CN115927384A - Laodelphax striatellus salivary gland specific gene LsSGSP and application thereof - Google Patents
Laodelphax striatellus salivary gland specific gene LsSGSP and application thereof Download PDFInfo
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Abstract
The invention discloses a specific gene LsSGSP of salivary glands of small brown planthoppers and application thereof, wherein the nucleic acid sequence of the specific gene LsSGSP of salivary glands of small brown planthoppers is shown in SEQ ID No.1, dsRNA of the gene LsSGSP is introduced into the small brown planthoppers, and after the gene LsSGSP is silenced, the feeding capacity of the small brown planthoppers can be obviously reduced, so that the survival rate of the small brown planthoppers is reduced, which shows that the specific gene LsSGSP of salivary glands of the small brown planthoppers plays a key role in the feeding process of the small brown planthoppers, and meanwhile, the specific gene LsSGSP of the small brown planthoppers has important value in biological control of the small brown planthoppers and good application prospect.
Description
Technical Field
The invention relates to the field of biological control, in particular to a specific gene LsSGSP of a salivary gland of a laodelphax striatellus, and also relates to an application of the LsSGSP in the control of the laodelphax striatellus.
Background
Laodelphax striatellus is an important pest in China and other rice planting areas in Asia. Since the last 60 s, laodelphax striatellus frequently outbreaks in multiple rice areas of China to cause disasters, and seriously threatens the food safety of China. Laodelphax striatellus is mainly harmed by directly sucking plant juice, laying eggs and spreading rice viruses. In the field, the laodelphax striatellus is usually clustered in rice clusters by adults and nymphs to absorb juice of stem and leaf tissues, plant nutrients are consumed, and the weight of the unsaturated grains is reduced. At present, the control of the laodelphax striatellus is mainly carried out by chemical pesticides, the dosage and the dosage frequency of the chemical pesticides are increased along with the continuous enhancement of the drug resistance of the laodelphax striatellus, and the serious threat is also caused to the ecological environment of farmlands and the quality safety of rice. In view of the severity of the damage, rice planthoppers including Laodelphax striatellus are listed in the category of crop pest and disease records in the Ministry of agricultural rural areas in 2020.
Salivary glands are important secretory organs of laodelphax striatellus. The colloidal saliva and the watery saliva secreted by the laodelphax striatellus during feeding are mainly secreted by salivary glands and injected into plant tissues through salivary ducts. Salivary glands play an important role in the processes of food intake, digestion, toxin transmission and the like of the laodelphax striatellus. Researches find that a plurality of genes related to the feeding of the laodelphax striatellus are specifically expressed in salivary glands, and the functions of the genes and the application of the genes in the control of the laodelphax striatellus are to be further explored.
RNA interference technology is a hot spot of life science research in recent years. The small-molecule double-stranded RNA specifically degrades or inhibits the expression of target gene mRNA, and further inhibits or closes a specific gene. The RNA interference technology has the advantages of high specificity, high efficiency, convenient operation and the like. At present, the technology is widely applied to prevention and control of various agricultural pests, namely, specific dsRNA is introduced into insect bodies, so that the purpose of controlling pest populations or reducing the pest toxicity transmission capability is achieved.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a laodelphax striatellus salivary gland-specific gene LsSGSP; the second purpose of the invention is to provide a primer for amplifying the specific gene LsSGSP of the laodelphax striatellus salivary gland; the invention also aims to provide an application of a reagent for silencing the specific gene LsSGSP of the salivary gland of the laodelphax striatellus in preparation of a control agent for the laodelphax striatellus.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the amino acid sequence of the specific gene LsSGSP of the saliva gland of the Laodelphax striatellus is shown in SEQ ID NO. 2.
Preferably, the nucleotide sequence of the Laodelphax striatellus LsSGSP gene is shown in SEQ ID No. 1.
2. And (3) amplifying primers of the specific gene LsSGSP of the salivary gland of the small brown planthopper, wherein the primers are shown as SEQ ID NO.3 and SEQ ID NO. 4.
3. The dsRNA of the saliva gland specific gene LsSGSP of the small brown planthopper.
Preferably, the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 5.
