Method for constructing drosophila strain for site-specific integration of exogenous gene

Abstract

A method for constructing a drosophila strain for site-specific integration of an exogenous gene. In the method, PAI of a large serine recombinase is used to specifically recognize an attP sequence and / or an attB sequence contained in a genome of a drosophila strain, so as to integrate an exogenous gene into a specific location within the genome of the drosophila strain. The amino acid sequence of the PAI is as shown in SEQ ID NO: 1; and / or the large serine recombinase is derived from Pseudomonas aeruginosa.

Description

A method for constructing a fruit fly strain for site-specific integration of foreign genes This application claims the priority of Chinese patent application No. 2023110817338 filed on August 25, 2023. This application cites the entire text of the above Chinese patent application. Technical Field The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a fruit fly strain for site-specific integration of exogenous genes. Background Art For a hundred years, Drosophila melanogaster has been an important genetic model organism in life science and biomedical research. Its small size, easy breeding, and short reproductive cycle make it an ideal material for genetic research. More than 75% of human disease genes have highly homologous corresponding genes in Drosophila, such as Notch, Wnt, Hedgehog and other important signaling pathways. Drosophila functional genes show obvious single gene-single phenotype relationships, which are very suitable for high-throughput functional genomics analysis. Therefore, the establishment of efficient Drosophila transgenic technology is of great significance for functional gene research and the construction of humanized disease models. At present, transgenic fruit fly technology mainly relies on the following three methods: (1) Using transposons such as P-element or PiggyBac to randomly mediate the integration of exogenous genes through transposase. (2) Through homologous recombination (or non-homologous end joining) pathways, the exogenous gene is precisely knocked into the preset site. (3) The phiC31 integrase system is used to mediate the site-specific integration of exogenous genes. However, the exogenous gene integration efficiency of these methods is low, resulting in a lot of time and manpower required to obtain transgenic fruit flies, which has become a bottleneck restricting the application of fruit flies in biomedical research. Summary of the invention The technical problem to be solved by the present invention is to overcome the defect of low integration efficiency when the existing Drosophila transgenic technology, such as using the phiC31 recombinase system to integrate foreign genes, so the present invention provides a method for constructing a Drosophila strain for site-specific integration of foreign genes. The fruit fly strain prepared by the method can realize efficient site-specific integration of exogenous genes, and the method can significantly improve the integration efficiency of exogenous genes in fruit flies. The present invention provides a method for constructing a fruit fly strain for site-specific integration of exogenous genes based on a large serine recombinase PAI (Pseudomonas aeruginosa Integrase) derived from Pseudomonas aeruginosa. (1) The expression unit (cassette) of the optimized encoded PAI enzyme (containing a nuclear localization signal) is integrated into the fruit fly X chromosome (or into chromosome 2 or 3) by conventional genetic manipulation means, and the vasa (or nanos) promoter is used to drive the specific expression of the PAI enzyme in female reproductive cells. (2) The PAI enzyme recognition sequence attP (or attB) is respectively integrated into a specific site of chromosome 2 or 3 of the fruit fly (or into chromosome X) by conventional genetic methods. (3) Through hybridization screening of the above fruit flies, a double transgenic fruit fly (starter line) containing both the PAI enzyme and the recognition sequence attP (or attB) is obtained. (4) Microinjecting the exogenous gene donor plasmid containing the exogenous gene and the PAI enzyme recognition sequence attB (or attP) into the above starter line embryos, and obtaining the transgenic individuals with the exogenous gene integration through hybridization screening. (5) Comparing the results with the phiC31 recombinase system in the prior art to evaluate the integration efficiency of the method for constructing a Drosophila strain for site-specific integration of exogenous genes based on PAI provided by the present invention. The present invention solves the above technical problems through the following technical solutions. In the present invention, the term "genome" refers to the sum of all genes in an organism. The Drosophila genome is the sum of all genes in Drosophila, which is composed of diploid chromosomes. The size of