CN114921477A

CN114921477A - Brown orange aphid carotenoid oxygenase gene and dsRNA thereof

Info

Publication number: CN114921477A
Application number: CN202210670159.9A
Authority: CN
Inventors: 丁碧月; 王进军; 尚峰; 张永特
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-06-14
Filing date: 2022-06-14
Publication date: 2022-08-19
Anticipated expiration: 2042-06-14
Also published as: CN114921477B

Abstract

The invention discloses a brown orange aphid carotenoid oxygenase gene which is shown as SEQ ID NO. 3; also discloses dsRNA of the carotenoid oxygenase gene of the brown orange aphid, and the sequence of the dsRNA is shown as SEQ ID NO. 6; also disclosed are methods of synthesizing dsRNA: extracting total RNA of the brown orange aphid, carrying out reverse transcription to form cDNA serving as an amplification template, carrying out PCR amplification by using an upstream primer with a sequence of SEQ ID NO. 4 and a downstream primer with a sequence of SEQ ID NO. 5, carrying out electrophoresis on a PCR amplification product, recovering the product, and synthesizing by using a gel recovery product as a template to obtain dsRNA of the brown orange aphid carotenoid oxygenase gene. The brown orange aphid carotenoid oxygenase gene sequence is identified and obtained for the first time, the provided dsRNA gene has high silencing efficiency, and the dsRNA gene has good application prospect in the aspect of researching and developing novel insecticides.

Description

Brown orange aphid carotenoid oxygenase gene and dsRNA thereof

Technical Field

The invention relates to the technical field of growth and development regulation and genetic engineering of insects, in particular to a brown orange aphid carotenoid oxygenase gene and dsRNA thereof.

Background

Aphis citricola is a major transmission vector for the Citrus tristeza virus, a Citrus tristeza virus, which causes serious economic losses to the Citrus industry in the world. Typical wing-shaped plasticity of the brown orange aphid occurs when the brown orange aphid responds to stress, such as: under crowded conditions, the brown orange aphids are differentiated into winged type, and under normal conditions, the brown orange aphids are wingless type. The wing-shaped plasticity is closely related to the outbreak and the spread of plant viruses as an important ecological countermeasure for the brown orange aphid. The brown orange aphid has the characteristics of field parthenogenesis, high winged rate (about 20 percent), strong wing plasticity, short generation period, high reproduction rate and the like, and is difficult to control by using chemical agents and easy to cause the problems of drug resistance and the like (shipts et al, 2018; Gao et al, 2021). The molecular regulation mechanism of the plasticity of the wing type of the brown orange aphid is clarified, a new target for preventing and controlling the aphid is explored, and a novel efficient green aphid prevention and control technology is found, which is a great scientific and technological demand at home and abroad.

RNA interference (RNAi), an important gene silencing means discovered in recent years, refers to a phenomenon of highly conserved evolution process, induced by Double-stranded RNA (dsRNA), and efficient and specific degradation of homologous RNA, and is the specific degradation of mRNA of a corresponding sequence through the mediation of dsRNA. Since the expression of a specific gene can be specifically inhibited using RNAi technology, the technology has been widely used in the fields of exploring gene functions and gene therapy.

Beta-carotene regulates important physiological processes such as insect blackening, visual body color, stress resistance and the like (Heath et al, 2013; Tougeron et al, 2021). Beta-carotene is the highest carotenoid content in insects (40 carbon-containing hydrocarbons and oxygenated derivatives) (Ding et al, 2020; Maoka, 2020). Beta-carotene participates in the blackening reaction of Trichoplusia ni and regulates its growth rate (Clark and Lampert, 2018). In addition, beta-carotene is converted into vitamin A through enzyme catalysis and regulates the visual function of insects, and the deficiency of beta-carotene causes the loss of the capability of single eyes and compound eyes of five-instar silkworm larvae to the electric reaction generated by light stimulation (Shimizu and Kato, 1981). Beta-carotene induces insect somatic plasticity in response to stress factors (Tsuchida, 2016; Wyboow et al, 2019; Sunxingxia et al, 2020). The content difference of beta-carotene between the red type and the green type of the pea aphid is larger, the content of the beta-carotene is obviously reduced along with the deterioration of host nutritional conditions, and the population suitability is reduced (Ding et al, 2020); locusta migratoria exhibits uniform green color, while the colony type exhibits black back plate and brown ventral surface. Further analysis shows that migratory locust regulates body color change mainly through separation and binding of beta-carotene and its binding protein (Yang et al, 2019). Previous studies by the applicant found that the insulin signaling pathway regulates brown orange aphid wing plasticity (Ding et al, 2017) and found its upstream regulatory factor miR-9b (Shang et al, 2020).

β -carotene is synthesized by aphids themselves by means of fungal level gene transfer (Horizontal gene transfer). Studies have found that most insects cannot synthesize beta-carotene de novo, and can only ingest food or be provided by symbiotic microorganisms in the body (Jeremy et al, 2013). For example, silkworm Bombyx mori eats mulberry leaves to obtain beta-carotene, pigment substances are absorbed by midgut and then enter blood, and then are absorbed by sericin through silk glands, so that silkworm cocoons are colored; bemisia tabaci primary symbiont Candidatus Poritia alisnodididarum genome contains beta-carotene biosynthesis key genes, regulating the biosynthesis of beta-carotene in self and symbiotic hosts (Santos-Garcia et al, 2012; Sloan and Moran, 2012). Recent studies have found that some piercing-sucking insects (mites) obtain the relevant gene synthesis β -carotene from fungi by horizontal gene transfer (Altincic et al, 2012; Nov a kov a and Moran, 2012; Cobbs et al, 2013; Bryon et al, 2017). Genes encoding dehydrogenase and synthetase/cyclase transferred horizontally by fungi are found in the genome of the pea aphid Acyrthosporin pisum, and are all necessary genes for beta-carotene biosynthesis (Moran and Jarvik,2010), and the discovery opens up a new field for insect beta-carotene biosynthesis and function research. The phenomenon of fungal transfer of genes involved in the biosynthesis of beta-carotene was subsequently found in the genomes of other aphids, Tetranychus urticae and Heisera nigricans Mayetiae detactor (Altincek et al, 2012; Cobbs et al, 2013; Bryon et al, 2017).

