CN111454959B

CN111454959B - Migratory locust Spinless gene dsRNA and application thereof

Info

Publication number: CN111454959B
Application number: CN202010243961.0A
Authority: CN
Inventors: 张学尧; 刘娇; 张建琴; 张婷婷; 张建珍; 吴海花
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2021-09-28
Anticipated expiration: 2040-03-31
Also published as: CN111454959A

Abstract

The invention provides a migratory locust Spinless gene dsRNA and application thereof. In particular to a migratory locust Spinless gene, the nucleotide sequence of which is SEQ ID NO: 1, based on SEQ ID NO: 1, designing and synthesizing a gene fragment SEQ ID NO: 7 and the corresponding dsRNA. When the designed and synthesized dsRNA is injected into a migratory locust body, the expression level of the migratory locust Spinless gene is remarkably reduced by 85.3-87.5%, and the lethality of the bromine phosphorus pine to the migratory locust is improved to 6.1 times.

Description

Migratory locust Spinless gene dsRNA and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to dsRNA (double-stranded ribonucleic acid) synthesized based on a migratory locust Spinless gene fragment and application thereof in improving the control effect of the brifosson on migratory locust.

Background

The bromaciphos is a broad-spectrum pesticide with contact poisoning and stomach poisoning functions. Has better control effect on various pests on corn, wheat, cotton, various vegetables and fruit trees. The bromophospone has no systemic effect, so that pesticide residue of agricultural products can be reduced, and the food safety of consumers can be guaranteed. For a long time, organic phosphorus pesticides represented by the bromine phosphorus pine are used in a large amount in emergency prevention and treatment work of locusts, so that the locusts have obvious resistance to organic phosphorus, the application amount of the pesticides is increased, the prevention and treatment cost of locusts is improved, and the excessive use of the bromine phosphorus pine can further cause great threat to public health and ecological balance. Although the traditional pesticide mixing and alternate use can reduce the pesticide resistance of locusta migratoria to a certain extent. But the problem of the resistance of locusta migratoria to organic phosphorus is still very serious at present. Therefore, a novel method for enhancing the locusta migratoria control effect of the brifosson is urgently needed to be developed.

RNA silencing refers to the phenomenon of highly specific degradation of homologous mRNA induced by endogenous or exogenous double-stranded RNA (dsrna). RNA silencing technology has the obvious advantages of high efficiency and specificity, high safety to environment and non-target organisms and the like, and is widely used for pest drug resistance control.

Through search, the currently available locust-resistant dsRNA related patent applications are as follows: patent application No. 201610316474.6 entitled "application of locusta migratoria intestinal nucleic acid hydrolase gene in pest control"; patent application No. 201610186141.6, entitled "migratory locust wing epidermal protein gene and its application in pest control"; patent application No. 201610186149.2, entitled "migratory locust wing specific epidermal protein gene and application of dsRNA thereof"; patent application No. 201510160191.2 entitled "application of Knickkopf gene dsRNA in pest control"; patent application No. 201510160194.6 entitled "Retroactive gene and its dsRNA for pest control; patent application No. 201510118799.9 entitled "an insect cytochrome P450 gene and uses thereof"; patent application No. 201510080636.6 entitled "insect chitin deacetylase gene 1 and its use in pest control"; the patent application No. 201410647729.8, the name is 'the application of ATP synthase beta subunit gene of migratory locust in east Asia and dsRNA thereof in pest control'; the patent application No. 201410665503.0, the name is 'the application of ATP synthase alpha subunit gene of migratory locust in east Asia and dsRNA thereof in pest control'; patent application No. 201410292658.4 entitled "use of insect type ii chitinase gene specific dsRNA"; the application is a patent application No. 201410069843.7, which is named as the sequence of locust I-type chitinase gene and the application of dsRNA thereof, and the application is a patent application No. 201310023385.9, which is named as the application of insect UDP-N-acetylglucosamine pyrophosphorylase gene and dsRNA thereof; patent application No. 201210356675.0 entitled "insect beta-N-acetylglucosaminidase gene and uses thereof"; patent application No. 201010163625.1 entitled "an insect chitin synthase 1 gene fragment and dsRNA and application thereof"; patent application No. 201010163645.9, entitled "an insect chitin synthase 1A gene fragment and dsRNA and application thereof"; patent application No. 201010163618.1 entitled "an insect chitin synthase 2 gene and uses"; the name of patent application No. 201010163634.0 is 'insect chitin synthetase 1B gene fragment and dsRNA and application thereof', and the name of patent application No. 201010136330.5 is 'insect chitinase gene and application of dsRNA'. The patent application No. 201710115802, entitled "cytochrome P450 reductase gene dsRNA of migratory locust" and its application ".

