CN117646005B - Locusta migratory E-cadherein gene and application of dsRNA thereof in locusta migratory prevention and control - Google Patents
Locusta migratory E-cadherein gene and application of dsRNA thereof in locusta migratory prevention and control Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a migratory locust E-cadherin (E-cad) gene and application of dsRNA thereof in control of migratory locust. After dsRNA of the synthesized E-cadherin gene is injected into the body cavity of the migratory locust, the E-cadherin gene of the migratory locust can be specifically silenced, so that a large number of lethal phenomena of the migratory locust occur, the lethality rate is 100%, the population number increase and migration hazard of the migratory locust are effectively inhibited, and the good effect of preventing and treating the migratory locust can be achieved by inhibiting the expression of the E-cadherin gene. Because of the high efficiency, specificity and safety of the invention, the invention has wide application prospect in the prevention and control of migratory locust and has good economic benefit for protecting agricultural production and ecological safety in China.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a migratory locust E-cadherein gene and application of dsRNA thereof in migratory locust control.
Background
Pest control based on RNA interference technology has the following advantages: (1) specificity: the dsRNA only degrades mRNA homologous to the dsRNA, reduces toxic effects on non-target organisms, and has higher safety particularly on higher animals and human beings; (2) high efficiency: a small amount of dsRNA can effectively inhibit the expression of the target gene, and entry of dsRNA into the pest can cause silencing of systemic and even progeny target genes. (3) security: RNA is extremely easy to degrade in nature, has no residue, has no pollution to ecological environment and crop products, and is relatively safe. At present, the basic technology for pest control based on RNA interference is to screen target sequences to obtain dsRNA with high lethal effect.
E-cadherein (E-cad) is a transmembrane, calcium-dependent cell adhesion protein that regulates cell-to-cell adhesion and maintains the structural and functional integrity of epithelial tissue. At present, no report on silencing of the adult migratory locust E-cadherein gene to realize control of the migratory locust exists.
Disclosure of Invention
The invention discovers that the migratory locust E-cadherein gene is used as a drug target spot, and can be used for prevention and control of the migratory locust. Specifically, the invention provides dsRNA synthesized based on the migratory locust E-cadherein gene, and the dsRNA is injected into the migratory locust body, so that the migratory locust can be killed, and the migratory locust is prevented and controlled to break down.
The ORF sequence of the migratory locust E-cadherein gene is shown as SEQ ID NO. 1, the full length of the ORF sequence of the E-cadherein gene is 4551bp, and 1516 amino acids are encoded.
The invention provides dsRNA synthesized based on the migratory locust E-cadherein gene, which is double-stranded RNA composed of sense strand nucleotide shown in SEQ ID NO. 9 and antisense strand nucleotide complementary to SEQ ID NO. 9.
The invention also provides a primer pair for synthesizing the dsRNA, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 5.
The invention also provides a synthesis method of dsRNA based on the migratory locust E-cadherein gene synthesis, which comprises the following steps:
Based on the E-cadherein gene sequence shown in SEQ ID NO. 1, using cDNA reverse transcribed from extracted total RNA of migratory locust fat body as a template, and performing PCR amplification by using the upstream and downstream primers shown in SEQ ID NO. 4 and SEQ ID NO. 5 to obtain a PCR amplification product shown in SEQ ID NO. 6;
and connecting the PCR amplification product shown in SEQ ID NO. 6 into a pGEM-T vector to obtain a recombinant plasmid pGEM-T-E-cadherein.
Then, PCR amplification is carried out by using recombinant plasmid pGEM-T-E-cadherein as a template and using a primer pair with a T7 promoter sequence as shown in SEQ ID NO. 7 and SEQ ID NO. 8 to obtain a DNA template (namely DNA with the T7 promoter sequence) for synthesizing dsRNA;
dsRNA was then transcribed in vitro using the T7RiboMAX TM Express RNAI SYSTEM (Promega) kit using DNA with the T7 promoter sequence as template.
The invention also provides the migratory locust E-cadherein gene and application of the dsRNA thereof in migratory locust control.
