CN113846073A

CN113846073A - Nilaparvata lugens NlLIPH gene and application thereof

Info

Publication number: CN113846073A
Application number: CN202111029782.8A
Authority: CN
Inventors: 袁龙宇; 张振飞; 肖汉祥; 李燕芳; 梁其昌; 张文庆
Original assignee: Plant Protection Research Institute Guangdong Academy of Agricultural Sciences
Current assignee: Plant Protection Research Institute Guangdong Academy of Agricultural Sciences
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2021-12-28
Anticipated expiration: 2041-09-03
Also published as: CN113846073B

Abstract

The invention discloses a nilaparvata lugens NlLIPH gene and application thereof, wherein dsRNA of the targeted silent nilaparvata lugens NlLIPH gene is designed and obtained by an inventor, the dsRNA is found to cause the feed intake of nilaparvata lugens to be reduced, the survival rate of the nilaparvata lugens is effectively reduced, the dsRNA can also prevent and treat the nilaparvata lugens by inhibiting the molting of the nilaparvata lugens, generating a food refusing effect and influencing the ovarian development of the nilaparvata lugens, and the dsRNA has extremely high and wide application prospect. Moreover, products such as the expression cassette, the recombinant vector or the recombinant bacterium obtained based on the method can also produce the same effect, and have important application value in the field of brown planthopper prevention and control.

Description

Nilaparvata lugens NlLIPH gene and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a Nilaparvata lugens NlLIPH gene and application thereof.

Background

Brown plant hoppers (BPH; Nilaparvata lugens Stal.), belonging to the order Homoptera (Homoptera), the family planthopper (Delphacidae), are among the leading pests of rice farming. Brown planthopper has long-distance migration habit, is a monophagic pest and can only take and breed offspring on rice and common wild rice. The piercing-sucking mouthparts of the brown planthopper can pierce into the plant body to suck phloem juice. However, the damage of the brown planthopper is not limited to eating phloem sap to cause direct damage, and the brown planthopper is also a transmission medium of rice straw-like dwarf virus and odontoblast dwarf virus to cause indirect damage. Brown planthoppers feed rice before the tillering stage, and the spike number and the grain number of the rice are reduced. Eating rice in the mature stage results in a reduction in the number of mature grains and weight. Therefore, when the brown planthopper is seriously damaged, the rice can die in a large area, and no grains are collected. According to statistics, in 2005 to 2007, more than 2500 million hectares of rice paddy fields have brown planthopper disasters each year, which causes 10 to 15 hundred million kilograms of rice to be reduced, and billions of yuan of economic loss is folded.

In the related art, infestation of brown planthopper is mainly controlled by using an insecticide. However, with the use of large quantities of insecticides, brown planthopper is rendered resistant to a number of insecticides, such as delphacidae, carbarfuran, bentazon, and even including the chemical synthetic insecticide pyrethrin. And the spraying of pesticide and insecticide is harmful to human and livestock, and can cause serious environmental pollution, so that the development of a method for scientifically and effectively controlling or killing brown planthopper has important significance for guaranteeing grain safety and environmental safety.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the inventor designs the dsRNA for silencing the Nilaparvata lugens NlLIPH gene, and finds that the dsRNA can reduce the feed intake of Nilaparvata lugens, effectively reduce the survival rate of the dsRNA, inhibit the reproductive capacity of Nilaparvata lugens, and play a role in effectively preventing and controlling Nilaparvata lugens.

In a first aspect of the present invention, there is provided a protein, wherein the protein is:

(1) protein with amino acid sequence shown as SEQ ID NO. 6; or

(2) The protein with the same function is obtained by substituting and/or deleting and/or inserting 1 or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO. 6.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding a protein according to the first aspect of the invention.

According to a second aspect of the invention, in some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No. 5.

In a third aspect of the present invention, there is provided a product for controlling brown planthopper, wherein any one of (1) to (8):

(1) a nucleic acid molecule according to the second aspect of the invention;

(2) an expression cassette comprising a nucleic acid molecule according to the second aspect of the invention;

(3) a recombinant vector comprising a nucleic acid molecule according to the second aspect of the invention;

(4) a recombinant vector comprising the expression cassette described in (2);

(5) a recombinant microorganism comprising a nucleic acid molecule according to the second aspect of the invention;

(6) a recombinant microorganism comprising the expression cassette described in (2);

(7) a recombinant microorganism comprising the expression cassette described in (3);

(8) a recombinant microorganism comprising the expression cassette described in (4).

Wherein the recombinant vector comprises a cell vector and a plasmid vector, and the cell vector does not comprise propagation material.

In a fourth aspect of the invention there is provided the use of a protein according to the first aspect of the invention and/or a product according to the third aspect of the invention for the control of brown planthopper.

According to a fourth aspect of the invention, in some embodiments of the invention, the brown planthopper control comprises:

(1) inhibiting the brown planthopper from taking food;

(2) killing brown planthopper;

(3) inhibiting molting of brown planthopper;

(4) inhibiting reproduction of brown planthopper.

In a fifth aspect of the present invention, there is provided a substance which inhibits or silences the expression of the nilaparvata lugens NlLIPH gene.

RNA interference (RNAi) refers to the phenomenon of double-stranded RNA mediated induction of highly specific degradation of homologous mrnas. Among them, a method of injecting dsRNA synthesized in vitro into a hemolymph of an insect or a specific site using a microinjection apparatus is called a microinjection method, and the microinjection method is one of the most commonly used methods of introducing dsRNA at present. The microinjection method has the following advantages: the injection amount can be accurately controlled; the influence of the reduced expression level of the specific gene on the single brown planthopper can be specifically observed, and the theoretical research make internal disorder or usurp on the gene function is greatly helpful. However, there are some limitations to the study of salivary proteins by RNA interference, for example, some genes of insects appear only in specific age, specific period or specific tissue of insects, or some genes of salivary proteins are indispensable for normal development of insects, if RNA interference is performed on these genes, the insects will die rapidly, and it is difficult to perform experiments on the subsequent action of the genes in insect feeding. Therefore, the selection of a suitable target site is crucial for the effectiveness of RNA interference and subsequent studies.