Preferably, the dsRNA is prepared by the following method: using a DNA template shown in SEQ ID NO.5 and a T7High Yield RNA transcription kit, in a Reaction system, 2 mul 10 × Reaction Buffer,2 mul ATP solution,2 mul UTP solution,2 mul CTP solution,2 mul GTP solution,2 mul enzyme mix and 1 mul DNA template, and no RNase water is supplemented to 20 mul, the Reaction system is mixed evenly and reacted at 37 ℃ for overnight; then adding TURBO DNase to react at 37 ℃ for 15min to eliminate DNA in the reaction system, and carrying out denaturation at 65 ℃ for 5min to obtain the DNA.
4. The application of the reagent for silencing the specific gene LsSGSP of the salivary gland of the laodelphax striatellus in preparing a control agent of the laodelphax striatellus.
In the invention, the preferable reagent for silencing the salivary gland specific gene LsSGSP of the small brown rice planthopper is dsRNA of the salivary gland specific gene LsSGSP of the small brown rice planthopper.
5. The application of the reagent for silencing the specific gene LsSGSP of the salivary gland of the laodelphax striatellus in reducing the secretion amount of the honeydew of the laodelphax striatellus.
6. A method for preventing and treating the laodelphax striatellus comprises the step of silencing the expression of a laodelphax striatellus salivary gland specific gene LsSGSP in the laodelphax striatellus, wherein a nucleotide sequence coded by the laodelphax striatellus salivary gland specific gene LsSGSP is shown as SEQ ID No. 2.
The invention has the beneficial effects that: the invention discloses a specific gene LsSGSP of salivary glands of laodelphax striatellus, wherein dsRNA of the obtained LsSGSP gene is introduced into the laodelphax striatellus, and after the LsSGSP gene is silenced, the feeding capacity of the laodelphax striatellus can be obviously reduced, so that the survival rate of the laodelphax striatellus is reduced, which shows that the specific gene LsSGSP of the salivary glands of the laodelphax striatellus plays a key role in the feeding process of the laodelphax striatellus, and meanwhile, the specific gene LsSGSP has important value in biological control of the laodelphax striatellus (transgenic rice and the like), and has good application prospect.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the result of PCR amplification of LsSGSP fragment (504 bp);
FIG. 2 shows the result of the experiment for synthesizing dsRNA of LsSGSP gene;
FIG. 3 shows the results of experiments on the survival rate of Laodelphax striatellus introduced with dsRNA of LsSGSP gene;
FIG. 4 shows the amount of the honeydew produced by Laodelphax striatellus.
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 cloning of LsSGSP Gene fragment of Laodelphax striatellus
Amplification of Laodelphax striatellus LsSGSP gene fragment
(1) Taking nymphs or adults of the laodelphax striatellus, and placing the nymphs or adults on ice for anesthesia; pre-cooled 1 XPBS (37mM NaCl,2.68mM KCl,8.1mM Na) was added to the slides 2 HPO 4 ，1.47mM KH 2 PO 4 pH 7.4), salivary glands of planthoppers were dissected immediately under the scope of the stereoscope;
(2) Dissected salivary glands were added to 1ml Trizol (Takara) and ground thoroughly; adding 400 μ l chloroform, mixing, centrifuging at 12,000rpm at 4 deg.C for 15min, carefully sucking the uppermost aqueous phase, and transferring to a new centrifuge tube; adding isopropanol with the same volume, uniformly mixing, and standing at room temperature; after 10 minutes, centrifuge at 12,000rpm for 10 minutes, discard the supernatant and add 1ml 75% ethanol to the pellet; centrifuging at 9600rpm for 5 minutes, removing the supernatant, and adding a proper amount of RNase-free water to the precipitate; determining the RNA concentration by using NanoDrop;
(3) Reverse transcription of extracted total salivary gland RNA of laodelphax striatellus was performed using the reverttra Ace q PCR RT Master with gDNA remover kit: first, total RNA was denatured at 65 ℃ for 5min; 1 μ g of total RNA was taken, added to 2 μ l of 4 XDN Master Mix, and made up to 8 μ l with RNase-free water; after mixing, incubating at 37 ℃ for 5min to remove the contaminating genomic DNA; then, 2. Mu.l of 5 XTT Master Mix was added to the system; incubating at 37 deg.C for 1h after mixing, and denaturing at 98 deg.C for 5min to inactivate reverse transcriptase and obtain Laodelphax striatellus salivary gland cDNA;
(4) Designing a primer according to an open reading frame (SEQ ID NO. 1) of the LsSGSP gene as shown in Table 1, wherein a coded amino acid is shown as SEQ ID NO.2, and the primer is synthesized by Hangzhou Youkang biological technology Co., ltd;
TABLE 1 LsSGSP Gene amplification primers
(5) Using the cDNA of the salivary gland of the laodelphax striatellus as a template, and using primers shown in table 1 to perform PCR amplification on a target gene, wherein the specific amplification system is as follows:
Max Buffer 25μl，dNTP Mix(10mM each)1μl，/>
max Super-Fidelity DNA Polymerase 1. Mu.l, upstream and downstream primers 1. Mu.l each, laodelphax striatellus salivary gland cDNA template 1. Mu.l, and ddH 2 The amount of O was adjusted to 50. Mu.l. The PCR amplification conditions were: 3 minutes at 95 ℃; 35 cycles of 95 ℃ for 15 seconds, 60 ℃ for 1 minute; 10 minutes at 72 ℃.
