CN114807156A - Application of brown planthopper NCS2 gene or encoded protein as target point in preparation of drugs for preventing and treating brown planthopper - Google Patents

Abstract

The invention discloses the application of a brown planthopper NCS2 gene or an encoded protein as a target in preparing a drug for controlling the brown planthopper, and relates to the technical field of plant genetic engineering. The present invention finds the role of the rice pest NCS2 gene in the control of N. lugens. Since N. lugens has efficient RNAi effect, this advantage can be used to interfere with the expression of N. lugens NCS2 gene in the insect. The dsRNA of NCS2 gene was obtained by RNAi technology, and the NCS2 gene was silenced by microinjection to reduce its transcription level. The ability to inhibit the reproduction of brown planthopper and realize the control of brown planthopper. During the research on the function of the NCS2 gene, the present invention finds that the use of RNAi technology to interfere with the expression of the gene in the 2-5 instar nymphs of N. lugens produces an obvious lethal effect.

Description

Application of brown planthopper NCS2 gene or encoded protein as target in preparation of brown planthopper medicine

technical field

The invention relates to the technical field of plant genetic engineering, in particular to the application of the NCS2 gene or the encoded protein of N. lugens as a target in preparing a drug for controlling N. lugens.

Background technique

Intracellular calcium ion is an important second messenger in eukaryotic cells. Calcium ion-binding proteins mediate intracellular calcium signaling. Most calcium-binding proteins contain one or more pairs of EF-hand domains. The EF-hand domain has a high affinity for calcium ions. The EF-hand domain is usually 29 amino acids and has a helix-loop-helix structure (Tsujimoto T, Jeromin A, Saitoh N, Roder JC, Takahashi T. 2002. Neuronal calciumsensor 1 and activity-dependent facilitation of P/Q- type calcium currents atpresynaptic nerve terminals. Science, 295(5563):2276-2279.). Members of the neural calcium receptor (NCS) family belong to calcium-binding proteins containing the EF-hand domain. NCS genes are often highly expressed in the nervous system, and their main functions are to regulate neurotransmitter release, cell surface receptors and ion channels, gene transcription, neuronal growth, development, and survival (McCue HV, Haynes LP, Burgoyne RD. 2010. The diversity of calciumsensor proteins in the regulation of neuronal function. Cold Spring Harbor perspectives in biology, 2(8):a004085.). NCS2 is a newly discovered member of the NCS family, which has so far only been present in nematodes and insects. The function of NCS2 has only been studied in C. elegans, and it has an important role in the balance of excitation and inhibition of neural synapses in C. elegans. However, unlike the nematode NCS2 protein structure, the insect NCS2 protein contains only three EF-hand domains. Since the function of insect NCS2 protein is unclear, we took the model insect N. lugens as the research object to analyze the physiological function of NCS2 protein. The brown planthopper (scientific name Nilaparvata lugens, English name brownplanthopper) only feeds on the sap of rice, causing the plants to wilt and die. It is an important pest of rice production in my country and even in Asia. The brown planthopper is often rampant intermittently, and the number of insects increases sharply in the outbreak year. At present, there is a lack of sufficient understanding of the occurrence regularity and catastrophic mechanism of brown planthopper outbreaks. In order to prevent the brown planthopper from harming rice crops, chemical control methods are generally adopted at this stage, but the results are often less effective. The application of pesticides not only kills the natural enemies of the brown planthopper, but also stimulates the reproduction of the pests, resulting in a rampant population situation. In view of the fact that the brown planthopper has become the most serious biological disaster that endangers the food security and ecological security of rice in my country, it is of great scientific significance to find effective pest target genes and reduce the economic losses caused by the target pests to rice crops through biological control methods. N. lugens has the phenomenon of systemic RNAi, which can silence the expression of host genes efficiently and for a long time. Therefore, N. lugens has become a new model insect to study gene function using RNAi. Since the NCS2 gene only exists in insects and nematodes, the use of this gene to produce transgenic plants will not produce toxic side effects on other animals including humans, so it will play a major role in ensuring food security.

SUMMARY OF THE INVENTION

The invention provides the application of the NCS2 gene of N. lugens as a target in preparing a drug for controlling N. lugens, and the dsRNA of the NCS2 gene of N. lugens and its application in preparing a drug for preventing and controlling N. lugens. The gene can realize an environment-friendly green pest control strategy and improve the Insect resistance of transgenic rice crops provides new genetic resources for breeding new varieties of rice pests.

The specific technical solutions are as follows:

The invention provides the application of the NCS2 gene or the encoded protein of N. lugens as a target in preparing a drug for controlling N. lugens.