The key enzyme systems of the β -carotene pathway vary widely among different species (Miziorko et al, 2011; Sun et al, 2018; Ma et al, 2022). Two geranylgeranyl pyrophosphate (GGPP) molecules produce phytoene under the action of a synthetase. In plants, algae and bacteria, the enzyme has only a synthetase function (Sieiro et al, 2003). In fungi, synthetases are bifunctional enzymes having both a synthetase function and a cyclase function (Velayos et al, 2000; Arach et al, 2001). The phytoene is dehydrogenated under the action of dehydrogenase to generate the lycopene. In fungi and eubacteria, this process is catalyzed by a dehydrogenase gene, and in plants and algae this is done separately by different enzyme systems (Sandmann et al, 2009). The cyclase can cyclize one or two ends of lycopene to generate beta-carotene, alpha-carotene and gamma-carotene (Maoka et al, 2020), and the beta-carotene forms a compound under the action of oxygenase (Ninab) and beta-carotene binding protein (beta-CBP) and is further metabolized into other pigments or energy substances (Clark et al, 2018; Chai et al, 2019; Yang et al, 2019). Analysis of 34 aphid-related sequences revealed that the dehydrogenase, synthetase and cyclase genes were obtained early in aphid evolution, and through continuous replication, recombination and screening, different genes expanded or contracted among different species of aphids, presenting a diverse β -carotene pathway (Nov a kov a and Moran, 2012; Takemura et al, 2021).

Carotenoid oxygenases (Carotenoid oxygenases) are capable of oxidative cleavage of carotenoids to produce Vitamin A (VA) species. The enzyme is an important enzyme soluble in cytoplasm, and has the highest activity in intestinal mucosa, especially jejunum mucosa cells. At present, related researches on carotenoid oxygenase protein of brown orange aphid are few, and no report on dsRNA of the carotenoid oxygenase gene of the brown orange aphid is found.

Disclosure of Invention

Aiming at the key scientific problem of the analysis of a brown orange aphid beta-carotene pathway and the regulation and control effect of the brown orange aphid beta-carotene on the plasticity of a wing, the pathway of the brown orange aphid beta-carotene is constructed at the living body level by applying RNAi on the basis of systematically identifying the gene of the pathway of the brown orange aphid beta-carotene; analyzing an expression mode of a brown orange aphid beta-carotene passage responding to adversity stress; analyzing the influence of the beta-carotene on the plasticity of the wing type of the orange aphid and the regulation and control of the beta-carotene pathway by the insulin.

According to the invention, the brown orange aphid carotenoid oxygenase gene (AcNinab gene) is identified for the first time through research and verification, dsRNA of the brown orange aphid carotenoid oxygenase gene is designed and synthesized, and experiments prove that the dsRNA gene silencing efficiency is high. Based on this, the invention protects the following technical scheme:

a carotenoid oxygenase gene of brown orange aphid has a nucleotide sequence shown in SEQ ID NO. 3.

The brown orange aphid carotenoid oxygenase gene or the protein coded by the brown orange aphid carotenoid oxygenase gene is used as a target in any one of the following applications:

1) the application in regulating and controlling the wing-shaped plasticity of the brown orange aphids or preparing products for regulating and controlling the wing-shaped plasticity of the brown orange aphids;

2) the application in regulating the growth and development of the brown orange aphids or preparing a regulating agent for regulating the growth and development of the brown orange aphids;

3) the application of the composition in preventing and treating the orange aphid brown or preparing products for preventing and treating the orange aphid brown.

The invention also protects a primer group for amplifying the brown orange aphid carotenoid oxygenase gene, wherein the nucleotide sequence of an upstream primer of the primer group is shown as SEQ ID NO. 1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2.

The invention also provides a PCR amplification method of the brown orange aphid carotenoid oxygenase gene, which comprises the following steps: taking cDNA obtained by reverse transcription of total RNA of the brown orange aphid as a template, and carrying out PCR amplification by using an upstream primer with a sequence of SEQ ID NO. 1 and a downstream primer with a sequence of SEQ ID NO. 2.

In the above-mentioned PCR amplification method, when the reaction system for PCR amplification is 25. mu.l, the 25. mu.l PCR reaction system includes: cDNA template 1 at a concentration of 200-700 ng/. mu.lMu.l of upstream and downstream primers with concentration of 0.15-0.25. mu.M, 0.25. mu.l of DNA polymerase LA Taq MIX, 10 Xbuffer (Mg) ²⁺ plus) 3. mu.l, dNTP 4. mu.l and enucleated enzyme water 14.75. mu.l;

the PCR conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min.

The invention also protects dsRNA of the carotenoid oxygenase gene of the brown orange aphid, which is obtained by taking the carotenoid oxygenase gene segment of the brown orange aphid shown as SEQ ID NO. 3 as a template for transcription.