Obtaining a single clone strain of the LsSGSP gene of the laodelphax striatellus:
(1) Separating PCR amplification product by 1% agarose gel electrophoresis, the result is shown in figure 1, cutting target fragment with a blade after separation;
(2) The target DNA was recovered using a DNA agarose gel recovery kit (shanghai bio-organism, SK 8131): cutting off agarose containing the target fragment from the agarose gel, and putting the agarose into a centrifuge tube; adding 5 times of glue dissolving solution Buffer B2, and carrying out water bath at 70 ℃ for 10 minutes; adding the dissolved glue solution into an adsorption column, centrifuging for 30 seconds at 8,000g, and discarding waste liquid; adding 500 mul wash Solution, centrifuging for 30 seconds at 9,000g, and discarding the waste liquid; centrifuging the empty adsorption column 9,000g for 1 minute; the adsorption column was placed in a new centrifuge tube and 30. Mu.l ddH was added to the center of the adsorption column 2 O; centrifuging for 2 minutes at 10,000g to finally obtain a purified DNA fragment of the LsSGSP gene of the laodelphax striatellus;
(3) The DNA fragment of the laodelphax striatellus LsSGSP gene was ligated to the pClone007 vector using a blunt-end cloning kit (beijing engine biology ltd): sequentially adding 1 mu l of Laodelphax striatellus LsSGSP gene DNA fragment and 1 mu l of pClone007 Blunt simple vector into a 0.2ml centrifuge tube; 1 μ l 10 × buffer, finally ddH 2 Supplementing the O to 10 mu l, uniformly mixing the samples, and reacting for 5 minutes at 25 ℃;
(4) Adding the ligation product into competence, flicking the tube wall by hand, and carrying out ice bath for 30 minutes; transferring the centrifuge tube to a 42 ℃ water bath for 90 seconds, quickly taking out and standing on ice for 2 minutes; adding 500 mul of non-resistant LB culture medium into a centrifuge tube, and shaking at 180rpm for 1 hour; coating 100-200 μ l of bacterial liquid on LB plate with ampicillin resistance;
(5) Picking a single colony to 1ml of LB liquid culture medium containing ampicillin resistance, shaking at 180rpm for 6 hours, and carrying out colony PCR on the bacterial liquid, wherein the reaction system is as follows:
Max Buffer 12.5μl，dNTP Mix(10mM each)0.5μl，
max Super-Fidelity DNA Polymerase 0.5. Mu.l, upstream and downstream primers0.5. Mu.l, 1. Mu.l of bacterial suspension, and finally ddH 2 Supplementing O to 25 μ l; the PCR amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 1 minute, and 35 cycles; extension at 72 ℃ for 10 min;
(6) Separating PCR products by using 1% agarose gel electrophoresis, selecting recombinant clones, sending the recombinant clones to Hangzhou Youkang biological technology limited company for sequencing to obtain 504bp Laodelphax striatellus LsSGSP gene sequence, wherein the obtained sequence is shown as SEQ ID NO. 5.