the Drosophila genome is about 140 million base pairs, which contains about 13,000 genes. In the present invention, normal fruit fly somatic cells have 8 chromosomes, 6 of which are autosomes and 2 are sex chromosomes, and the sex is determined by XY type. Normal fruit fly germ cells have 4 chromosomes. The first aspect of the present invention provides a method for constructing a fruit fly strain for site-specific integration of exogenous genes, using a large serine recombinase PAI that can specifically recognize attP sequences and / or attB sequences contained in the genome of the fruit fly strain, thereby integrating the exogenous gene into a specific position of the genome of the fruit fly strain; wherein the amino acid sequence of the PAI is as shown in SEQ ID NO: 1; and / or the large serine recombinase is derived from Pseudomonas aeruginosa. In some embodiments of the present invention, the Drosophila strain comprises Drosophila cells or Drosophila embryos; Drosophila cells include somatic cells and germ cells. In some embodiments of the present invention, the chromosome of the Drosophila strain contains a nucleotide sequence encoding the PAI. In some embodiments of the present invention, the PAI or the nucleotide sequence encoding the PAI is introduced into the Drosophila strain so that the Drosophila strain contains the PAI, and the method of introduction is, for example, microinjection. In some embodiments of the present invention, the genome of the Drosophila strain comprises one or more attP sequences or attB sequences. In some preferred embodiments of the present invention, the fruit fly strain is Drosophila melanogaster. In some embodiments of the present invention, the attP sequence has a nucleotide sequence as shown in SEQ ID NO:3; and / or, the attB sequence has a nucleotide sequence as shown in SEQ ID NO:4. In some embodiments of the present invention, the nucleotide sequence encoding the PAI and the attP sequence and / or attB sequence recognized by PAI are located on chromosome X, chromosome 2 or chromosome 3. In some specific embodiments of the present invention, the nucleotide sequence encoding the PAI is shown as SEQ ID NO:2. In some embodiments of the present invention, the nucleotide sequence encoding the PAI is integrated into a Drosophila transgenic plasmid to obtain a PAI expression plasmid, whereby the PAI expression plasmid can express the PAI in Drosophila cells, such as female germ cells. In some embodiments of the present invention, the PAI enzyme expression plasmid further comprises a nuclear localization signal and a promoter, and the promoter is, for example, a Drosophila vasa promoter or a nanos promoter; In some specific embodiments of the present invention, the sequence of the vasa promoter is shown as SEQ ID NO:5; and / or the sequence of the nanos promoter is shown as SEQ ID NO:6. In some embodiments of the present invention, the fruit fly strain is a PAI-attP2 fruit fly strain or a PAI-attP40 fruit fly strain; the X chromosome of the PAI-attP2 fruit fly strain contains a nucleotide sequence encoding PAI that can be stably expressed, and chromosome 3 contains an attP sequence; the X chromosome of the PAI-attP40 fruit fly strain contains a nucleotide sequence encoding PAI that can be stably expressed, and chromosome 2 contains an attP sequence. sequence. In the present invention, the insertion position of the PAI, attP or attB sequence in the Drosophila genome is not limited to the above. The second aspect of the present invention provides a method for preparing transgenic fruit flies using the fruit fly strain constructed by the method described in the first aspect of the present invention. In some embodiments of the present invention, the exogenous gene donor plasmid includes an exogenous gene and an attB sequence and / or attP sequence recognized by PAI; when the genome of the fruit fly strain contains an attP sequence, the exogenous gene donor plasmid contains an attB sequence; when the genome of the fruit fly strain contains an attB sequence, the exogenous gene donor plasmid contains an attP sequence. In some embodiments of the present invention, the exogenous gene donor plasmid is introduced into the PAI-attP2 fruit fly strain or the PAI-attP40 fruit fly strain, and transgenic fruit flies carrying the exogenous gene in their genome are obtained by screening. On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. The reagents, tools, fruit flies and other raw materials used in the present invention are all commercially available. The positive and progressive effects of the present invention are: The present invention provides a method for constructing a fruit fly strain for site-specific integration of exogenous genes. The fruit fly strain constructed by the method achieves efficient site-specific integration of exogenous genes. Compared with the method based on the phiC31 recombinase system in the prior art, the integration efficiency