptera exigua are carried out on the same 3 groups, namely a gene 1, a gene 2 and a negative control group, and 10 groups are respectively carried out. Genes P1 and P2 of plutella xylostella Plutella xylostella caused mortality rates of 40% and 50%, respectively; whereas the mortality of the two groups S1 and S2 of asparagus caterpillar Spodoptera exigua was 30% and 50%, respectively. Similarly, the undelivered insects observe obvious dysplasia symptoms, the body type is obviously thin, the cocoon setting time is longer, the final pupa rate of P1 of the plutella xylostella Plutella xylostella is 50%, and the P2 is 40%; the final pupa rate of the asparagus caterpillar Spodoptera exigua is 30% and 10%. From the figure, it can be seen that Ace 2 is smaller than Ace 1 in all types, and it can also be shown that the second-stage genes have a greater influence on the growth and development of larvae.
5. Analysis and summary
Firstly, in the aspect of preparation of engineering bacteria, double-stranded RNA of three pests is successfully expressed in recombinant engineering bacteria, so that the stability of double-stranded RNA fragments in the environment and in insects is enhanced, and the double-stranded RNA fragments are protected by one layer of escherichia coli more than double-stranded RNA synthesized directly in vitro, and the mode of producing double-stranded RNA by using escherichia coli is universal in applicability and can be applied to other different insects or different genes in the future.
In addition, by utilizing the characteristic that bacteria can be continuously cultured, the method effectively and greatly improves the yield of double-stranded RNA, and particularly greatly reduces the production cost compared with an in-vitro synthesis kit, and all the factors enable the double-stranded RNA to be applied to pesticide production, but not only in a laboratory.
In the aspect of RNA interference technology, the novel recombinant strain is introduced into a pest body in a feeding way, and three acetylcholinesterase genes all show a certain gene silencing phenomenon, so that RNA interference is conservative.
The inventor tries to prepare double-stranded RNA of different pests into wettable powder, screens the double-stranded RNA of each pest with different auxiliary materials, and the result is approximate, and the auxiliary material screening process of the target RNA of the cotton bollworms is illustrated by taking examples 6-9 as an example:
Example 6: screening of wettable powder-carriers
1. Types and amounts of carriers
The carrier is kaolin, bentonite, diatomite or white carbon black.
2. Dosage of
2.1 drug loading 10%
Taking different carriers, adding the raw materials (the freeze-dried powder prepared in the example 5) in a proportion of 90%, wherein the mass ratio of the raw materials to the carriers is 90:10, after fully mixing, freeze-drying to prepare the mother powder.
The lyophilization process was freeze-dried, as in example 5, step 4.
2.2 drug loading 4.8%:
the raw materials (the freeze-dried powder prepared in the example 5) and different carriers are uniformly mixed according to the mass ratio of 20:1 (100 parts of raw materials, 5 parts) and freeze-dried to prepare the mother powder.
2.3 different proportions:
according to the screening result, determining a carrier, proportioning the carrier and the raw materials according to different mass ratios, wherein the weight ratio of the carrier to the raw materials is 15:1, 12:1, 9:1, 6:1 and 3:1, fully mixing by a grinder, and freeze-drying to prepare the mother powder.
3. Detecting the index:
the prepared mother powder is stored for 3d at normal temperature, and then the experiment is carried out:
3.1 wettability measurement; the specific method for measuring the wetting time is as follows by adopting national standard GB/T5451-2001:
(l) Placing 100mL plus or minus 1mL of hard water in a 250mL beaker, and keeping the liquid level in the beaker flush with the two liquid levels of the external liquid all the time under the condition of constant-temperature water bath at 25 ℃;
(2) When the temperature of hard water in the beaker reaches 25+/-1 ℃, weighing 5g of sample powder by weighing paper, uniformly pouring all samples into the hard water along the position flush with the beaker mouth, adding the samples while timing immediately until the samples are all wetted, stopping timing, and recording the wetting time;
(3) The sample wetting process is repeated for 5 times, and the wetting time is precisely the average value of seconds, so that the wetting time can be obtained.
