CN114921477B - Brown orange aphid carotenoid oxygenase gene and dsRNA thereof - Google Patents

Abstract

The invention discloses a brown orange aphid carotenoid oxygenase gene, as shown in SEQ ID NO: 3; also discloses the dsRNA of the brown orange aphid carotenoid oxygenase gene, whose sequence is shown in SEQ ID NO: 6 Shown; Also disclose the synthetic method of dsRNA: extract the total RNA of brown orange aphid, reverse transcribe into cDNA as amplification template, be the upstream primer of SEQ ID NO:4 with the sequence and be the upstream primer of SEQ ID NO:5 with the sequence The downstream primers were amplified by PCR, and the amplified PCR products were electrophoresed to recover the products, and the dsRNA of the brown orange aphid carotenoid oxygenase gene was synthesized using the gel recovered products as templates. The present invention identifies and obtains the brown orange aphid carotenoid oxygenase gene sequence for the first time, and the dsRNA gene silencing efficiency provided has high efficiency, and has a good application prospect in the research and development of new insecticides.

Description

Carotenoid Oxygenase Gene and dsRNA of Brown Orange Aphid

technical field

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

Background technique

The brown orange aphid, Aphis citricidus, is the main vector of Citrus tristeza virus, which causes severe economic losses to the world's citrus industry. The brown orange aphid showed typical plasticity of wing type when it responded to adversity stress. For example, under crowded conditions, the brown orange aphid differentiated into a winged type, but under normal conditions, the brown orange aphid was a wingless type. Wing plasticity, as an important ecological strategy of the brown orange aphid, is closely related to its outbreak and plant virus transmission. Brown orange aphid has the characteristics of parthenogenesis in the field, high wing rate (about 20%), strong plasticity of wing shape, short generation period, and high reproduction rate. It is difficult to control it with chemical agents and it is easy to cause problems such as drug resistance ( Shilts et al., 2018; Gao et al., 2021). Clarifying the molecular regulation mechanism of wing-shaped plasticity of brown orange aphids, exploring new targets for aphid control, and finding new and efficient green control technologies for aphids are major scientific and technological needs at home and abroad.

RNA interference (RNA interference, RNAi) is an important gene silencing method discovered in recent years. The phenomenon of high-efficiency and specific degradation of homologous RNA is through the mediation of dsRNA to specifically degrade the mRNA of the corresponding sequence. Since the expression of a specific gene can be specifically inhibited using RNAi technology, this technology has been widely used to explore gene function and the field of gene therapy.

β-carotene regulates important physiological processes such as insect melanization, visual body color, and stress resistance (Heath et al., 2013; Tougeron et al., 2021). β-carotene is the most abundant carotenoid (hydrocarbons and oxidized derivatives containing 40 carbons) in insects (Ding et al., 2020; Maoka, 2020). β-carotene participates in the melanization response of Trichoplusia ni and regulates its growth rate (Clark and Lampert, 2018). In addition, β-carotene is converted into vitamin A through enzymatic catalysis and regulates the visual function of insects. The lack of β-carotene will lead to the loss of the electrical response ability of the fifth instar larvae of silkworm and adult compound eyes to light stimulation (Shimizu and Kato, 1981). β-carotene induces body color plasticity in insects in response to adversity stress factors (Tsuchida, 2016; Wybouw et al., 2019; Sun Mingxia et al., 2020). There is a large difference in β-carotene content between the red type and the green type of pea aphid. As the nutritional conditions of the host become poor, the β-carotene content decreases significantly, and the population fitness decreases (Ding et al., 2020); Locusta migratory locust Migratoria is uniformly green in solitary form, and black in back and brown ventral in gregarious form. Further analysis found that migratory locusts mainly regulate body color changes through the separation and binding of β-carotene and its binding protein (Yang et al., 2019). The applicant's previous research found that the insulin signaling pathway regulates the wing plasticity of the brown orange aphid (Ding et al., 2017), and found its upstream regulator miR-9b (Shang et al., 2020).

