Arma chinensis dopamine D2 receptor AcDD2R gene and activator and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to an arma chinensis dopamine D2 receptor (AcDD2R) gene and application thereof. The invention provides a nucleotide sequence of an arma chinensis dopamine D2 receptor gene as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2; the invention further provides an activator of the AcDD2R gene and application of the activator in regulation of insect diapause. After expression of the AcDD2R gene is inhibited through an RNA interference technology, lipid accumulation in the diapause period can be remarkably increased, the survival rate and reproductive capacity after diapause is relieved are promoted, and dsRNA of the AcDD2R gene can be used as a related preparation and product for regulating and controlling arma chinensis diapause. In addition, lipid accumulation in the diapause period can be inhibited by activating the AcDD2R through a specific agonist quinpirole. The invention has important significance for understanding the insect diapause regulation mechanism, and has wide application prospects in the aspects of natural enemy insect preservation and application.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a coleoptera dopamine D2 receptor AcDD2R gene and an activator and application thereof. Background Art

[0002] The caterpillar stink bug (Arma chinensis) is widely distributed throughout my country. It is a typical natural enemy of predatory agricultural pests and is of great significance in green biological control. As an excellent predatory natural enemy insect in biological control, the caterpillar stink bug A. chinensis has a large egg-laying capacity, strong colonization, long survival time, and a wide distribution range. Both adults and nymphs can prey on more than 40 common agricultural and forestry pests such as Lepidoptera, Coleoptera, Hymenoptera and Hemiptera. It has been widely used in agricultural production at home and abroad, and has achieved large-scale commercial expansion. Diapause is a physiological state in which the growth and development of insects are inhibited when responding to adverse environmental conditions. The most obvious feature is that once entering diapause, the development is extremely slow, reproduction is inhibited, metabolic activity is downregulated, and stress resistance is enhanced. The use of insect diapause can allow caterpillar stink bugs to resist adverse environmental conditions, prolong survival time, and solve the problem of long-term storage and shelf life extension of caterpillar stink bug products.

[0003] RNA interference (RNAi) is a mechanism that can precisely regulate gene expression. This technology specifically degrades target mRNA molecules through double-stranded RNA (dsRNA, Double-stranded RNA, double-stranded ribonucleic acid), thereby inhibiting the activity of specific genes. This technology can specifically inhibit the expression of specific genes, so it has been widely used in the field of exploring gene function and gene therapy. RNAi can efficiently screen target genes required for biological control, laying a solid foundation for the development and application of economic insect products such as pest biological control and natural enemy insects. At present, this technology has become a new means of pest control in the field of plant protection. This technology uses insects' own gene fragments to inhibit the expression of key genes in insect growth and development or biochemical metabolism through RNAi, thereby controlling pest damage or regulating the development progress of economic insects to meet the needs of production applications. RNAi technology has broad development prospects in the field of agricultural plant protection and control. The key issue in achieving effective control of pests based on RNAi is to screen target genes that are pest-specific and have a significant inhibitory effect on their growth and development.

[0004] Dopamine and its receptors play a key role in regulating physiological and developmental processes in insects, and can affect important life activities such as growth, development, molting and reproduction. Based on this regulatory property, dopamine and its receptors have been widely studied for regulating the physiological processes of insects as a potential biological control method. Summary of the invention

[0005] The present invention aims to provide a dopamine D2 receptor (AcDD2R) gene of a stink bug and a protein encoded by the gene, as well as a regulator of the AcDD2R gene, wherein the regulator can be used to regulate the diapause state of insects.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention first provides a dopamine D2 receptor gene of a coleoptera stink bug, which comprises any one of the following nucleotide sequences:

[0008] (1) the nucleotide sequence shown in SEQ ID NO.1;

[0009] (2) A nucleotide sequence encoding a protein having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.1.

[0010] Furthermore, the present invention provides a protein encoding the above-mentioned coleoptera dopamine D2 receptor gene, having an amino acid sequence as shown in SEQ ID NO. 2, or having an amino acid sequence having at least 95% homology with the amino acid sequence as shown in SEQ ID NO. 2. This illustrates that those skilled in the art can replace, delete and / or add one or more amino acids based on the amino acid sequence disclosed in the present invention without affecting its activity, to obtain a mutant sequence of the protein.

[0011] Furthermore, the present invention provides two methods for regulating the AcDD2R gene:

[0012] (1) Inhibitors: RNA interference technology is used to inhibit the expression of AcDD2R gene;

[0013] (2) Activator: Use the specific agonist quinpirole to activate the AcDD2R receptor.

[0014] Furthermore, the dsRNA can be amplified by the forward primer shown in SEQ ID NO.3 and the reverse primer shown in SEQ ID NO.4.

[0015] Furthermore, the present invention provides the use of the AcDD2R gene regulator in regulating insect diapause. The present invention proves through experiments that:

[0016] (1) Inhibition of AcDD2R gene expression by RNA interference significantly increased lipid accumulation during diapause, improved survival rate during the late diapause period, shortened the pre-spawning period, and increased egg production.