The invention provides the application of the NCS2 gene or the encoded protein of N. lugens as a target in preparing a medicine for reducing the egg production of N. lugens.

The nucleotide sequence of the NCS2 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the NCS2 gene is shown in SEQ ID NO.2.

Preferably, the brown planthopper is a 2-5 instar nymph.

The present invention provides a dsRNA of the NCS2 gene of N. lugens, the dsRNA is composed of two complementary sequences, the nucleotide sequence of the sense strand is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.3 The reverse complement of the sequence shown in ID NO.3.

Nucleotide sequences that can still achieve substantially the same inhibitory effect as the sequences shown in the present invention after the above sequences are transformed by simple deletion, modification, etc., shall also belong to the protection scope of the present invention.

The present invention also provides a kind of medicine for preventing and treating brown planthopper, and its active component is one of following (1) or (2):

(1) dsRNA or siRNA for reducing NCS2 gene expression in N. lugens;

(2) The gene encoding the siRNA in (1).

The present invention also provides a method for preventing and treating N. lugens, using the medicine.

The brown planthopper is a 2-5 instar nymph.

No matter whether young (2nd instar) nymphs or old (5th instar) nymphs are disturbed, only about 20% of the nymphs develop into adults.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention discovers the role of the NCS2 gene of the rice pest N. lugens in controlling N. lugens. Since N. lugens has an efficient RNA interference (RNAi) effect, this advantage can be used to interfere with the expression of the N. planthopper NCS2 gene in the insect. The dsRNA of NCS2 gene is obtained by RNAi technology, and the NCS2 gene is silenced by microinjection to reduce its transcription level, which can effectively inhibit the ontogeny, ovary development and egg production of N. lugens, thereby inhibiting the growth, development and reproduction of the pest. The ability to inhibit the reproduction of brown planthopper and realize the control of brown planthopper.

(2) The present invention finds that the use of RNAi technology to interfere with the expression of the NCS2 gene in the 2-5 instar nymphs of N. lugens produces a significant lethal effect.

Description of drawings

Fig. 1 is a PCR identification diagram of the NCS2 gene of the present invention.

Fig. 2 is the identification diagram of the double-stranded dsNCS2 synthesized by the present invention.

Fig. 3 is a mortality chart of the 2nd-5th instar nymphs in which the NCS2 gene is interfered by the present invention.

Fig. 4 is a graph showing the oviposition situation of the first-emergent female adults that interfered with the NCS2 gene according to the present invention; ** means p<0.01.

Fig. 5 is a graph showing the formation of oocytes in the ovary with the interference of NCS2 gene of the present invention.

Detailed ways

Example 1

Rice pest brown planthopper NCS2 gene.

Step 1: Extraction of total RNA from N. lugens and cloning of NCS2 gene

The adult N. lugens (100 mg) was placed in a grinding tube, an appropriate amount of silica grinding beads and 1 mL of Trizol reagent were added, and the tissue grinder was shaken and ground three times at 60 Hz for 30 seconds each time, and stood on ice for 3 minutes. The sample was centrifuged at 12,000 rpm and 4°C for 10 minutes, and 800 μL of the supernatant was transferred to a new 1.5 mL RNase free EP tube; 500 μL of chloroform was added, vigorously shaken for 30 seconds, and allowed to stand at room temperature for 3 minutes. Centrifuge at 12000rpm, 4°C for 10 minutes, pipette the supernatant into a new 1.5mL RNase freeEP tube, add an equal volume of isoacetone, gently invert and mix for 30 seconds, and let stand at room temperature for 20 minutes. Centrifuge at 12,000 rpm, 4°C for 20 minutes, a precipitate can be seen at the bottom of the centrifuge tube, and the supernatant is discarded. Wash by slowly adding 1 mL of 75% RNase-free ethanol (v/v) along the tube wall. 7500rpm, 4°C, centrifuge for 5 minutes, discard the supernatant, open the lid and let it stand in a fume hood. After the sediment at the bottom of the tube becomes translucent, add 20 μL RNase free ddH ₂ O to fully dissolve, and use NanoDrop 2000 to determine the RNA concentration and quality .

Step 2: RNA reverse transcription

Pipette 1000 ng of the RNA prepared in step 1 into an RNase free PCR tube and make up to 12 μL with RNase free ddH ₂ O. Add 4 μL of 4×gDNA wiper Mix, mix well, place in a PCR machine and heat at 42°C for 2 minutes. Add 4 μL of 5×HiScript II qRT SuperMix II, mix well, and place in the PCR machine. The reaction program is: 50°C, 15 minutes; 85°C, 1 minute. 180 μL of ddH ₂ O was added to the product for dilution and stored at -20°C.

