Method for screening albino pathogenic gene by using fruit fly and application thereof

Abstract

The application discloses a method for screening albino pathogenic genes by using fruit flies and application thereof, and the method is characterized in that: GAL4 strain fruit flies and USA-X strain fruit flies are used as parents to cross to obtain F1 generation newly-emerged fruit flies, then male fruit flies of the F1 generation newly-emerged fruit flies are selected to mate with female virgin fruit flies of the Sp;Dr / SM6B-TM6B strain to obtain a stable preservation strain, and then the fruit flies are collected and the pigment changes of the fruit fly body surface and eyes are observed to screen the albino pathogenic genes. The method can effectively use the fruit fly model to study the albino related genes and process, and provide new experimental basis for the pathogenesis and treatment of the albino, thereby promoting the development and clinical application of related drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for screening albinism pathogenic genes using fruit flies and its application. Background Technology

[0002] Albinism is a genetic disorder characterized by a lack of melanin production, resulting in a deficiency of pigment in the skin, hair, and / or eyes. It is often accompanied by ocular symptoms such as photophobia, nystagmus, strabismus, and decreased vision; some patients also experience abnormalities in other systems. The global incidence is approximately 1 in 17,000, while the incidence in China is estimated at 1 in 20,000 to 1 in 10,000. Currently, there is no effective treatment; management primarily involves symptomatic and supportive therapies. Prevention is the only truly effective medical strategy, and early prenatal genetic testing is the main route of prevention.

[0003] In recent years, with the development of genetic engineering technology, several albinism-related genes have been discovered. However, there is still a lack of animal models for studying albinism, making it impossible to verify phenotypes and functions. A few studies have used models such as miniature pigs or macaques, which have drawbacks such as multiple genetic factors and complex phenotypic expressions, as well as high cost, long processing time, and inability to conduct large-scale screening. Drosophila, as a classic model organism for genetic research, has homologous genes for approximately 75% of human disease genes. Furthermore, Drosophila has advantages such as rapid reproduction, ease of rearing, small chromosome number, small genome, and ease of manipulation. The well-known binary expression system UAS-GAL4 in Drosophila can precisely regulate the spatiotemporal expression of genes.

[0004] Based on this, the present invention uses the model organism Drosophila to study the relevant pathogenic genes and processes of albinism, providing a new approach for the construction of albinism models. Summary of the Invention

[0005] One objective of this invention is to provide a method for screening albino pathogenic genes using fruit flies, comprising the following steps:

[0006] Step 1: Select GAL4 strain fruit flies and USA-X strain fruit flies as parents, and cross the two strains to obtain F1 generation newly emerged fruit flies;

[0007] The GAL4 strain of fruit flies is either tub-GAL4 / Cyo; Dr / TM6B or act-GAL4 / Cyo; Dr / TM6B.

[0008] The USA-X strain of Drosophila is Sp / Cyo; the UAS-X-RNAi transgenic strain of Drosophila, with the X gene being the gene to be screened.

[0009] Step 2: Select newly emerged male fruit flies from the F1 generation and mate them with female virgin flies of the Sp; Dr / SM6B-TM6B strain to obtain a stable and preserved strain.

[0010] Step 3: After preserving the fruit flies from Step 2 for 3 generations, collect and observe the pigmentation changes on the fruit fly's body surface and eyes.

[0011] Furthermore, the culture medium formulas in steps 1, 2, and 3 are as follows: each 100 ml of culture medium contains 6.0 g of brown sugar, 0.725 g of white sugar, 1.0 g of agar, 5.0 g of corn flour, 2.45 g of yeast, 0.4 ml of propionic acid, and 0.15 g of methylparaben.

[0012] Furthermore, in step 1, yeast granules are added to the culture medium during hybridization to encourage female fruit flies to lay eggs and collect a sufficient number of offspring. Specifically, 20 yeast granules are added per 10 ml of culture medium.

[0013] Furthermore, the rearing conditions in steps 1, 2, and 3 are as follows: the animals are raised in an incubator at a temperature of 24–26°C and a humidity of 50%–60%.

[0014] A second objective of this invention is to provide the application of the above method in screening for albinism-causing genes.

[0015] In one specific embodiment of the present invention, tub-GAL4 / Cyo; Dr / TM6B Drosophila and Sp / Cyo; UAS-blos2-RNAi Drosophila were mated, and the expression of the blos2 gene was downregulated by the GAL4 / UAS system. Newly emerged male tub-GAL4 / Sp; UAS-blos2-RNAi / Dr Drosophila from the F1 generation were selected as the experimental group and mated with Sp; Dr / SM6B-TM6B Drosophila to obtain a stably preserved tub-GAL4; UAS-blos2-RNAi / SM6B-TM6B strain. After preservation for 3 generations, Drosophila with reduced pigment content and lighter color in the body surface and eyes were collected, which can be used as a Drosophila albinism model for subsequent use.