According to a fifth aspect of the present invention, in some embodiments of the present invention, the substance that inhibits or silences the nilaparvata lugens nliphh gene expression comprises at least one of (1) to (3):

(1) dsRNA, and an expression cassette, a recombinant vector and a recombinant microorganism containing the dsRNA;

(2) siRNA, and an expression cassette, a recombinant vector and a recombinant microorganism containing the siRNA;

(3) dsRNA induces a silencing complex.

NlLIPH is expressed at the time points of different stages of insect states of brown planthopper. In the adult stage, the expression level of NlLIPH is slightly higher than that in the nymph stage, which is probably related to the relatively low food intake and energy metabolism of the brown planthopper nymphs in the 1-3L stage, and in the 4-5L stage, the expression level of NlLIPH is not obviously different from that in the adult stage, so that the NlLIPH can be used as an effective silencing target.

In some preferred embodiments of the invention, the nucleotide sequence of the dsRNA is as shown in SEQ ID NO. 11.

According to a fifth aspect of the invention, in some embodiments of the invention, the brown planthopper comprises a high-causing rice planthopper population and a low-causing rice planthopper population.

In some preferred embodiments of the invention, the brown planthopper comprises at least one of an RH brown planthopper population, a TN1 brown planthopper population.

Through verification of the inventor, the dsRNA can effectively prevent and control the brown planthopper, including high-harmful varieties and weak-harmful populations of the brown planthopper, and has high applicability.

In a sixth aspect of the invention, there is provided the use of a substance according to the fifth aspect of the invention in the manufacture of a product for the control of brown planthopper.

According to a sixth aspect of the present invention, in some embodiments of the present invention, the brown planthopper-controlling product comprises at least one of (1) to (4):

(1) brown planthopper antifeedant;

(2) brown planthopper insecticides;

(3) molting inhibitors of brown planthopper;

(4) brown planthopper reproduction inhibitor.

The inventor finds that dsNlLIPH silencing NlLIPH gene can effectively control the feed intake of brown planthopper, and can also have the same effect on the brown planthopper with resistance gene. Moreover, the survival rate of dsNlLIPH injected under different rice feeding conditions is remarkably reduced, the death rate of the dsNlLIPH is directly related to the NlLIPH gene, and the fact that the survival rate of brown planthopper is directly influenced by the silencing of dsNlLIPH is shown. In addition, through mechanism research, the method is found to prevent and control the brown planthopper mainly through inhibiting molting of the brown planthopper, generating a food refusal effect and influencing the ovarian development of the brown planthopper, so that the method has extremely high and extremely wide application prospect.

According to a sixth aspect of the invention, in some embodiments of the invention, the product further comprises an adjuvant.

In some preferred embodiments of the present invention, the adjuvant comprises at least one of a plant essential oil, a surfactant, a pesticide synergist, a transdermal absorbent, and a bactericide.

Of course, one skilled in the art can also choose to add any adjuvants that are beneficial to their product performance.

In a seventh aspect of the present invention, there is provided a method for producing a transgenic plant, comprising the steps of: introducing any one of the nucleic acid molecules of the following (1) to (2) into a target plant to obtain a transgenic plant;

(1) dsRNA is prepared according to the full length of the coding gene of the protein of the first aspect of the invention or any fragment thereof;

(2) the dsRNA of the fifth aspect of the invention.

The invention has the beneficial effects that:

1. the invention discloses a nucleotide sequence of a Nilaparvata lugens NlLIPH gene for the first time at home and abroad, and firstly discovers that the function of the Nilaparvata lugens on eating can be obviously influenced by inhibiting the expression of the Nilaparvata lugens NlLIPH gene.

2. The dsRNA for silencing the Nilaparvata lugens NlLIPH gene is obtained through design, and is verified to reduce the feed intake of the Nilaparvata lugens through RNAi technology and microinjection, so that the survival rate of the Nilaparvata lugens is effectively reduced, and the dsRNA can be used for preventing and treating the Nilaparvata lugens by inhibiting the moulting of the Nilaparvata lugens, generating a food refusal effect and influencing the development of the ovary of the Nilaparvata lugens and has extremely high and wide application prospect.

3. Products such as an expression cassette, a recombinant vector or recombinant bacteria obtained based on NlLIPH genes and dsRNA for silencing Nilaparvata lugens NlLIPH genes can produce the effects of reducing feed intake of Nilaparvata lugens, reducing the survival rate of Nilaparvata lugens and inhibiting the reproductive capacity of Nilaparvata lugens, and have important application value in the field of Nilaparvata lugens control.

Drawings

FIG. 1 shows the expression of NlLIPH in different tissues of Nilaparvata lugens in the examples of the present invention;

FIG. 2 shows the NlLIPH expression levels of nymphs and adults of brown planthopper at different ages in the example of the present invention;

FIG. 3 is a graph showing the temporal expression pattern of the NlLIPH gene after gene silencing in examples of the present invention, the ordinate being a multiple;

fig. 4 is a schematic diagram of the detection of the feeding behavior of the brown planthoppers and the wave patterns in the embodiment of the invention, wherein a is the wave pattern change in the feeding process of the brown planthoppers, B is a statistical graph of the wave patterns of TN1 rice fed by the brown planthoppers in different treatment groups, C is a statistical graph of the wave patterns of IR56 rice fed by the brown planthoppers in different treatment groups, and D is a statistical graph of the wave patterns of ASD7 rice fed by the brown planthoppers in different treatment groups;