The core content of the locust-related dsRNA patent mainly relates to introducing dsRNA into a locust-like fly, silencing a certain key gene/lethal gene of the locust-like fly to cause disorder of an important metabolic pathway in a body, and further triggering a locust-like fly lethal effect. However, the lethal effect caused by the key gene/lethal gene of the locusta migratoria is slow to take effect, most locusta migratoria can still continue to develop for several days and even molt to the next age, which is not favorable for the emergency control of locusta and the loss reduction of locusta. More importantly, locusta migratoria is not only an important agricultural pest but also an important link of the food chain of the farmland. In the production process of important key genes/lethal genes dsRNA of migratory locust, the key genes/lethal gene sequences of migratory locust are inevitably required to be artificially amplified or introduced into crop genomes through a plant transgenic technology, so that the potential risks of leakage and transfer of the key genes/lethal genes of migratory locust exist, the excessive reduction of the number of migratory locust and even population extinction are further caused, natural enemy organisms of farmlands are reduced, the ecological balance of the farmlands is threatened, and secondary insect disasters of the farmlands are induced.

Spinless protein is an important signal molecule for insects to adapt to external environmental stress. Spinless protein is distributed in cytoplasm under physiological conditions, and enters insect cell nucleus after being stimulated by exogenous compounds represented by pesticides, so that the expression of a series of metabolic detoxification genes including cytochrome P450 genes, glutathione-S-transferase genes and the like is activated. Therefore, the silent pest Spinless gene can reduce the response of the insect metabolism detoxification system to the pesticide, further reduce the metabolism detoxification capability of the insect to the pesticide, and further improve the control effect of the pesticide to the pest. The migratory locust only contains one Spinless gene, and the silencing of the Spinless gene can not cause the migratory locust to die directly. The method greatly facilitates researchers to regulate a plurality of metabolism detoxification related genes by interfering the migratory locust Spinless gene, thereby improving the sensitivity of the migratory locust to the pesticide.

Disclosure of Invention

Aiming at the problems, the invention provides dsRNA synthesized based on a migratory locust Spinless gene fragment and application thereof in improving the control effect of the brifosson on the migratory locust.

The invention provides a migratory locust Spinless gene, the nucleotide sequence of which is SEQ ID NO: 1; the nucleotide sequence of the gene is obtained by a bioinformatics analysis method, and comprises the steps of firstly comparing and annotating a migratory locust transcriptome database by blastx software, then obtaining the undetermined nucleotide sequence of the migratory locust Spinless gene by searching the transcriptome annotation database, and designing a full-length upstream primer SEQ ID NO:3 and full-length downstream primer SEQ ID NO: 4; using total cDNA of migratory locust as a template, and obtaining a DNA fragment with the size of a target fragment by reverse transcription PCR amplification; separating a PCR product by agarose gel electrophoresis, cutting and recovering a target fragment, purifying a gel recovery kit, inserting the purified DNA fragment into a pGEM-T easy Vector, transferring an escherichia coli DH5 alpha competent cell by a heat shock method, selecting an independent and full white colony on a screening plate of X-gal, IPTG and ampicillin, inoculating the colony into an LB liquid culture medium containing ampicillin, carrying out overnight oscillation amplification culture, extracting a plasmid by a plasmid miniprep kit, and carrying out DNA sequencing by a biological engineering (Shanghai) member company Limited; the sequencing result is completely consistent with the undetermined sequence of the migratory locust Spinless gene, and the result proves that the total length of the migratory locust Spinless gene is 2529bp, and the nucleotide sequence of the migratory locust Spinless gene is SEQ ID NO: 1 is shown.

The invention provides an amino acid sequence coded by a migratory locust Spinless gene, which is represented by SEQ ID NO: 2; the amino acid sequence was obtained by converting SEQ ID NO: 1, translating into corresponding amino acid by using Translate tool software, predicting the relative molecular weight and the isoelectric point by using ProtParam software, wherein the prediction result shows that the migratory locust Spinless gene encodes 842 amino acids, the protein prediction relative molecular weight is 91.65KD, and the theoretical isoelectric point is 8.13.