[0017] (2) Activation of AcDD2R by the specific agonist quinpirole can inhibit lipid accumulation during diapause.

[0018] The AcDD2R gene and the regulator thereof of the present invention can be used to regulate the lipid metabolism and fecundity of the diapause of the stink bug, and have important application value in the preservation and application of natural enemy insects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the spatiotemporal expression pattern of the AcDD2R gene in the caterpillar, where A to D are the transcription levels of the AcDD2R gene in the head, ovary, midgut, and fat body under diapause conditions;

[0020] Figure 2 is the multiple sequence alignment of AcDD2R;

[0021] Figure 3 The effect of AcDD2R gene on lipid accumulation of female caterpillars, where A and B respectively show the effect of GFP and DD2R double chain injection on abdominal lipid accumulation 10 days and 20 days after injection; C and D respectively show the total lipid content and triglyceride content 10 days and 20 days after GFP and DD2R double chain injection;

[0022] Figure 4 The effect of quinpirole, an activator of AcDD2R gene, on lipid accumulation of female stink bugs, where A and B are the effects of water and quinpirole injection on abdominal lipid accumulation 10 days and 20 days after injection, respectively; C and D are the total lipid content and triglyceride content 10 days and 20 days after water and quinpirole injection, respectively;

[0023] Figure 5 The survival time and fecundity of the stink bugs after diapause release after injection of DD2R double chain. A is the survival statistics after diapause release after injection of GFP and DD2R double chain; B is the pre-oviposition statistics after diapause release after injection of GFP and DD2R double chain; C is the total egg-laying amount after diapause release after injection of GFP and DD2R double chain; D is the hatching rate statistics after diapause release after injection of GFP and DD2R double chain. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below in conjunction with the drawings of the specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific technology or conditions are not indicated in the embodiments, they are all conventional methods or carried out according to the technology or conditions described in the literature of the field, or according to the product manual. The reagents and instruments used, etc., which do not indicate the manufacturer, are all conventional products that can be purchased through regular channels.

[0025] The following table shows the dsRNA of genes produced in Examples and the RT-qPCR primers of genes.

[0026] The specific primer sequences used in the following examples are shown in Table 1. Table 1 - Primer sequences and their functions Example 1

[0027] Acquisition of ORF sequence of dopamine D2 receptor (AcDD2R) gene of caterpillar stink bug The transcriptome data of caterpillar stink bug in this experiment was used to search the database annotation files by keywords, and the unigene sequences found were compared on the NCBI website to screen the sequence of Ac-DD2R gene. The forward primer of AcDD2R gene set by Primer premier 5.0 software was used to

[0028] The reverse primer 5'-ATGTACCCGTCGCCATGAACCT-3' and the reverse primer 5'-TCATGAACCGAAGGACATTAT-3' were cloned from the star bug into Escherichia coli and sequenced.

[0029]

[0030] The sequence of the Ac-DD2R gene obtained by screening the sequencing results was compared to verify and obtain the nucleotide sequence encoding the gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

[0031] Example 2

[0032] Analysis of tissue-specific expression of AcDD2R gene in Asclepias spp.

[0033] Takara's TRIzol reagent was used to extract RNA from different stages (5, 10, 15 and 20 days after eclosion) and different tissues (including head, fat body, midgut and ovary) of female coccinella reared under post-eclosion diapause conditions. Three biological replicates were performed for each treatment, with 12 female insects in each replicate. cDNA was obtained by reverse transcription using the first-strand synthesis kit. Using the cDNA obtained by reverse transcription as a template, real-time quantitative fluorescence PCR (Quantitative Real time RT-PCR) was used to determine the expression level of the Ac-DD2R gene in different developmental stages and different tissues of Coccinella septempunctata. The real-time fluorescence quantitative PCR experiment was performed on a StepOne thermocycler (ABI, USA) instrument using Takara's SYBR Premix Ex TaqTMII (TliRNaseH Plus) was used to detect the fluorescence signal. The reaction system was as follows: the reaction parameters were two-step method: 95°C for 3 min, hot start; 95°C for 15 s, 60°C for 30 s, and 40 cycles. The RT-qPCR data were analyzed using the 2-ΔΔCt method, ΔΔCt = (Ct target gene - Ct housekeeping gene) experimental group - (Ct target gene - Ct) control group, and the gene expression was standardized and plotted using GraphPad Prism 7.00 software. The above-mentioned Ac-DD2R gene of the stink bug was highly expressed in the head under diapause conditions and was lowest in the midgut. Figure 1 The RT-qPCR primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, wherein SEQ ID NO.5 is a forward primer and SEQ ID NO.6 is a reverse primer.