Aphids synthesize β-carotene by fungal horizontal gene transfer. Studies have found that most insects cannot synthesize β-carotene de novo and can only be ingested from food or provided by endosymbiotic microorganisms (Jeremy et al., 2013). For example, the silkworm Bombyx mori eats mulberry leaves to obtain β-carotene. The pigment substance is absorbed by the midgut and enters the blood first, and then is absorbed by sericin through the silk gland, thereby coloring the cocoon; the primary symbiotic bacterium Candidatus Portiera The aleyrodidarum genome contains key genes for β-carotene biosynthesis, which regulate the biosynthesis of β-carotene in itself and symbiotic hosts (Santos-Garcia et al., 2012; Sloan and Moran, 2012). In recent years, studies have found that some piercing-sucking insects (mite) obtain related genes from fungi to synthesize β-carotene through horizontal gene transfer (Altincicek et al., 2012; Nováková and Moran, 2012; Cobbs et al., 2013; Bryon et al., 2017). In the genome of the pea aphid Acyrthosiphon pisum, fungal horizontally transferred genes encoding dehydrogenase and synthesis/cyclase were found, both of which are essential for β-carotene biosynthesis (Moran and Jarvik, 2010). - Carotene biosynthesis and function research has opened up a new field. Subsequently, the phenomenon of fungal transfer of genes related to β-carotene biosynthesis was also found in the genomes of other aphids, the two-spotted spider mite Tetranychus urticae and the Hessian gall midge Mayetiola destructor (Altincicek et al., 2012; Cobbs et al., 2013; Bryon et al., 2017).

The key enzymes of the β-carotene pathway vary greatly among different species (Miziorko et al., 2011; Sun et al., 2018; Ma et al., 2022). Two geranylgeranyl pyrophosphate (GGPP) molecules are synthesized into phytoene under the action of synthetase. In plants, algae and bacteria, the enzyme has only synthetase function (Sieiro et al., 2003). In fungi, however, synthetases are bifunctional enzymes with both synthetase and cyclase functions (Velayose et al., 2000; Arrach et al., 2001). Phytoene is dehydrogenated by dehydrogenase to produce lycopene. In fungi and eubacteria, this process is catalyzed by a dehydrogenase gene, while in plants and algae, this process is performed by different enzyme systems (Sandmann et al., 2009). Cyclase can cyclize one or both ends of lycopene to generate β-carotene, α-carotene and γ-carotene (Maoka et al., 2020), and β-carotene is oxygenated Under the action of enzyme (NinaB) and β-carotene binding protein (β-CBP), a complex is formed and 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 found that dehydrogenase, synthetase and cyclase genes were acquired in the early evolution of aphids, and after continuous replication, recombination and screening, different genes were expanded or contracted in different species of aphids, presents a diverse β-carotene pathway (Nováková and Moran, 2012; Takemura et al., 2021).

Carotenoid Oxygenase (Carotenoid Oxygenase) can oxidatively crack carotenoids to produce vitamin A (Vitamin A, VA) substances. As an important enzyme soluble in cytoplasm, this enzyme has the highest activity in intestinal mucosa, especially in jejunal mucosal cells. At present, there are few related studies on the carotenoid oxygenase protein of the brown orange aphid, and there is no report on dsRNA targeting the carotenoid oxygenase gene of the brown orange aphid.

Contents of the invention

Aiming at the key scientific problem of brown orange aphid β-carotene pathway analysis and its regulating effect on wing shape plasticity, the present invention uses RNAi to construct on the basis of systematic identification of brown orange aphid β-carotene pathway gene at the living level The β-carotene pathway of the brown orange aphid; analysis of the expression pattern of the brown orange aphid β-carotene pathway in response to adversity stress; analysis of the effect of β-carotene on the brown orange aphid wing plasticity and the regulation of insulin on the β-carotene pathway .

The present invention identifies the brown orange aphid carotenoid oxygenase gene (AcNinaB gene) for the first time through research and verification, and designs and synthesizes the dsRNA thereof. It is verified by experiments that the dsRNA gene has high silencing efficiency. Based on this, the present invention protects the following technical solutions:

A brown orange aphid carotenoid oxygenase gene, the nucleotide sequence of which is shown in SEQ ID NO:3.

The application of the above brown orange aphid carotenoid oxygenase gene or the protein encoded by the brown orange aphid carotenoid oxygenase gene as a target in any of the following:

1) Application in regulating the plasticity of the brown orange aphid wing shape or preparing products for regulating the plasticity of the brown orange aphid wing shape;

2) Application in regulating the growth and development of the brown orange aphid or preparing a regulator for regulating the growth and development of the brown orange aphid;

3) Application in preventing and treating brown orange aphid or preparing products for preventing and controlling brown orange aphid.

The present invention also protects the primer set for amplifying the above-mentioned brown orange aphid carotenoid oxygenase gene, the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer As shown in SEQ ID NO:2.

The present invention also protects the PCR amplification method of the above-mentioned brown orange aphid carotenoid oxygenase gene, comprising the following steps: using the cDNA obtained by reverse transcription of the brown orange aphid total RNA as a template, and using the sequence as SEQ ID NO:1 The upstream primer and the downstream primer whose sequence is SEQ ID NO:2 carry out PCR amplification.