The beneficial effects of the invention are as follows:
The invention discovers that the dsRNA based on the synthesis of the migratory locust E-cadherin gene can effectively inhibit the gene expression of the E-cadherin and generate a lethal effect, the dsRNA has small dosage (15 mug), quick response (the death of the migratory locust is caused after 3 days of treatment), the mortality rate can reach 100 percent, and the effect is obvious. In addition, the dsRNA is used for preventing and controlling the locust, the species specificity is strong, and the gene interference to other species can not be caused; meanwhile, the dsRNA is easy to degrade in natural environment and is friendly to the environment. Therefore, in view of the high efficiency, specificity and safety of the invention, the invention has wide application prospect in the prevention and control of migratory locust and has good economic benefit for protecting agricultural production and ecological safety in China.
Drawings
FIG. 1 shows the efficiency of interference of the E-cadherein gene in adipose tissue after double-stranded RNA injection. P < 0.001.
FIG. 2 is a statistical plot of the survival rate of adult locusts after interfering with E-cadherein expression.
FIG. 3 shows the lethal phenotype of migratory locust after injection of synthetic dsRNA, wherein FIG. 3a shows the phenotype of dsE-cadherein treated group (a 1: lateral side, a2: ventral side), and 3b shows the phenotype of control group dsGFP (b 1: lateral side, b2: ventral side).
Detailed Description
The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.
Embodiment one: sequence of migratory locust E-cadherein gene and obtaining of dsRNA thereof
1. Obtaining of the E-cadherein Gene sequence of migratory locust
1.1 Dissection of the migratory locust tissue
Selecting healthy migratory grasshopper male and female adults, shearing off the heads of the adult grasshoppers by using an anatomic shear sterilized by alcohol in advance, shearing off the head along the abdomen from the sheared-off parts, taking out fat bodies in the abdominal cavity by using an anatomic forceps sterilized by alcohol, putting the fat bodies into a 2mL centrifuge tube, rapidly putting the centrifuge tube into liquid nitrogen to enable the centrifuge tube to be frozen instantly, and storing the centrifuge tube in a refrigerator at the temperature of minus 80 ℃ for later use.
1.2 Total RNA extraction
(1) Placing the obtained adipose body tissue on ice, adding 300 μl of TRIzol (Tiangen), and sufficiently grinding on ice with an electric grinding rod; and standing the submerged tissue suspension for 30 minutes at room temperature to ensure the tissue to be fully cracked.
(2) Centrifuge at 12000rpm,4 ℃ for 5 minutes, remove most of the fat, transfer the lower clear lysate to a new centrifuge tube.
(3) Adding 500. Mu.L of chloroform, shaking vigorously by hand for 2 minutes, ice-bathing for 5 minutes, centrifuging at 12000rpm at 4 ℃ for 15 minutes; the supernatant was taken into a new centrifuge tube.
(4) Adding 500. Mu.L of acid phenol chloroform (phenol: chloroform: isoamyl alcohol=25:24:1), shaking vigorously by hand for 2 minutes, ice-bath for 5 minutes, centrifuging at 12000rpm at 4℃for 10 minutes; the supernatant was taken to a new centrifuge tube.
(5) 500. Mu.L of isopropanol was added, and the mixture was turned upside down until it was thoroughly mixed, overnight at-20 ℃.
(6) RNA precipitated overnight was centrifuged at 12000rpm at 4℃for 10 minutes, and the supernatant was discarded to retain the precipitate.
(7) To the pellet was added 500. Mu.L of pre-chilled 75% glacial ethanol, centrifuged at 12000rpm at 4℃for 10 min, and the supernatant was discarded.
(8) Repeating the step (7) to obtain RNA precipitate.
(9) The RNA pellet was air dried for 30 minutes on an ultra clean bench to dry the RNA.
(10) An appropriate amount of ultrapure water was added to the tube containing RNA, and the tube was left at 4℃to dissolve the RNA sufficiently.
(11) After the RNA was sufficiently dissolved, the mixture was gently shaken and mixed, the integrity of the RNA was checked by 1% agarose gel electrophoresis, and the concentration of the RNA was measured by NanoDrop 2000, followed by diluting the RNA to 500 ng/. Mu.L with ultrapure water for use.
1.3 Reverse transcription
The total RNA extracted above was reverse transcribed into a first cDNA as follows:
(1) Mu.g of total RNA was taken, made up to 8. Mu.L with water, incubated in a PCR instrument for 5 minutes at 65℃and rapidly cooled on ice.
(2) Mu.L of 5 XgDNA Buffer was added, gently mixed and centrifuged briefly, incubated in a PCR apparatus at 42℃for 3 minutes, and rapidly cooled on ice.
