CN112831506B - Yellow phyllotreta striolata cytochrome P450 gene and application thereof - Google Patents

Abstract

The invention discloses a phyllotreta striolata cytochrome P450 gene and application thereof, wherein the nucleotide sequence of the phyllotreta striolata cytochrome P450 gene is shown as SEQ ID NO:1, wherein the similarity between the gene sequence and other known P450 homologous genes is lower than 80%; the invention selects a 250bp gene fragment as a target point, and the nucleotide sequence of the gene fragment is shown as SEQ ID NO:2, the similarity between the fragment sequence and other homologous genes in NCBI database is lower than 73%. Cloning a phyllotreta striolata cytochrome P450 gene fragment, connecting the fragment with an L4440 vector, transferring the recombinant plasmid into an escherichia coli HT115 competent cell, and obtaining dsRNA corresponding to a target fragment through IPTG induction, wherein the phyllotreta striolata can be effectively killed by feeding food containing the dsRNA, the phyllotreta striolata mortality rate can reach 61.5%, and the method can be applied to the prevention and treatment of Huang Qutiao phyllotreta striolata.

Description

A kind of Cytochrome P450 gene and its application

technical field

The invention relates to the field of biotechnology, in particular to a cytochrome P450 gene and application thereof.

Background technique

Phyllotreta striolata (Fabricius) is a pest of the family Coleoptera (Coleoptera), which mainly harms cruciferous vegetables, such as cabbage, kale, rape, and Chinese cabbage. It goes through four stages in its life, which are egg, larva, adult and pupa. The larva lives in the soil and mainly feeds on the root bark of plants, which often causes the phenomenon of lack of seedlings and broken ridges. Feeds on young leaves of cruciferous plants.

At present, domestic and foreign researches on the control of flea beetle mainly focus on chemical control, physical control, biological control and transgenic technology. By using the plant secondary substances of the non-food-loving host as a direction to control the flea beetle, and by extracting some other plant secondary substances and chemical pesticides as a mixed reagent, it has a certain effect on the control of the flea beetle . In crop cultivation, crop rotation between cruciferous plants and non-cruciferous plants is also used as a common method in the field to control the flea beetle, but the control effect of these methods is not ideal. Spraying pesticides on cruciferous vegetables can only target the pests on the ground, but cannot affect the larvae in the soil. Not only can it not kill the larvae, pupae and eggs in the soil, it will treat the symptoms but not the root cause, and it will also cause serious environmental pollution. , reduce the quality of vegetables and affect people's health. At the same time, the abuse of insecticides will lead to the resistance of the flea beetle. Therefore, in order to effectively control the flea beetle without causing harm to the environment, new technologies for prevention and control have become a top priority. Transgenic insect-resistant technology, through the expression of insect-resistant toxin genes in plants to improve crop insect-resistant traits, although it has the advantages of broad-spectrum, high resistance, etc., but it will also induce resistance in pests. In addition, the specificity is not high, which is harmful to the environment and The safety hazard of biological groups is relatively large, and it has not been unanimously recognized by consumers.

With the development of molecular biotechnology, RNAi technology has been applied more and more in the field of insect control. At present, good control effects have been achieved in experiments on insects such as Diptera and Hymenoptera. In Diptera mosquitoes, RNAi technology was used to silence the antimicrobial peptide gene Defensin of Anopheles gambiae, and the function of the gene was identified, which opened a new prelude to the application of RNAi technology to study the gene function of mosquitoes. The most studied Hymenoptera insect using RNAi technology is the Italian bee, and the expression of adult genes of the Italian honeybee has been successfully reduced by constructing dsRNA of related genes.