FIG. 5 is a graph comparing the effect of NlLIPH silencing on feed intake of Nilaparvata lugens in the present example, where A is the effect of different resistant rice plants on Nilaparvata lugens secretion and B is the effect of NlLIPH silencing on Nilaparvata lugens secretion;

FIG. 6 is a graph comparing the effect of NlLIPH silencing on Nilaparvata lugens mortality in examples of the present invention, where A is the effect of NlLIPH silencing on TN1 rice on Nilaparvata lugens mortality, B is the effect of NlLIPH silencing on IR56 rice on Nilaparvata lugens mortality, and C is the effect of NlLIPH silencing on ASD7 rice on Nilaparvata lugens mortality;

FIG. 7 is an electrophoresis chart of NlLIPH proteins of brown planthopper populations TN1-P and RH-P in different treatment groups according to the example of the present invention;

FIG. 8 is a diagram of two dead brown planthoppers in an example of the present invention;

FIG. 9 is an image of ovaries following injection with GFP dsRNA (A) and NlLIPH dsRNA (B).

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Experimental Material

The brown planthopper used in the following examples was bred at the plant protection institute of agricultural academy of Guangdong province in a conventional breeding manner at a breeding temperature of 26 + -2 deg.C.

RH-P represents the highly-harmful population of brown planthopper.

TN1-P represents a low-virulent population of brown planthopper.

Wherein RH-P is the brown planthopper population of the rice with high pest resistance obtained by screening after brown planthopper is propagated on RH rice (carrying Bph3 insect-resistant gene) for multiple generations. TN1-P is the brown planthopper population of low-harm-resistant rice obtained by screening after TN1 rice (insect-susceptible rice) is bred for multiple generations.

The screening method adopts a honeydew method.

Acquisition of the genome of Nilaparvata lugens

Randomly selecting 20 RH-P brown planthoppers, and extracting total RNA.

The method for extracting the total RNA of the brown planthopper adopts a Trizol method, and comprises the following specific operations:

1) the brown planthopper samples were individually placed in grinding tubes, snap frozen in liquid nitrogen, added 100 μ L Trizol reagent and immediately ground well on ice.

2) Adding 900 μ L Trizol reagent, blowing, cleaning residual brown planthopper tissue on grinding rod, mixing well, and standing at room temperature for 10 min.

3) Adding 200 mu L of trichloromethane, quickly shaking for 15s, and standing at room temperature for 10 min.

4) Centrifuging at 12000g for 15min at 4 deg.C, sucking supernatant, adding 500 μ L dimethyl methanol, shaking rapidly for 15s, and standing at room temperature for 10 min.

5) Centrifuging at 12000g for 15min at 4 deg.C, discarding supernatant, and adding 400 μ L75% ethanol to wash precipitate.

6) Centrifuging at 7500g for 5min at 4 deg.C, discarding supernatant, air drying precipitate to obtain total RNA.

Adding 30 μ L diethyl pyrocarbonate (DEPC water) to dissolve total RNA, quickly freezing in liquid nitrogen for 5s, and storing at-80 deg.C.

The total RNA extracted was reverse transcribed using the PrimeScript RT reagent Kit with the gDNAeraser first strand cDNA Synthesis Kit from Takara to obtain a cDNA template, for the details of the procedure described in the specification.

The resulting cDNA template was stored at-20 ℃ for future use.

Cloning of Nilaparvata lugens NlLIPH (Nilaparvata lugens H-type lipase) gene

The open reading frame of the NlLIPH gene was amplified using the following open reading frame primers, using the cDNA templates of the above examples.

The nucleotide sequence of the NlLIPH gene open reading frame primer is as follows:

NLLIPH-CDS-F1：5’-CCAGAGCCGAGAAAAGAACAATG-3’(SEQ ID NO.1)；

NLLIPH-CDS-F2：5’-CCAGAGCCGAGAAAAGAACAATGAATGC-3’(SEQ ID NO.2)；NLLIPH-CDS-R1：5’-TTTCTCAACGCCGGTAAATAGGA-3’(SEQ ID NO.3)；

NLLIPH-CDS-R2：5’-ATTGATGTATTTTTGATGGAAATAAATTGA-3’(SEQ ID NO.4)。

the nucleotide sequence of the amplified product (ORF of NlLIPH) obtained by amplification is as follows:

5'-ATGTTTTTATTTTTCAGACACAATCCAACCAGAGCCGAGAAAAGAACAATGAATGCCTCCTGTCTGCCAGTGTTCAAGTACTTCAAGGCAGGCAGACCTTCCAAGATGTTGATTCATGGCTTCGGAGACAGTGTGCAAGACTCCATCATGTATCCGATACTCAGAGATGCATTCCTGACGAAAGGTGACTACAACATATTTCTGTTGGACTGGTCGGATTTGGCGGCGACACCCTGGTACAACAGGGCGATGCGAAACACTGAGACAGTGGCACGACAGGCGGCCGGCCTCATAGACCACCTGTGCAGGTCTACCGGAGCTGAAGCCTCCAGCTTCCACCTGGTTGGCTTCAGTCTGGGGTCGCACATTGCCGGCATGATCGGCCAGTTTGTCAAGACCGGCAAAATCAAACGGATCACCGCTCTTGATCCGGCCCAAGTCCTATTTACCGGCGTTGAGAAAAGCAGGAGATTGGATGAAACTGACGCTCATTTAGTCGAAGTGGTGCATACCTCGGGAGGATATCTCGGCTTCCAGGATCCAATTGGACATAGAGATATATTTCCCAACGGAGGTTCCTGGCCACAGCCTGGCTGTTTCCTAGATTATGCTGCTGTTTGCAGTCACAGAAGAGCCTACTATTATTACGGCGAAGCCGTGAGGAACAAGAGAGGATTTCGAGCGATTCCTTGCAGCAACTGGCCTGACTACATGGCCGGATCGTGTAAAAATAACATTGAAAACTCGTTTGAATTGGCAAATGATCACAATACAAAAAAATACAGAGGAAAGTATTTCTTGAACACCAACAGTGATGAGCCTTTTGGGATAATTGATAAACCAGTCAGGGAGACCAACACCAACGATTATGACTATTTTGGAATTTTGTTTGATGAATTCCAGAACTACGTT-3'(SEQ ID NO.5)。

the amino acid sequence is as follows:

MFLFFRHNPTRAEKRTMNASCLPVFKYFKAGRPSKMLIHGFGDSVQDSIMYPILRDAFLTKGDYNIFLLDWSDLAATPWYNRAMRNTETVARQAAGLIDHLCRSTGAEASSFHLVGFSLGSHIAGMIGQFVKTGKIKRITALDPAQVLFTGVEKSRRLDETDAHLVEVVHTSGGYLGFQDPIGHRDIFPNGGSWPQPGCFLDYAAVCSHRRAYYYYGEAVRNKRGFRAIPCSNWPDYMAGSCKNNIENSFELANDHNTKKYRGKYFLNTNSDEPFGIIDKPVRETNTNDYDYFGILFDEFQNYV(SEQ ID NO.6)。

preparation of dsNlLIPH

(1) Obtaining of recombinant plasmid:

the PCR amplification product obtained in the above example was ligated with Takara pMD18-T vector and transformed into competent cells. Culturing and subculturing the cells without the transformation problems, cracking the cells, and extracting the recombinant plasmid.

(2) Synthesis of dsNlLIPH:

taking the recombinant plasmid and the GFP recombinant plasmid (the preparation method is the same as the recombinant plasmid) to carry out cross PCR (overlap PCR) amplification, and the specific steps are as follows:

the A reaction system in the cross PCR is as follows:

TABLE 1A reaction System

Components	Dosage of
		GFP recombinant plasmid	1.0μL
NlLIPH-RNAi-F(10μM)	1.0μL
		NlLIPH-RNAi-RT7(10μM)	1.0μL
10 XPCR buffer (with magnesium salt)	2.5μL
		dNTP mix (2.5 mM each)	2.0μL
HiFi Taq(5U/μL)	0.2μL
		dH₂O	17.3μL

The B reaction system in the cross PCR is as follows:

TABLE 2B reaction System

The PCR reaction program of the A system and the B system is as follows: pre-denaturation at 94 ℃ for 5 min; the reaction stage is as follows: circulating for 35 times at 94 deg.C for 30s, 57 deg.C for 30s, and 72 deg.C for 1 min; extension at 72 ℃ for 10min and storage of the amplification product at 4 ℃.

Wherein the NlLIPH-RNAi-F sequence is as follows: 5'-AATTGGGCGAATTACAGGTTTGG-3' (SEQ ID NO. 7);

the NlLIPH-RNAi-R sequence is as follows: 5'-TGAGCACGTAAGTGAGAACAAGT-3' (SEQ ID NO. 8);

NlLIPH-RNAi-F (forward primer): 5' -GGATCCTAATACGACTCACTATAGGAATTGGGCGAATTACAGGTTTGG-3'(SEQ ID NO.9)；

NlLIPH-RNAi-R (reverse primer): 5' -GGATCCTAATACGACTCACTATAGGTGAGCACGTAAGTGAGAACAAGT-3'(SEQ ID NO.10)。

The underlined region is the T7 RNA polymerase promoter sequence.

The amplification products of the above A system and B system were transcribed in vitro using methods conventional in the art.

Mixing the transcription products of the A system and the B system, carrying out constant temperature water bath at 70 ℃ for 10min, standing at room temperature for 20min, slightly shaking and centrifuging, adding 2 mu L of RQ1 RNase Free DNase and 2 mu L of RNase A solution diluted by 200 times, and carrying out constant temperature water bath at 37 ℃ for 30min to obtain dsNlLIPH.

(3) Purification of dsNlLIPH:

to the obtained dsNlLIPH, 4.4. mu.L of sodium 3M acetate (pH5.2) and 110. mu.L of 95% ethanol were added, mixed by pipetting, and allowed to stand on ice for 5 min. Centrifuge at 14000rpm for 10min and discard the supernatant. The precipitate was washed with 0.5mL of 70% glacial ethanol. Centrifuge at 14000rpm for 10min and discard the supernatant. Drying for 15min, adding 50 μ L DEPC water to dissolve the precipitate to obtain purified dsNlLIPH.

The nucleotide sequence of the resulting dsrna (dsnlliph) is:

5'-CAUUGCCGGCAUGAUCGGCCAGUUUGUCAAGACCGGCAAAAUCAAACGGAUCACCACUGACGCUCAUUUAGUCGAAGUGGUGCAUACCUCGGGAGGAUAUCUCGGCUUCCAGGAUCCAAUUGGACAUAGAGAUAUAUUUCCCAACGGAGGUUCCUGGCCACAGCCUGGCUGUUUCCUAGAUUAUGCUGCUGUUUGCAGUCACAGAAGAGCCUACUAUUAUUACGGCGAAGCCGUGAGGAACAAGAGAGGAUUUCGAGCGAUUCCUUGCAGCAACUGGCCUGACUACAUGGCCGGAUCGUGUAAAAAUAACAUUGAAAACUCGUUUGAAUUGGCAAAUGAUCACAAUACAAAAAAAUACAGAGGAAAGUAUUUCUUGAACACCAACAGUGAUGAGCCUUUUGGGAUAAUUGAUAAACCAGUCAGGGAGACCAACA-3'(SEQ ID NO.11)。

expression of Nilaparvata lugens NlLIPH gene in different tissues of Nilaparvata lugens

The expression level of the NlLIPH gene was examined in 7 different tissues of brown planthopper using Trizol method.