Preferably, the nucleotide sequence of the dsRNA is shown in SEQ ID NO. 6.

The dsRNA of the carotenoid oxygenase gene of the brown orange aphid can be applied to any one of the following parts:

3) the application of the composition in preventing and treating the brown orange aphid or preparing a product for preventing and treating the brown orange aphid.

The invention also provides a synthesis method of dsRNA of the carotenoid oxygenase gene of the brown orange aphid, which comprises the following steps: taking cDNA obtained by reverse transcription of total RNA of brown orange aphid as a template, carrying out PCR amplification by using an upstream primer with a sequence of SEQ ID NO. 4 and a downstream primer with a sequence of SEQ ID NO. 5, carrying out electrophoresis on a PCR amplification product, recovering the product, and taking a gel recovered product as a template to synthesize dsRNA of the carotenoid oxygenase gene of the brown orange aphid.

In the technical scheme of the synthesis method of dsRNA of the carotenoid oxygenase gene of the brown orange aphid, the dsRNA synthesis reaction system is 20 mul, and the synthesis reaction system comprises the following steps: mu.l (about 1. mu.g) of fragment template obtained by gel recovery, 4. mu.l of 5 × Transcriptaid Reaction Buffer, 8. mu.l of ATP/CTP/GTP/UTP Mix at a concentration of 100mM, 2. mu.l of Transcriptaid Enzyme Mix, and 2. mu.l of DEPC-sequenced water; the reaction conditions are as follows: incubate at 37 ℃ for 4 h.

The invention has the beneficial effects that: according to the invention, the gene sequence of the carotenoid oxygenase of the brown orange aphid is identified and obtained for the first time, experiments verify that the gene participates in regulating wing-type plasticity of the brown orange aphid, and dsRNA of the carotenoid oxygenase gene is obtained according to the sequence and can be applied to RNA interference on the brown orange aphid so as to achieve the effect of preventing and treating the brown orange aphid. Experiments prove that the dsRNA gene silencing efficiency is high, the brown orange aphid has obvious phenotype change after gene interference, the problem that the brown orange aphid does not have effective dsRNA of carotenoid oxygenase gene at present is solved, and the dsRNA has good application prospect in the aspect of researching and developing novel insecticides.

Drawings

FIG. 1 is a Protein Blast alignment chart of the AcNinab gene of Aphis citricola in NCBI.

FIG. 2 is a tree diagram of phylogenetic tree of the amino acid sequence of AcNiNaB gene of Aphis citricola and AcNiNaB protein of other insects.

Fig. 3 is a device for feeding dsRNA in the examples of the present invention.

FIG. 4 shows the composition and content analysis of the winged and wingless carotenoids of the brown orange aphids.

FIG. 5 shows the difference in expression of the beta-carotene pathway gene of Aphis citricola between population density change (A) and type II wings (nymph (B) and adult (C)).

FIG. 6 shows the effect of the interference of insulin receptor gene 2(InR2) on the expression level (A) and the content of the beta-carotene pathway gene (B).

FIG. 7 shows the relative expression of AcNinab gene after Aphis citricola is fed with dsRNA of AcNinab gene, wherein dsGFP represents a control group, and dsAcNinab represents an experimental group.

FIG. 8 shows the winged rate of Aphis citricola after feeding dsRNA of AcNinab gene, wherein dsGFP represents the control group and dsAcNinab represents the experimental group.

FIG. 9 shows the result of detecting the content of beta-carotenoid produced by feeding dsRNA of AcNinab gene by Aphis citricola.

FIG. 10 is a phenotype representation of Aphis citricola after feeding with dsRNA derived from the AcNinab gene; wherein 1 is the normal brown orange aphid of the control group, and 2 is the brown orange aphid with abnormal wing development of the experimental group.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The experimental procedures in the following examples are all conventional procedures unless otherwise specified; the chemical and biological reagents used are conventional reagents in the field unless otherwise specified.

The main chemical reagent sources in the embodiment of the invention are as follows:

LA Taq MIX (Takara, Japan)

TRIzol kit (Invitrogen corporation, USA)

RNeasy Plus Micro Kit (QIAGEN, Germany)

PrimeSTAR Max Premix (Takara, Japan)

Gel recovery purification kit (Takara, Japan)

Transcript Aid T7 High Yield transformation Kit (Thermo fisher Scientific Co., USA)

Perfect real time RT reagent (Takara Co., Japan)

qPCR Master Mix (Promega corporation, USA)

TranscriptAId Enzyme Mix (Thermo fisher Scientific, USA)

ATP/CTP/GTP/UTPMix (Thermo fisher Scientific, USA)

PrimeScriptRT Enzyme Mix I (Thermo fisher Scientific, USA)

Oligo dT Primer (Thermo fisher Scientific, USA, AM5730G)

Random 6mers (Thermo fisher Scientific, USA, SO142)

Example one identification of Gene sequence of Carotenoid oxygenase from Aphis citricola

1) The potential carotenoid oxygenase sequences were looked up from the brown orange aphid genome data (internal database of the university of southwest entomology and pest control focus laboratory), one sequence was looked up in the genome by tBlastn, using the online software https:// www.ncbi.nlm.nih.gov/, primers were designed by Primer-BLAST:

PCR product size: min: 250, Max: 500, a step of; organic: aphidoidea, and obtaining a primer sequence. Self-complementarity test and free energy test were performed in software DNAMAN by the primers designed: the minimum free energy of the primer structure is 0.00 Kcal/mol, self-complementarity does not exist, and in the complementarity of the two primers, Max complementarity in continuity: 4bp, free energy ═ 1.60Kcal/mol and Max complementarity in discontinuity: 7bp obtain PCR primers AcNinaB-A (shown as SEQ ID NO: 1) and AcNinaB-S (shown as SEQ ID NO: 2).