3.2 sample suspension rate: the specific method for measuring the suspension rate by adopting national standard GB/T14825-2006 measurement is as follows:
(l) Weighing a balance, weighing a proper amount of sample, recording the mass as W1g (accurate to 0.0001 g), adding the sample into a 200mL beaker containing 50mL of hard water, oscillating for 2min to form a suspension, placing the suspension in a water bath with the temperature of 30+/-1 ℃ for 13min, and completely washing the suspension into a measuring cylinder with the temperature of 30+/-1 ℃ to fix the volume;
(2) Taking the bottom of the measuring cylinder as the axis, covering a measuring cylinder cover, turning over the measuring cylinder for 30 times within 1min, opening a measuring cylinder plug, vertically placing into a constant-temperature water bath, and standing for 30min;
(3) The 9/10 (i.e., 225 mL) suspension was removed with a pipette over a period of 10-15 seconds (care was taken to avoid aspiration of sediment into the cylinder), and the remaining 1/l0 was transferred to a drying oven for drying, and its dry weight was measured and recorded as W2g.
(4) Calculating the weight suspension rate of the sample:
Sample weight suspension ratio (%) =10/9× (sample mass W1-dry mass W2)/sample mass w1×100%
3.3 Activity investigation of lyophilized cells
And (3) electrophoresis verification: adding 0.1g of mother powder prepared by 2.1 g, 2.2 g and 2.3 g into a 5mL centrifuge tube, adding enzyme-free water and the like for redissolution, centrifuging, pouring out the supernatant, carrying out Trizol method on sediment in the centrifuge tube to extract double-stranded RNA, and carrying out electrophoresis experiment on the obtained RNA solution part to verify that the double-stranded RNA target band exists (the method is the same as that of experiment example 1);
RNA concentration comparison: and taking part of the obtained RNA solution, and using a Nanodrop nucleic acid concentration detector to determine the RNA concentration of the mother powder prepared by different carriers with the same mass ratio so as to determine the RNA content of different samples. The Nanodrop sampling arm is opened, 2 mu L of sample is added into the sampling arm each time, data are read and recorded, residual sample liquid in the sampling arm needs to be wiped off between sample replacement, and cross interference among samples (namely reading of the Nanodrop nucleic acid concentration detector) is prevented.
4. Experimental results
4.1 physicochemical Properties of 10% drug loading: see Table 3
Table 3: influence of different carriers on physicochemical properties of the preparation
| Content of original drug (%) | Carrier body | Wetting time(s) | Suspension percentage (%) |
| 10 | Kaolin clay | 39 | 43 |
| 10 | Bentonite clay | 38 | 62 |
| 10 | Diatomite | 35 | 71 |
| 10 | White carbon black | 29 | 69 |
As can be seen from table 3:
From the aspect of wetting, the wetting time of the white carbon black preparation is shortest, 29s is needed, the wetting time of the diatomite preparation is 35s, and the wetting time difference between the kaolin and the bentonite corresponding preparation is not great;
from the aspect of suspension rate, the suspension rate of the preparation corresponding to the white carbon black and the diatomite is relatively high, and is close to the national standard value which is more than or equal to 70 percent.
In general, the wetting effect of the white carbon black is best, the suspending rate of the diatomite is highest, but the suspending rate of the white carbon black is close to that of the diatomite and shows good performance, so that the primary selection of the white carbon black as a carrier has advantages.
4.2 drug loading 5%
1) The electrophoresis chart is shown in fig. 14:
in the graph, in samples corresponding to four carriers of kaolin, bentonite, diatomite and white carbon black, double-stranded RNA bands appear at 500bp (the length of a target gene fragment of plutella xylostella is between 400 and 500bp and slightly lower than 500 bp), wherein the sample bands corresponding to the kaolin are relatively shallow, the kaolin is used as a carrier, the adsorption performance of the kaolin on the original medicine is not good from the bands, and the double-stranded RNA content in samples corresponding to the other three carriers is higher.
2) RNA concentration comparison: see FIG. 15
As can be seen from fig. 19, the control group (control group, no added carrier) had the lowest RNA concentration of only 21ng/μl due to lack of carrier protection, but among the samples corresponding to the four carriers, the concentration of RNA solution corresponding to the sample with kaolin was the lowest, less than 50ng/μl, the concentration of RNA solution corresponding to the sample with bentonite and diatomaceous earth was not greatly different, and the concentration of RNA solution corresponding to white carbon black was the highest, up to 370ng/μl, higher than that of the control group, indicating that white carbon black as a carrier can effectively protect the sample components.
The above analysis of carrier suspension rate, wetting time, electrophoresis band and RNA concentration was combined, so that white carbon black was selected as the optimal carrier, and the following is a discussion of the results of the optimal addition mass ratio of white carbon black.