In the above-mentioned PCR amplification method, when the PCR amplification reaction system is 25 μl, the 25 μl PCR reaction system includes: 1 μl of cDNA template with a concentration of 200-700 ng/μl, upstream and downstream primers with a concentration of 0.15-0.25 μM 1 μl, DNA polymerase LA Taq MIX 0.25 μl, 10×Buffer (Mg ²⁺ plus) 3 μl, dNTP 4 μl and nuclease-free water 14.75 μl;

The PCR conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, a total of 35 cycles; extension at 72°C for 10 min.

The present invention also protects a dsRNA of the brown orange aphid carotenoid oxygenase gene, which is obtained by transcribing the brown orange aphid carotenoid oxygenase gene fragment as shown in SEQ ID NO:3 as a template.

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

The application of the dsRNA of the above-mentioned brown orange aphid carotenoid oxygenase gene in any of the following:

1) Application in regulating the plasticity of the brown orange aphid wing shape or preparing products for regulating the plasticity of the brown orange aphid wing shape;

2) Application in regulating the growth and development of the brown orange aphid or preparing a regulator for regulating the growth and development of the brown orange aphid;

3) Application in preventing and treating brown orange aphid or preparing products for preventing and controlling brown orange aphid.

The present invention also protects a dsRNA synthesis method of the brown orange aphid carotenoid oxygenase gene, comprising the following steps: using the cDNA obtained by reverse transcription of the brown orange aphid total RNA as a template, and using the sequence as SEQ ID NO:4 The upstream primer and the downstream primer whose sequence is SEQ ID NO: 5 were amplified by PCR, and the PCR amplified product was recovered after electrophoresis, and the dsRNA of the brown orange aphid carotenoid oxygenase gene was synthesized using the product recovered from the gel as a template.

In the above-mentioned technical scheme of the dsRNA synthesis method of the brown orange aphid carotenoid oxygenase gene, the dsRNA synthesis reaction system is 20 μl, which includes: 4 μl (about 1 μg) of the fragment template obtained by gel recovery, 5×TranscrptAid Reaction Buffer 4 μl, 8 μl of ATP/CTP/GTP/UTP Mix at a concentration of 100 mM, 2 μl of TranscriptAid Enzyme Mix and 2 μl of DEPC-treated water; the reaction conditions are: incubate at 37°C for 4 hours.

The beneficial effects of the present invention are: the present invention first identified and obtained the gene sequence of the brown orange aphid carotenoid oxygenase, and verified through experiments that the gene participates in regulating the plasticity of the brown orange aphid wing, and obtained the carotenoid oxygenase gene according to the sequence The dsRNA can be applied to the RNA interference of the brown orange aphid so as to control the brown orange aphid. It has been verified by experiments that the dsRNA gene silencing efficiency is high, and the brown orange aphid has obvious phenotypic changes after gene interference, which solves the problem that the brown orange aphid currently does not have an effective dsRNA of the carotenoid oxygenase gene, and is developing a new type of insecticide It has a good application prospect.

Description of drawings

Figure 1 is the Protein Blast comparison of the brown orange aphid AcNinaB gene in NCBI.

Fig. 2 is a phylogenetic tree diagram of the amino acid sequence of the AcNinaB gene of the brown orange aphid and the AcNinaB protein of other insects.

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

Figure 4 shows the composition and content analysis of carotenoids in winged and apterous brown orange aphids.

Figure 5 shows the differences in the expression of the brown orange aphid β-carotene pathway genes in the population density change (A) and the two types of wings (nymph (B) and adult (C)).

Fig. 6 Effect of interfering insulin receptor gene 2 (InR2) on the expression (A) and content (B) of β-carotene pathway genes.

Fig. 7 shows the relative expression level of AcNinaB gene after the brown orange aphid was fed the dsRNA of AcNinaB gene, wherein dsGFP represents the control group, and dsAcNinaB represents the experimental group.

Figure 8 shows the winged rate of the brown orange aphid fed with the dsRNA of the AcNinaB gene, wherein dsGFP represents the control group, and dsAcNinaB represents the experimental group.

Fig. 9 is the detection result of β-carotenoid content after the brown orange aphid is fed with the dsRNA of the AcNinaB gene.

Fig. 10 is a graph showing the phenotypic expression of the brown orange aphid fed with the dsRNA of the AcNinaB gene; wherein 1 is the normal brown orange aphid in the control group, and 2 is the brown orange aphid with abnormal wing development in the experimental group.