PCR product size: min: 150, Max: 300, and (c) a step of cutting; organic: aphidoidea, and obtaining a primer sequence. Self-complementarity and free energy tests were performed in software DNAMAN by designing the resulting primers: the minimum free energy of the primer structure is 0.00 Kcal/mol, self-complementarity does not exist, and in the complementarity of the two primers, Max complementarity in continuity: 4bp, free energy ═ 3.30Kcal/mol and Max complementarity in discontinuity: 7bp obtain primers of AcNinaB-L (shown as SEQ ID NO: 9) and AcNinaB-R (shown as SEQ ID NO: 10).

2) Total RNA of brown orange aphid is extracted by using an RNA extraction Kit RNeasy Plus Micro Kit according to the use instruction, and then 1 mu g of total RNA is reversely transcribed into cDNA by using a reverse transcription Kit Perfect real time RT reagent according to the use instruction.

3) Designing a full-length amplification primer by taking the obtained cDNA as a template, and carrying out PCR amplification by using upstream and downstream primers AcNinaB-A and AcNinaB-S; the PCR conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 10s, and extension at 72 ℃ for 2min for 35 cycles; extension for 10min at 72 ℃. A total of 25. mu.l of PCR reaction was included: mu.l of cDNA template with concentration of 400-500 ng/. mu.l, 1. mu.l of each of upstream and downstream primers with concentration of 0.15-0.25. mu.M, 0.25. mu.l of DNA polymerase LA Taq MIX, 10 XBuffer (Mg) ²⁺ plus) 3. mu.l, dNTP 4. mu.l and enucleated enzyme water 14.75. mu.l; the sequences of primers AcNiNaB-A (shown as SEQ ID NO: 1) and AcNiNaB-S (shown as SEQ ID NO: 2) are as follows:

AcNinaB-A：TCGCTGCTAAGAAACGGTCC，

AcNinaB-S：ACAATACCAGCCATCCGAGC。

4) the PCR product was separated in 1% agarose gel electrophoresis to obtain a band of about 1830bp, and the PCR product was sent to a sequencing company for sequencing, with the sequencing result shown in SEQ ID NO. 3.

5) The sequencing result is manually corrected, the sequence is spliced, and Protein Blast similarity comparison is carried out in NCBI, the comparison result is shown in figure 1, and the homology of the gene fragment obtained by amplification and cotton aphid (Aphis gossypii) Ninab gene (carotenoid oxygenase gene) is the highest and is up to 96%. The comparison result with other insects also shows that the gene obtained by the clone is the brown orange aphid AcNinB gene when being compared with the NinB gene of other insects.

6) Through the construction of the amino acid phylogenetic tree, as shown in fig. 2, phylogenetic tree analysis shows that the ninaB gene of the brown orange aphid is aggregated with the ninaB genes of other insects of the order hemiptera, which indicates that the ninaB gene of the brown orange aphid is highly homologous with the ninaB genes of other insects of the order hemiptera.

Example two Synthesis of dsRNA for Pholiota citricola Carotenoid oxygenase Gene

1) Adding a T7 promoter sequence at the 5' end of the sequences of AcNiNaB-A (shown as SEQ ID NO: 1) and AcNiNaB-S (shown as SEQ ID NO: 2), designing and amplifying an upstream primer T7-AcNiNaB-A1 (shown as SEQ ID NO: 4) and a downstream primer T7-AcNiNaB-S1 (shown as SEQ ID NO: 5) for synthesizing a carotenoid oxygenase (NiNaB) gene, synthesizing primers, wherein the primer sequences are respectively:

T7-AcNinaB-A1：TAATACGACTCACTATAGGGTCGCTGCTAAGAAACGGTCC，

T7-AcNinaB-S1：TAATACGACTCACTATAGGGACAATACCAGCCATCCGAGC。

2) extracting total RNA of brown orange aphid by using an RNA extraction Kit RNeasy Plus Micro Kit according to an instruction, and then performing reverse transcription on 1 mu g of total RNA into cDNA by using a reverse transcription Kit Perfect real time RT reagent according to the instruction, wherein the reverse transcription system is 20 mu l, and the reverse transcription system comprises: total RNA 1. mu.l (ca. 1. mu.g), 5 XPrimeScript Buffer 4. mu.l, PrimeScriptRT Enzyme Mix I1. mu.l, Oligo dT Primer(50μM)1μl，Random 6mers(100μM)1μl，RNase Free ddH ₂ O 12μl。

3) Carrying out PCR amplification on the cDNA obtained in the step 2), wherein the reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 10s, and extension at 72 ℃ for 2min for 35 cycles; extending for 10min at 72 ℃; a total of 25. mu.l of PCR reaction was included: 0.5. mu.l of cDNA template with a concentration of 400-500 ng/. mu.l, 1. mu.l of each of the upstream and downstream primers (T7-AcNinanB-A1, T7-AcNinanB-S1) with a concentration of 0.15-0.25. mu.M, 12.5. mu.l of PrimeSTAR Max Premix, and 10. mu.l of enucleated enzyme water.