4.3 different drug loading rates
1) Electrophoresis verification result: as shown in fig. 16
In the figure, the RNA solutions extracted under different mass ratios are all provided with bands (the length of the target gene fragment of the plutella xylostella is between 400 and 500bP and slightly lower than 500 bP) at 500bP, the depths of the bands are slightly different, wherein when the mass ratio of white carbon black to the raw medicine is 6:1, the bands are most obvious, the double-stranded RNA content is highest, when the mass ratio of the white carbon black to the raw medicine is 3:1, the bands are shallower compared with the bands, the double-stranded RNA content is reduced, and when the mass ratio of the white carbon black to the raw medicine is 15:1, the bands are shallowest.
2) RNA concentration comparison: see FIG. 17
In the graph, when the mass ratio of the white carbon black to the raw medicine is 15:1 and 12:1, the RNA concentration is lower than that of other groups, the RNA concentration is about 200 ng/. Mu.L under the condition of the mass ratio of 12:1, the RNA concentration is the lowest, the RNA concentration is obviously increased when the mass ratio is 9:1, wherein the RNA concentration reaches the highest at the mass ratio of 6:1 and is 630 ng/. Mu.L, and the RNA concentration starts to decrease at the mass ratio of 3:1.
After comprehensively considering the electrophoresis strip and the RNA concentration graph, the optimal adding ratio of the carrier to the raw material in the mass ratio of 6:1 is finally selected.
Example 7: screening of wettable powder-RNase inhibitors
Considering that double-stranded RNA is very easy to degrade in the environment, in order to prolong the storage of wettable powder, RNA degradation can be relieved by adding an RNase inhibitor into the wettable powder, the experiment uses common nuclease inhibitor such as urea and Sodium Dodecyl Sulfate (SDS) as preparation auxiliary agents to be added into the prepared mother powder (the mass ratio of white carbon black to raw material is 6:1, a grinding instrument is fully mixed to prepare the mother powder, and the experiment is carried out after the mixture is stored for 3 days at normal temperature), and the optimal inhibitor and the optimal mass ratio of the inhibitor are selected.
1. Rnase inhibitors:
the RNase inhibitor is urea or sodium dodecyl sulfate.
2. The dosage is as follows:
2.1 mixing the RNase inhibitor and the mother powder uniformly in a mass ratio of 5:1 to prepare a sample.
2.2 respectively and uniformly mixing urea, sodium dodecyl sulfate and the mother powder (the mass ratio of white carbon black to raw material is 6:1) prepared in the example 6 according to the mass ratio of 3%, 5% and 7%, so as to prepare a preparation sample.
3. The detection method comprises the following steps:
after the sample is stored for 3 days at normal temperature, adding enzyme-free water with equal concentration for redissolution, centrifuging, pouring out supernatant, carrying out Trizol method on sediment in a centrifuge tube to extract double-stranded RNA, and detecting according to the 'activity of freeze-dried bacteria' detection method of the embodiment 6, wherein the detection indexes comprise: electrophoresis verification and detection by a Nanodrop nucleic acid concentration detector.
4. Experimental results
4.1 electrophoresis:
1) The test inhibitor and the mother powder are evenly mixed according to the mass ratio of 5:1, double-stranded RNA is extracted, the electrophoresis result is shown in figure 18, the SDS corresponding sample in the figure has no band at 500bp, the bacterial cells are destroyed by SDS and are not selected because the SDS is cell lysate, and the urea has obvious band at 500 bp.
2) After urea is used as an RNase inhibitor and mixed with the mother powder in different addition amounts of 3%, 5% and 7%, the mixed double-stranded RNA bands are verified, and as shown in a result of FIG. 19, when the mother powder is added with 5% and 3% of urea, the corresponding bands at 500bp are relatively obvious, and 7% of urea is not found, so that the addition amount can not well inhibit RNase.
4.2RNA concentration: as a result of measuring the RNA concentrations of the respective samples in which SDS and urea were added at 3%, 5% and 7%, respectively, as shown in FIG. 20, the RNA concentrations of the respective samples in which SDS and urea were added were not greatly different at the addition of 3%, but the RNA concentration of the respective samples in which urea was added was highest at the addition of 5%, was 1040 ng/. Mu.L, whereas the RNA concentration of the respective samples in which SDS was not greatly changed, and at the addition of 7%, the RNA concentration of the respective samples in which urea was decreased as compared with the previous one.
Finally, screening out that the optimal RNase inhibitor is urea according to the analysis results of the electrophoresis chart and the histogram data, wherein the optimal addition amount of the urea is 5%, and the mass of the urea, the white carbon black and the raw materials after conversion is 5:60:10.
