Construction method and application of bactrocera cucurbitae genetic selection marker based on pupa color change

Abstract

The invention provides a construction method and application of a bactrocera cucurbitae genetic selection marker based on pupa color change. The construction method comprises the following steps: amplifying a plasmid skeleton from an Addgene 24353 vector by using primer sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2; the method comprises the following steps: amplifying 5 'UTR (Untranslated Region) and CDS (Coding Sequence) of an XM054231212 gene sequence by using primer sequences as shown in SEQ ID NO.3 and SEQ ID NO.4; the method comprises the following steps: amplifying an upstream 2000bp sequence of a 5 'UTR (Untranslated Region) of the LOC105216239 gene sequence by using primer sequences as shown in SEQ ID NO.5 and SEQ ID NO.6; carrying out seamless cloning, assembling and amplifying on the obtained fragments to obtain a WPRP vector, and screening to obtain a WPRP plasmid; the method comprises the following steps: mixing a WPRP plasmid with a transposon-assisted enzyme plasmid pHelper to obtain a plasmid solution; the plasmid solution is injected into eggs of a bactrocera cucurbitae white pupa strain, and after the eggs develop into imagoes, brown pupae appear when bred offspring pupate. By adopting the method disclosed by the invention, the bactrocera cucurbitae pupa color recovery can be used as a selection marker, and the positive transformant can be distinguished only through visual inspection, so that the screening efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of transgenic engineering of Bactrocera cucurbitae, and in particular to a construction method and application of a genetic screening marker of Bactrocera cucurbitae based on pupa color change. Background Art

[0002] In the field of genetics and biotechnology, research on gene transformation and genetic screening markers has always been an important topic. The melon fruit fly (Bactrocera cucurbitae) is an insect of the family Tephritidae in the order Diptera, widely distributed in tropical and subtropical regions. It mainly causes damage by feeding on the inside of fruits and vegetables as larvae, causing them to rot and deteriorate, causing serious economic losses to the fruit and vegetable planting industry. The melon fruit fly has strong reproductive capacity and adaptability, a short life cycle, and serious generation overlap, which makes its prevention and control work face huge challenges.

[0003] It is of great significance to construct genetic screening markers for melon fruit flies. First of all, accurate and effective screening markers can help researchers accurately identify and isolate melon fruit fly individuals with specific genetic characteristics, so as to deeply explore their genetic variation laws and evolutionary mechanisms. This plays a key role in revealing the population structure, diffusion pattern and adaptability to the environment of melon fruit flies. Secondly, screening markers can quickly locate genes related to important biological characteristics such as drug resistance and fertility, providing important theoretical basis and technical support for the development of more efficient and environmentally friendly pest control strategies. Furthermore, in the study of gene function, screening markers can help identify and analyze the role of key genes, and promote a deeper understanding of the growth and development, behavioral habits and other aspects of melon fruit flies. In addition, screening markers can also help monitor the dynamic changes of melon fruit fly populations in the field, and provide a decision-making basis for timely prevention and control measures, thereby effectively reducing its harm to agricultural production.

[0004] Insect genetic engineering requires screening true positive individuals that are successfully inserted into the genome from a large number of samples. The workload is huge and complex, so it is necessary to use visible screening markers to reduce the burden. In the study of Cucurbita cucurbita, there are many problems with previous genetic screening marker methods. The traditional fluorescent protein labeling method requires specific instruments and equipment to detect fluorescence, which not only increases the cost of the experiment, but also has high requirements for the experimental environment. In the screening process, since the fluorescent protein labeling needs to be observed under specific excitation light, this limits the convenience and flexibility of observation. Moreover, for experimenters, observing fluorescence in a dark room for a long time has an adverse effect on eye health. In addition, some traditional genetic screening marker methods also have difficulties in the selection of screening period. Cucurbita cucurbita in the larval stage is in an active growth and movement state, making it difficult to observe and screen in a centralized manner; and the Cucurbita cucurbita in the adult stage has a large range of activities, which increases the difficulty of screening. In addition, the phenotypic differences of some labeling methods are not obvious enough, making it difficult to accurately distinguish transformants, thereby affecting the accuracy and efficiency of screening. Some marker genes may interfere with the normal expression and function of the target gene, or recombinate with the target gene, etc., thereby affecting the accuracy and reliability of the experimental results. As a potential screening marker, pupal color change has not been effectively applied in genetic screening of Bactrocera melonensis due to limitations in technical means and research directions.

[0005] In summary, research on genetic screening markers for Bactrocera melonensis still needs further exploration and innovation to overcome the limitations of existing methods and improve screening efficiency and accuracy. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for constructing a genetic screening marker for Bactrocera cucurbitae based on pupal color change and its application.