Detailed ways

The present invention will be further described below in conjunction with embodiment, but does not limit the present invention thereby.

The experimental methods in the following examples, unless otherwise specified, are conventional methods; the chemical and biological reagents used, unless otherwise specified, are conventional reagents in the art.

Main chemical reagent sources are as follows in the embodiment of the present invention:

LA Taq MIX (Takara Company, Japan)

TRIzol kit (Invitrogen, USA)

RNeasy Plus Micro Kit (QIAGEN, Germany)

PrimeSTAR Max Premix (Takara, Japan)

Gel Recovery and Purification Kit (Takara, Japan)

Transcript Aid T7 High Yield Transcription Kit (Thermo fisher Scientific, USA)

Perfect real time RT reagent (Takara, Japan)

qPCR Master Mix(Promega公司，美国)

qPCR Master Mix (Promega, USA)

TranscriptAid Enzyme Mix (Thermo fisher Scientific, USA)

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

PrimeScriptRT Enzyme Mix I (Thermo fisher Scientific, USA)

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

Random 6mers (Thermo fisher Scientific company, the United States, SO142)

Example 1. Sequence Identification of Brown Citrus Aphid Carotenoid Oxygenase Gene

1) Find the possible carotenoid oxygenase sequence from the brown orange aphid genome data (the internal database of the Key Laboratory of Entomology and Pest Control, Southwest University), and find a sequence in the genome through tBlastn, using the online software https: http://www.ncbi.nlm.nih.gov/, design primers by Primer-BLAST:

PCR product size: Min: 250, Max: 500; Organism: Aphidoidea, get the primer sequence. Carry out self-complementarity test and free energy test on the designed primers in the software DNAMAN: the minimum free energy of the primer structure = 0.00 kcal/mol, there is no self-complementarity, and the Max complementarity in the complementarity of the two primers is continuous: 4bp, free energy=-1.60Kcal/mol, Max complementarity indiscontinuous: 7bp obtained primer AcNinaB-A (as shown in SEQ ID NO: 1) and AcNinaB-S (as shown in SEQ ID NO: 2) PCR primers.

PCR product size: Min: 150, Max: 300; Organism: Aphidoidea, get the primer sequence. Carry out self-complementarity test and free energy test on the designed primers in the software DNAMAN: the minimum free energy of the primer structure = 0.00 kcal/mol, there is no self-complementarity, and the Max complementarity in the complementarity of the two primers is continuous: 4bp, free energy=-3.30Kcal/mol, Max complementarity indiscontinuous: 7bp obtained primers are AcNinaB-L (as shown in SEQ ID NO: 9) and AcNinaB-R (as shown in SEQ ID NO: 10).

2) Use the RNA extraction kit RNeasy Plus Micro Kit to extract the total RNA of the brown orange aphid according to the instructions, and then use the reverse transcription kit Perfect real time RT reagent to reverse transcribe 1 μg of the total RNA into cDNA according to the instructions.

3) Using the cDNA obtained above as a template, design full-length amplification primers, and use the upstream and downstream primers AcNinaB-A and AcNinaB-S for PCR amplification; the PCR conditions are: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 30 seconds, 60°C Annealing for 10s, extension at 72°C for 2min, 35 cycles in total; extension at 72°C for 10min. A total of 25 μl of PCR reaction system, including: 1 μl of cDNA template with a concentration of 400-500 ng/μl, 1 μl of upstream and downstream primers with a concentration of 0.15-0.25 μM, 0.25 μl of DNA polymerase LA Taq MIX, 10×Buffer (Mg ²⁺ plus ) 3 μl, dNTP 4 μl and nuclease-free water 14.75 μl; 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. The sequencing result is shown in SEQ ID NO:3.

5) The sequencing results were manually proofread and sequence spliced, and the Protein Blast similarity comparison was carried out in NCBI. The comparison results are shown in Figure 1. It can be seen that the amplified gene fragment is similar to the cotton aphid (Aphis gossypii) NinaB gene ( Carotenoid oxygenase gene) has the highest homology, up to 96%. Compared with other insects, it is also found that it can be compared with the NinaB gene of other insects, indicating that the gene obtained by this clone should be the AcNinaB gene of the brown orange aphid.

6) Through the construction of the amino acid phylogenetic tree, as shown in Figure 2, the phylogenetic tree analysis shows that the NinaB gene of the brown orange aphid is clustered with the NinaB genes of other insects of the order Hemiptera, indicating that the NinaB gene of the brown orange aphid is closely related to that of the hemiptera. The NinaB genes of other insect orders and other insect orders are highly homologous.