The method comprises the following specific steps:

female brown planthoppers were dissected In Trizol solution, 7 different tissues (midgut (Mg), salivary gland (Sg), adipose body (Fb), muscle (Tm), ovary (Ov), foot (Lg), and epidermis (In)) were taken, total RNA was extracted by the Trizol method In the above example, and after reverse transcription into cDNA, nliphh expression amount In each tissue was detected using nliphh primer (nliphh-qPCR-fniliphh-qPCR-R) using qPCR technique.

Wherein the nucleotide sequence of NlLIPH-qPCR-F is as follows: 5'-ACTGACGCTCATTTAGTCGAAGT-3' (SEQ ID NO. 12); the nucleotide sequence of NlLIPH-qPCR-R is as follows: 5'-ATCGCTCGAAATCCTCTCTTGTT-3' (SEQ ID NO. 13).

The results are shown in FIG. 1.

It was found that NlLIPH was expressed in each tissue of brown planthopper, and the expression level of NlLIPH in muscle was the lowest, only about 0.5 times. NlLIPH is expressed in the ovary at the highest level, and then in the fat body (about 1.25 times), the midgut (about 1.21 times), the salivary gland (about 1.11 times), while in the epidermis (about 0.77 times) and the foot (about 0.83 times) at lower levels.

Expression condition of Nilaparvata lugens NlLIPH gene in Nilaparvata lugens in different age stages

The method comprises the steps of taking RH-P populations of brown planthoppers as experimental objects, respectively extracting total RNA (adopting a Trizol method) of the brown planthoppers in different age stages (1-5 instar nymphs and short-wing female brown planthoppers after eclosion for 1, 3, 5, 7, 9, 11, 13 and 15 days), carrying out reverse transcription to obtain cDNA, and detecting the expression amount of NlLIPH in the brown planthoppers in different age stages by using a qPCR (quantitative polymerase chain reaction) technology and NlLIPH primers (sequences shown in SEQ ID NO.12 and 13).

The results are shown in FIG. 2.

Quantitative qPCR detection shows that NlLIPH is expressed at the time points of different stages of insect states of brown planthopper. In the adult stage, the expression level of NlLIPH is slightly higher than that in the nymph stage, which is probably related to the relatively low food intake and energy metabolism of the brown planthopper nymphs in the 1-3L stage, but the expression level of NlLIPH is not obviously different from that in the adult stage in the 4-5L stage.

Silencing Effect of dsNlLIPH

RH-P brown planthopper four-year nymphs are taken as test objects.

RH-P brown planthopper four-year nymphs and CO with similar random growth vigor₂Anesthesia is performed for 20 s. It should be noted that CO was introduced₂The insect is not directly blown into the test chamber, so that collision damage caused by flying of the insect is avoided, and the test accuracy is not influenced. The anaesthetized worms were poured onto a 1.5% agar powder plate (obtained by mixing 1.5g agar powder with 100mL water, boiling, pouring into a glass plate and solidifying) with the abdomen facing up. dsRNA (dsNlLIPH, from the above examples) was injected using a Nanoliter 2010 microinjector according to the instructions, with the injection site between the anterior and medial thorax and an injection volume of 46. mu.L (5. mu.g/. mu.L). Taking the day of completion of dsRNA injection as day 0, sampling was started from day 1 after injection, and three samples were taken every day for three days. Meanwhile, brown planthopper which was not injected and injected with dsGFP (GFP dsRNA) was used as a control, and the change in gene expression amount was verified by RT-PCR.

The detection steps of RT-PCR are as follows:

after sampling, RNA was extracted as described in the above example, and reverse transcription was performed using the PrimeScript RT reagent Kit of TAKARA with gDNA Eraser according to the instructions to obtain cDNA. Taking 5. mu.L of the prepared cDNA as a template, ddH₂O diluted 10-fold and amplified on CFX96touch TM Real-Time PCR Detection System (Bio-Rad).

The amplification system is as follows:

TABLE 3 real-time quantitative PCR

Components	Dosage of
		DNase/RNase-Free ddH₂O	2.9μL
2×Supermix	4μL
		NlLIPH-qPCR-F(5mM)	0.6μL
NlLIPH-qPCR-R(5mM)	0.6μL
		cDNA template	0.5μL

The amplification procedure was: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5-10 s, annealing and extension at 55-65 ℃ for 30s, and repeating for 30 cycles. The preservation temperature is 10 ℃, and the internal reference gene actin is used as a reference.

The nucleotide sequence of the reference gene actin primer is as follows:

qPCR-actin-F:5'-ATGAAACCGTCTACAACTCG-3'(SEQ ID NO.14)；

qPCR-actin-R:5'-GCATCCTGTCGGCAATAC-3'(SEQ ID NO.15)。

dsRNA (dsNlLIPH) is injected into RH-P brown planthopper in the same way, and the change of gene expression quantity is verified by using the method when 2 brown planthoppers survive randomly at 12h, 24h, 48h and 72h after injection respectively.

The results are shown in FIG. 3.

It was found that RH-Pbrown planthopper produces a relatively significant effect 12h after dsRNA (dsNlLIPH) injection. The reduction in NlLIPH expression levels was more pronounced after 24h injection. After 48h of injection, the expression level of the RH-plnilla planthopper nliphh injected with dsnliph was only 47.3% of the control (GFP dsRNA injected), and the NlLIPH expression level was further reduced to a significant level (P < 0.05). The NlLIPH expression level was further reduced to very significant levels (P <0.01) 72h after injection. From the above results, it can be seen that the injection of dsnliph in this example can reduce the expression level of NlLIPH to a significant level within 48h after the injection, and therefore, it can be considered that the dsRNA fragment can successfully induce target gene silencing in brown planthopper.