[0007] In order to achieve the above purpose, the scheme of the present invention is as follows:

[0008] A method for constructing a genetic screening marker for Bactrocera melonensis based on pupal color change comprises the following steps:

[0009] The plasmid backbone was amplified from the Addgene 24353 vector using the primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2;

[0010] The 5'UTR and CDS sequence of the XM_054231212 gene sequence were amplified using the primer sequences shown in SEQ ID NO.3 and SEQ ID NO.4;

[0011] The primer sequences shown in SEQ ID NO.5 and SEQ ID NO.6 were used to amplify the 2000 bp sequence upstream of the 5'UTR of the LOC105216239 gene sequence;

[0012] The fragments obtained by seamless cloning and assembly were amplified to obtain the WPRP vector, and the WPRP plasmid was obtained by screening;

[0013] Mixing the WPRP plasmid with the transposition assistant enzyme plasmid pHelper to obtain a plasmid solution;

[0014] The plasmid solution was injected into the eggs of the melon fruit fly white pupa strain. After the eggs developed into adults, the offspring appeared brown pupae when they pupated.

[0015] Furthermore, the mass ratio of the WPRP plasmid to the transposition helper enzyme plasmid pHelper is 1 to 3:1.

[0016] Furthermore, the progeny produced include: progeny obtained by self-pollination of the G0 generation or heterosexual hybridization with the white pupa strain.

[0017] The method for constructing a genetic screening marker for Bactrocera melonensis based on pupal color change is used in judging whether transgenic technology is successful.

[0018] Furthermore, the gene includes the GAL4 gene.

[0019] Furthermore, the application comprises the following steps:

[0020] The GAL4 gene is constructed into the WPRP plasmid to obtain a transgenic plasmid, and the plasmid is transferred into the genome of the white-pupaed melon fruit fly using the PiggyBac transgenic technology. If brown pupae appear in the offspring, it means that the GAL4 gene transgenic is successful.

[0021] Furthermore, the progeny produced include: progeny obtained by self-pollination of the G0 generation or heterosexual hybridization with the white pupa strain.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention creatively uses the restoration of the pupal color of the melon fruit fly as a screening marker, and positive transformants can be distinguished by naked eye observation, which greatly improves the screening efficiency. The melon fruit fly white pupa color restoration strain we constructed overcomes the defects of fluorescent protein labeling: (1) It locates the screening period in the pupal period between larvae and adults, and the melon fruit fly will not move after pupation, which is convenient for observation and screening; (2) The dominant phenotype of the pupal color of the melon fruit fly is brown pupa, and the recessive phenotype is white pupa. The phenotypic difference is obvious, and the transformants are easy to distinguish; (3) The phenotype can be distinguished by naked eye observation without causing damage to the insect; (4) It is convenient and fast, and is not limited by the assistance of instruments; (5) Researchers do not need to observe fluorescence in a dark room for a long time, which is beneficial to the eye health of researchers. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :WPRP vector map.

[0025] Figure 2 : White pupa strain after wp knockout.

[0026] Figure 3 : Pupa color phenotype of G1 generation after wp complementation.

[0027] Figure 4 : Molecular detection of wp complemented G1 generation brown pupae. 1-11: 11 brown pupae; wp(-): white pupae individuals; +: positive control; -: negative control. WP-F1 and WP in vitro validation-R primers were used to amplify a sequence of about 874 bp on the plasmid. This band can only be amplified when the wp complemented sequence is inserted into the genome of the individual gDNA.

[0028] Figure 5 : PiggyBac-WPRP-Gal4 transgenic plasmid.

[0029] Figure 6 : GAL4 transgenic verification. DETAILED DESCRIPTION

[0030] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0031] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0032] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0033] Example 1

[0034] The specific process of constructing the strain for restoring the pupal color of Bactrocera melonensis white pupa is as follows:

[0035] 1. Construction of the complement vector for white pupa

[0036] 1.1 Primer design

[0037] Download the mRNA sequence (XM_054231212) and genome sequence (LOC105216239) of Bactrocera melonensis white pupae from the NCBI database. Extract the 5'UTR region and CDS coding sequence from the mRNA sequence, and extract the first 2000bp sequence of the 5'UTR region from the genome sequence, which is the wp upstream regulatory sequence. Extract the plasmid backbone sequence containing the piggyBac 5' and 3' reverse terminal repeat sequences from the Addgene #24353 # vector (a commercial product, product information link: https: / / www.addgene.org / 24353 / , full name of the plasmid: pBac-GH146-QF). Use snapgene software to construct an electronic clone of White Pupae Rescue Plasmid ( Figure 1 ).