4) Purifying and recycling the PCR product in the step 3) by using a gel recycling and purifying Kit according to an instruction and then using the PCR product as a Transcription template for synthesizing dsRNA, using an in vitro synthesis RNA Kit Transcript Aid T7 High Yield Transcription Kit to transcribe and purify according to the instruction to obtain a dsRNA (shown as SEQ ID NO: 6) solution of the Aphis citricola AcNinab gene, and diluting the concentration of the dsRNA solution to 200 ng/mu l for later use. The dsRNA synthesis reaction system is 20 mu l, and comprises: mu.l (about 1. mu.g) of the fragment template obtained by gel recovery, 4. mu.l of 5 × Transcriptaid Reaction Buffer, 8. mu.l of ATP/CTP/GTP/UTP Mix at a concentration of 100mM, 2. mu.l of Transcriptaid Enzyme Mix, and 2. mu.l of DEPC-treated water; the reaction conditions are as follows: incubate at 37 ℃ for 4 h.

Meanwhile, a dsRNA solution of GFP (green fluorescent protein) is synthesized and purified according to the method and is used as a negative control, the sequences of upstream and downstream primers used when the cDNA obtained by reverse transcription is subjected to PCR amplification are GFP-ds-T7F (shown as SEQ ID NO: 7) and GFP-ds-T7R (shown as SEQ ID NO: 8), and the sequences are: GFP-ds-T7F:

TAATACGACTCACTATAGGGCAGTTCTTGTTGAATTAGATG；

GFP-ds-T7R：

TAATACGACTCACTATAGGGTTTGGTTTGTCTCCCATGATG。

example III introduction of dsRNA of Carotenoid oxygenase Gene of Aphis citricola into Aphis citricola

Feeding dsRNA to orange aphid brown by using a device as shown in figure 3, and operating according to the following steps:

1) taking a clean 50mL centrifuge tube, shearing off the conical bottom of the centrifuge tube 1 by using scissors, taking 250 microliter of PCR tube 2, shearing off the tube bottom, and sticking the tube opening of the PCR tube 2 and the inner wall of the tube cover of the centrifuge tube 1 together by using a double-sided adhesive tape so that the PCR tube 2 is sleeved in the centrifuge tube 1;

2) a liquid-transferring gun is extended into the bottom of the PCR tube 2 to inject the prepared dsRNA solution, and the bottom of the centrifugal tube 1 is vertically placed upwards;

3) taking fresh citrus shoots 3, cleaning the fresh citrus shoots with nuclease-free water, sucking off excessive water, inserting the fresh citrus shoots into a dsRNA solution of a PCR tube 2, and then winding the PCR tube 2 with a sealing glue to cut off a gap left after the bottom of the tube, so as to prevent brown citrus aphids placed subsequently from entering the PCR tube 2;

4) picking 20 heads of brown orange aphids into a centrifugal tube 1, covering the tube bottom of the centrifugal tube 1 with a 5 cm-5 cm 80-mesh gauze 4, and tightly binding the gauze 4 with a rubber band to ensure normal respiration of the brown orange aphids and prevent the brown orange aphids from escaping out of the centrifugal tube 1; the whole dsRNA feeding device is put into an incubator and cultured for 72h under the conditions of 25 +/-1 ℃, humidity of 75 +/-5 percent and illumination of 14h in darkness and 10 h.

Every 20 brown orange aphids are one biological repetition, and 4 biological repetitions fed with AcNinab gene dsRNA solution are set as an experimental group. Treatment with a dsRNA solution fed with GFP was also set as a control group.

EXAMPLE IV study of AcNinab on Fin differentiation of Aphis citricola

Winged plasticity is the response of type II winged insects to adverse habitat (Vellichimammal et al, 2017; Parker and Brisson, 2019; Reyes et al, 2019; Hammelman et al, 2020), such as long and short wing types of planthoppers and crickets, wing and wingless types of aphids, etc. (Yuan Yang et al, 2020; Parker et al, 2021; Zhang et al, 2021). Population density variation is an important adversity stress factor affecting the plasticity of insect wings (Hayes et al, 2019; Richard et al, 2019; Zhang et al, 2019; rhenig et al, 2021).

Analysis of beta-carotene content difference of winged and non-winged brown orange aphids

Reference is made to the method of (Ding et al, 2021, scientific relating to a horizontal transformed cardiac gene uses a reduction of red fragment and a five in the pea aphid, Pest Management Science,76: 2423-. Picking up winged aphids (three-year old and four-year old), winged aphids (raised with high population density) and wingless aphids (three-year old and four-year old) and wingless aphids (raised with low population density), weighing, extracting beta-carotene, detecting and comparing content difference of the winged type two-brown orange aphids beta-carotene, setting 4 biological repeats in each group, and repeating for 30 times. And (3) putting the weighed brown orange aphid sample into a 1.5mL centrifuge tube, adding 500 mu L of 60% absolute ethyl alcohol, fully grinding, and carrying out ultrasonic treatment for 30min in a dark condition. 500. mu.L of n-hexane was added to extract the beta-carotene. After vigorous shaking, 12,000g of the n-hexane layer containing beta-carotene and the lower ethanol extract layer were thoroughly separated by centrifugation at 4 ℃. Transferring the upper-layer extracting solution into a new centrifugal tube, concentrating by using a drying concentrator in the dark, adding 50 mu of LMTBE to fully dissolve beta-carotene, centrifuging, absorbing supernatant, filtering and sterilizing by using a filter membrane, detecting the content change of the beta-carotene by HPLC, adopting a special chromatographic column of YMC (Wilmington, NC, USA) C30 beta-carotene, carrying out gradient elution by using a sample injection volume of 10 mu L, a column temperature of 40 ℃, a flow rate of 0.3mL/min, a mobile phase A of acetonitrile, methanol, and MTBE at a ratio of 3:1, and a mobile phase B of 100% MTBE, and calculating the content of the beta-carotene according to a peak area and a standard curve of a beta-carotene standard product.