Control and killing effect of dsNlLIPH on Nilaparvata lugens

(1) Effect of dsNlLIPH on feed intake of brown planthopper:

the feed intake and the pest-causing ability of brown planthopper, the piercing-sucking insect, are closely related, and the amount of honeydew secretion is usually used for measuring the pest-causing ability.

Selecting 10 heads of brown planthopper primary emergence imagoes of TN1-P and RH-P populations with similar growth vigor, starving for half an hour in advance, respectively connecting the imagoes to 4 types of rice, namely sensitive rice TN1, resistant rice RH (the resistant gene is Bph3), resistant rice ASD7 (the resistant gene is Bph2) and resistant rice IR56 (the resistant gene is Bph3), and recording honeydew secretion amount within 24 hours.

The results are shown in FIG. 4.

It can be found that the TNI-P population can normally feed on TN1 rice, but the feed intake of 3 resistant rice is reduced, and the reduction degree reaches a remarkable level. Wherein, the food intake of the TNI-P population on the RH rice is only half of that of TN1 rice, which indicates that TN1-P can not normally take food on the RH rice. The RH-P population can normally take food on 3 resistant rice and TN1 rice, the food taking amount on the RH rice is the same as that of TN1 rice, and the food taking amount on IR56 rice containing a Bph3 resistance gene is not obviously different, which shows that the brown planthopper of the RH-P population can adapt to the rice containing the Bph3 resistance gene and can normally take food. However, the food intake of the RH-P population on RH rice is obviously different from that of rice ASD7 containing Bph2 resistance gene, which shows that the brown planthopper of the RH-P population has weaker capability of adapting to the Bph2 resistance gene than that of the Bph3 resistance gene.

Based on the above results, dsNlLIPH was further introduced into the experiment.

dsNlLIPH was injected into RH-P population brown planthopper female adults according to the above example, and three days after the injection of dsNlLIPH, they were transferred to sensitive rice TN1, ASD7 rice containing Bph2 resistance gene and IR56 containing Bph3 resistance gene, respectively, and their honeydew secretion amount within 24h was observed.

The results are shown in FIG. 5.

It can be found that, for the brown planthopper three days after the dsnllihh injection, the honeydew secretion amount on TN1 rice is 77.17% of that of the dsGFP in the control group, and no significant difference exists, which indicates that the brown planthopper can normally take food on non-resistant rice after the silencing of the brown planthopper nllihh gene. On the other hand, in rice IR56 containing the resistance gene Bph3, after the NlLIPH gene is silenced by dsNlLIPH, compared with a control group, the honeydew secretion amount of brown planthopper is reduced by 85.75%, and the difference is very obvious. After NlLIPH is silenced on rice ASD7 containing Bph2 resistance genes, compared with a control group, the honeydew secretion amount of brown planthoppers is reduced by 22.38%, and the obvious difference exists because the NlLIPH silence can obviously reduce the resistance of the brown planthoppers to Bph3 and also reduce the resistance to Bph 2. In conclusion, it can be considered that the NlLIPH gene is not only closely related to the food intake of the brown planthopper, but also related to the capability of the NlLIPH gene to damage rice containing the Bph3 resistance gene to the brown planthopper, and the damage degree of the brown planthopper to the rice containing the Bph3 resistance gene can be effectively controlled by silencing the NlLIPH gene through dsNlLIPH.

(2) Effect of dsNlLIPH on mortality of brown planthopper:

in order to explore the death condition of brown planthopper caused by killing NlLIPH gene when different resistant rice is damaged, the RH-Pbrown planthopper population is specially used as a test object for testing, and the specific detection steps are as follows:

randomly selecting a proper amount of RH-P brown planthopper population with similar growth vigor to perform different treatments on the initial emergence female imagoes: the experimental group was injected with dsNlLIPH and the control group with GFP dsRNA using the methods described in the examples above. After the RNAi interference is confirmed to be successful, the surviving brown planthoppers are respectively inoculated to RH rice, ASD7 rice and TN1 rice for feeding, and the survival rates of the brown planthoppers on different resistant rice are observed and recorded. The experiment was repeated 3 times.

The results are shown in FIG. 6.

For RH-P population primary eclosion female adults bred 1 day after dsNlLIPH injection and inoculated on TN1 rice, the survival rate is always remarkably lower than that of a dsGFP control group within 3-10, and the death amplitude of brown planthopper in the test is the largest on the 6 th day. For brown planthopper which is injected for 1 day after dsNlLIPH and is connected to ASD7 rice, the survival rate of the brown planthopper is always lower than that of a dsGFP control group within 4-10 days, and the survival rate is obviously different, and in the test, the death of the brown planthopper is concentrated on the 4-6 days after injection. When experiments were performed on IR56 rice, the survival rate of the dsNlLIPH experimental group was consistently significantly lower than the dsGFP control group at day 3-10 post-injection, and brown planthopper mortality was mainly concentrated on day 4 post-injection. In summary, it can be found that, on day 4 basis, NlLIPH silencing by dsNlLIPH injection reduced the survival rate of brown planthopper 32.22% on TN1 pest-resistant rice, 31.11% on ASD7 pest-resistant rice and 51.11% on IR56 pest-resistant rice.

(3) Effect of dsNlLIPH on protein expression patterns of different nociceptive brown planthopper populations:

respectively randomly taking 20 TN1 and RH-P brown planthopper female adults with similar growth vigor to be inoculated on RH rice stalks for eating for 24 hours. And extracting the total protein of the brown planthopper by using PBS according to a conventional method, and concentrating. Western blot was then performed on the concentrated protein samples with NlLIPH antibody.

The results are shown in FIG. 7.

From the electrophorogram, it was found that all bands were uniform in size (34.55kDa), wherein

lanes

1 and 2 in FIG. 7 represent the bands of NlLIPH protein detected after feeding RH rice from the TN1 population of brown planthopper.