[0038] The principle of seamless cloning was used to introduce recombination regions at both ends of the above fragments. The primers were designed as shown in Table 1. The synthesis of primers was completed by Guangzhou Aiji Biotechnology Co., Ltd. The underlined part is the linker sequence added by seamless cloning.

[0039] Table 1

[0040]

[0041]

[0042] 1.2 PCR amplification

[0043] Each fragment was amplified using Takara's high-fidelity enzyme PrimeSTAR Max DNA Polymerase (R045). The reaction system was as follows (50 μL):

[0044] Table 2

[0045]

[0046] The above solutions were mixed evenly. The PCR reaction conditions were: 98°C for 10 sec, 55°C for 15 sec, and 72°C for 5 sec / kb, for a total of 35 cycles.

[0047] 1.3 Agarose gel electrophoresis and gel recovery

[0048] ① Add 6× loading buffer to the PCR product (10 μL per 50 μL system) and mix well.

[0049] ② Use a pipette to add the PCR product and DNA marker into the gel wells respectively, and perform electrophoresis at 120V for 20 minutes.

[0050] ③Observe the fluorescent bands under ultraviolet irradiation to confirm the position of the PCR product bands.

[0051] ④ Cut out the single target DNA band from the agarose gel (try to remove the excess part) and put it into a clean centrifuge tube, and use TIANGEN Universal DNA Purification and Recovery Kit (DP214) to recover the PCR product.

[0052] 1.4 Seamless cloning to construct expression vector

[0053] The fragments were seamlessly cloned and assembled using the ABclonal 2X MultiF Seamless Assembly Mix (RK21020) kit to construct the WPRP vector.

[0054] Table 3

[0055]

[0056] The solution was mixed by gentle pipetting, incubated at 50°C for 60 min, and immediately placed on ice.

[0057] 1.5 Seamless cloning product transformation into E. coli

[0058] ① Thaw the Top10 competent cells on ice, add 20 μl of seamless cloning product to the competent cell suspension, tap the tube wall to mix the solution, and place on ice for 30 min.

[0059] ②The reactants were warmed at 42°C for 90 seconds and then immediately placed in an ice bath for 2 minutes.

[0060] ③ Add 1 mL of antibiotic-free LB medium to the centrifuge tube and culture at 37°C, 180 rpm, with shaking for 1 h.

[0061] ④ Take 300 μL of culture medium and spread it on the LB plate containing Amp. After drying, culture it at 37℃ overnight for 12-16 hours.

[0062] 1.6 Screening of positive clones and sequencing

[0063] On the second day, 8 single colonies were randomly picked from the plate and dissolved in 20 μl sterile water as templates. Colony PCR was performed using TIANGEN 2×Taq PCR Mix (KT201) and primers M13-20 and M13-26. The reaction system was as follows:

[0064] Table 4

[0065]

[0066] The above solutions were mixed evenly. The PCR reaction conditions were as follows: 94°C for 3 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 5 min, for a total of 35 cycles; and final extension at 72°C for 10 min.

[0067] Place the single colony solution that is correct by electrophoresis into a shaking tube, add 3 mL of LB liquid culture medium containing Amp, and shake and culture at 37°C for 16 hours. Take 1 mL of the bacterial solution and send it to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing.

[0068] 1.7 Endotoxin-free plasmid extraction

[0069] The correctly sequenced bacterial solution was added to 60 mL of liquid culture medium for overnight culture, and the plasmid was extracted by removing endotoxin using the OMEGA Endo-Free Plasmid Mini Kit II-fast kit (D6950B).

[0070] 2. Microinjection of Plasmids

[0071] The WPRP plasmid and the transposition auxiliary enzyme plasmid pHelper are mixed at a ratio of 1:1 (in another embodiment, the WPRP plasmid and the transposition auxiliary enzyme plasmid can be mixed at a ratio of 1 to 3:1) to prepare a solution with a final concentration of 500 ng / μl each. Use a micro-injector to inject 3 μL of the plasmid solution into the drawn capillary glass tube, and touch the tip of the tube lightly on the glass slide under a microscope to provide pressure. If liquid slowly flows out of the tip, the needle is successfully broken.

[0072] Collect wp knockout strain of melon fruit fly ( Figure 2 ) Eggs laid within 15 minutes were softened by treatment with sodium hypochlorite and Triton-X 100, and the plasmids were injected into the eggs one by one using an Eppendorf FemtoJet 4i microinjector. After the injection, the eggs were placed in a humidified box and waited for the eggs to hatch into larvae.

[0073] 3. Screening of white pupa color restoration strains

[0074] After the injection, the G0 generation of melon flies pupated, and the pupae were all white. After they emerged as adults, they were self-fertilized, and the offspring G1 generation had two pupae colors during pupation, brown and white ( Figure 3 We conducted quantitative statistics on the pupal colors of the G0 and G1 generations (Table 5).