Example 8: screening of wettable powder-surfactant
The surfactant is one of important components for preparing the wettable powder, and the addition of the surfactant directly influences whether the wettable powder can be uniformly covered on applied crops and control objects, so that the addition of the surfactant can increase the adhesion force to the applied crops and control objects, and therefore, proper surfactant needs to be selected, and the principle is selected: the preparation method comprises the steps of carrying out preliminary screening by combining factors such as wettability, dispersibility and price, and further selecting a proper surfactant according to the activity influence of the surfactant on the preparation samples (urea, white carbon black and raw materials with the mass of 5:60:10) prepared in the example 7.
1. And (2) a surfactant:
sodium Dodecyl Sulfate (SDS), nekal (BX), sodium lignin Sulfonate (SL), calcium lignin sulfonate (CL).
2. Preparation and investigation of wettable powder samples:
2.1 mixing four surfactants with the preparation samples (the mass ratio of urea to white carbon black to raw materials is 5:60:10) prepared in the example 7 according to the mass ratio of 3%, 5% and 7% to obtain a wettable powder sample.
After the wettable powder sample is stored for 3d, RNA concentration comparison is carried out according to the methods of wettability measurement, sample suspension rate and activity investigation of freeze-dried bacteria in the example 6, the wetting time, suspension rate and RNA concentration are measured, and the effect of different surfactants on the physicochemical properties of the preparation is judged repeatedly.
2.2 examining the physicochemical properties (wettability measurement and sample suspension ratio) of the four surfactants 2.1, adding the preparation sample prepared in example 7 (the mass ratio of urea, white carbon black and raw materials is 5:60:10) in an amount of 5% to prepare a wettable powder sample, storing for 3 days, and performing electrophoresis verification and RNA concentration comparison according to the method of "activity examination of freeze-dried cells" in example 6.
3.3 optimization of the optimum ratio of surfactant to RNase inhibitor
And (3) selecting the surfactant with good performance and the RNase inhibitor from the above materials for compounding, measuring the suspension rate and wetting time of the compounded preparation, and screening the optimal proportion.
4. Experimental results
4.1 physicochemical Properties (wetting time and suspension ratio): see Table 4
Table 4: influence of surfactant on physicochemical Properties of formulations
| Surface active agent | Additive amount (%) | Wetting time(s) | Suspension percentage (%) |
| Sodium Dodecyl Sulfate (SDS) | 3 | 103 | 89 |
| Nekal (BX) | 3 | 67 | 51 |
| Sodium lignin Sulfonate (SL) | 3 | 85 | 47 |
| Calcium lignin sulfonate (CL) | 3 | 72 | 69 |
As shown in Table 4, the wetting time of the nekal preparation is shortest, 67s is needed, the wetting time of the calcium lignosulfonate CL preparation and the sodium dodecyl sulfate SDS preparation is longest, 103s is needed, but the wetting time of all four samples is less than or equal to 120s in the national standard range; from the aspect of suspension rate, the suspension rate of Sodium Dodecyl Sulfate (SDS) is highest and reaches 89%, and secondly, calcium lignosulfonate has better performance and is close to the national standard which is more than or equal to 70%.
4.2 Activity investigation
1) And (3) electrophoresis verification: the inspection results of the same mass ratio and different surfactants are shown in FIG. 21, the sodium lignin sulfonate SL and the calcium lignin sulfonate CL have brighter bands at 500bp corresponding to the samples, the SDS does not have bands at the 500bp corresponding to the samples, and the nekal does not have visible bands at the 500bp corresponding to the samples.
2) RNA concentration investigation: inspection of different mass ratios of surfactants is shown in fig. 22, the concentration of RNA in a corresponding preparation sample of SDS at three addition levels is not too high, and the ability of SDS to inhibit RNase is weak; the RNA content of the 5% nekal and 5% sodium lignin sulfonate SL corresponding samples was relatively high in their range, but far less than 5% calcium lignin sulfonate; under the condition that the sodium lignin sulfonate SL and the calcium lignin sulfonate CL both detect the corresponding target bands, the RNA concentration corresponding to the calcium lignin sulfonate CL is obviously higher than that of the sodium lignin sulfonate SL, and the highest RNA concentration corresponding to 5% of the calcium lignin sulfonate is 1700 ng/. Mu.L.
In conclusion, according to the experimental results, calcium lignosulfonate CL is selected as the optimal surfactant, and the optimal addition amount is 5%.