(3) The reaction system mixture as shown in Table 1 was prepared, and the components and amounts thereof were as shown in the following Table:
TABLE 1 reaction System for reverse transcription into first cDNA
| Composition of components | Volume (mu L) |
| 10×Fast RT Buffer | 2 |
| RT Enzyme Mix | 1 |
| FQ-RT Primer Mix | 1 |
| Ultrapure water | 5 |
| Total volume of | 10 |
(4) To the system (3), 10. Mu.L of the above-mentioned mixed solution was added, gently mixed and centrifuged briefly.
(5) The cDNA was obtained by incubating at 42℃for 15 minutes, at 95℃for 3 minutes in a PCR apparatus, and then placing the resultant on ice.
1.4 Acquisition of ORF sequence of E-cadherein Gene
Searching in a migratory locust transcriptome by utilizing homology comparison according to the sequence information of known E-cadherein protein of the insect to obtain a migratory locust E-cadherein transcript, and further combining the migratory locust genome information to splice to obtain a complete migratory locust E-cadherein gene. The upstream and downstream primer sequences (shown as SEQ ID NO:2 and SEQ ID NO: 3) were designed using PRIMER PREMIER 5.0.0 software and sent to Beijing Liuhua big Gene technologies Co.
The sequence of the primer for amplifying the E-cadherein gene and the downstream primer for amplifying the E-cadherein gene by PCR is as follows:
E-cadherin-F(SEQ ID NO:2)：5’-ATGCGACGTGGACAAATCTC-3’；
E-cadherin-R(SEQ ID NO:3)：5’-TCAGCACCACGATTCTGATG-3’。
The cDNA obtained by reverse transcription in the step 1.3 is used as a template, an ORF fragment of the E-cadherin gene is obtained through PCR amplification by combining with an upstream primer and a downstream primer shown as SEQ ID NO. 2 and SEQ ID NO. 3, the fragment is sent to Beijing Liuhua big Gene science and technology Co., ltd for sequencing (all subsequent primer synthesis and sequencing are completed in Beijing Liuhua big Gene science and technology Co., ltd.), the ORF sequence of the E-cadherin gene is shown as SEQ ID NO. 1, the full length of the ORF sequence is 4551bp, and 1516 amino acids are encoded.
2. Synthesis of dsRNA
2.1 Amplification of DNA templates for dsRNA
Based on the E-cadherin gene sequence shown in SEQ ID NO. 1, PRIMER PREMIER 5.0.0 is adopted to design and synthesize an upstream primer and a downstream primer of a DNA molecular template sequence of dsE-cadherin (the sense strand is shown as SEQ ID NO. 6), and the primer sequences are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 5.
The cDNA reverse transcribed in step 1.3 is used as a template, and the PCR amplification is carried out by using the upstream and downstream primers shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the reaction system is shown in Table 2.
dsE-cadherin-F(SEQ ID NO:4)：5’-CACAATGCTTGCTGGAACACA-3’；
dsE-cadherin-R(SEQ ID NO:5)：5’-TCATCTTGATGCTCTGCCACTA-3’。
TABLE 2 reaction System for PCR amplification Using cDNA as template
The reaction conditions are as follows: (1) 95℃for 3 min; (2) 95℃for 30 seconds; 56 ℃ for 30 seconds; 72 ℃,30 seconds; 35 cycles; (3) extension at 72℃for 5 min; preserving at 4 ℃.
And (3) connecting a single product obtained by PCR amplification into the pGEM-T (Tiangen) vector to obtain the recombinant plasmid pGEM-T-E-cadherein. Sequencing results show that the size of the PCR amplification product is 405bp, and the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 6.
The recombinant plasmid pGEM-T-E-cadherin obtained above is used as a template, and a primer pair dsE-cadherin-T7-F/dsE-cadherin-T7-R (shown as SEQ ID NO:7 and SEQ ID NO: 8) with a T7 promoter sequence is used for PCR amplification to obtain a DNA template for synthesizing dsRNA (namely, the two ends of the sequence shown as SEQ ID NO:6 are provided with T7 promoters). The PCR reaction system and conditions were as in Table 2. UsingSV GEL AND PCR CLEAN-Up System (Promega) kit for purification of the fragment of interest. NanoDrop 2000 was measured for concentration and determined to be in the range of 125 ng/. Mu.l to 1 mg/. Mu.l.