[0075] Table 5. Color and proportion of melon fruit fly pupae after wp filling

[0076]

[0077] gDNA was extracted from brown pupae for molecular verification. Hangzhou Xinjing 3101250 Animal Tissue DNA Kit was used to extract gDNA. After extraction, PCR amplification was performed using primers that verified the insertion to detect whether the wp complement plasmid was inserted into the genome. The PCR results showed that the white pupa was successfully complemented and the pupa color returned to brown ( Figure 4 ).

[0078] The G1 generation brown pupae were crossed with homozygous white pupae male and female adults, and the ratio of brown pupae to white pupae in the G2 generation was 1:1, which conforms to Mendel's law of inheritance. The G2 generation brown pupae are the complement strains of the melon fruit fly white pupae, indicating that the restoration of pupal color can be successfully used as a genetic screening marker.

[0079] Example 2

[0080] Using the change of pupa color as a transgenic screening marker, referring to the method of Example 1, using the principle of seamless cloning, the GAL4 gene was constructed into the WPRP vector to obtain the PiggyBac-WPRP-Gal4 transgenic plasmid, which contains the WPRP complement sequence (SEQ ID NO.11), the actin 5C promoter and the GAL4 gene (GenBank: K01486.1). The plasmid elements were transferred into the genome of the white pupa Bactrocera melonensis using the PiggyBac transgenic technology, and the brown pupa individuals obtained by selfing in the G0 generation were transgenic individuals.

[0081] To further verify that the brown pupae were transgenic individuals, we selected wild type (WT), white pupae (WP) and transgenic brown pupae (GAL4-1, GAL4-2), extracted their genomic DNA, and then PCR amplified the GAL4 gene to verify whether the GAL4 gene was successfully transferred into the genome. Figure 6 .

[0082] As can be seen from the figure, the GAL4 gene cannot be amplified in WT and WP individuals, but can be amplified in brown pupa individuals. The wp gene does not affect the expression of the GAL4 gene, indicating that wp can be used as a marker gene and pupa color changes can be used as a transgenic screening marker for the GAL4 gene.

[0083] Further explanation: The GAL4 gene described in this embodiment is only an example, and those skilled in the art can replace it with other genes such as GAA gene (GenBank: M34424.1), GAL80 (NCBI Reference Sequence: XM_033912378.1), RHR2 (NCBI Reference Sequence: XM_449883.1), beg1 gene (GenBank: U60830.2), etc.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a genetic screening marker for Bactrocera cucurbitae based on pupal color change, characterized in that: The following steps are involved: The plasmid backbone was amplified from the Addgene 24353 vector using the primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2; The 5'UTR and CDS sequence of the XM_054231212 gene sequence were amplified using the primer sequences shown in SEQ ID NO.3 and SEQ ID NO.4; The primer sequences shown in SEQ ID NO.5 and SEQ ID NO.6 were used to amplify the 2000 bp sequence upstream of the 5'UTR of the LOC105216239 gene sequence; The fragments obtained by seamless cloning and assembly were amplified to obtain the WPRP vector, and the WPRP plasmid was obtained by screening; Mixing the WPRP plasmid with the transposition assistant enzyme plasmid pHelper to obtain a plasmid solution; The plasmid solution was injected into the eggs of the melon fruit fly white pupa strain. After the eggs developed into adults, the offspring appeared brown pupae when they pupated.

2. The method for constructing a genetic screening marker for Bactrocera cucurbitae based on pupa color change according to claim 1, characterized in that: The mass ratio of WPRP plasmid to transposition helper enzyme plasmid pHelper is 1 to 3:

1.

3. The method for constructing a genetic screening marker for Bactrocera cucurbitae based on pupa color change according to claim 1, characterized in that: The offspring produced include offspring obtained through self-pollination of the G0 generation or heterosexual hybridization with the white pupa strain.

4. Application of the method for constructing a genetic screening marker for Bactrocera cucurbitae based on pupal color change as claimed in claim 1 in determining whether transgenic technology is successful.

5. The use according to claim 4, characterized in that: The genes include the GAL4 gene.

6. The use according to claim 5, characterized in that: The following steps are involved: The GAL4 gene is constructed into the WPRP plasmid of claim 1 to obtain a transgenic plasmid, and the plasmid is transferred into the genome of the white-pupaed melon fruit fly using the PiggyBac transgenic technology. If brown pupae appear in the offspring, it means that the GAL4 gene transgenic is successful.

7. The use according to claim 6, characterized in that: The offspring produced include offspring obtained through self-pollination of the G0 generation or heterosexual hybridization with the white pupa strain.

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