Step 3: NCS2 Gene Fragment Cloning

First, the following specific PCR primers containing the T7 promoter were synthesized:

Forward:

Reverse:

The underlined sequence is the T7 promoter sequence.

PCR reaction system

reagent volume cDNA 1μL PCR Forward Primer (10μM) 1μL PCR Reverse Primer (10μM) 1μL Green Taq Mix 25μL ddH₂O 22μL

PCR reaction parameters: 95°C, pre-denaturation for 3 minutes; 95°C, denaturation for 30 seconds; 60°C, annealing for 1 minute, 72°C, extension for 1 minute, a total of 40 cycles; total extension at 72°C for 10 minutes. The PCR products were confirmed by agarose gel electrophoresis, and the corresponding fragments were recovered with DNA gel recovery kits, and then the TA cloning kits were used to construct the target genes recovered from the gels into the pMD19-T vector.

Step 4: Transform E. coli

Take out the E. coli competent cells from the -80°C refrigerator, thaw on ice for 5 minutes, add the recombinant pMD19-T vector to the competent cells, gently shake and mix, and bathe on ice for 30 minutes. The above samples were placed in a water bath at 42°C for 90s, and then immediately ice-bathed for 2min. Add 1 mL of anti-LB liquid medium and incubate for 2 hours at 37°C on a shaker at 220 rpm. The cultured bacterial liquid was centrifuged at 6000 rpm for 10 min, part of the liquid was discarded, the remaining 100 μL of liquid was resuspended and precipitated, and spread on the LB plate containing Amp ⁺ resistance; in a bacterial incubator at 37 °C, invert for 12 hours . A single colony was picked into 1 mL of LB liquid medium containing Amp ⁺ resistance, and incubated at 37°C with a shaker at 220 rpm for 3 hours. Aspirate 500 μL of turbid bacterial liquid and send it to a biological company for sequencing (ABI3730XL automatic sequencer; Zhejiang Shangya Biotechnology Co., Ltd.) to obtain a 6421bp gene NCS2 (the nucleotide sequence is shown in SEQ ID NO. The amino acid sequence is shown in SEQ ID NO. 2), and the extracted plasmid was named pMD-NCS2.

Example 2

1. Synthesis of dsRNA

The following specific primers containing the T7 promoter were used:

Forward:

Reverse:

The underlined sequence is the T7 promoter sequence.

dsGFP primer sequences are as follows

dsGFP-F:

dsGFP-R:

Using the recombinant plasmid pMD-NCS2 extracted in Example 1 as a template, a high-concentration DNA fragment of about 550 bp was amplified by PCR with the above-mentioned primers (the nucleotide sequence is shown in SEQ ID NO. 4), and this was used as a template. Synthesize dsNCS2 (the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is the reverse complement of the sequence shown in SEQ ID NO.3), and the template synthesized by dsGFP is a well-known sequence .

Prepare the double-stranded RNA synthesis reaction system:

ATP solution, UTP solution, CTP solution, GTP solution each 2μL, 10×Reaction buffer 2μL, T7 RNAPolimerase mix 2μL, DNA template 8μL. The reaction system was mixed and incubated in a constant temperature incubator at 37°C for 14h. Add 1 μL DNase I and incubate in a metal bath at 37°C for 15 minutes to remove the DNA template. Take 1 μL of dsRNA product, use agarose gel electrophoresis to detect the quality of dsRNA, and store it at -80°C.

2. dsRNA microinjection

Microinjection needle preparation. Use a microelectrode needle puller (P-97 Micropipette Puller) to make a 0.6 mm diameter capillary glass tube into a microinjection needle. The needle puller program is: Heat=750, Pull=300, Velovcity=150, Time=150. dsRNA microinjection. Anesthetize N. lugens with CO ₂ for 15-30 seconds, pick N. lugens with a writing brush and place them on a pre-cooled 2% agarose shallow groove with the worm body ventral side up. The synthetic dsRNA was added to the microinjection needle, and the needle was fixed on the mobile robotic arm; according to the age of N. lugens, pinch into a suitable size with dissecting forceps, and the microinjector (FemtoJet 4×) set the parameters as: injection The pressure was 900 pah, the injection time was 0.3 seconds, and the compensation pressure was 10 pah; the manipulator was manipulated to inject dsRNA into the worms from the soft place between the bases of the second and third pairs of feet of the thorax of N. lugens. The injected brown planthopper was put into a transparent jar with fresh rice seedlings, sealed with gauze, and placed in an artificial climate room for observation.