The sequences of the upstream and downstream primers of the DNA template for synthesizing the dsRNA are the sequence SEQ ID NO. 4 and the 5' -end of the sequence SEQ ID NO. 5 plus a T7 promoter sequence, specifically as follows (underlined T7 promoter sequence):
dsE-cadherin-T7-F(SEQ ID NO:7)：
5’-TAATACGACTCACTATAGGCACAATGCTTGCTGGAACACA-3’；
dsE-cadherin-T7-R(SEQ ID NO:8)：
5’-TAATACGACTCACTATAGGTCATCTTGATGCTCTGCCACTA-3’。
2.2 Synthesis of dsRNA
DsRNA was transcribed in vitro using the T7RiboMAX TM Express RNAI SYSTEM (Promega) kit using the DNA with the T7 promoter sequences at both ends obtained in step 2.1 as a template.
The in vitro transcription reaction system is shown in Table 3.
TABLE 3 in vitro transcription reaction System
| Composition of components | Volume (mu L) |
| RiboMAX Express T7 2×Buffer | 10 |
| Linear DNA template | 1～8 |
| Enzyme Mix,T7 Express | 2 |
| Nuclease-Free Water | Up to20 |
| Total volume of | 20 |
The reaction conditions are as follows: 37 ℃ for 60 minutes; 70 ℃ for 10 minutes; 25℃for 30 min.
2.3 Purification of dsRNA
(1) Adding 160 mu L of Nuclease-Free water and 200 mu L of acidic phenol chloroform into the reaction system in the step 2.2, fully and uniformly mixing, and then placing on ice for 5 minutes;
(2) Centrifuging at 4 ℃ for 10 minutes with 13000g, sucking the supernatant and placing the supernatant into a new 1.5mL centrifuge tube;
(3) Adding 0.1 times volume of sodium acetate (3.0M, pH 5.2) and 2.5 times volume of absolute ethyl alcohol into the supernatant fluid, fully and uniformly mixing, and placing on ice for 5 minutes;
(4) Centrifuging for 10 minutes at 4 ℃ and 13000g, and discarding the supernatant;
(5) 500. Mu.L of precooled 75% ethanol was added, centrifuged at 13000g for 10 min at 4℃and the supernatant was discarded;
(6) Repeating the step (5), and washing with 75% ethanol again;
(7) Drying in an ultra-clean bench for 20 min, adding 40 mu LNuclease-Free water, dissolving in ice for 20 min, mixing, measuring concentration with NanoDrop 2000, and placing at-20deg.C.
The dsRNA obtained was sequenced. Sequencing results showed that: the dsRNA of the migratory locust E-cadherein gene is double-stranded RNA, and consists of a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO. 9, and the nucleotide sequence of the antisense strand is the reverse complementary sequence of SEQ ID NO. 9.
Embodiment two: experiments on killing migratory locust by synthesizing dsRNA from migratory locust E-cadherein gene
1. DsRNA injection of migratory locust
Adults of migratory locust within 12 hours after eclosion are selected as experimental injection materials and randomly divided into dsE-cadherein treatment group and dsGFP control group (the migratory locust injected with dsGFP), and each group is repeated for 3 times with 20 heads. 15 μg dsRNA (at a concentration of about 5 μg/μl, about 3 μl) was aspirated with a10 μl-sized microsyringe and injected into the body from the second, third internode of the abdomen of the migratory locust. The locusts after injection are placed in a metal cage (25 cm multiplied by 25 cm) with good ventilation for feeding, the temperature is 30+/-2 ℃, the photoperiod is 14L:10D, and fresh wheat seedlings are fed twice a day. Adult mortality was observed and counted daily after injection.
E-cadherein gene interference efficiency detection
After 48h of dsRNA injection, 7 heads were randomly picked at dsE-cadherein treatment and dsGFP control groups, respectively, to test the interference efficiency of E-cadherein.
The procedure of example one was followed by grinding of the fat bodies of the worms, followed by extraction of total RNA and inversion of the total RNA into the first cDNA. According to the instruction method of a fluorescent quantitative premix reagent enhancement (SYBR Green) kit (PF 205) of radix angelicae SuperReal, the relative expression amounts of a target gene (E-cadherin) and an internal reference gene (beta-actin) are detected respectively by adopting an E-cadherin upstream primer and a E-cadherin downstream primer shown as SEQ ID NO. 10 and SEQ ID NO. 11, and a beta-actin upstream primer and a beta-actin downstream primer shown as SEQ ID NO. 12 and SEQ ID NO. 13, respectively carrying out qRT-PCR reaction on a LightCycler 96 (Roche), and then calculating according to a2 -ΔΔCt method to analyze the gene silencing efficiency. As a result, as shown in FIG. 1, the E-cadherin expression level of dsE-cadherin-treated group was significantly reduced by 83.7% compared to dsGFP control group, indicating that E-cadherin was effectively silenced.