Embodiment two, the synthesis of the dsRNA of brown orange aphid carotenoid oxygenase gene

1) Add T7 promoter sequence at the 5' end of AcNinaB-A (as shown in SEQ ID NO: 1) and AcNinaB-S (as shown in SEQ ID NO: 2) sequence, design and amplify synthetic carotenoid oxygenase The upstream and downstream primers T7-AcNinaB-A1 (as shown in SEQ ID NO: 4), T7-AcNinaB-S1 (as shown in SEQ ID NO: 5) of the enzyme (NinaB) gene, synthetic primers, and the primer sequences are respectively:

T7-AcNinaB-A1: TAATACGACTCACTATAGGGTCGCTGCTAAGAAACGGTCC,

T7-AcNinaB-S1: TAATACGACTCACTATAGGGACAATACCAGCCATCCGAGC.

2) Use the RNA extraction kit RNeasy Plus Micro Kit to extract the total RNA of the brown orange aphid according to the instructions, and then use the reverse transcription kit Perfect real time RT reagent to reverse transcribe 1 μg of the total RNA into cDNA according to the instructions, reverse transcribe The system is 20 μl, including: total RNA 1 μl (about 1 μg), 5×PrimeScriptBuffer 4 μl, PrimeScriptRT Enzyme Mix I 1 μl, Oligo dT Primer (50 μM) 1 μl, Random6mers (100 μM) 1 μl, RNase Free ddH ₂ O 12 μl.

3) Perform PCR amplification on the cDNA obtained in step 2), and the reaction conditions are: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 2 minutes, a total of 35 cycles; extension at 72°C for 10 minutes ; A total of 25 μl of PCR reaction system, including: 0.5 μl of cDNA template with a concentration of 400-500 ng/μl, 1 μl of upstream and downstream primers (T7-AcNinaB-A1, T7-AcNinaB-S1) with a concentration of 0.15-0.25 μM, PrimeSTAR Max Premix 12.5μl and nuclease-free water 10μl.

4) Purify and recover the PCR product in step 3) with the Gel Recovery and Purification Kit according to the instructions and use it as a transcription template for dsRNA synthesis. Use the in vitro synthesis RNA kit Transcript Aid T7 High Yield Transcription Kit to transcribe and purify according to the instructions to obtain brown orange dsRNA (as shown in SEQ ID NO: 6) solution of aphid AcNinaB gene, the concentration of the dsRNA solution is diluted to 200-300ng/μl for later use. The dsRNA synthesis reaction system is 20 μl, including: 4 μl (about 1 μg) of the fragment template recovered from the gel, 4 μl of 5×TranscrptAid Reaction Buffer, 8 μl of ATP/CTP/GTP/UTP Mix with a concentration of 100 mM, 2 μl of TranscriptAid Enzyme Mix and DEPC- Treated water 2μl; reaction conditions: 37 ℃ incubation for 4h.

Simultaneously according to above-mentioned method synthesis and purification obtains the dsRNA solution of GFP (green fluorescent protein) as negative control, the upstream and downstream primer sequence that the cDNA that reverse transcription obtains carries out PCR amplification uses respectively is GFP-ds-T7F (as SEQ ID NO: 7), GFP-ds-T7R (as shown in SEQ ID NO:8), the sequence is: GFP-ds-T7F:

TAATACGACTCACTATAGGGCAGTTCTTGTTGAATTAGATG;

GFP-ds-T7R:

TAATACGACTCACTATAGGGTTTGGTTTGTCTCCCATGATG.

Embodiment 3, the dsRNA of the brown orange aphid carotenoid oxygenase gene is introduced into the brown orange aphid

Utilize the device shown in Figure 3 to feed dsRNA to the brown orange aphid, follow the steps below:

1) Take a clean 50mL centrifuge tube, cut off the conical bottom of centrifuge tube 1 with scissors, take 250μl PCR tube 2 and cut off the bottom of the tube, use double-sided tape to connect the mouth of PCR tube 2 to the cap of centrifuge tube 1. The inner walls are glued together so that the PCR tube 2 is set in the centrifuge tube 1;

2) Extend the pipette gun from the bottom of the PCR tube 2 to inject the dsRNA solution prepared above, and place the centrifuge tube 1 upright with the bottom up;

3) Take fresh citrus shoots 3, wash them with nuclease-free water and absorb excess water, then insert them into the dsRNA solution of PCR tube 2, then wrap the PCR tube 2 with sealing glue and cut off the gap left by the bottom of the tube, Prevent the brown orange aphid that puts in subsequently into PCR tube 2;

4) Pick 20 brown orange aphids into the centrifuge tube 1, cover the bottom of the centrifuge tube 1 with a 5cm*5cm 80-mesh gauze 4, and tighten the gauze 4 with a rubber band to ensure the normal breathing of the brown orange aphid. Prevent it from escaping from the centrifuge tube 1; put the entire dsRNA-fed device into an incubator, and incubate for 72 hours at 25±1° C., humidity 75±5% and light:dark ratio of 14h:10h.