Example 9: wettable powder-surfactant and RNase inhibitor ratio optimization result
As can be seen from fig. 20 in example 7: the RNA content of the corresponding formulation samples was relatively high at both 5% and 7% urea addition, as can be seen from fig. 22 in example 8: when the addition amount of the calcium lignosulfonate is 5% and 7%, the corresponding preparation sample has higher RNA content, so that the urea and the calcium lignosulfonate are selected to be respectively compounded into preparations according to the addition amounts of 5%, 6% and 7%, the wetting time and the suspension rate of the preparation sample are measured (the method is the same as that of the example 5), and the optimal formula is screened, and the result is shown in the following table 5:
Table 5: influence of different additive ratios on physicochemical properties of the preparation
( And (3) injection: the data in the table are mean ± standard error, the same column with different letters indicates significant differences at 0.05 level )
The results from the data in table 5 show that:
when the urea addition amount is 7% and the calcium lignosulfonate addition amount is 5%, the suspension rate of the sample is the highest and is 81.8%;
when the urea addition was 5% and the calcium lignosulfonate addition was 7%, the wetting time of the sample was the shortest, 76.4s.
Overall, the wetting times of the samples were not significantly different and were not significantly different.
Considering comprehensively, the urea addition is 6%, the calcium lignosulfonate addition is 7%, the corresponding suspension rate is 76.8%, the suspension rate is slightly lower than the highest suspension rate, the corresponding wetting time is 77.6s, and the shortest wetting time is 76.4s.
Example 10: optimal wettable powder formulation
According to the experiments of examples 6-9, the optimal wettable powder formulation was determined as: based on the mass ratio of the white carbon black to the raw material in 6:1, 6% of urea and 7% of calcium lignosulfonate are sequentially added.
1. The composition is as follows: 100mg of white carbon black, 10mg of raw material, 4.2mg of urea and 4.9mg of calcium lignosulfonate.
2. The preparation method comprises the following steps: mixing well to obtain the final product.
Experimental example 5: experimental results on pest control of wettable powder
1. Experimental materials
The cotton bollworms, plutella xylostella and asparagus caterpillar and foods thereof to be tested are purchased from Henan province family cloud biopesticide limited company;
vegetable seeds are purchased from Xingshun vegetable seed company;
du Bangkang A wide (20% chlorantraniliprole) was purchased from Fumei plant protection Co., ltd. And Bacillus thuringiensis was purchased from Hubei Kangxin agricultural pharmaceutical Co., ltd.
2. Test sample
Drug administration group: the wettable powder provided in example 10 was diluted 300-fold with water for use.
3. Quality index of wettable powder
3.1 the main quality indexes of the wettable powder include suspension rate, wetting time, pH value, moisture content, fineness and the like. Wherein the wetting time and the suspension rate were measured according to the "wettability measurement" and the "sample suspension rate" of example 6, and the pH value was measured according to GB/T1601-1993 and the moisture content was measured according to the oven drying weight-reduction method. The fineness is more than or equal to 95 percent through a 325-target standard sieve.
3.2 validity period of wettable powder: sampling every other month under normal temperature storage condition, electrophoresis to determine target band corresponding to double-stranded RNA, and Nanodrop to determine residual RNA concentration, repeating for 3 times.
4. Formulation spraying experiments
The preparation corresponding to the three insects and the mixed preparation for jointly controlling the two or three insects are prepared respectively, and are sprayed for multiple times, so that the corresponding control effect of each insect and the common control effect of the mixed preparation on the three insects are observed.
(illustration: 1) either plutella xylostella or spodoptera exigua can be selected as a representative, and experimental investigation proves that any plutella xylostella RNA is effective against plutella xylostella, and optionally one of the plutella exigua is also effective against spodoptera exigua; 2) Two or three insect mixed preparations can be compounded aiming at insect types, and the proportioning relation between the preparations is 1:1: 1)
4.1 spray test method:
(1) Plutella xylostella spraying experiment:
the method comprises the steps of selecting green vegetables as a food source for picking up plutella xylostella, cultivating soil in a laboratory, placing two-year-old plutella xylostella larvae on leaf surfaces of the vegetables after the green vegetables grow for 2-3 weeks, placing 3-4 larvae on each leaf, setting a pesticide application group and a control group, spraying the leaf surfaces each day, spraying the prepared wettable powder solution by the pesticide application group, spraying clear water by the control group, and spraying for one week.
(2) Cotton bollworm spraying experiment:
selecting green vegetables as food sources for bollworms, planting the green vegetables in indoor soil, placing two-year-old bollworm larvae on leaf surfaces of the green vegetables until the number of the sprouting grows Zhou Zhouhou, placing 2-3 larvae on each leaf, setting a pesticide application group and a control group, spraying the prepared wettable powder solution on the pesticide application group, and spraying clear water on the control group for about 7 days.