The invention provides a migratory locust Spinless gene fragment with T7 promoter sequences fused at two ends, dsRNA synthesized based on the fragment and application of the dsRNA synthesized based on the fragment in improving the migratory locust control effect of the brifossilizid; based on a migratory locust Spinless gene sequence, an upstream primer SEQ ID NO:5 and 5' end containing downstream primer SEQ ID NO:6, using locusta migratoria spinoss gene DNA as a template, and obtaining a target fragment SEQ ID NO: 7; separating the target fragment by agarose gel electrophoresis, cutting and recovering the target fragment, purifying the target fragment by a gel recovery kit, and synthesizing dsRNA by using a T7RNA polymerase in-vitro transcription kit; and then injecting the dsRNA into the second abdominal segment of the locusta migratoria by using a micro-injector, and detecting the reduction degree of the mRNA expression level of the Spinless gene of the locusta migratoria and the capability of the dsRNA for improving the control effect of the bromophenon on the locusta migratoria.

The invention has the beneficial effects that: the dsRNA synthesized based on the migratory locust Spinless gene fragment is injected into the second abdominal segment of the migratory locust by a micro-injector, the mRNA expression level of the migratory locust Spinless gene can be detected to be reduced by 85.3-87.5%, and the lethality of the bromophenophos to the migratory locust of 3 years is improved to 6.1 times.

Drawings

FIG. 1: effect of injection of dsRNA on locust fliss gene expression. (dsGFP is a control group injected with dsGFP, dsSS is a treatment group injected with locust Spinless gene dsRNA), beta-actin is an internal reference gene, wherein P is less than 0.01.

FIG. 2: the effect of injection of dsRNA on the lethality of brifosson on migratory locusts. (dsGFP is a control group injected with dsGFP, dsSS is a treatment group injected with locust Spinless gene dsRNA), wherein P < 0.01.

Detailed Description

In order to further illustrate the technical solution of the present invention, the present invention is further illustrated by the following examples.

Example 1: obtaining full-length cDNA of the migratory locust Spinless gene and predicting an amino acid sequence.

1. And (3) carrying out bioinformatics search on the migratory locust Spinless gene.

Firstly, the Unigene sequence database sequence in the migratory locust transcriptome database and the sequence in the non-redundant protein database are compared pairwise by using Blastx software, and the 'Spinless' is used as a key word to search in an annotation result to obtain the undetermined sequence of 1 migratory locust Spinless gene.

2. And obtaining the full-length cDNA of the migratory locust Spinless gene.

2.1) designing full-length primers required by PCR full-length amplification:

respectively designing a full-length upstream primer ATGAGCCAGC TGGGGACGGT GTACGCC (SEQ ID NO:3) and a full-length downstream primer TCACCTCAGA GATTGACAGC AAAGGAC (SEQ ID NO:4) by using Oligo software; the designed full-length primer is synthesized by the generation of biological engineering (Shanghai) GmbH.

2.2) preparing a template required by PCR amplification:

selecting 3-year nymphs of migratory locusts with good development condition and normal activity, grinding the nymphs in liquid nitrogen, and extracting RNA of the total migratory locusts by referring to a TRIzol-chloroform method; extracting Kit and RevertAId by adopting Oligotex mRNA Mini Kit^TMH Minus Reverse Transcriptase prepares migratory locust total cDNA, thereby obtaining the template required by PCR amplification reaction.

2.3) PCR amplification reaction:

using a full-length primer and using total cDNA of the migratory locust as a template, carrying out PCR amplification to obtain a migratory locust Spinless gene sequence, and then carrying out separation and detection by agarose gel electrophoresis (figure 1); cutting and recovering the target band, purifying the DNA agarose gel recovery kit, inserting the purified DNA agarose gel recovery kit into a pGEM-T easy Vector, transferring escherichia coli DH5 alpha competent cells by a heat shock method, culturing and picking full white colonies on agar plates of X-gal, IPTG and ampicillin, inoculating the colonies into an LB liquid culture medium containing the ampicillin for amplification culture, extracting plasmids by adopting a small plasmid extraction kit, and sequencing the plasmids by a company Limited in engineering (Shanghai); the sequencing result shows that the DNA sequence of the insert is 2529bp in length, and the nucleotide sequence is SEQ ID NO: 1, or a fragment thereof.