Cytochrome P450 is widely distributed, and most of the reported studies on P450 genes focus on economical insects such as housefly, silkworm, cotton bollworm, trichosporum, Drosophila melanogaster, Aedes aegypti, North American black-tailed swallowtail butterfly, aphids, etc. Sanitary insects and agricultural insects. However, there is no report on the cytochrome P450 gene and its RNAi of T. flavus. The present invention obtains a highly expressed cytochrome P450 gene in the flea beetle by sequencing the transcriptome of the flea beetle, and finds that the similarity between the gene sequence and other known P450 homologous genes is low by performing BLAST analysis on the NCBI database at 80%. We selected a 250bp gene fragment as a target to construct an RNAi expression vector. Through sequence comparison analysis, it was found that the similarity between the fragment sequence and other homologous genes in the NCBI database was less than 73%, indicating that the target had very high specificity. By cloning the cytochrome P450 gene fragment of the flea beetle and constructing its RNAi expression vector, the expression conditions of dsRNA were further optimized, and it was proved by feeding that it can effectively kill the flea beetle, and the death rate of the flea beetle can reach 61.5%. , was significantly higher than that of the control group, so it can be applied to the prevention and treatment of flea beetle.

Contents of the invention

In order to solve the problems raised in the above-mentioned background technology, the object of the present invention is to provide a cytochrome P450 gene and its application.

In order to achieve the above-mentioned purpose, the technical solution adopted in the present invention is: a cytochrome P450 gene of Leprosy japonica, the nucleotide sequence of which is shown in SEQ ID NO:1.

A cytochrome P450 gene fragment of Leprosy japonica, the nucleotide sequence of which is shown in SEQ ID NO:2.

A method for obtaining a cytochrome P450 gene fragment of Leprosy japonica, using a plasmid containing a nucleotide sequence as shown in SEQ ID NO: 1 as a template, and using an upstream primer SEQ ID NO: 3 with a restriction site Kpn I Perform PCR amplification with the downstream primer SEQ ID NO: 4 with restriction site Sac I to obtain the cytochrome P450 gene fragment of Leprosy japonica.

A kind of dsRNA expression vector containing the cytochrome P450 gene fragment of Leprosy japonica, the construction method of the expression vector is: using Fast digest Kpn I and Fast digest Sac I, under the condition of 37 degrees, the vector plasmid that can produce dsRNA Carry out double enzyme digestion with the cytochrome P450 gene fragment of Leprosy japonica;

Perform 1.2% agarose gel electrophoresis on the digested product of the vector plasmid that can produce dsRNA, and then cut the gel to recover the large fragment and remove the small fragment;

Purify the digested product of P450 using a common DNA product purification kit;

The dsRNA-producing vector plasmid product recovered by gel cutting and the purified P450 product were ligated at 25°C;

Preferably, the carrier plasmid capable of producing dsRNA is L4440 plasmid.

A high-efficiency expression system obtained by constructing the above-mentioned expression vector.

The preparation method of the above-mentioned high-efficiency expression system includes transfecting the expression vector into host cells, and obtains it.

Further, the host cell is an RNaseIII-deleted host cell, preferably, the RNaseIII-deleted host cell is a HT115 (DE3) competent cell.

A method for synthesizing a dsRNA of a cytochrome P450 gene fragment of Leprosy japonica, in which the above-mentioned expression system or the prepared expression system is induced by IPTG to obtain the dsRNA corresponding to the target fragment.

The dsRNA of the Cytochrome P450 gene fragment of the flea beetle beetle beetle synthesized by the above-mentioned method.

Application of the dsRNA of the cytochrome P450 gene fragment of the flea beetle mentioned above in killing the flea beetle.

The beneficial effects of the present invention are as follows: the nucleotide sequence of the cytochrome P450 gene of Leprosy japonica is shown in SEQ ID NO: 1, and the similarity between the gene sequence and other known P450 homologous genes is less than 80%; The invention selects a 250 bp gene fragment as a target, its nucleotide sequence is shown in SEQ ID NO: 2, and the similarity between this fragment sequence and other homologous genes in the NCBI database is less than 73%. By cloning the cytochrome P450 gene fragment of the flea beetle beetle and connecting it into the L4440 vector, the recombinant plasmid was transformed into Escherichia coli HT115 competent cells, and the dsRNA corresponding to the target fragment was obtained by IPTG induction. Effectively kill the flea beetle, the mortality rate of the flea beetle can reach 61.5%, and it can be applied to the prevention and control of the flea beetle.

In this paper, the stability of dsRNA at room temperature was studied, and the results showed that it did not degrade after being placed at room temperature for 72 hours, which solved the problem of how to obtain dsRNA that is rich in content and easy to produce and use. Efficient and large-scale preparation of dsRNA.