of the method can be increased by 5 to 10 times. This provides a more efficient method for the integration of large fragments of exogenous genes and has important application value for constructing transgenic fruit flies. BRIEF DESCRIPTION OF THE DRAWINGS FIG1 is a transgenic expression plasmid map of the large serine recombinase PAI (WF-036). FIG. 2 is a map of a transgenic plasmid containing the PAI enzyme recognition site attP (WF-038). FIG3 is a schematic diagram of site-directed integration of vasa-PAI expression elements on chromosome X mediated by the phiC31 system. FIG4 is a diagram showing the construction of a transgenic fruit fly containing a PAI enzyme expression unit on the X chromosome. FIG. 5 shows the construction of a transgenic fruit fly with a PAI enzyme recognition site attP inserted into chromosome 3 (the RFP selection marker is removed). FIG6 is a schematic diagram of constructing a double transgenic Drosophila PAI-attP2 hybrid (X chromosome contains PAI, chromosome 3 contains attP site). FIG. 7 shows the construction of a transgenic fruit fly with a PAI enzyme recognition site attP inserted into chromosome 2 (the RFP selection marker is removed). FIG8 is a schematic diagram of constructing a double transgenic Drosophila PAI-attP40 hybrid (X chromosome contains PAI, chromosome 2 contains attP site). FIG. 9 is a schematic diagram of transgenic integration of exogenous genes on chromosome 3 mediated by PAI-attP2. FIG. 10 is a schematic diagram of transgenic integration of exogenous genes on chromosome 2 mediated by PAI-attP40. DETAILED DESCRIPTION The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications. Example 1 In this example, Drosophila melanogaster is used as an example to describe how to construct a starter line of Drosophila containing PAI-attP double transgenes. (1) Drosophila melanogaster is a classic genetic model organism. The fruit fly breeding and genetic manipulation experiments in this study were all carried out on standard fruit fly culture medium. The culture medium is mainly composed of corn flour, sucrose, yeast powder, agar, and preservatives p-hydroxybenzoic acid and propionic acid. The specific preparation method is: weigh each component in a certain proportion, add water to boil and dissolve, add preservatives and propionic acid after cooling, mix well, and fill into plastic tubes for later use. The life cycle of fruit flies is 12 days, and it goes through four stages: fertilized eggs, third-instar larvae, pupae, and adults. All experiments were conducted at 25°C and 50% humidity. (2) In this experiment, CO2 is used to anesthetize fruit flies when collecting virgin flies and performing hybridization. The operation steps are to first inject CO2 into the culture tube to anesthetize the fruit flies, then move the fruit flies to a plate with CO2 flowing through it, and gently pick and collect them with a brush. (3) The genetic hybridization of fruit flies in the experiment mainly follows the preset hybridization strategy, selecting virgin flies of a specific genotype and male fruit flies of a specific genotype for hybridization. The offspring are identified by the differences in the balancer phenotype and specific markers of the individual offspring. (4) This experiment designed a hybridization strategy to obtain fruit fly offspring with the target gene composition. Among the four chromosomes of fruit flies, this experiment mainly involved the X chromosome, chromosome 2 and chromosome 3 for hybridization operations. In order to ensure the stability of the genotype of the hybrid offspring, tool fruit flies with X chromosome balancers Fm7a or Fm7b (bar eyes), chromosome 2 balancer CyO (curled wings) and chromosome 3 balancer TM6B (short pupa) were used during the hybridization. When several genetic elements are integrated into the same fruit fly, they can be identified based on the label of the offspring and the phenotypic changes of the balancer. (5) The tool fruit fly strains used in the establishment of the double transgenic fruit fly starter line of the present invention are available in the fruit fly public library, including: ①W 1118 Wild-type fruit flies, used for routine transgenic hybrid screening, were kept in the laboratory; ②w; Sco / Cyo, w; Sco / Cyo; Sb / Tm6B, FM7a; Sco / cyo, FM7b; Sp / Cyo; Tm2 / Tm6B balanced lines of fruit flies, cyo has a warped wing phenotype, FM7a and FM7b have a bar-eye phenotype, and Tm6B has a short pupa phenotype, which are from the laboratory; ③P{CaryP}attP18 genotype fruit flies: the X chromosome contains the phiC31 recognition sequence attP, which is from the Bloomington Drosophila Stock Center in the United States, with the number 32107; ④P{CaryIP}su(Hw)attP8 genotype fruit flies: the X chromosome contains the phiC31 recognition sequence attP, which is from the Bloomington Drosophila Stock Center in the United States, with the number 32233; ⑤P{y[+t7.7]=nanos-phiC31\int.NLS}X,y[1]sc[1]v[1]sev