[0016] Current research lacks animal models of albinism, making it impossible to verify phenotypes and functions. A few studies have used models such as miniature pigs or macaques, but these suffer from drawbacks such as high cost, long processing times, and inability to conduct large-scale screening. The method of this invention can effectively utilize the Drosophila model to study albinism-related genes and processes, providing new experimental evidence for the pathogenesis and treatment of albinism, thereby promoting the development and clinical application of related drugs.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. Fruit flies have a short lifespan and high reproductive capacity, making them a classic model organism for genetics research;

[0019] 2. The number of genes in the fruit fly genome is known and easy to manipulate, which helps in studying the function of specific genes;

[0020] 3. Phenotypic verification of potential albinism-causing genes using fruit flies;

[0021] 4. It enables high-throughput gene function verification and drug screening. Attached Figure Description

[0022] Figure 1 This is the screening process of the present invention.

[0023] Figure 2 The results show the expression levels of the blos2 gene in Drosophila tub-GAL4 / Sp; UAS-blos2-RNAi / Dr.

[0024] Figure 3 Photographs of the appearance of fruit flies representing the control group and those with the blos2 gene knocked down. Detailed Implementation

[0025] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0028] like Figure 1 As shown, the screening process in this embodiment includes the following steps:

[0029] Step 1: Select female tub-GAL4 / Cyo; Dr / TM6B and male Sp / Cyo; UAS-blos2-RNAi fruit flies for hybridization. The sex of the parents can be interchanged. Culture in an incubator at 24–26℃ and 50%–60% humidity. Approximately 2–3 days after mating, check the mating bottle for egg production. Remove the parent fruit flies from the mating bottle, and continue culturing the culture bottle containing eggs to obtain the F1 generation. Select F1 generation fruit flies with the genotype tub-GAL4 / Sp; UAS-blos2-RNAi / Dr, and use quantitative RT-PCR to verify whether the blos2 gene expression is decreased. Figure 2 As shown, the results indicate that the expression of the blos2 gene in Drosophila tub-GAL4 / Sp;UAS-blos2-RNAi / Dr was significantly decreased.

[0030] Step 2: The newly emerged male tub-GAL4 / Sp; UAS-blos2-RNAi / Dr fruit flies of the F1 generation were mated with female virgin flies of the Sp; Dr / SM6B-TM6B strain to obtain a stable preservation strain of tub-GAL4; UAS-blos2-RNAi / SM6B-TM6B.

[0031] Step 3: After preserving this strain of fruit flies for three generations, collect and observe the pigment changes on the body surface and eyes of the fruit flies. For example... Figure 3 As shown, compared with the control group, the blos2 gene knockdown fruit flies exhibited reduced pigment content and paler color in their body surface and eyes. This indicates that the blos2 gene knockdown flies may be associated with albinism, and this fruit fly model can serve as an albinism model.

Claims

1. A method for screening albino pathogenic genes using fruit flies, characterized in that, Includes the following steps: Step 1: Select GAL4 strain fruit flies and USA-X strain fruit flies as parents, and cross the two strains to obtain F1 generation newly emerged fruit flies; The GAL4 strain of fruit flies is either tub-GAL4 / Cyo; Dr / TM6B or act-GAL4 / Cyo; Dr / TM6B. The USA-X strain of Drosophila is Sp / Cyo; the UAS-X-RNAi transgenic strain of Drosophila, with the X gene being the gene to be screened. Step 2: Select newly emerged male fruit flies from the F1 generation and mate them with female virgin flies of the Sp; Dr / SM6B-TM6B strain to obtain a stable and preserved strain. Step 3: After preserving the fruit flies from Step 2 for 3 generations, collect and observe the pigmentation changes on the fruit fly body surface and eyes. The culture medium formulas for steps 1, 2, and 3 are as follows: per 100 ml of culture medium, there are 6.0 g of brown sugar, 0.725 g of white sugar, 1.0 g of agar, 5.0 g of corn flour, 2.45 g of yeast, 0.4 ml of propionic acid, and 0.15 g of methylparaben. In step 1, yeast granules are added to the culture medium during hybridization culture, with 20 yeast granules added per 10 ml of culture medium. The rearing conditions in steps 1, 2 and 3 are as follows: cultured in an incubator with a temperature of 24-26℃ and a humidity of 50%-60%.

2. The application of the method described in claim 1 in screening for albinism pathogenic genes.

Images