Every 20 brown orange aphids was a biological replicate, and 4 biological replicates fed with AcNinaB gene dsRNA solution were set up as the experimental group. At the same time, the treatment of feeding the dsRNA solution of GFP was set as the control group.

Example 4, AcNinaB affects the differentiation of brown orange aphid wing type

Wing plasticity is a response of wing type II insects to adverse habitat conditions (Vellichirammal et al., 2017; Parker and Brisson, 2019; Reyes et al., 2019; Hammelman et al., 2020), such as planthoppers and crickets. Winged and short-winged types, winged and wingless types of aphids, etc. (Yuan Yiyang et al., 2020; Parker et al., 2021; Zhang et al., 2021). Population density change is an important stress factor affecting insect wing plasticity (Hayes et al., 2019; Richard et al., 2019; Zhang et al., 2019; He Yanbiao et al., 2021).

1. Difference analysis of β-carotene content between winged and apterous brown orange aphids

Refer to (Ding et al., 2021 Parental silencing of a horizontally transferred carotenoid desaturase gene causes a reduction of red pigment and fitness in the pea aphid, Pest Management Science, 76:2423-2433). The winged nymphs (third and fourth instars), winged adults (raised at high population density), wingless nymphs (third and fourth instars), and wingless adults (raised at low population density) were picked respectively, and called After replanting, β-carotene was extracted, and the difference in β-carotene content of the brown orange aphid type II was detected and compared. Four biological replicates were set up in each group, with 30 heads in each replicate. Put the weighed brown orange aphid sample into a 1.5mL centrifuge tube, add 500μL of 60% absolute ethanol to fully grind, and ultrasonicate for 30min in the dark. Add 500 μL n-hexane to extract β-carotene. After vigorous shaking, centrifuge at 12,000g at 4°C to completely separate the n-hexane layer containing β-carotene and the ethanol extract of the lower layer. Transfer the upper layer extract to a new centrifuge tube, use a dry concentrator to concentrate in the dark, then add 50 μL MTBE to fully dissolve β-carotene, absorb the supernatant after centrifugation, filter and sterilize with a filter membrane, and detect the change of β-carotene content by HPLC. Adopt YMC (Wilmington, NC, USA) C30 β-carotene special chromatographic column, injection volume is 10 μ L, column temperature is 40 ℃, and flow rate is 0.3mL/min, mobile phase A is acetonitrile:methanol=3:1, mobile phase B is 100% MTBE, gradient elution is used, and the content of β-carotene is calculated according to the peak area and the standard curve of β-carotene standard.

Comparing the difference in composition and content of carotenoids between winged and apterous forms of brown orange aphids by HPLC, it was found that two carotenoids in apterous aphids (peaking times of 20.49 and 37.14) were significantly higher than those in winged aphids; Combined with the peak spectrum of the existing β-carotene standard product, it can be seen that the substance with a peak time of 37.14 is β-carotene, and the other substance has not been identified yet. The results showed that there was a significant difference in the abundance of β-carotene between the two winged brown orange aphids, indicating that it was involved in regulating the wing-like plasticity of the brown orange aphid (Fig. 4).

2. Analysis of the regulation of adversity stress on β-carotene pathway genes

Refer to (Shang et al., 2020 The miR-9b microRNA mediates dimorphism and development of wing in aphids, Proceedings of the National Academy of Sciences. 117(15): 8404-8409). On the basis of the applicant's previous discovery that changes in population density can significantly induce wing-type plasticity in brown orange aphids (Shang et al., 2020), this experiment uses population density changes as the key adversity stress factor, and selected freshly molted wingless adult aphids start an experiment. According to the population density: 10 heads/dish (low density) and 80 heads/dish (high density), samples were collected after 24 hours of treatment, with 4 biological replicates for each treatment. After grinding, the total RNA was extracted using the Trizol method, and the reverse-transcribed cDNA was used as a template, and NovoStart SYBR qPCR SuperMix was used for RT-qPCR amplification, and EF1α and β-act were used as internal references, and the effect of population density changes on β-carotene was calculated using qBASE Transcriptional regulation of biosynthesis genes (5 synthesis/cyclase genes (CscA, CscB, CscC, CscD, CscE), 2 dehydrogenase genes (CdeA and CdeB).