Example 2 dsRNA Synthesis of Laodelphax striatellus LsSGSP Gene
PCR amplification and purification of T7 primer
(1) Using recombinant plasmid or bacterial liquid containing Laodelphax striatellus LsSGSP gene as template, amplifying target gene by using primer (shown in Table 2) with T7 promoter sequence (LsSGSP-F primer underlined sequence), wherein the primer is synthesized by Youkang Biotechnology Ltd, hangzhou, and the amplification system is
Max Buffer 100μl，dNTP Mix(10mM each)4μl，
Max Super-Fidelity DNA Polymerase 4. Mu.l, upstream and downstream (SEQ ID NO.6 and SEQ ID NO. 7) 4. Mu.l each, recombinant plasmid or bacterial solution 4. Mu.l, and ddH 2 The volume of O is up to 200. Mu.l. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 1 minute, 35 cycles; extension was carried out at 72 ℃ for 10 minutes.
TABLE 2 primers for T7 promoter sequences
(2) Separating the amplified product by agarose gel electrophoresis, and recovering by a DNA agarose gel recovery kit to finally obtain a large amount of single LsSGSP gene fragments containing the T7 promoter.
Synthesis and purification of dsRNA
The dsRNA of the LsSGSP gene is synthesized and purified by using a T7High Yield RNA transcription kit of Novozan biotechnology limited, and the specific method is as follows:
(1) Synthesizing dsRNA by taking an LsSGSP gene segment containing a T7 promoter obtained by PCR amplification as a DNA template, wherein the reaction system is as follows: 2 mul 10 × Reaction Buffer,2 mul ATP solution,2 mul UTP solution,2 mul CTP solution,2 mul GTP solution,2 mul enzyme mix,1 mul DNA template, and using RNase-free water to make up to 20 mul, mixing the Reaction system after the Reaction system is prepared, reacting overnight at 37 ℃;
(2) Adding 1 μ l of TURBO DNase into the reaction system to eliminate DNA in the reaction system, and reacting for 15min at 37 ℃;
(3) Denaturing the reacted sample at 65 ℃ for 5min;
(4) dsRNA concentration was determined by Nanodrop and dsRNA quality was determined by 1% agarose gel electrophoresis (FIG. 2);
(5) The dsRNA of the LsSGSP gene was stored at 80 ℃ until use.
Example 3 introduction of dsRNA of LsSGSP Gene into Laodelphax striatellus
The dsRNA of the LsSGSP gene is introduced into the laodelphax striatellus body by a microinjection method, which comprises the following steps:
(1) Collecting three-age (for mortality statistics) and four-age (for honeydew determination) nymphs of Laodelphax striatellus, and applying the nymphs to the nymphs with CO 2 Anaesthetizing for 10s;
(2) Glass capillaries (Wuhan microprobe scientific instruments, inc.) were drawn to size using a capillary probe puller (P97, letter Instrument) using program parameters set to: heat =800pull =150vel =150time =80;
(3) Respectively injecting dsRNA of the LsSGSP gene into a glass capillary by using a micro-sample application head (Eppendorf);
(4) The glass capillary with the sample added is mounted to a microinjector (Eppendorf), and the dsRNA is introduced into the Laodelphax striatellus under a stereoscope, and the parameters of the microinjector are set as follows: the injection pressure is 1300pah, the injection time is 0.3s, and the compensation pressure is 10pah;
(5) By the same method, dsRNA of an Aequorea victoria green fluorescent protein Gene (GFP) is introduced into the body of the small brown planthopper to serve as a negative control;
(6) And after the Laodelphax striatellus introduced with the dsRNA revives, transferring the Laodelphax striatellus to a rice plant.
Example 4 Laodelphax striatellus mortality statistics
Feeding the injected Laodelphax striatellus nymphs on rice seedlings, placing about 30 Laodelphax striatellus on each group of rice, repeating the steps for 3 groups, and counting the death rate every day, wherein the feeding conditions of the Laodelphax striatellus are as follows: the temperature is 26 +/-0.5 ℃, the relative humidity is 50% +/-5%, and the photoperiod is 1697 h;
the experimental result is shown in fig. 3, the survival curve of laodelphax striatellus (dsLsSGSP) introduced with the LsSGSP gene dsRNA has a significant difference (p < 0.01) compared with that of a control (dsGFP), and the LsSGSP gene dsRNA has a potential application value in controlling the laodelphax striatellus.