3. The effect of RNAi on the growth and development of brown planthopper

2, 3, 4 and 5 instar nymphs reared on rice seedlings were selected and injected with about 100 ng of dsNCS2 per head. After injection, they were placed in rice seedlings for observation for 24 hours, and then transferred to new rice seedlings to observe the growth and development of N. lugens and count the mortality. The results showed that the 2-5th instar nymphs injected with dsNCS2 had a survival rate of over 76% on the second day after the disturbance, and then declined rapidly thereafter to only 16-24% on the 14th day. In contrast, the 2-5th instar nymphs of the control group injected with dsGFP had a survival rate of more than 88% on the second day after interference, and still reached 67-86% on the 14th day. It showed that the survival rate of N. lugens injected with dsNCS2 and dsGFP was significantly different.

4. The effect of RNAi on the reproduction of brown planthopper

4th instar nymphs reared on rice seedlings were selected and injected with about 100 ng of dsNCS2 per head. After injection, it was placed on rice seedlings, and after eclosion, the male and female insects were mated, and the mating was carried out for 4 days. Then remove the male and female adults from the rice seedlings, observe the hatching situation of the nymphs on the rice seedlings and count the hatching rate. The results showed that after mating of the male and female adults with dsNCS2 interference, the females did not lay eggs, that is, the egg laying rate was 0; Three females produced an average of 18 eggs. After mating, dsGFP-injected male and female adults laid an average of 84 eggs per female, of which 77% successfully hatched into nymphs. It showed that the difference in egg laying rate of N. lugens females injected with dsNCS2 and dsGFP was extremely significant. Most of the eggs produced by dsGFP-injected female adults had typical eyespots, whereas dsNCS2-injected females produced eggs without typical eyespots or inversions. Dissection of the ovaries on day 5 after eclosion of dsNCS2-injected female adults revealed no mature oocytes in the ovaries. On the fifth day after eclosion, the ovaries of dsGFP-injected female adults were filled with typical banana-shaped mature eggs. It indicated that interfering with NCS2 gene inhibited the maturation of oocytes in female ovary.

No matter whether young (2nd instar) nymphs or old (5th instar) nymphs are disturbed, only about 20% of the nymphs develop into adults. In addition, interfering with the expression of the NCS2 gene in the embryonic female adult worms failed to lay eggs after mating with the male adult worms that interfered with the NCS2 gene, resulting in reproductive failure. The results showed that NCS2 gene plays an important role in the growth, development and reproduction of N. lugens. Therefore, the NCS2 gene can be applied in the field of rice crop breeding, which is of great significance for the screening and cultivation of rice planthopper-resistant crops. To cultivate a transgenic rice line that can express the double-stranded RNA of the NCS2 gene of N. lugens, the nymphs and adults of N. lugens can produce RNAi effect when feeding on rice, so as to achieve the goal of inhibiting the development, mutation and egg-laying of N. lugens.

sequence listing

<110> Zhejiang University

<120> Application of NCS2 gene or encoded protein of N. lugens as a target in the preparation of drugs for controlling N. lugens

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ctttcagttg atgaatgtcc aatgttattg taaatttgaa tcgaacacta tgtgtttaat 6300

aaaataattc aatctacagt gacttgtgtt catcatttgt cgaatattta ctacagttcc 6360

aataagttac tcatatttta ttttcattct cattgcgttt tatattacct attaatgtat 6420

caatttgaaa tgatgtgaat tacattaatc tgtggaa 6457

<210> 2

<211> 186

<212> PRT

<213> Brown planthopper (Nilaparvata lugens)