When the population density of the mother brown orange aphid increased, the winged rate of its offspring increased significantly. Using population density as an environmental stress factor to study the transcriptional regulation of β-carotene pathway genes, and analyzing the expression differences between winged and apterous nymphs and winged and apterous adults, it was found that both CscE and CdeB of the brown orange aphid were in the crowded stage and Highly expressed in winged adult aphids, while CscD was down-regulated in the crowded stage and in winged nymphs. At the same time, the expressions of β-carotene oxygenase (NinaB) and β-carotene binding protein (β-CBP) were up-regulated in crowded and winged aphids (Fig. 5). Combined with the results of HPLC, the β-carotene pathway is closely related to the plasticity of the brown orange aphid's wing shape.

3. The regulation of insulin activity on gene expression and content of β-carotene pathway

Refer to (Takemura et al., 2021, Elucidation of the whole carotenoid biosynthetic pathway of aphids at the gene level and arthropodal food chain involving aphids and the red dragonfly. BMC Zoology.6:1-13. and Sun et al., 2 021, Biosynthesis of Aphid alarm pheromone is modulated in response to starvationstress under regulation by the insulin, glycolysis and isoprenoid pathways. Journal of Insect Physiology. 128, 104174.) method. The RNA interference key gene InR2 was used as an indicator of insulin activity. The dsRNA of InR2 was synthesized and diluted to 800 ng/mL with nuclease-free water. Insert the young shoots of citrus into the 1.5mL centrifuge tubes equipped with the above, and collect the test insects after continuous feeding for 24 and 48 hours, and detect the changes of gene expression and β-carotene content, with feeding dsGFP as the control, 4 in each group biological duplicates.

Successfully interfered with the brown aphid insulin receptor gene 2 (InR2) and found that the expression of β-carotene pathway genes changed, NinaB and β-CBP were significantly up-regulated, resulting in accelerated metabolism and decreased content of β-carotene (Figure 6), indicating that The β-carotene pathway is indeed regulated by insulin activity.

Example 5. Detection of silencing efficiency and phenotype

After the dsRNA feeding device in Example 3 was cultured in the incubator for 72 hours, the brown orange aphids of the experimental group treated with the dsRNA solution of the above-mentioned AcNinaB gene and the control group treated with the dsRNA solution of GFP were collected, and were extracted using the TriZol method. After the RNA was reverse transcribed using Perfect real time RT reagent to obtain the corresponding cDNA, the reverse transcription system was 20 μl, which included: 1 μl of total RNA (about 1 μg), 4 μl of 5×PrimeScript Buffer, 1 μl of PrimeScriptRTEnzyme Mix I, Oligo dT Primer (50 μM ) 1 μl, Random 6mers (100 μM) 1 μl, RNase FreeddH ₂ O 12 μl. The reaction conditions are: 37°C for 15 minutes, 85°C for 5s.

Using qRT-PCR technology, the relative expression of AcNinaB gene in the experimental group and the control group was detected. The upstream and downstream primers of the experimental group in qPCR are AcNinaB-L (as shown in SEQ ID NO: 9) and AcNinaB-R (as shown in SEQ ID NO: 10), and the upstream and downstream primers of the control group are GFP-ds-F ( As shown in SEQ ID NO:11) and GFP-ds-R (as shown in SEQ ID NO:12), the primer sequences are as follows:

AcNinaB-L: CCATTAAGTGTCGGTGGCCT;

AcNinaB-R: TGCTCCGTTATGCCGAAAGA;

GFP-ds-F:CAGTTCTTGTTGAATTAGATG;

GFP-ds-R: TTTGGTTTGTCTCCCATGATG.

qPCR Master Mix 10μl。选用褐色橘蚜的EF1A基因为内参基因进行相对定量分析，分析方法则采用国际上惯用的2^-ΔΔCt法。内参基因EF1A的qPCR引物序列：The qRT-PCR reaction conditions were: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, a total of 40 cycles. The PCR reaction system is 20 μl, including: 1 μl (250ng) of cDNA template, 1 μl of the aforementioned upstream and downstream qPCR primers, 7 μl of nuclease-free water and fluorescent dye

qPCR Master Mix 10μl. The EF1A gene of the brown orange aphid was selected as the internal reference gene for relative quantitative analysis, and the analysis method used the 2 ^-ΔΔCt method commonly used in the world. qPCR primer sequence of internal reference gene EF1A:

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

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

The results of qRT-PCR detection showed that the relative expression of the carotenoid oxygenase gene (AcNinaB gene) of the brown orange aphid in the experimental group fed with the corresponding dsRNA was significantly down-regulated compared with the control group (GFP), and the down-regulation was as high as 50%. % or so, as shown in Figure 7. Simultaneously, found by observation, the brown orange aphid progeny wing rate of the experimental group treated with the dsRNA of the carotenoid oxygenase gene was reduced to 3.75%, while the wing rate of the control group was 43% (results as shown in Figure 8) , the content of β-carotenoids increased by about 3 times (the results are shown in Figure 9). Among Fig. 10, 1 is the situation that the offspring of the brown orange aphid of the control group (dsGFP) has wings, and 2 is the situation that the offspring of the brown orange aphid of the dsNinaB treatment group has wings, and the experiment has verified the carotenoid oxygenase gene of the brown orange aphid of the experimental group The expression of AcNinaB was suppressed, and the AcNinaB gene was involved in the wing-type differentiation of aphids. The experimental results also show that the method of the present invention is used to feed the dsRNA of the AcNinaB gene to the brown orange aphid, and the dsRNA can be effectively introduced to achieve the effect of RNA interference.

sequence listing

<110> Southwest University

<120> Carotenoid Oxygenase Gene and dsRNA of Brown Orange Aphid

<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> AcNinaB gene of brown orange aphid

<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 tattataccca 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 the AcNinaB gene of the brown orange aphid

<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 (9)

1. A brown orange aphid carotenoid oxygenase gene, characterized in that its nucleotide sequence is as shown in SEQ ID NO:3. 2. The brown orange aphid carotenoid oxygenase gene or the protein encoded by the brown orange aphid carotenoid oxygenase gene as claimed in claim 1 is used as a target in regulating the plasticity of the brown orange aphid wing type or preparing and regulating the brown orange aphid wing type Applications in plastic products. 3. the application of a primer set in the brown orange aphid carotenoid oxygenase gene described in amplification claim 1, is characterized in that: the nucleotide sequence of the upstream primer of described primer set is as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2. 4. the PCR amplification method of brown orange aphid carotenoid oxygenase gene described in claim 1 is characterized in that, comprises the following steps: the cDNA that obtains with brown orange aphid total RNA reverse transcription is template, with sequence as The upstream primer of SEQ ID NO:1 and the downstream primer with sequence as SEQ ID NO:2 carry out PCR amplification. 5. PCR amplification method as claimed in claim 4, is characterized in that: when the reaction system of PCR amplification is 25 μ l, the PCR reaction system of 25 μ l comprises: concentration is the cDNA template 1 μ l of 200-700ng/μl, and concentration is 0.15-0.25μM upstream and downstream primers 1μl each, DNA polymerase LA Taq MIX 0.25μl, 10×Buffer 3μl, dNTP 4μl and nuclease-free water 14.75μl; The PCR conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, a total of 35 cycles; extension at 72°C for 10 min. 6. A dsRNA of the brown orange aphid carotenoid oxygenase gene, characterized in that, the brown orange aphid carotenoid oxygenase gene fragment as shown in SEQ ID NO:3 is obtained by template transcription, and the dsRNA The nucleotide sequence is shown in SEQID NO:6. 7. The application of the dsRNA of the brown orange aphid carotenoid oxygenase gene according to claim 6 in regulating the plasticity of the brown orange aphid wing or preparing a product for regulating the brown orange aphid wing plasticity. 8. A synthetic method of dsRNA of brown orange aphid carotenoid oxygenase gene, is characterized in that, comprises the steps: the cDNA that obtains with brown orange aphid total RNA reverse transcription is template, is SEQ ID NO with sequence: The upstream primer of 4 and the downstream primer whose sequence is SEQ ID NO: 5 were used for PCR amplification, and the PCR amplification product was subjected to electrophoresis to recover the product, and the product recovered from the gel was used as a template to synthesize the brown orange aphid carotenoid oxygenase gene dsRNA. 9. The method for synthesizing the dsRNA of the brown orange aphid carotenoid oxygenase gene as claimed in claim 8, wherein the dsRNA synthesis reaction system is 20 μl, which includes: 4 μl of fragment templates obtained by gel recovery, 5×TranscrptAidReaction Buffer 4μl, ATP/CTP/GTP/UTP Mix 8μl with a concentration of 100mM, TranscriptAid EnzymeMix 2μl and DEPC-treated water 2μl; the reaction conditions were: incubate at 37°C for 4h.

Images