3. And predicting the amino acid sequence of the migratory locust Spinless gene.

Translation and prediction are carried out on the amino acid coded by the migratory locust Spinless gene by adopting a transfer tool, and the result shows that the migratory locust Spinless gene codes 842 amino acids, and the amino acid sequence of the migratory locust Spinless gene is SEQ ID NO: 2, the relative molecular weight of Spinless protein is predicted to be 91.65KD by adopting ProtParam software, and the theoretical isoelectric point is 8.13. The functional domain of the migratory locust Spinless gene is predicted by using SMART software, and the result shows that the migratory locust Spinless gene contains typical domains such as a basic residue domain (basic residues domain), an HLH domain (helix-loop-helix domain), a PAS domain (Period/ARNT/Single minded domain) and a transcription activation domain.

Example 2: and (3) synthesizing dsRNA of the migratory locust Spinless gene.

1. Preparing a template required by synthesizing locusta migratoria cytochrome gene dsRNA.

Nucleotide sequence SEQ ID NO: 1, designing a pair of primers required for synthesizing dsRNA; sequence showntaatacgactcactatagggCTTATGGAGGAGGAGGGTCGCGATC (SEQ ID NO:5) andtaa tacgactcactatagggGCGGCGTACATGTTGTTGAGGTTGG (SEQ ID NO:6) are upstream and downstream primers required for dsRNA synthesis (the underlined part is the T7 promoter sequence); the designed dsRNA primer is synthesized by the generation of biological engineering (Shanghai) GmbH.

Carrying out PCR amplification on the primers (the 5' tail ends of which contain a T7 promoter sequence) for synthesizing the dsRNA to obtain a segment, wherein both ends of the segment are provided with T7 promoters, and the total length of the segment is 423 bp; the DNA sequence is SEQ ID NO: 7; after agarose gel electrophoresis separation, cutting of a target band and purification of a DNA agarose gel recovery kit, a NanoDrop2000 micro-spectrophotometer is used for quantification and detection, and the obtained product is used as a template for synthesizing dsRNA.

2. And (3) synthesizing dsRNA of the migratory locust Spinless gene.

Based on the template for synthesizing dsRNA of the above steps, dsRNA was synthesized by in vitro Transcription with T7RNA polymerase using HiScribe RNAi Transcription Kit (NEB). The purified dsRNA was quantified by a NanoDrop2000 microspectrophotometer to a final concentration of 1.5. mu.g/. mu.L and stored in a freezer at-20 ℃ for future use.

Example 3: silencing of Spinilus gene.

1. And (3) injecting dsRNA of the Spinless gene of the migratory locust.

And selecting the nymphs of 120 migratory locusts at 3 rd day 2-3, and using the nymphs for dsRNA injection experiment. Mu.g of dsRNA fragments were injected into the second abdominal segment of locusta migratoria using a microinjection needle. Migratory locusts injected with GFP gene dsRNA are used as a control group, and migratory locusts injected with Spinless gene dsRNA are used as a treatment group. After the injection is finished, the locusts of the control group and the locusts of the treatment group are placed in an artificial climate box for raising. The breeding conditions are as follows: the lighting period is 14 hours and the darkness is 10 hours, the temperature is 28-32 ℃, the relative humidity is 30-60 percent, and enough fresh wheat seedlings are fed.

2. And (3) detecting the expression level of the mRNA of the migratory locust Spinless gene.

Continuously feeding a control group migratory locust injected with dsGFP gene dsRNA and a treatment group migratory locust injected with Spinless gene dsRNA for 24 hours and 48 hours respectively in an artificial climate box, and reserving samples in a liquid nitrogen tank for storage; crushing a migratory locust sample by using a tissue grinder in a liquid nitrogen environment, extracting total RNA of the migratory locust by using a TRIzol reagent, and performing reverse transcription by using a RevertAID First Strand cDNA Synthesis Kit (ThermoFisher) to synthesize cDNA; detecting the mRNA relative expression level of Spinless gene and beta-actin gene by real-time quantitative PCR technology, thereby determining the silencing efficiency of Spinless gene of migratory locust; the results are shown in FIG. 1: compared with a control group migratory locust, the mRNA level of the Spinless gene of the experimental group migratory locust is remarkably reduced by 85.3% -87.5%.