Example 5 measurement of the amount of Trapa acori Hance honeydew secretion
The feed intake of the rice planthopper is positively correlated with the honeydew secretion. Therefore, the feeding condition of the laodelphax striatellus is confirmed by measuring the honeydew secretion amount of the laodelphax striatellus treated differently.
Taking rice seedlings in the 4-5 leaf stage, and fixing parafilm on rice stalks by a double-layer clamping method. The dsRNA-treated 5-year-old Laodelphax striatellus nymphs are placed into a parafilm raising chamber according to 10 heads/group, and a certain gap is ensured in the raising chamber, so that the Laodelphax striatellus can contact rice and can move freely. After the Parafilm raising chamber is sealed, the Parafilm raising chamber is placed in an artificial climate chamber for 24 hours, the temperature is controlled to be 26 +/-0.5 ℃, the relative humidity is 50 +/-5%, and the photoperiod is 16h (day: night).
After 24h in the climatic chamber, the plant hopper was carefully removed from the Parafilm rearing chamber and the Parafilm containing the honeydew was weighed out on an electronic balance (sensitivity 0.0001 g). And wiping off the honeydew by using absorbent paper, weighing the Parafilm without the honeydew again, wherein the difference of the mass of the two is the amount of the honeydew secreted by the laodelphax striatellus. Each treatment was performed in 20 independent biological replicates.
The experimental result is shown in fig. 4, the honeydew secretion amount of the laodelphax striatellus (dsLsSGSP) introduced with the dsrssgsp gene dsRNA is obviously lower than that of the control (dsGFP) (p is less than 0.01), which indicates that the LsSGSP gene can influence the feeding of the laodelphax striatellus to further cause the phenotypic abnormality of the laodelphax striatellus.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. The Laodelphax striatellus salivary gland specific gene LsSGSP is characterized in that: the amino acid sequence of the specific gene LsSGSP of the laodelphax striatellus is shown in SEQ ID No. 2.
2. The laodelphax striatellus salivary gland-specific gene LsSGSP as claimed in claim 1, wherein: the nucleotide sequence of the specific gene LsSGSP of the salivary gland of the laodelphax striatellus is shown in SEQ ID No. 1.
3. The primer for amplifying the laodelphax striatellus salivary gland specific gene LsSGSP of claim 1, which is characterized in that: the primers are shown as SEQ ID NO.3 and SEQ ID NO. 4.
4. The dsRNA of the laodelphax striatellus salivary gland-specific gene LsSGSP according to claim 1.
5. The dsRNA of the laodelphax striatellus salivary gland-specific gene LsSGSP as claimed in claim 4, wherein: the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 5.
6. The dsRNA of the laodelphax striatellus salivary gland-specific gene LsSGSP according to claim 4 or 5, wherein: the dsRNA is prepared by the following method: using a DNA template with a sequence described in SEQ ID NO.5 and a T7High Yield RNA transcription kit, and using a Reaction system of 2 mul 10 × Reaction Buffer,2 mul ATP solution,2 mul UTP solution,2 mul CTP solution,2 mul GTP solution,2 mul enzyme mix and 1 mul DNA template, supplementing no RNase water to 20 mul, uniformly mixing the Reaction system after the Reaction system is prepared, and reacting at 37 ℃ for overnight; then adding TURBO DNase to react at 37 ℃ for 15min to eliminate DNA in the reaction system, and carrying out denaturation at 65 ℃ for 5min to obtain the DNA.
7. Use of a reagent for silencing the salivary gland-specific gene LsSGSP of the Laodelphax striatellus of claim 1 in preparation of a control agent for the Laodelphax striatellus.
8. Use according to claim 7, characterized in that: the reagent for silencing the specific gene LsSGSP of the saliva gland of the small brown rice planthopper is dsRNA of the specific gene LsSGSP of the saliva gland of the small brown rice planthopper.
9. Use of an agent for silencing the salivary gland-specific gene LsSGSP of the plant hopper as claimed in claim 1 in reducing the amount of honeydew secretion of the plant hopper.
10. A method of controlling laodelphax striatellus, comprising: the expression of the specific gene LsSGSP of the salivary gland of the small brown planthopper is silenced in the small brown planthopper, and the nucleotide sequence coded by the specific gene LsSGSP of the salivary gland of the small brown planthopper is shown as SEQ ID NO. 2.
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