[0034] Example 3

[0035] Acquisition of dsRNA of AcDD2R gene of caterpillar stink bug

[0036] 1. Based on the ORF sequence of the Ac-DD2R gene obtained in Example 1, dsRNA primers were designed using Primer premier5.0 software, and dsRNA was synthesized by in vitro transcription using the T7 RiboMAX™ Express RNAi System (Promega) kit, named dsAc-DD2R. Green fluorescent protein GFP was used as a negative control to synthesize dsGFP. After the concentration of the synthesized dsRNA was determined using NanoDrop 2000 (Invitrogen), it was diluted with DEPC water to a final concentration of 2 μg / μL and stored in a -80°C refrigerator for standby use. The dsAc-DD2R primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10. Among them, SEQ ID NO.9 is a forward primer and SEQ ID NO.10 is a reverse primer; the dsGFP primer sequences are shown in SEQ ID NO.11 and SEQ ID NO.12. Among them, SEQ ID NO.11 is a forward primer and SEQ ID NO.12 is a reverse primer.

[0037] Example 4

[0038] Effects of AcDD2R interference and activators on lipid accumulation in the star bug

[0039] 1. Effects of AcDD2R and its activators on abdominal lipid accumulation

[0040] Female crickets 48 hours after eclosion were selected for microinjection. 1 μg of dsAcDD2R was injected into the thorax and abdomen of the crickets. Female crickets injected with the same amount of dsGFP were used as controls and raised under diapause conditions. The abdominal tissues were dissected and observed in PBS buffer. The results showed that compared with the injection of dsGFP, the abdominal fat accumulation was more significant after the injection of dsAcDD2R, such as Figure 3 As shown in A and B, the total lipid content was detected by chloroform-methanol extraction method. The dsAcDD2R group was significantly higher than the control group at 10 and 20 days. Figure 3 C; the triglyceride content was detected using a TG detection kit, and the results showed that the content in the dsAcDD2R group was significantly increased. Figure 3 D. Further verification by agonist experiment, QNP (experimental group) and equal amount of water (control group) were injected into the newly emerged female insects. Compared with the control group, the abdominal fat accumulation in the QNP treatment group was significantly reduced, as shown in Figure 4 As shown in A and B; the total lipid content and triglyceride content also decreased significantly, such as Figure 4 As shown in C and D. These results indicate that the AcDD2R gene plays a negative regulatory role in regulating lipid metabolism during diapause, and its expression level is negatively correlated with lipid accumulation, which is consistent with the energy storage regulation mode during diapause.

[0041] 2. Effect of dsAcDD2R injection on female survival rate

[0042] The female insects injected with dsAcDD2R were raised under diapause conditions for 40 days and then transferred to diapause release conditions (light cycle of 16:8; humidity of 70%; temperature of 26°C) for single feeding, and the survival rate was counted. The female insects injected with dsGFP were used as controls. The survival status was monitored by survival curve analysis. Compared with the female insects injected with dsGFP, the survival rate of the female insects injected with dsAcDD2R was significantly improved, as shown in Figure 2. Figure 5 As shown in A.

[0043] 3. Effects of dsAcDD2R injection on female fertility

[0044] The females injected with dsAcDD2R were kept under diapause conditions for 40 days and then transferred to diapause release conditions and paired with newly emerged males to measure the pre-oviposition period, egg production, and hatching rate. Figure 5 As shown in B, it dropped from 14.5 days in the control group to 11.2 days; the total egg production increased significantly. Figure 5 As shown in C, the number of eggs increased from 190 in the control group to 220; the egg hatching rate also increased significantly, as shown in Figure 5As shown in Figure D, it increased from 48% in the control group to 58%. These results indicate that knocking down the AcDD2R gene can significantly improve the survival and fecundity of the stink bug, which is consistent with the phenotype of enhanced fitness under diapause conditions.

Claims

1. A dopamine D2 receptor in a starfrog AcDD2R A gene characterized by Having any of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) A nucleotide sequence encoding a protein having the same function as the nucleotide sequence shown in SEQ ID NO.1 obtained by replacing, deleting or inserting one or more nucleotides.

2. Encoding of claim 1 AcDD2R Gene protein.

3. The protein according to claim 2, characterized in that It has an amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence having at least 95% homology with the amino acid sequence as shown in SEQ ID NO.

2.

4. A AcDD2R A gene regulator, characterized in that include: (1) Inhibitors: used to inhibit the expression of the gene described in claim 1 or reduce the level of the protein described in claim 2 or 3; (2) Activator: used to activate the protein described in claim 2 or 3.

5. According to claim 4 AcDD2R A gene inhibitor, characterized in that The inhibitor is dsRNA.

6. The inhibitor according to claim 5, characterized in that The dsRNA is a double-stranded RNA obtained by amplifying the forward primer shown in SEQ ID NO.3 and the reverse primer shown in SEQ ID NO.

4.

7. According to claim 4 AcDD2R A gene activator, characterized in that The activator is quinpirole.

8. The method according to any one of claims 4 to 7 AcDD2R Application of gene regulators in regulating insect diapause.

9. The use according to claim 8, characterized in that: The regulating agent is introduced into the insect body by injection.

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