<400> 2

Met Gly Cys Phe Gly Ser Lys Asp Lys Leu Ser Lys Glu Asp Met Asp

1 5 10 15

Phe Leu Lys Ser Gln Thr Arg Tyr Asp Glu Ala Thr Ile Lys Glu Trp

20 25 30

Tyr Lys Gly Phe Lys Gln Asp Cys Pro Asn Gly Arg Leu Thr Pro Ala

35 40 45

Lys Phe Val Asp Met Tyr Lys Met Phe Phe Pro Ser Gly Asn Ala Glu

50 55 60

Glu Phe Cys Asp His Val Phe Arg Thr Phe Asp Met Asp Lys Asn Gly

65 70 75 80

Tyr Ile Asp Phe Lys Glu Phe Leu Leu Ala Ile Asp Val Thr Ser Ser

85 90 95

Gly Thr Pro Glu Glu Lys Leu Lys Trp Ala Phe Arg Met Tyr Asp Val

100 105 110

Asp Gly Asn Gly Val Ile Asp Ile Gln Glu Met Thr Lys Ile Val Gln

115 120 125

Ala Ile Tyr Asp Met Leu Gly Ala Cys Ser Ser Asn Arg Pro Ala Asp

130 135 140

Ser Ala Glu Glu Arg Ala Lys Asn Ile Phe Ala Lys Met Asp Glu Asn

145 150 155 160

Asn Asp Gly Gln Leu Thr Gln Glu Glu Phe Leu Lys Gly Cys Leu Gln

165 170 175

Asp Glu Glu Leu Ser Lys Met Leu Ala Pro

180 185

<210> 3

<211> 556

<212> RNA

<213> Artificial Sequence

<400> 3

gguguuucgg gagcaaagac aaauuaucca aagaggauau ggauuuccuc aaaucacaaa 60

cacgauacga cgaagcuaca auuaaagaau gguacaaagg uuuuaagcaa gacuguccaa 120

acgguagacu aacgccggcc aaguucguag auauguacaa gauguucuuu cccaguggua 180

augccgaaga guucugcgac cacguauuua ggacauucga cauggacaag aacggaauua 240

ucgauuucaa agaguucuug cuugcuauug augugacauc gucgggcacc ccagaagaaa 300

aacuaaaaug ggcguuuagg auguacgacg uugacggcaa uggugucauc gacauccagg 360

aaaugacaaa gauuguucag gcgaucuacg acaugcuggg cgcaugcucg ucgaacagac 420

cggccgacuc ggccgaggag cgcgccaaga acaucuuugc caagauggac gagaacaacg 480

acggucagcu cacgcaggag gaguuccuga aggguugucu ccaggacgag gagcugucca 540

aaaugcuggc gccaua 556

<210> 4

<211> 556

<212> DNA

<213> Artificial Sequence

<400> 4

ggtgtttcgg gagcaaagac aaattatcca aagaggatat ggatttcctc aaatcacaaa 60

cacgatacga cgaagctaca attaaagaat ggtacaaagg ttttaagcaa gactgtccaa 120

acggtagact aacgccggcc aagttcgtag atatgtacaa gatgttcttt cccagtggta 180

atgccgaaga gttctgcgac cacgtattta ggacattcga catggacaag aacggatata 240

tcgatttcaa agagttcttg cttgctattg atgtgacatc gtcgggcacc ccagaagaaa 300

aactaaaatg ggcgtttagg atgtacgacg ttgacggcaa tggtgtcatc gacatccagg 360

aaatgacaaa gattgttcag gcgatctacg acatgctggg cgcatgctcg tcgaacagac 420

cggccgactc ggccgaggag cgcgccaaga acatctttgc caagatggac gagaacaacg 480

acggtcagct cacgcaggag gagttcctga agggttgtct ccaggacgag gagctgtcca 540

aaatgctggc gccata 556

<210> 5

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 5

taatacgact cactataggg agaggtgttt cgggagcaaa gac 43

<210> 6

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 6

taatacgact cactataggg agatatggcg ccagcatttt gga 43

<210> 7

<211> 47

<212> DNA

<213> Artificial Sequence

<400> 7

taatacgact cactataggg agaatgagta aaggagaaga acttttc 47

<210> 8

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 8

taatacgact cactataggg agatttgtat agttcatcca tgccatgt 48

Claims (8)

1. The application of the NCS2 gene or the encoded protein of the brown planthopper as a target in the preparation of a drug for controlling the brown planthopper. 2. The application of the NCS2 gene or the encoded protein of the brown planthopper as a target in the preparation of a drug for reducing the egg production of the brown planthopper. 3. The application according to claim 1 or 2, wherein the nucleotide sequence of the NCS2 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the NCS2 gene is shown in SEQ ID NO.2 . 4. The application according to claim 1 or 2, wherein the brown planthopper is a 2-5 instar nymph. 5. the dsRNA of a brown planthopper NCS2 gene, it is characterised in that the dsRNA is made up of two complementary sequences, the nucleotide sequence of the sense strand is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is The reverse complement of the sequence shown in SEQ ID NO.3. 6. a medicine for the prevention and treatment of brown planthopper, is characterized in that, its active component is a kind of in following (1) or (2): (1) dsRNA or siRNA for reducing NCS2 gene expression in N. lugens; (2) The gene encoding the siRNA in (1). 7. A method for preventing and treating brown planthopper, characterized in that, the medicine of claim 6 is used. 8. The method of claim 7, wherein the brown planthopper is a 2-5 instar nymph.

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