(3) Beet leaf moth spraying experiment:
the method comprises the steps of selecting green vegetables as food sources for asparagus caterpillar, cultivating the green vegetables in indoor soil, putting two-year asparagus caterpillar larvae on leaf surfaces after the green vegetables grow for a period of time, putting 2-3 larvae on each leaf surface, setting a pesticide application group and a control group, spraying the prepared wettable powder solution by the pesticide application group, and spraying clear water by the control group for about 7 days.
(4) Three insect mixed spray experiments:
vegetable is selected as food source for three insects, vegetable is planted and cultivated in indoor soil, and when each vegetable seedling grows to 10-15 cm, two-stage larvae of the three insects are placed on rape leaves, and one larva is placed on each leaf. Setting an administration group, a blank control, a negative control and a positive control, wherein:
drug administration group: the wettable powder prepared in example 10 was diluted 300 times.
Blank control: spraying only clean water.
Negative control: spraying common coliform bacteria liquid.
Positive control (chemical pesticide): spray diluted 3000 times Du Bangkang wide (20% chlorantraniliprole).
Positive control (biopesticide): spraying and diluting the bacillus thuringiensis 300 times.
The leaves were sprayed according to the above group, and the experiment was performed for about one week.
4.2, examining indexes: and observing the growth conditions of different groups of pests after the pesticide application, and analyzing and calculating the leaf surface predation rate of the green vegetables.
Vegetable leaf surface predation rate = leaf surface area to be eaten/initial leaf surface area
5. Detection index
5.1 quality inspection results of wettable powder: see Table 6
Table 6: quality index of wettable powder
| National standard value | Measurement value | |
| Wetting time | ≤120s | 77.6s |
| Suspension rate | ≥70％ | 76.8％ |
| pH | 5～8 | 7.4 |
| Moisture content | ≤2.5％ | 0.09％ |
| Fineness of | ≥95％ | 98％ |
5.2 validity period detection:
the double-chain engineering bacteria corresponding to the target gene of the cotton bollworm are used as raw materials, the mass ratio of the carrier to the raw materials is 6:1, then 6% urea is added as an RNase inhibitor, and finally 7% nekal BX, sodium lignin sulfonate SL, calcium lignin sulfonate CL and sodium dodecyl sulfate SDS are respectively added as surfactants to prepare four wettable powders, and the freeze-dried raw materials are used as a control group for five samples to be tested. The electrophoresis bands were measured under normal temperature conditions for one month and six months, and the results are shown in FIG. 23:
as can be seen from the graph, the calcium lignosulfonate CL stored for about one month corresponds to the brightest band at 300bp, and other samples have no obvious band; after six months of storage, the corresponding target band of the calcium lignin sulfonate CL sample still exists, the brightness is not remarkably reduced, and the preparation still has effectiveness.
The physical and chemical index data of the RNA interference preparation developed for the experiment are the following production quality control indexes of the preparation in consideration of the fact that the RNA interference preparation is applied to actual production in the later period:
bacterial liquid fermentation OD600 value range: 0.8 to 1.2;
RNA purity: A260/A280 is 1.7-2.0;
formulation CFU value: 3.34×10 33 CFU/g
6. Formulation spraying experiments
(1) Spraying results of plutella xylostella:
comparing the growth of the two groups of plutella xylostella after the administration for a few days, it can be seen that the plutella xylostella in the administration group is inhibited by a certain degree of growth, and the insect bodies are generally smaller than those of the plutella xylostella in the control group. The plutella xylostella in the lower right panel causes RNA interference death four days after dosing.
(2) Cotton bollworm spraying results:
the cotton bollworms are placed on the green vegetables, and after a period of time, the cotton bollworms start to gnaw the green vegetables, and obvious worm damage appears on leaf surfaces.
Comparing the growth of the bollworms of the control group with the growth of the bollworms of the seven-day dosing group, it can be seen that the growth inhibition of the bollworms of the dosing group is started to appear under the influence of the drug from the third day, the body of the bollworms is slightly smaller than the control group, the growth of the bollworms of the dosing group is stopped after five to seven days of dosing, and compared with the bollworms of the control group which normally grow, the body length of the bollworms is only one half of that of the bollworms of the dosing group, which indicates that the bollworms of the dosing group are greatly influenced by the RNA interference, and the preparation shows better insecticidal effect.
(3) Beet armyworm spray results:
the asparagus caterpillar of the control group which takes the green vegetables by spraying the edible clear water is fed normally, the insect body is not affected by any matter, the growth is normal, and the insect body length is obviously longer than that of the asparagus caterpillar of the dosing group.