3. Screening of migratory locust differential expression genes after migratory locust Spinless gene silencing

Total RNA of a control group migratory locust and a treatment group migratory locust is extracted, migratory locust mRNA is purified by a poly-T magnetic bead method, then a reverse transcription kit is used for synthesizing first strand cDNA, and DNA polymerase is used for synthesizing second strand cDNA. After the cDNA tail sequence was completed with DNA polymerase, adenine nucleotides were added to the 3' -end of the cDNA tail, and adapter sequences were added to both ends of the cDNA sequence. The size of the cDNA sequence fragment is interrupted to 150-200bp by a User enzyme method, and then high-fidelity DNA polymerase is used for amplification to obtain an RNA-seq library. And (3) performing machine sequencing on the RNA-seq library, removing low-quality sequences and error sequences, and performing redundancy removal and splicing by a Trinity program to obtain a transcriptome database. Screening of the differential expression genes of the control group and the treatment group is completed by a DESeq program package, the screening threshold value is that P is less than or equal to 0.05, and the difference multiple is more than or equal to 1.5 times. The results show that: compared with the locusta migratoria in a control group, the locusta migratoria in the treatment group has no significant difference in expression level of 16652 genes, and 67 genes are significantly up-regulated and 145 genes are significantly down-regulated.

Example 4: the migratory locust Spinless gene dsRNA promotes the death rate of the brifosson on migratory locust.

A control group migratory locust injected with dsGFP gene dsRNA and a treatment group migratory locust injected with Spinless gene dsRNA are raised in an artificial climate box for 24 hours, and 3 microliters of aqueous solution containing 30 micrograms/milliliter of brifossils bromide is dripped into the abdomen of the migratory locust. After continuously raising the locusts in the artificial climate box for 24 hours, counting the lethality rate of the briphos pines to locusts migratoria; the results are shown in FIG. 2: after the dsRNA of the Spinless gene is injected, the lethality of the briphos to 3-year migratory locust can be improved to 6.1 times.

While there have been shown and described what are at present considered to be the essential features and advantages of the invention, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> university of Shanxi

<120> migratory locust Spinless gene dsRNA and application thereof

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785 790 795 800

His Gln Tyr His Gln Tyr Tyr Pro Tyr Thr Thr Pro Tyr His His His

805 810 815

Ala Pro His Thr Ser Val Asn Asp Ser Gly Tyr Pro Gln Ile Asn Arg

820 825 830

Thr Gly Val Leu Cys Cys Gln Ser Leu Arg

835 840

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgagccagc tggggacggt gtacgcc 27

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcacctcaga gattgacagc aaaggac 27

<210> 5

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taatacgact cactataggg cttatggagg aggagggtcg cgatc 45

<210> 6

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

taatacgact cactataggg gcggcgtaca tgttgttgag gttgg 45

<210> 7

<211> 423

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

taatacgact cactataggg cttatggagg aggagggtcg cgatctgctg ggaaagcgca 60

ccatggactt caaggtgagt tacctggacg cggggctgac gaacagctac ttcgcggacg 120

cggaccagct ggtggtgacg tcgacgccgc agcacgcggg gtccccgtcg gcgacgtcga 180

cgacgccgca gcgcgtcaac cggcgctaca agacgcagct gcgcgacttc ctgtcgacgt 240

gccgcaccaa gcgcaagctg tcggcggcgg cggcggctgc ggcgtccacg tcgcccgcgc 300

agctgcccgc ggccgcgccc gccatcgagt acgtggacac gtcgagcgcg gccgccgccg 360

ccgtcgccgc cgcctactcc aacctcaaca acatgtacgc cgcccctata gtgagtcgta 420

tta 423

Claims

1. A migratory locust Spinless gene, the nucleotide sequence of which is SEQ ID NO: 1, or a fragment thereof.

2. A migratory locust Spinless gene fragment with two ends fused with a T7 promoter has a nucleotide sequence shown as SEQ ID NO: 7, or a sequence shown in the figure.

3. The application of dsRNA synthesized by the migratory locust Spinless gene segment with the two ends fused with the T7 promoter according to claim 2 in improving the migratory locust control effect of the brifosson.