[0021] ; P{y[+t7.7]=CaryP}attP2 genotype fruit flies (abbreviated as: phiC31-attP2): Its X chromosome contains the phiC31 enzyme expression unit driven by the nanos promoter, which realizes the specific expression of the phiC31 enzyme in female reproductive cells. At the same time, the fruit fly chromosome 3 contains the attP site recognized by the phiC31 enzyme (attP2 "2" indicates that the attP sequence is located at a specific position on chromosome 3). The fruit fly was from the Bloomington Drosophila Stock Center in the United States and is numbered 25710; ⑥y[1]v[1]P{y[+t7.7]=nanos-phiC31\int.NLS}X;P{y[+t7.7]=CaryP}Msp300[attP40] genotype fruit fly (abbreviated as: phiC31-attP40), whose X chromosome contains the phiC31 enzyme expression unit driven by the nanos promoter, realizing the specific expression of the phiC31 enzyme in female germ cells. At the same time, the chromosome 2 of the fruit fly contains the attP site recognized by the phiC31 enzyme (the "40" in attP40 indicates that the attP sequence is located at a specific position on chromosome 2). The fruit fly comes from the Bloomington Drosophila Stock Center in the United States, with the number 25709; ⑦y+,w(Cre);Sco / Cyo and y+,w(Cre);Dr / Tm6B genotype fruit flies: X chromosome contains elements expressing Cre enzyme (Cre recombinase can mediate loxP sequence exchange), which are preserved in the laboratory; (6) The method for constructing transgenic fruit flies in this study is as follows: First, according to the preset hybridization strategy, virgin flies and male flies of specific genotypes are collected for genetic hybridization. Secondly, the desired transgenic fruit fly lines are obtained by screening based on the observable traits of the hybrid offspring (such as eye color or fluorescent markers and balancer phenotypes). Finally, homozygous lines of transgenic fruit flies are obtained by self-pollination. Parent fruit fly lines not otherwise specified are selected according to the preset scheme. Please refer to the instructions for the specific preparation and hybridization process. (7) The various transgenic cloning plasmids constructed in the present invention are carried out according to conventional molecular cloning methods, and the reagents are all commercially available, as described below: ①Construct the expression plasmid WF-036 of PAI enzyme (as shown in Figure 1): This plasmid contains a 3P3_RFP eye selection marker, which contains FRT sites on both sides, and the selection marker can be removed by FLP enzyme. The nucleotide sequence of PAI in the plasmid has been optimized by Drosophila codons, and can drive specific expression in female germ cells under the control of vasa promoter; in order to prevent the PAI enzyme expression unit from being affected by the surrounding sequences of the Drosophila genome, the gypsy isolation element is used to encapsulate the vasa-PAI expression unit; at the same time, the plasmid also contains the phiC31 enzyme recognition site attB (phiC31-attB) element, which can embed all elements of the exogenous plasmid into a specific position of the genome under the action of phiC31 integrase. ②Construction of transgenic plasmid WF-038 containing PAI enzyme recognition sequence attP (Figure 2): This plasmid contains 3P3_RFP selection marker, with loxP sites at both ends of the marker, which can be removed by Cre enzyme-mediated trans-action; in addition, the plasmid also contains phiC31 enzyme recognition site attB (phiC31-attB) Elements, under the action of phiC31 integrase, all elements of the exogenous plasmid can be inserted into a specific position of the genome. (8) Construction of transgenic fruit flies containing the PAI expression unit on the X chromosome: As shown in Figures 3 and 4, the PAI expression plasmid WF-036 (Figure 1) and phiC31-mRNA (preserved in the laboratory) were mixed at a ratio of 600ng:300ng per microliter and microinjected into P{CaryP}attP18 or P{CaryIP}su(Hw)attP8 embryos for transgenic. After the injected embryos were incubated as adults, they were incubated with W 1118 After hybridization, inbred lines were selected from the RFP-marked offspring, and finally a fruit fly line was obtained in which all X chromosomes were homozygous for the PAI transgene, and the female individual germ cells could stably express PAI integrase. (9) Construction of a double transgenic Drosophila model: a double transgenic starter line containing both the PAI enzyme and its recognition site attP, and the PAI enzyme and the recognition sequence attP are located on different chromosomes. A. Model establishment (PAI-attP2: chromosome X contains PAI enzyme, chromosome 3 contains attP site) ①Construction of transgenic fruit flies with PAI enzyme recognition site attP inserted into chromosome 3 (removing the selection marker). As shown in Figure 5, the WF-038 plasmid containing PAI recognition site attP was microinjected into phiC3-attP2 