(4) Spraying results of the mixed preparation: see FIG. 24
The growth of five groups of green vegetables is compared before and after the pesticide spraying, the green vegetables grow normally and have intact leaves before the pesticide spraying, and after the pesticide spraying and the pesticide spraying are carried out for several days, the green vegetables in the blank control group and the negative control group are eaten by insects in a large quantity, and almost only channels and collaterals and a few residual leaves are left; the foliar insects of the dosing group of the experimental preparation are reduced or stopped eating under the interference of RNA, and the number of insects is reduced, so that the foliar insects of the green vegetables are relatively intact, and the foliar insects of the chemical pesticide control group are not damaged; after chemical pesticide is sprayed, the foliar insects die and fall off quickly, the biopesticide control group insects are also affected by the foliar medicament, the pesticide effect volatilizes after feeding, and the insects die quickly.
And comparing the growth inhibition degrees of the three insects, wherein the negative control group sprayed with the common escherichia coli solution and the blank control group sprayed with the clear water are normal in growth and development of the insects, the dosing group sprayed by the mixed preparation is slow in growth, the three insects show obvious RNA interference phenomenon after seven days, and various insect prevention effects of the mixed preparation are verified.
Fig. 24 shows that, in the case of leaf surface predation, the leaf surface survival rate of both the blank control group and the negative control group was 70% on the third day, and the leaf surface predation of the blank control group and the negative control group was serious with the increase of the number of days. By the fourth day, leaf insects enter a three-year overeating period, the food intake is increased, the residual rate of leaves of the blank green vegetables is only 40%, the residual rate of green vegetables of the negative control group is only 30%, and two groups of green vegetables almost only leave residual leaf channels after one week. In contrast, the growth of the other three groups of green vegetables is not greatly changed, the drug effect is exerted quickly after the broad group of Du Bangkang is applied, the insects die quickly less than half a day, the larvae on the leaf surfaces of the Bacillus thuringiensis group die quickly after the application, and the larvae drop from the leaf surfaces. The survival rate of the two groups of leaves after seven days is 100% and 98% respectively, the insects in the RNA interference preparation group eat a small amount of green vegetables, and after eating for 2-3 days, the insects are affected by RNA interference, so that eating is reduced or stopped, and then the insects die, and the survival rate of the leaves after seven days is about 90%.
7. Summary of the experiment:
the physical and chemical indexes of the wettable powder provided by the application meet the national standard, and the validity period of the wettable powder can be maintained for at least six months at normal temperature.
Subsequently, verification of the control effect of the wettable powder after amplification production is carried out, indoor potted leaf surface spraying experiments are carried out on cotton bollworms, plutella xylostella and asparagus caterpillar successively, three insects all show RNA interference effect in the third day after application, the growth of the insects is limited, the insects die to different degrees within seven days after application, and the death rate is 85%.
After the independent spraying experiment is successful, the preparation for specifically controlling the three insects is considered to be uniformly mixed to prepare a mixed preparation capable of simultaneously controlling the three insects, and the experimental spraying is carried out. In this period, other control groups are set to compare the control effect of the RNA interference preparation, and the results show that the chemical control effect of spraying 20% chlorantraniliprole is optimal, the protection effect of spraying the chlorantraniliprole on the applied green vegetables is up to 100%, the green vegetables sprayed with the bacillus thuringiensis are protected by about 98%, the preparation provided by the application also has up to 95% protection on the green vegetables, and the RNA interference preparation has good effect on crop protection compared with the green vegetables which are not applied.
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. An RNA that causes gene silencing of a pest AChE, which is a cotton bollworm primer pair, or a combination of a cotton bollworm primer pair with one or both of a plutella xylostella primer pair, a asparagus caterpillar primer pair, wherein:
Cotton bollworm a: an upstream primer F GTGGAGACTCAACGAAGATC, seq_1;
a downstream primer R CTCTTAGACCACATAATGAACTC, seq_2;
plutella xylostella P1: an upstream primer F ATTGTTTGGAGAATCGTCCG, seq_3;
a downstream primer R GGATTCAGTTCTCTGACCGC, seq_4; or (b)
Plutella xylostella P2: an upstream primer F GGCAAGAATTACTCACCCGA, seq_5;
a downstream primer R CGGAAAAGCGAGATTCAAAC, seq_6;
beet armyworm S1: an upstream primer F GGCTGTGTCGGTTTCATTG, seq_7;
a downstream primer R GATTCAACTCTCTAACTGCCTGC, seq_8; or (b)
Beet armyworm S2: an upstream primer F GGGAGAAGAAATGTGGAATCC, seq_9;
downstream primer R GCCGACTCACCAAATAATGTTA, seq_10.