Lanes

3 and 4 represent the in vivo protein content of brown planthopper after intake of RH rice after silencing NlLIPH, and there is no band in both lanes indicating that there is no NlLIPH protein.

Lanes

5 and 6 show the NlLIPH protein bands detected after feeding RH rice by the brown planthopper in the RH-P population. When all the samples were tested with the secondary antibody only, no bands were detected in the Western blot results (data not shown). The results prove that the expression amount of the NlLIPH protein in the RH-P population of the brown planthopper is higher than that of the TN1 population, and the expression of the NlLIPH protein can be obviously reduced after the NlLIPH protein is interfered. The results show that the NlLIPH protein can improve the resistance of the brown planthopper to RH rice, and the NlLIPH expression inhibition can reduce the resistance of the brown planthopper, so that the effect of controlling the number of the brown planthopper population is achieved.

(4) Effect of dsNlLIPH on molting of brown planthopper:

to further explore the effect of NlLIPH on the mortality of brown planthoppers, the inventors collected the dead brown planthopper populations after dsNlLIPH injection in the above examples and examined them under microscope.

The results are shown in FIG. 8.

It was found that the dead brown planthopper phenotype after dsNlLIPH injection was mainly concentrated in two. The first is dsNlLIPH injection, which presents a significant ecdysone disorder compared to the control group, which is mainly manifested 3 days before RNAi intervention. The other death phenotype is mainly the belly of brown planthopper is shriveled, and the phenomenon mainly occurs in 4-10 days after dsNlLIPH injection. This indicates that silencing NlLIPH inhibits molting of brown planthopper, thereby achieving the effect of controlling brown planthopper.

(5) Effect of dsNlLIPH on brown planthopper ovary:

the NlLIPH has an indispensable function in the lipid metabolism of the brown planthopper, and the lipid plays an important role in the feeding and digestion of the brown planthopper and also plays a key role in the reproduction of the brown planthopper. Therefore, the inventors further investigated the role of NlLIPH in reproductive capacity of brown planthoppers.

And selecting the primary eclosion female adults in the RH-P brown planthopper population with similar growth vigor, injecting dsNlLIPH, and injecting GFP dsRNA to the control group. After 7 days of injection, the ovaries of the surviving brown planthoppers were selected for dissection.

As a result, in the control group injected with GFP dsRNA, the ovary development was significantly better than that of the brown planthopper in the experimental group injected with dsNlLIPH. The experimental group injected with dsNlLIPH had insufficiently developed and plump ovaries of brown planthoppers, more silk threads, fewer eggs and more empty ovaries (FIG. 9), which indicates that NlLIPH genes can influence the development of the ovaries of the brown planthoppers. Therefore, the dsNlLIPH can also achieve the purpose of controlling the brown planthopper by inhibiting the ovarian development of the brown planthopper.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> institute for plant protection of academy of agricultural sciences of Guangdong province

<120> Nilaparvata lugens NlLIPH gene and application thereof

<130>

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> DNA

<213> Artificial sequence

<400> 1

ccagagccga gaaaagaaca atg 23

<210> 2

<211> 28

<212> DNA

<213> Artificial sequence

<400> 2

ccagagccga gaaaagaaca atgaatgc 28

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence

<400> 3

tttctcaacg ccggtaaata gga 23

<210> 4

<211> 30

<212> DNA

<213> Artificial sequence

<400> 4

attgatgtat ttttgatgga aataaattga 30

<210> 5

<211> 912

<212> DNA

<213> NlLIPH

<400> 5

atgtttttat ttttcagaca caatccaacc agagccgaga aaagaacaat gaatgcctcc 60

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ttcggagaca gtgtgcaaga ctccatcatg tatccgatac tcagagatgc attcctgacg 180