Comparing the composition and content difference of winged carotenoids and wingless carotenoids of brown orange aphids by HPLC, finding that 2 carotenoids (the peak time is 20.49 and 37.14) in the wingless adult aphids are obviously higher than that of the winged adult aphids; the peak spectrogram combining the existing beta-carotene standard product shows that the substance with the peak time of 37.14 is the beta-carotene, and the other substance is not identified. The result shows that the abundance of the beta-carotene is remarkably different between two leafy orange aphids, which indicates that the beta-carotene participates in regulating the leafy plasticity of the leafy orange aphids (figure 4).

Second, regulation and analysis of beta-carotene pathway gene by adversity stress

Reference is made to The method of Shang et al, 2020The miR-9b microRNA mediators dimorphism and maintenance of winding in applications, Proceedings of The National Academy of sciences.117(15): 8404-. On the basis that the change of population density can obviously induce the plasticity of the wing shape of the brown orange aphid (Shang et al, 2020) in the earlier period of the applicant, the experiment takes the change of population density as a key adversity stress factor, and picks the just ecdysed wingless adult aphid to carry out the experiment. According to the population density: samples were collected after 24h of 10 heads/dish (low density) and 80 heads/dish (high density) treatments, 4 biological replicates per treatment. After grinding, total RNA was extracted by Trizol method, RT-qPCR amplification was performed using NovoStart SYBR qPCR Supermix with reverse transcribed cDNA as template, and qBASE was used to calculate the transcriptional regulation of the population density change on the beta-carotene biosynthesis genes (5 synthetase/cyclase genes (CsccA, CscB, CscC, CscD, CscE) and 2 dehydrogenase genes (CdeA and CdeB) with EF1 alpha and beta-act as internal references.

When the population density of the parent generation brown orange aphid is increased, the wing rate of the later generation is obviously increased. The population density is used as an environmental stress factor to research the transcriptional regulation of a beta-carotene pathway gene, and the expression difference of winged and wingless aphids and winged and wingless adult aphids is analyzed to find that the brown orange aphids CscE and CdeB are highly expressed in a crowding stage and winged adult aphids, and CscD is down-regulated in the crowding stage and winged adult aphids. Meanwhile, both beta-carotene oxygenase (Ninab) and beta-carotene binding protein (beta-CBP) were up-regulated in the expression level in crowded and aphid winged aphids (FIG. 5). The combination of HPLC results shows that the beta-carotene pathway is closely related to the wing plasticity of the orange aphid.

Thirdly, regulation and control of beta-carotene pathway gene expression and content by insulin activity

Reference is made to the methods of (Takemura et al, 2021, emulsification of the porous carbonaceous approach and adhesion of the pore food in the impregnation of the approaches and the red adhesion. BMC Zoology.6:1-13. and Sun et al, 2021, Biosynthesis of the adhesion of inorganic phenol modified in deposition to regulation of the properties of the tissue by the tissue, glycosylation and isopropyl adhesion pathways. journal of the absorption physiology.128, 104174). The RNA interference key gene InR2 was used as an indicator of insulin activity. dsRNA for InR2 was synthesized and diluted to 800ng/mL with ribozyme-free water. And (3) respectively inserting the tender tips of the oranges into the centrifuge tubes with the volume of 1.5mL, continuously feeding for 24h and 48h, collecting test insects, detecting the gene expression amount and the content change of beta-carotene, and taking dsGFP feeding as a control, wherein each group has 4 biological repeats.

Successfully interfering with an insulin receptor gene 2(InR2) of Aphis citricola, the discovery shows that the expression level of a beta-carotene pathway gene is changed, Ninab and beta-CBP are obviously up-regulated, so that the metabolism of beta-carotene is accelerated, and the content is reduced (figure 6), which indicates that the beta-carotene pathway is really regulated and controlled by the activity of insulin.

EXAMPLE V silencing efficiency and phenotypic testing

In the third embodiment, after the device for feeding dsRNA is cultured in an incubator for 72 hours, the experimental group treated by the dsRNA solution of the acnina b gene and the brown orange aphid of the control group treated by the dsRNA solution of GFP are collected, after RNA is extracted by using TriZol method, the corresponding cDNA is obtained by reverse transcription with Perfect real time RT reagent, the reverse transcription system is 20 μ l, which includes: mu.l of total RNA (ca. 1. mu.g), 4. mu.l of 5 XPrimeScript Buffer, 1. mu.l of PrimeScriptRT Enzyme Mix I, 1. mu.l of Oligo dT Primer (50. mu.M), 1. mu.l of Random 6mers (100. mu.M), RNase Free ddH ₂ O12. mu.l. The reaction conditions are as follows: 15min at 37 ℃ and 5s at 85 ℃.