2. The engineering bacterium for expressing RNA is characterized in that the raw materials are as follows: the RNA, vector, and competent cell of claim 1.
3. The engineered bacterium of claim 2, wherein the carrier isT Easy vector; the competent cells were E.coli.
4. A method for preparing the engineering bacterium according to claim 2 or 3, comprising the steps of:
1) Preparation of total RNA of pests: extracting intestinal and brain tissues of corresponding pests by using an RNA prepure animal tissue total RNA extraction kit to obtain total RNA of the pests;
2) Preparation of template DNA:
step 1) preparing a total RNA solution of the pests, synthesizing single-stranded cDNA by using an M-MuLV first-strand cDNA synthesis kit, amplifying the single-stranded cDNA solution into double strands with a primer pair of the corresponding pests according to claim 1 by using a polymerase chain reaction (Polymerase chain reaction, PCR) technology, and simultaneously positioning template DNA corresponding to target double-stranded RNA by using the primer;
3) Preparation of plasmids: 1. Mu.L of vector, 2. Mu.L of template DNA prepared in step 2), 1. Mu.L of T4 DNA Ligase, 2X Rapid Ligation Buffer. Mu.L, ddH 2 O1. Mu.L; standing overnight at 4deg.C to obtain plasmid;
4) Transformation of plasmids: preparing escherichia coli into competent cells, adding the competent cells into the plasmid prepared in the step 3), carrying out heat shock reaction after ice bath, resuscitating in an LB liquid medium, concentrating, discarding supernatant, culturing the precipitate on a ampicillin-containing flat plate overnight, picking single bacterial colony, placing the single bacterial colony in the LB liquid medium containing ampicillin, and culturing to obtain the engineering bacteria expressing RNA.
5. A double-stranded RNA produced by an engineering bacterium, characterized by induced expression by: the engineered bacterium of claim 2 or 3, which produces double-stranded RNA under IPTG induction.
6. The double-stranded RNA according to claim 5, wherein the method of inducing expression comprises the steps of: and (3) picking single bacterial colony, placing the single bacterial colony into ampicillin-containing LB liquid culture medium solution for culturing overnight, taking overnight culture bacterial liquid into ampicillin-containing 2 XYT culture medium solution, carrying out shake culture until the OD600 is more than 0.8, adding IPTG solution, continuing shake culture to obtain double-stranded RNA, centrifuging the double-stranded RNA, removing supernatant, and drying bacterial sludge.
7. A wettable powder comprising double stranded RNA comprising: the double-stranded RNA, vector, surfactant and RNase inhibitor of claim 5.
8. The wettable powder of claim 7, wherein the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 30-150 parts of a carrier, 2-9 parts of a surfactant and 2-9 parts of an RNase inhibitor;
preferably, the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 60-120 parts of a carrier, 2-5 parts of a surfactant and 2-6 parts of an RNase inhibitor;
further preferably, the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 60-90 parts of a carrier, 3.5-5 parts of a surfactant and 3.5-5 parts of an RNase inhibitor;
optimally, the wettable powder comprises the following components in parts by mass: 10 parts of double-stranded RNA, 60 parts of a carrier, 4.2 parts of a surfactant and 4.9 parts of an RNase inhibitor.
9. The wettable powder of claim 7, wherein the surfactant is sodium dodecyl sulfate, nekal, sodium lignin sulfonate or calcium lignin sulfonate, preferably calcium lignin sulfonate;
the carrier is kaolin, bentonite, diatomite or white carbon black, preferably white carbon black;
The rnase inhibitor is urea or sodium dodecyl sulfate, preferably urea.
10. Use of a wettable powder according to any one of claims 7 to 9 in the manufacture of a pesticide for the prevention and control of lepidopteran pests.
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| WO2016110691A1 (en) * | 2015-01-06 | 2016-07-14 | The University Court Of The University Of Aberdeen | Enhanced rnai mediated gene regulation |
| CN108024542A (en) * | 2015-06-19 | 2018-05-11 | 昆士兰大学 | Composition |
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| WO2016110691A1 (en) * | 2015-01-06 | 2016-07-14 | The University Court Of The University Of Aberdeen | Enhanced rnai mediated gene regulation |
| CN108024542A (en) * | 2015-06-19 | 2018-05-11 | 昆士兰大学 | Composition |
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