aaaggtgact acaacatatt tctgttggac tggtcggatt tggcggcgac accctggtac 240

aacagggcga tgcgaaacac tgagacagtg gcacgacagg cggccggcct catagaccac 300

ctgtgcaggt ctaccggagc tgaagcctcc agcttccacc tggttggctt cagtctgggg 360

tcgcacattg ccggcatgat cggccagttt gtcaagaccg gcaaaatcaa acggatcacc 420

gctcttgatc cggcccaagt cctatttacc ggcgttgaga aaagcaggag attggatgaa 480

actgacgctc atttagtcga agtggtgcat acctcgggag gatatctcgg cttccaggat 540

ccaattggac atagagatat atttcccaac ggaggttcct ggccacagcc tggctgtttc 600

ctagattatg ctgctgtttg cagtcacaga agagcctact attattacgg cgaagccgtg 660

aggaacaaga gaggatttcg agcgattcct tgcagcaact ggcctgacta catggccgga 720

tcgtgtaaaa ataacattga aaactcgttt gaattggcaa atgatcacaa tacaaaaaaa 780

tacagaggaa agtatttctt gaacaccaac agtgatgagc cttttgggat aattgataaa 840

ccagtcaggg agaccaacac caacgattat gactattttg gaattttgtt tgatgaattc 900

cagaactacg tt 912

<210> 6

<211> 304

<212> PRT

<213> NlLIPH

<400> 6

Met Phe Leu Phe Phe Arg His Asn Pro Thr Arg Ala Glu Lys Arg Thr

1 5 10 15

Met Asn Ala Ser Cys Leu Pro Val Phe Lys Tyr Phe Lys Ala Gly Arg

20 25 30

Pro Ser Lys Met Leu Ile His Gly Phe Gly Asp Ser Val Gln Asp Ser

35 40 45

Ile Met Tyr Pro Ile Leu Arg Asp Ala Phe Leu Thr Lys Gly Asp Tyr

50 55 60

Asn Ile Phe Leu Leu Asp Trp Ser Asp Leu Ala Ala Thr Pro Trp Tyr

65 70 75 80

Asn Arg Ala Met Arg Asn Thr Glu Thr Val Ala Arg Gln Ala Ala Gly

85 90 95

Leu Ile Asp His Leu Cys Arg Ser Thr Gly Ala Glu Ala Ser Ser Phe

100 105 110

His Leu Val Gly Phe Ser Leu Gly Ser His Ile Ala Gly Met Ile Gly

115 120 125

Gln Phe Val Lys Thr Gly Lys Ile Lys Arg Ile Thr Ala Leu Asp Pro

130 135 140

Ala Gln Val Leu Phe Thr Gly Val Glu Lys Ser Arg Arg Leu Asp Glu

145 150 155 160

Thr Asp Ala His Leu Val Glu Val Val His Thr Ser Gly Gly Tyr Leu

165 170 175

Gly Phe Gln Asp Pro Ile Gly His Arg Asp Ile Phe Pro Asn Gly Gly

180 185 190

Ser Trp Pro Gln Pro Gly Cys Phe Leu Asp Tyr Ala Ala Val Cys Ser

195 200 205

His Arg Arg Ala Tyr Tyr Tyr Tyr Gly Glu Ala Val Arg Asn Lys Arg

210 215 220

Gly Phe Arg Ala Ile Pro Cys Ser Asn Trp Pro Asp Tyr Met Ala Gly

225 230 235 240

Ser Cys Lys Asn Asn Ile Glu Asn Ser Phe Glu Leu Ala Asn Asp His

245 250 255

Asn Thr Lys Lys Tyr Arg Gly Lys Tyr Phe Leu Asn Thr Asn Ser Asp

260 265 270

Glu Pro Phe Gly Ile Ile Asp Lys Pro Val Arg Glu Thr Asn Thr Asn

275 280 285

Asp Tyr Asp Tyr Phe Gly Ile Leu Phe Asp Glu Phe Gln Asn Tyr Val

290 295 300

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence

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<211> 23

<212> DNA

<213> Artificial sequence

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tgagcacgta agtgagaaca agt 23

<210> 9

<211> 48

<212> DNA

<213> Artificial sequence

<400> 9

ggatcctaat acgactcact ataggaattg ggcgaattac aggtttgg 48

<210> 10

<211> 48

<212> DNA

<213> Artificial sequence

<400> 10

ggatcctaat acgactcact ataggtgagc acgtaagtga gaacaagt 48

<210> 11

<211> 434

<212> RNA

<213> Artificial sequence

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cauugccggc augaucggcc aguuugucaa gaccggcaaa aucaaacgga ucaccacuga 60

cgcucauuua gucgaagugg ugcauaccuc gggaggauau cucggcuucc aggauccaau 120

uggacauaga gauauauuuc ccaacggagg uuccuggcca cagccuggcu guuuccuaga 180

uuaugcugcu guuugcaguc acagaagagc cuacuauuau uacggcgaag ccgugaggaa 240

caagagagga uuucgagcga uuccuugcag caacuggccu gacuacaugg ccggaucgug 300

uaaaaauaac auugaaaacu cguuugaauu ggcaaaugau cacaauacaa aaaaauacag 360

aggaaaguau uucuugaaca ccaacaguga ugagccuuuu gggauaauug auaaaccagu 420

cagggagacc aaca 434

<210> 12

<211> 23

<212> DNA

<213> Artificial sequence

<400> 12

actgacgctc atttagtcga agt 23

<210> 13

<211> 23

<212> DNA

<213> Artificial sequence

<400> 13

atcgctcgaa atcctctctt gtt 23

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

atgaaaccgt ctacaactcg 20

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence

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gcatcctgtc ggcaatac 18

Claims

1. A protein, wherein said protein is:

(1) protein with amino acid sequence shown as SEQ ID NO. 6; or

2. A nucleic acid molecule encoding the protein of claim 1;

the nucleotide sequence of the nucleic acid molecule is preferably shown as SEQ ID NO. 5.

3. A product for controlling brown planthopper, which is characterized by comprising any one of (1) to (8):

(1) the nucleic acid molecule of claim 2;

(2) an expression cassette comprising the nucleic acid molecule of claim 2;

(3) a recombinant vector comprising the nucleic acid molecule of claim 2;

(4) a recombinant vector comprising the expression cassette described in (2);

(5) a recombinant microorganism comprising the nucleic acid molecule of claim 2;

4. Use of a protein according to claim 1 and/or a product according to claim 3 for the control of brown planthopper.

5. A substance for suppressing or silencing the expression of the NlLIPH gene of the brown planthopper, which is characterized by comprising at least one of (1) to (3):

(3) dsRNA induces a silencing complex.

6. The agent according to claim 5, wherein the dsRNA has the nucleotide sequence shown in SEQ ID No. 11.

7. The substance as claimed in claim 5, wherein the brown planthopper comprises a high-and low-pest-causing rice planthopper population, and the brown planthopper preferably comprises at least one of an RH brown planthopper population and a TN1 brown planthopper population.

8. The use of the substance of claim 5 for the preparation of a product for controlling brown planthopper;

the brown planthopper control product comprises at least one of (1) to (4):

(1) brown planthopper antifeedant;

(2) brown planthopper insecticides;

(3) molting inhibitors of brown planthopper;

(4) brown planthopper reproduction inhibitor.

9. The use as claimed in claim 8, wherein the product for controlling brown planthopper further comprises an auxiliary agent, and the auxiliary agent comprises at least one of plant essential oil, surfactant, pesticide synergist, transdermal absorbent and bactericide.

10. A method of making a transgenic plant comprising the steps of:

introducing any one of the nucleic acid molecules of the following (1) to (2) into a target plant to obtain a transgenic plant;

(1) dsRNA produced by using the full length of the gene coding for the protein according to claim 1 or any fragment thereof;

(2) the dsRNA of claim 6.