fruit flies (BDSC: 25710) embryos at a concentration of 600ng / μL for transgenic. After the injected embryos were incubated into adults, they were compared with wild-type fruit flies W 1118 Hybridize, select inbred lines from the RFP-marked offspring, and obtain a transgenic line with attP homozygous for PAI enzyme recognition on chromosome 3 (abbreviated as: attP2(PAI), where "2" indicates that the PAI recognition site attP and attP2 recognized by phiC31 are at the same position on the genome), and its female individual germ cells can stably express PAI enzyme. To obtain the attP2(PAI) transgenic line without RFP marker: as shown in Figure 5, firstly, attP2(PAI) male fruit flies with RFP label are hybridized with FM6B / X; Sp / Cyo; Tm2 / Tm6B virgin flies, and select RFP-positive male flies with Cyo curled wings, FM6b bar eyes and Tm6B short pupae. At the same time, use X Cre ; Dr / Tm6B female flies were crossed with FM6B / Y; Sp / Cyo; Tm2 / Tm6B male flies, and virgin flies with Cyo and Tm6B phenotypes were selected. The above two groups of hybrid offspring were crossed, and the offspring with short pupae with warped wings, heart-shaped eyes and no RFP marker were selected under the action of Cre recombinase, thus obtaining the target line fruit fly attP2 (PAI) with RFP marker removed. ② As shown in Figure 6, firstly, the X chromosome transgenic female flies containing PAI expression were crossed with X / Y; Sco / Cyo; The Sb / Tm6B balanced lines were crossed to screen male flies that simultaneously contained the PAI gene on the X chromosome and the phenotypes of Cyo 2 and Tm6B 3. These male flies were then crossed with the attP2 (PAI) transgenic female flies from which the RFP tag had been removed to screen for double transgenic female flies that simultaneously contained PAI on the X chromosome and the attP2 (PAI) site on chromosome 3 (i.e., non-Tm6B phenotype). Finally, these female flies were crossed with the double transgenic male flies obtained in the previous step to establish a double transgenic homozygous Drosophila starterline (named: PAI-attP2). B. Model establishment (PAI-attP40: chromosome X contains PAI enzyme, chromosome 2 contains attP site) ① Construction of transgenic fruit flies with PAI recognition site attP inserted into chromosome 2 (removing the selection marker). As shown in Figure 7, the WF-038 plasmid containing PAI recognition site attP was microinjected into phiC3-attP40 fruit flies (BDSC: 25709) embryos at a concentration of 600 ng / μL for transgenic. After the injected embryos were incubated as adults, they were compared with wild-type fruit flies W 1118 Hybridize, select inbred lines from the RFP-marked offspring, and obtain a transgenic line with attP homozygous for PAI enzyme recognition on chromosome 2 (abbreviated as: attP40 (PAI), where "40" means that the PAI recognition site attP and attP40 recognized by phiC31 are at the same position on the genome), whose female germ cells can stably express PAI enzyme. To obtain the attP40 (PAI) transgenic line with the RFP marker removed: As shown in Figure 7, firstly, the attP40 (PAI) transgenic male flies with RFP tags are hybridized with FM7a / Fm7a; Sco / Cyo balanced line virgin flies to screen male fly offspring with both RFP markers and Cyo curled wing phenotypes. At the same time, FM7a / Y; Sco / Cyo male flies are hybridized with X Cre Sco / Cyo female flies were crossed, and virgin flies with both Cyo wing-curling phenotype and heart-shaped eyes were selected. The above two groups of hybrid offspring were crossed, and offspring with Sco phenotype, heart-shaped eyes and no RFP marker were selected under the action of Cre recombinase, thus obtaining the target line fruit fly attP40 (PAI) with the RFP marker removed. ② As shown in Figure 8, firstly, the transgenic female flies with PAI expression on the X chromosome were hybridized with the X / Y; Sco / Cyo balanced fruit flies to screen for transgenic male flies containing both the X chromosome PAI gene and the balancer Cyo on chromosome 2. Then, these male fruit flies were hybridized with the transgenic female fruit flies containing the PAI recognition site attP40 (RFP tag removed) on chromosome 2 to screen for transgenic male flies containing both the PAI on the X chromosome and the attP40 (PAI) site on chromosome 2. Transgenic female flies. Finally, these female fruit flies were crossed with the double transgenic male flies obtained in the previous step to establish a double transgenic homozygous fruit fly starter line (named: PAI-attP40). Example 2 In this example, Drosophila melanogaster is used as an example to introduce how to use the double transgenic PAI-attP2 fruit fly model of the present invention to achieve efficient site-specific integration of exogenous genes on chromosome 3 of fruit flies, thereby achieving rapid transgene acquisition. The efficiency of transgenic acquisition in the experiment will be