The qRT-PCR reaction primer sequences were as follows:
E-cadherin-qRT-F(SEQ ID NO:10)：5’-CGCCAGATGAAGTGCCTGATA-3’；
E-cadherin-qRT-R(SEQ ID NO:11)：5’-AATGAGCCTGACGAGTTACCAT-3’。
β-actin-qRT-F(SEQ ID NO:12)：5’-AATTACCATTGGTAACGAGCGATT-3’；
β-actin-qRT-R(SEQ ID NO:13)：5’-TGCTTCCATACCCAGGAATGA-3’。
the qRT-PCR reaction system is shown in Table 4.
TABLE 4qRT-PCR reaction System
| Composition of components | Volume (mu L) |
| 2×SuperReal PreMix Plus | 10 |
| Forward primer (8. Mu.M) | 0.4 |
| Reverse primer (8. Mu.M) | 0.4 |
| CDNA template | 2 |
| Ultrapure water | 7.2 |
| Total volume of | 20 |
The reaction conditions are as follows: (1) pre-denaturation at 95℃for 10 min; (2) 95℃for 10s;58 ℃ for 30s;72 ℃,30s, 40 cycles total; (3) dissolution profile analysis, 95 ℃,60s;65 ℃ for 30s;95℃for 30s.
3. Death rate statistics and phenotypic observation of migratory locust after dsRNA injection
After the adult is injected with dsRNA, the physical state vitality of the control group of the injected dsGFP is normal, and has no obvious difference with the adult which normally develops. The survival rate statistics of the dsE-cadherein treated group injected totally for 60 heads are shown in Table 5 and FIG. 2, and it can be seen that death starts on day 3, a lot of deaths start on day 4, the death rate reaches 77% and all deaths start on day 5, and the death rate is 100%. The phenotype maps of injection dsE-cadherein and injection dsGFP are shown in FIG. 3.
TABLE 5 statistical results of survival of migratory locust after dsRNA injection
| Time after injection (Tian) | 0 | 1 | 2 | 3 | 4 | 5 |
| dsGFP(％) | 100±0.00 | 100±0.00 | 96.226±0.00 | 96.226±0.00 | 96.226±0.00 | 96.226±0.00 |
| dsE-cadherin(％) | 100±0.00 | 100±0.00 | 100±0.00 | 90.56±0.00 | 22.642±1.51 | 0.00±0.00 |
The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.
Claims (5)
1. The application of the migratory locust E-cadherein gene in the control of the migratory locust is characterized in that the adult migratory locust E-cadherein gene is silenced to realize the control of the migratory locust; the ORF sequence of the E-cadherein gene is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the adult migratory locust E-cadherein gene is silenced by RNA interference technology.
3. Use of dsRNA synthesized based on the migratory locust E-cadherin gene according to claim 1 for control of migratory locust, characterized in that the dsRNA consists of a sense strand nucleotide shown in SEQ ID No. 9 and an antisense strand nucleotide complementary to the sequence shown in SEQ ID No. 9.
4. The use according to claim 3, wherein the nucleotide sequences of the primer pair for synthesizing the dsRNA are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.
5. The use according to claim 3, characterized in that the method of synthesis of dsRNA comprises the steps of:
Using cDNA reverse transcribed from extracted total RNA of migratory locust fat body as a template, and performing PCR amplification by using the upstream and downstream primers shown in SEQ ID NO. 4 and SEQ ID NO. 5 to obtain a PCR amplification product shown in SEQ ID NO. 6;
Connecting the PCR amplification product shown in SEQ ID NO. 6 into a vector to obtain a recombinant plasmid;
Then, the recombinant plasmid is used as a template, and a primer pair with a T7 promoter sequence shown in SEQ ID NO. 7 and SEQ ID NO. 8 is used for carrying out PCR amplification to obtain DNA with the T7 promoter sequence;
Then, the DNA with the T7 promoter sequence is used as a template to synthesize dsRNA through in vitro transcription.
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