And detecting the relative expression quantity of the AcNinab gene in an experimental group and a control group by utilizing a qRT-PCR technology. The upstream and downstream primers of the experiment group in qPCR are AcNiNaB-L (shown as SEQ ID NO: 9) and AcNiNaB-R (shown as SEQ ID NO: 10), the upstream and downstream primers of the control group are GFP-ds-F (shown as SEQ ID NO: 11) and GFP-ds-R (shown as SEQ ID NO: 12), and the sequences of the primers are as follows:

AcNinaB-L：CCATTAAGTGTCGGTGGCCT；

AcNinaB-R：TGCTCCGTTATGCCGAAAGA；

GFP-ds-F：CAGTTCTTGTTGAATTAGATG；

GFP-ds-R：TTTGGTTTGTCTCCCATGATG。

the qRT-PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s and annealing at 60 ℃ for 30s for 40 cycles. The PCR reaction system was 20. mu.l, and included: mu.l of cDNA template (250ng), 1. mu.l of each of the upstream and downstream qPCR primers, 7. mu.l of enucleated enzyme water and fluorescent dye

qPCR Master Mix 10. mu.l. Selecting EF1A gene of brown orange aphid as reference gene to make relative quantitative analysis, and adopting 2 which is customary internationally as analysis method ^-ΔΔCt The method is carried out. qPCR primer sequence for reference gene EF 1A:

EF1A-S: GATGCACCTGGTCACAGAGA as shown in SEQ ID NO: 13;

EF1A-A: CCATCTTGTTCACACCAACG, shown as SEQ ID NO: 14.

The qRT-PCR detection result shows that the relative expression amount of the carotenoid oxygenase gene (AcNinab gene) of the orange aphid brown in an experimental group fed with corresponding dsRNA is obviously reduced compared with that of a control Group (GFP), and the reduction is as high as about 50%, as shown in figure 7. Meanwhile, through observation, the progeny of the brown orange aphid treated by dsRNA of the carotenoid oxygenase gene has the wing rate reduced to 3.75%, while the wing rate of the control group is 43% (the result is shown in figure 8), and the content of beta-carotenoid is increased by about 3 times (the result is shown in figure 9). In fig. 10, 1 is the winged condition of the brown orange aphid progeny of the control group (dsGFP), and 2 is the winged condition of the brown orange aphid progeny of the dsNinaB treatment group, and experiments verify that the carotenoid oxygenase gene expression of the brown orange aphid of the experimental group is inhibited, and the acnnab gene participates in winged differentiation of aphid. The experimental result also shows that dsRNA of AcNinab gene is fed to the orange aphid brown by using the method disclosed by the invention, the dsRNA can be effectively introduced, and the RNA interference effect is achieved.

Sequence listing

<110> university of southwest

<120> brown orange aphid carotenoid oxygenase gene and dsRNA thereof

<160> 14

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<223> AcNinaB-A

<400> 1

tcgctgctaa gaaacggtcc 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<223> AcNinaB-S