Description of drawings

Figure 1 shows the results of P450 amplification, where M: DL5000 DNA marker; 1: cytochrome P450 gene fragment;

Figure 2 is the L4440 plasmid map, multiple cloning sites and digestion results, in which Figure 2A is the L4440 plasmid map and multiple cloning sites; Figure 2B is the result of Sac Ⅰ and Kpn Ⅰ digestion of the L4440 plasmid, where M: DL5000 DNA marker; 1, 2, 3: L4440 plasmid;

Figure 3 is the colony PCR identification, where M: DL2000 DNA marker; 1, 2, 3, 4, 5, 6, 7: recombinant plasmid L4440-P450; 8: negative control;

Figure 4 shows the analysis results of dsRNA and digestion with DNase Ⅰ and RNase A, where M: DL2000 DNA Marker; 1: the extract was digested with DNase Ⅰ; 2: the extract was digested with RNase A; 3: the extract was digested with DNase Ⅰ and RNase A successively treatment; 4: untreated extract control group;

Figure 5 shows the effect of different conditions of IPTG on the induced dsRNA expression, where M: DL2000 DNA Marker; 1, 2, 3: 0mM IPTG induction time is 2h, 3h, 4h; 4, 5, 6: 0.2mM IPTG induction time respectively 2h, 3h, 4h; 7, 8, 9: 0.4mM IPTG induction time is 2h, 3h, 4h; 10, 11, 12: 0.8mM IPTG induction time is 2h, 3h, 4h;

Figure 6 shows the stability test results of dsRNA, where M: DL2000 DNA Marker; 1-6: dsRNA were placed at room temperature for 0h, 12h, 24h, 36h, 48h, and 72h; arrows indicate the position of the dsRNA band;

Figure 7 is the statistics of dsRNA insecticide mortality.

Detailed ways

In order to better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following examples.

Experimental Materials

The adult flea beetles were collected from Longhua Base, Shenzhen, mainly in the heart leaves and leaf surfaces of cruciferous vegetables. They were placed in the cultivation room (the temperature was kept at 28°C, the relative humidity was 75%, the light intensity was set to 60% Lx, and the cycle L:D=14:10), and they were fed with fresh young leaves of Chinese cabbage.

The main reagents needed in the experiment include Trizol; DNA Marker DL2000; DNA Marker DL5000; 11×DNA/RNA Loading buffer; T4 DNA Ligase; 10×T4 DNA Ligase buffer; 10×FastDigase green buffer; (FastDigase); Isopropanol; 75% Ethanol; Chloroform; DEPC Water; Deionized Water (ddH2O); Liquid Nitrogen; EasyScript One-Step gDNA Removal and cDNASynthesis SuperMix Kit; General DNA Product Purification Kit; HT115 (DE3) competent cells.

Because wild-type E. coli has RNase III in its own gene, the enzyme can recognize and cut dsRNA, so the method of inducing expression and producing dsRNA through wild-type E. coli is not feasible. On the contrary, HT115 (DE3) is a defective Escherichia coli and does not have RNase III, so it can be used to express dsRNA. In addition, L4440 contains a bidirectional T7 promoter and a lac lactose operon, so this study uses HT115 competent cells to transform recombinant Plasmid L4440-P450, to express dsRNA.

Example 1 The extraction of the flea beetle total RNA and the synthesis of cDNA

About 30 adult beetle beetles were collected, rinsed with ddH2O, blotted dry with filter paper, and ground quickly with liquid nitrogen until white powder was obtained. Total RNA of flea beetle was extracted by Trizol method. The single-stranded cDNA was obtained by reverse-transcribing the total RNA of the flea beetle by using the EasyScript One-StepgDNA Removal and cDNA Synthesis SuperMix kit, and its nucleotide sequence is shown in SEQ ID NO:1. The cytochrome P450 gene (c13467_g1_i1) of the flea beetle has a high expression level, and the similarity between this gene and other known P450 homologous genes is less than 80%. It has strong specificity and is a specific gene of the flea beetle. The efficient expression of the gene is very important for the metabolism, growth and development of the flea beetle. Silencing the gene by RNAi reduces the expression level of the gene and affects the normal metabolism, growth and development of the flea beetle, so the effect of killing the pest can be achieved. And because of the strong specificity of the gene, generally it will not cause harm to other insects or other organisms.