compared with the phiC31 recombination system. (1) Transgenic plasmids containing exogenous genes of different sizes were constructed, including Expt.1', Expt.2', Expt.3', Expt.4' and Expt.5'. In addition to the target exogenous genes, these plasmids also contain the PAI recognition site attB and the white red eye gene screening marker. As a control, control plasmids containing the phiC31 enzyme recognition site attB and insert fragments of different sizes corresponding to PAI were also constructed, including Expt.1, Expt.2, Expt3, Expt.4 and Expt.5. These plasmids also contain the white red eye screening marker. (2) As shown in FIG9 , the plasmids containing exogenous genes of different sizes (including Expt.1', Expt.2', Expt.3', Expt.4' and Expt.5') were microinjected into PAI-attP2 Drosophila embryos at a concentration of 600 ng / μL for transgenic. 1118 Crossbreeding and screening of red-eyed offspring can achieve site-specific integration of exogenous genes. Transgenic fruit flies carrying the integrated exogenous genes will be stably passed on to the next generation. (3) As shown in FIG10 , the control plasmids containing exogenous genes of different sizes (including Expt.1, Expt.2, Expt.3, Expt.4 and Expt.5) were microinjected into phiC31-attP2 Drosophila (BDSC:25710) embryos at a concentration of 600 ng / μL for transgenic. 1118 Crossbreeding and screening of red-eyed offspring can achieve site-specific integration of exogenous genes. Transgenic fruit flies carrying the integrated exogenous genes will be stably passed on to the next generation. (4) By counting the number of fruit fly tubes with screening markers produced by hybrid fruit flies, the integration efficiency of the PAI and phiC31 recombination systems at the same position on the fruit fly genome was compared. The results showed that compared with the phiC31 system, the efficiency of exogenous gene integration mediated by the PAI system was increased by 5 to 10 times, up to 52%, as shown in Tables 1 and 2. Table 1 Integration efficiency of exogenous genes on chromosome 3 mediated by PAI-attP2 Table 2 Integration efficiency of exogenous genes on chromosome 3 mediated by phiC31-attP2 These results indicate that the PAI recombination system can mediate site-specific integration of exogenous genes on Drosophila chromosome 3 in the Drosophila animal model, and its integration efficiency is much higher than that of the existing phiC31 recombination system. Example 3 In this example, Drosophila melanogaster is used as an example to describe how to use the double transgenic PAI-attP40 fruit fly of the present invention to achieve efficient site-specific integration of exogenous genes on Drosophila chromosome 2. The efficiency of transgenic acquisition in the experiment will be compared with the phiC31 recombination system. (1) As shown in FIG. 9 , the plasmids containing exogenous genes of different sizes constructed in Example 2 (including Expt.1′, Expt.2′, Expt.3′, Expt.4′ and Expt.5′) were expressed at a concentration of 600 ng / μL. Microinjection of PAI-attP40 into Drosophila embryos for transgenics. After the injected embryos were incubated as adults, they were compared with wild-type Drosophila W 1118 Crossbreeding and screening of red-eyed offspring can achieve site-specific integration of exogenous genes. Transgenic fruit flies carrying integrated exogenous genes will be stably passed on to the next generation. (2) As shown in FIG. 10 , the control plasmids containing exogenous genes of different sizes constructed in Example 2 (including Expt.1, Expt.2, Expt.3, Expt.4 and Expt.5) were microinjected into phiC31-attP40 Drosophila (BDSC: 25709) embryos at a concentration of 600 ng / μL for transgenic. After the injected embryos were incubated as adults, they were compared with wild-type Drosophila W 1118 Crossbreeding and screening of red-eyed offspring can achieve site-specific integration of exogenous genes. Transgenic fruit flies carrying integrated exogenous genes will be stably passed on to the next generation. (4) By counting the number of fruit fly tubes with screening markers produced by hybrid fruit flies, the integration efficiency of the PAI and phiC31 recombination systems at the same position on the fruit fly genome was compared. The results showed that compared with the phiC31 system, the efficiency of exogenous gene integration mediated by the PAI system was increased by 5 to 10 times, up to 61.9%, as shown in Tables 3 and 4. Table 3 Integration efficiency of exogenous genes on chromosome 2 mediated by PAI-attP40 Table 4 Integration efficiency of exogenous genes on chromosome 2 mediated by phiC31-attP40 These results indicate that the PAI recombination system can mediate site-specific integration of exogenous genes on Drosophila chromosome 2 in the Drosophila animal model, and that the integration efficiency is much higher than that of the existing phiC31 recombination system.