<400> 2

acaataccag ccatccgagc 20

<210> 3

<211> 1809

<212> DNA

<213> Artificial sequence

<223> Aphis citricola AcNinab gene

<400> 3

atgaaaaaaa cagctcggta tttttcaaag aagaaatact tattaaaaac aatattgacc 60

tctaaaatca aatcaactat taaccggtat aaaaaatgtc aattttggtt gactaagggt 120

ttgtggcgcg tcgcaaataa ttggaaagag actgcaccgg cggcgagcag aaaaacttct 180

ggaattgacg gtatgtttca aggattggac gaaaacagag acgtgttagt gaaaaggttg 240

aatgctggtc agaaattata tcccaattgt gatacatcgg tctggctaag aaactgtacg 300

catgacatac caactcccgt ccaagggaaa atcgaaggta agataccaga atggttatcg 360

ggatcgctgc taagaaacgg tccaggaagt acacaagtgg gaaattatga atttaaacat 420

atatttgaca gctctgcttt actgcacagg tttgcattca aaaatggagc agtttcatat 480

caatgtagat ttttagaatc aaatacatat aaacaaaata aagcagccca aagaattgtt 540

attaccgaat ttggaaccag agcatgtcct gatccttgta aaacaatttt tcacagggtg 600

tctaatgtat ttaaatgggg agatgatcaa tcagataatg cgatgatatc tatttaccca 660

atcggcgatg aatattatgc atttaccgag aatccaataa tgatcaaaat taatcctaca 720

actctcgaaa cacttaatac tatagatata gctcggatgg ctggtattgt tcaccatact 780

gctcacccac atatggcagc cgatggtgcg gttttcaatt tagcaactgt tccaaaaatt 840

gatggacctc attattgcgt ggttaaattt cctcgggttg attcagaaag tggatatcag 900

tattcgacgg acgaaatgtt tgggcgaatg tgcatcgtgg ctaccattaa gtgtcggtgg 960

cctctgcacc ctgggtatat gcattctttc ggcataacgg agcattattt catcatagtc 1020

gaacaacctc taagtatttc attgtcaacc gccatgatta atagattcaa aggagatcca 1080

atgtatagtg cactaaaatg gtttcaagac tgccctactc taatttactt gatttcgcga 1140

tctgatggca aaacggtgaa gacatttaaa tcagatgcat tcttttacct acatataata 1200

aaccaatatg aagaagacga taacgtagtg atcgatattt gttgttaccg agatccttcc 1260

atgattgact gcatgttcat cgaagcatta caaaatctca ataaaaaccc agattatgca 1320

gccatgtttc gcagcaggcc tttgagattt gtgttgccag ttaatcgtaa tcctactagt 1380

ggcgatatcg tgaacgagca cccgtatgtc agccccgaaa agctatgtga tttaggttgt 1440

gaaacaccca gaattaatga ctttaaaatt ggcacaaaat acagattttt ctatgcaata 1500

tcatcagatg ttgatgctga aaaccctggg acgctcatta aagtggatac gtacaataaa 1560

acttgtaaaa catggtgcga aaagaacgta tatcctagtg aaccaatttt tgtttcttcg 1620

ccggacgctg aagacgaaga cgatggtgta attttgtcat caattatttg gggtggatcg 1680

gagtgtacac acaaagctgg agtaatagtt ttggatgcaa agagttggac agagataggg 1740

cgagctattt ttatcactca gtcaccagtt cctaaatgct tacatggatg gtatgctgat 1800

gctgtttaa 1809

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence

<223> T7-AcNinaB-A1

<400> 4

taatacgact cactataggg tcgctgctaa gaaacggtcc 40

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence

<223> T7-AcNinaB-S1

<400> 5

taatacgact cactataggg acaataccag ccatccgagc 40

<210> 6

<211> 407

<212> DNA

<213> Artificial sequence

<223> dsRNA of AcNinab gene of orange aphid brown

<400> 6

tcgctgctaa gaaacggtcc aggaagtaca caagtgggaa attatgaatt taaacatata 60

tttgacagct ctgctttact gcacaggttt gcattcaaaa atggagcagt ttcatatcaa 120

tgtagatttt tagaatcaaa tacatataaa caaaataaag cagcccaaag aattgttatt 180

accgaatttg gaaccagagc atgtcctgat ccttgtaaaa caatttttca cagggtgtct 240

aatgtattta aatggggaga tgatcaatca gataatgcga tgatatctat ttacccaatc 300

ggcgatgaat attatgcatt taccgagaat ccaataatga tcaaaattaa tcctacaact 360

ctcgaaacac ttaatactat agatatagct cggatggctg gtattgt 407

<210> 7

<211> 41

<212> DNA

<213> Artificial sequence

<223> GFP-ds-T7F

<400> 7

taatacgact cactataggg cagttcttgt tgaattagat g 41

<210> 8

<211> 41

<212> DNA

<213> Artificial sequence

<223> GFP-ds-T7R

<400> 8

taatacgact cactataggg tttggtttgt ctcccatgat g 41

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<223> AcNinaB-L

<400> 9

ccattaagtg tcggtggcct 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<223> AcNinaB-R

<400> 10

tgctccgtta tgccgaaaga 20

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<223> GFP-ds-F

<400> 11

cagttcttgt tgaattagat g 21

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<223> GFP-ds-R

<400> 12

tttggtttgt ctcccatgat g 21

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<223> EF1A-S

<400> 13

gatgcacctg gtcacagaga 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<223> EF1A-A

<400> 14

ccatcttgtt cacaccaacg 20

Claims

1. A brown orange aphid carotenoid oxygenase gene is characterized in that the nucleotide sequence is shown in SEQ ID NO. 3.

2. The use of the brown citrus aphid carotenoid oxygenase gene or the protein encoded by the brown citrus aphid carotenoid oxygenase gene according to claim 1 as a target in any of:

1) the application of the compound in regulating the plasticity of the wing type of the orange aphid or preparing a product for regulating the plasticity of the wing type of the orange aphid;

3. The primer group for amplifying the carotenoid oxygenase gene of brown orange aphid as claimed in claim 1, is characterized in that: the nucleotide sequence of the upstream primer of the primer group is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.

4. The PCR amplification method of the brown orange aphid carotenoid oxygenase gene is characterized by comprising the following steps: taking cDNA obtained by reverse transcription of total RNA of the brown orange aphid as a template, and carrying out PCR amplification by using an upstream primer with a sequence of SEQ ID NO. 1 and a downstream primer with a sequence of SEQ ID NO. 2.

5. The method of PCR amplification of claim 4 wherein: when the reaction system for PCR amplification is 25. mu.l, 25. mu.l of the reaction system for PCR comprises: mu.l of 200-700 ng/mu.l cDNA template, 1. mu.l of each of upstream and downstream primers with concentration of 0.15-0.25. mu.M, 0.25. mu.l DNA polymerase LA Taq MIX, 10 XBuffer (Mg) ²⁺ plus) 3. mu.l, dNTP 4. mu.l and enucleated enzyme water 14.75. mu.l;

6. The dsRNA of the brown orange aphid carotenoid oxygenase gene is characterized by being obtained by taking a brown orange aphid carotenoid oxygenase gene segment shown as SEQ ID NO:3 as a template for transcription.

7. The dsRNA of a brown orange aphid carotenoid oxygenase gene according to claim 6, characterized in that: the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 6.

8. The use of the dsRNA of a Pholiota citrifolia carotenoid oxygenase gene of claim 6 in any one of:

2) the application of the compound in regulating the growth and development of the green orange aphids or preparing a regulating agent for regulating the growth and development of the green orange aphids;

9. A synthetic method of dsRNA of a carotenoid oxygenase gene of brown orange aphid is characterized by comprising the following steps: taking cDNA obtained by reverse transcription of total RNA of brown orange aphid as a template, carrying out PCR amplification by using an upstream primer with a sequence of SEQ ID NO. 4 and a downstream primer with a sequence of SEQ ID NO. 5, carrying out electrophoresis on a PCR amplification product, recovering the product, and taking a gel recovered product as a template to synthesize dsRNA of the carotenoid oxygenase gene of the brown orange aphid.

10. The method for synthesizing dsRNA of a chazus citricola carotenoid oxygenase gene according to claim 9, wherein the dsRNA synthesis reaction system is 20 μ l, and comprises the following steps: mu.l (about 1. mu.g) of the fragment template obtained by gel recovery, 4. mu.l of 5 × Transcriptaid Reaction Buffer, 8. mu.l of ATP/CTP/GTP/UTP Mix at a concentration of 100mM, 2. mu.l of Transcriptaid Enzyme Mix, and 2. mu.l of DEPC-treated water; the reaction conditions are as follows: incubate at 37 ℃ for 4 h.