Example 2 Cloning of Cytochrome P450 Gene

Use Primer5.0 software to design primers, and add Kpn I and Sac I restriction sites to the 5' end of the upstream primer and the 5' end of the downstream primer respectively (the P450 primers with restriction sites are shown in the table below, GGTACC is the Kpn I restriction site, GGTACC is the Sac I restriction site). Using the cDNA obtained by reverse transcription as a template, PCR amplification with primers containing enzyme cleavage sites (Kpn I, SacI) yielded a fragment of the Cytochrome P450 gene of Leprosy japonica, with a size of about 250bp, as shown in Figure 1 , the similarity between this gene segment and other known homologous genes is less than 73%. The PCR reaction system was: 25 µL 2×TransTaqHiFiPCR SuperMix II, 1 ul of upstream and downstream primers, 2 ul of template, and 50 ul of ddH2O. The amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s s, 72°C extension for 1 min, 36 cycles; 72°C extension for 5 min.

P450-F GG GGTACC ACTCGAAGGATCTTGAGTACAT SEQ ID NO: 3 P450-R C GGTACC ATGACGGCAACTGTAGGTCCCGA SEQ ID NO: 4

Example 3 Construction of flea beetle P450 interference vector

Using Fast digest Kpn I and Fast digest Sac I, the L4440 plasmid and the P450 PCR recovery product were subjected to double enzyme digestion at 37 degrees for 2 hours. Perform 1.2% agarose gel electrophoresis on the digested product of the L4440 plasmid, and then cut the gel to recover the large fragment and remove the small fragment; use the ordinary DNA product purification kit to purify the digested product of P450. The L4440 product recovered by gel cutting and the purified P450 product were reacted at 25°C for 2 hours, and the ligation product was transformed into HT115 (DE3) competent cells, cultured on a constant temperature shaker at 37°C, 250 r/min for 60 minutes, and then spread on LB solid medium (100mg/L AMP), cultivate overnight in a constant temperature incubator at 37°C until colonies grow. Select the white colonies, carry out colony PCR identification on the white single colonies, sequence the positive single clones that have been screened and identified successfully, compare the sequencing results, and select the successfully constructed L4440-P450 recombinant plasmids.

The size of the L4440 plasmid is 2790bp, and it has a bidirectional T7 promoter, as shown in Figure 2A. In this experiment, Sac Ⅰ and Kpn Ⅰ were selected for double digestion at the multiple cloning site. Agarose gel electrophoresis analysis showed that the L4440 plasmid produced two fragments after double enzyme digestion, the size of the large fragment was about 2700bp, and the size of the small fragment was about 138bp. Well, the enzyme-digested product is recovered by gel cutting, the large fragment product is retained, and the small fragment product is removed, as shown in Figure 2B.

The P450 PCR amplification product was subjected to double enzyme digestion, and the product was purified. Then, the two products were connected under the ligation system, and then the ligated product was transformed into Escherichia coli HT115 competent cells, and the resistance screening and sequencing comparison were carried out, as shown in Figure 3, after colony PCR identification, the transformed ligation plasmid It was in line with the expected results, indicating that the recombinant plasmid was constructed successfully, and the recombinant plasmid was named L4440-P450 plasmid.

Example 4 Induced expression and purification of dsRNA

The L4440-P450 recombinant plasmid was transformed into HT115. Take 20µL to 10mL of fresh LB liquid medium (containing 100mg/L AMP), shake and culture it in a constant temperature culture shaker at 37°C and 220r/min for 16h, absorb 2mL of the cultured bacterial liquid, and add it to 100mL of fresh liquid medium (containing 100mg/L AMP), cultivated to logarithmic growth phase, at this time, OD600=0.5, and the shaking culture time is generally 3 to 4 hours. Add IPTG for induction, the final concentration of IPTG is 0.4mM, put it back into the constant temperature culture shaker, and continue to shake and culture at 37°C and 220r/min for 4h. dsRNA was extracted by Trizol method, and 30µLddH2O was added. An appropriate amount of the above-mentioned samples was drawn, digested and treated with DNase I and RNase A respectively, and dsRNA was identified. The results of agarose gel electrophoresis are shown in FIG. 4 . DNase I is mainly used to degrade DNA molecules in the extract, while RNase A can digest single-stranded RNA. The dsRNA extracted by Trizol remained stable after being treated with DNase I and RNase A successively, indicating that the dsRNA was successfully obtained.