Claims

1. A method for constructing a Drosophila strain for site-specific integration of exogenous genes, characterized in that: The large serine recombinase PAI is used to specifically recognize the attP sequence and / or attB sequence contained in the genome of the fruit fly strain, thereby integrating the exogenous gene into a specific position of the genome of the fruit fly strain; wherein the amino acid sequence of the PAI is shown in SEQ ID NO: 1; and / or the large serine recombinase is derived from Pseudomonas aeruginosa.

2. The method according to claim 1, characterized in that The fruit fly strain includes fruit fly cells or fruit fly embryos; the fruit fly cells include somatic cells and germ cells; and / or, the chromosome of the fruit fly strain contains a nucleotide sequence encoding the PAI; and / or, the PAI or the nucleotide sequence encoding the PAI is introduced into the fruit fly strain so that the fruit fly strain contains the PAI, and the introduction method is, for example, microinjection; and / or, the fruit fly strain genome contains one or more attP sequences or attB sequences.

3. The method according to claim 1 or 2, characterized in that The fruit fly strain is Drosophila melanogaster.

4. The method according to any one of claims 1 to 3, characterized in that: The attP sequence has the nucleotide sequence shown in SEQ ID NO:3; and / or, the attB sequence has the nucleotide sequence shown in SEQ ID NO:

4.

5. The method according to any one of claims 1 to 4, characterized in that: The nucleotide sequence encoding the PAI and the attP sequence and / or attB sequence recognized by PAI are located on chromosome X, chromosome 2 or chromosome 3.

6. The method according to any one of claims 1 to 5, characterized in that: The nucleotide sequence encoding the PAI is shown in SEQ ID NO:

2.

7. The method according to any one of claims 1 to 6, characterized in that: The nucleotide sequence encoding the PAI is integrated into a Drosophila transgenic plasmid to obtain a PAI expression plasmid, whereby the PAI expression plasmid can express the PAI in Drosophila cells.

8. The method according to claim 7, characterized in that The Drosophila cells are female reproductive cells.

9. The method according to claim 7 or 8, characterized in that The PAI expression plasmid also includes a nuclear localization signal and a promoter.

10. The method according to claim 9, characterized in that The promoter is a vasa promoter or a nanos promoter.

11. The method according to claim 10, characterized in that The sequence of the vasa promoter is shown as SEQ ID NO:5; and / or the sequence of the nanos promoter is shown as SEQ ID NO:

6.

12. The method according to any one of claims 1 to 11, characterized in that: The fruit fly strain is a PAI-attP2 fruit fly strain or a PAI-attP40 fruit fly strain; the X chromosome of the PAI-attP2 fruit fly strain comprises a nucleotide sequence encoding PAI that can be stably expressed, and chromosome 3 comprises an attP sequence; the X chromosome of the PAI-attP40 fruit fly strain comprises a nucleotide sequence encoding PAI that can be stably expressed, and chromosome 2 comprises an attP sequence.

13. A method for preparing transgenic fruit flies using the fruit fly strain constructed by the method according to any one of claims 1 to 12.

14. The method according to claim 13, characterized in that The exogenous gene donor plasmid includes an exogenous gene and an attB sequence and / or attP sequence recognized by PAI; when the genome of the fruit fly strain contains an attP sequence, the exogenous gene donor plasmid contains an attB sequence; when the genome of the fruit fly strain contains an attB sequence, the exogenous gene donor plasmid contains an attP sequence.

15. The method according to claim 14, characterized in that The exogenous gene donor plasmid is introduced into the PAI-attP2 fruit fly strain or the PAI-attP40 fruit fly strain, and the transgenic fruit flies carrying the exogenous gene in the genome are obtained by screening.

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