Example 5 Optimization of dsRNA induction conditions

dsRNA is induced by IPTG, so gradient experiments can be carried out in terms of IPTG concentration and culture time to select induction conditions suitable for high-efficiency expression of dsRNA. The first is the concentration of IPTG. Take the HT115 bacterial solution transformed with the L4440-P450 recombinant vector and add it to 4 Erlenmeyer flasks at a ratio of 1:50. Each bottle contains 100mL fresh LB liquid medium (100mg/L AMP). , and set the final concentrations of IPTG as 0 mM, 0.2 mM, 0.4 mM, and 0.8 mM, respectively. Then, when OD600=0.5, add IPTG, set the induction culture time of IPTG, and set the time as 2h, 3h, 4h respectively. Different concentrations of IPTG were induced and cultured for three different times. Other conditions remain unchanged.

The dsRNA extracted at different concentrations and at different times was first stored in a -20°C refrigerator, and after all extraction was completed, the expression of dsRNA induced by different IPTG concentrations at different times was detected by agarose gel electrophoresis.

It can be seen from Figure 5 that the bacteria solution without IPTG induction cannot produce dsRNA; when the IPTG concentration is low (0.2-0.4 mM), under the same IPTG concentration condition, different induction times will lead to different brightness As the time increases, the brightness of the band increases, which indicates that the expression of dsRNA will increase with time, and the expression of dsRNA is the largest at 4h (6 and 9 in Figure 5), but When the concentration of IPTG was high (0.8 mM), the expression of dsRNA was the largest at 3 hours of induction (11 in Figure 5); at the same induction time of IPTG, the expression of dsRNA induced by different concentrations of IPTG was different, at 0.2-0.4mM IPTG The expression level of dsRNA is relatively high under induction. Therefore, according to the results of optimization of dsRNA induction conditions, it is considered that 0.2-0.4mM IPTG induction for 4h will produce relatively high expression dsRNA.

Example 6 dsRNA Stability Detection

Use a high-pressure cell disruptor to crush the IPTG-induced bacterial liquid, and extract the dsRNA roughly by isopropanol precipitation, and then place it at room temperature for 0h, 12h, 24h, 36h, 48h, and 72h, and absorb a part of the dsRNA at each time period. Stored at 20°C, and finally tested by agarose electrophoresis to compare and judge the stability of dsRNA at room temperature.

From Figure 6, it can be found that dsRNA basically does not degrade at room temperature, indicating that dsRNA can remain stable at room temperature. Therefore, the follow-up research on anti-flea beetle experiment can detect the insecticidal effect by spraying the broken bacterial solution.

Embodiment 7 dsRNA insecticidal test

The dsRNA feeding method is mainly by feeding artificial feed to insects, crops expressing dsRNA, strains with the effect of inducing dsRNA, or directly spraying dsRNA on the surface of crops. The feeding method is simple to operate, low in input cost, and easy to implement. It enters the pest body in the most primitive and natural way, and achieves the purpose of silencing genes.

Prepare a P450 dsRNA solution with a concentration of 200ng/ml by diluting with sterile water, soak the young cabbage leaves in the P450dsRNA solution and the control solution (ddH2O) for one to two minutes, so that the surface is fully in contact with the dsRNA solution, every 24 hours , update the soaked leaves for feeding the flea beetle, observe and record the growth status and statistics of the flea beetle. Three repetitions were set up in the control group and the test group respectively, and 20 flea beetle adults were used in the feeding experiment in each repetition.

By feeding young leaves of Chinese cabbage soaked with ddH2O and P450-dsRNA, it was observed that in the first three to four days, the growth and development of the adult flea beetles in the control group and the test group were not significantly affected, and on the fifth day the test group The RNAi effect began to show, and the death of flea beetles appeared one after another. In about 15 days, P450 dsRNA showed a significant inhibitory effect, and the mortality rate of the tested insects reached more than 50%, and the average mortality rate reached 61.5%. significantly higher than that of the control group, as shown in Figure 7.

The process of RNAi is completed by the following steps. The first step is the initial stage of RNAi, the exogenously introduced dsRNA is cut into 21-25 bp siRNA by the endonuclease Dicer; the second step is the assembly stage of siRNA, siRNA is loaded under the action of Argonaute and other enzymes To the RISC complex, one strand of the double-stranded RNA is degraded after the complex is formed, and the single-stranded RNA loaded and activated on the RISC complex is called the guide strand; the third step is the effector stage of RNAi, entering the RISC complex The siRNA of the body finds the target mRNA sequence through sequence complementation and binds to it, resulting in the translation inhibition of the target mRNA or cutting it into short fragments of RNA through the action of some endonucleases, thereby causing the silencing effect on the target gene.

RNAi control of pests is mainly a non-cell-autonomous application of RNAi. Non-cell autonomous RNAi includes environmental RNAi and systemic RNAi. Environmental RNAi is the absorption of dsRNA by cells from the external environment, and systemic RNAi is the transfer of silencing signals in multicellular organisms between cells or tissues. The dsRNA of the target gene is sprayed on the leaves of the crops, and the pests eat the leaves to make the dsRNA reach the midgut of the pest and be absorbed by the pest. This process is called environmental RNAi; after the midgut cells absorb the dsRNA, the silencing signal passes through systemic RNAi The translocation process reaches the expression cells or tissues of the target gene, and induces the specific silencing of the target gene.

The above is only the specific implementation mode of the present invention, not all the implementation modes. Any equivalent transformation taken by those of ordinary skill in the art to the technical solution of the present invention by reading the description of the present invention is covered by the claims of the present invention. .

SEQUENCE LISTING

<110> Shenzhen University

<120> A Cytochrome P450 Gene and Its Application

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Claims (3)

1. The application of the dsRNA of the phyllotreta striolata cytochrome P450 gene fragment in killing the phyllotreta striolata is characterized in that,

the synthesis method of the dsRNA of the Huang Qutiao flea beetle cytochrome P450 gene fragment comprises the following steps: constructing a dsRNA expression vector containing a phyllotreta striolata cytochrome P450 gene fragment to obtain a high-efficiency expression system, and inducing the expression system by IPTG to obtain dsRNA corresponding to the target fragment;

the nucleotide sequence of the Huang Qutiao flea beetle cytochrome P450 gene fragment is shown in SEQ ID NO:2 is shown in the figure;

the nucleotide sequence of the Huang Qutiao flea beetle cytochrome P450 gene is shown in SEQ ID NO:1 is shown in the specification;

the method for obtaining the Huang Qutiao flea beetle cytochrome P450 gene fragment comprises the following steps: to contain a nucleotide sequence as shown in SEQ ID NO:1, by means of an upstream primer with an enzyme cleavage site Kpn I as a template, the sequence of SEQ ID NO:3 and a downstream primer with a cleavage site Sac I SEQ ID NO:4, carrying out PCR amplification to obtain a phyllotreta striolata cytochrome P450 gene fragment;

the construction method of the dsRNA expression vector containing the phyllotreta striolata cytochrome P450 gene fragment comprises the following steps: double enzyme digestion is carried out on vector plasmid capable of producing dsRNA and phyllotreta striolata cytochrome P450 gene fragment respectively under the conditions of Fast digest Kpn I and Fast digest Sac I and 37 ℃;

carrying out 1.2% agarose gel electrophoresis on a vector plasmid enzyme cutting product capable of generating dsRNA, then cutting gel to recover large fragments, and removing small fragments;

purifying the enzyme digestion product of P450 by using a common DNA product purification kit;

ligating the vector plasmid product recovered by cutting gel at 25 ℃ and capable of producing dsRNA with the P450 product obtained by purification;

the vector plasmid capable of producing the dsRNA is L4440 plasmid;

the preparation method of the high-efficiency expression system comprises the step of transfecting the expression vector into a host cell.

2. The use of claim 1, wherein the host cell is an RNaseIII-deleted host cell.

3. The use of claim 2, wherein the RNaseIII-deleted host cell is an HT115 (DE 3) competent cell.