CN116326549A - Construction and identification method of transgenic drosophila model with abnormal intestinal acidity - Google Patents

Abstract

The invention relates to a method for constructing and identifying transgenic fruit flies capable of inducing abnormal intestinal acidity. The fruit fly transgenic carrier pValium20-RagA RNAi is constructed, and the RagA RNAi transgenic fruit flies are obtained by microinjection. (Double balancer) Drosophila SP/SM6; MKRS/TM6 was crossed for two generations to obtain a UAS‑RagA RNAi transgenic Drosophila SP/SM6; UAS‑RagA RNAi/TM6 with a balancer gene that can be stably inherited. Crosses of NP1‑GAL4 flies that drive gene expression in epithelial cells yield NP1‑GAL4; UAS‑RagA RNAi flies that knockdown RagA in the gut. Through phenotypic detection, it was found that the intestinal morphology of the fruit fly was abnormal; by feeding bromophenol blue and cresyl violet, it was found that the acidity in the intestine of RagA knockdown fruit flies was reduced; by immunofluorescence detection of copper cells, it was determined that the fruit fly The phenotype of abnormal gastrointestinal acidity was associated with a reduced number of hydrogen ion-secreting cells. These results confirm abnormal intestinal acidity in RagA knockout flies. The invention can be used for faster and more convenient screening of targeted drugs for gastrointestinal acidity deficiency diseases.

Description

Construction and identification method of a transgenic Drosophila model with abnormal intestinal acidity

technical field

The invention relates to a construction method and an identification method of a transgenic fruit fly model, in particular to a construction and identification method of a transgenic fruit fly model with abnormal intestinal acidity, belonging to the field of biotechnology.

Background technique

Drosophila melanogaster has a highly developed nervous system and a short life cycle. It is easy to raise and easy to operate and analyze genetics, cell biology, and molecular biology. It is a very practical model organism. Therefore, Drosophila is widely used in the construction and research of disease models.

RagA is a component of the Rag complex, which can sense the nutritional status outside the cell. When the cell is starved, RagA is inactivated by combining with GDP. When nutrients are sufficient, RagA combines with GTP to become an active form, promoting cell growth and metabolism. We used the dsRNA sequence targeting Rag gene based on RNA interference technology to construct transgenic Drosophila, and found that the specific knockdown of RagA in the gut can make the Drosophila gut thicker; Inhibition of the gut thickening phenotype.

The Drosophila gut and the mammalian gut are highly similar in terms of genetic regulation and cell composition. The Drosophila midgut can be divided into the anterior midgut, the copper cell zone, and the midgut. The copper cell zone is located in the middle of the midgut. Acidic like the stomach of mammals, with a pH less than 4. After feeding the bromophenol blue acid-base indicator, the area turns yellow. In recent years, relevant studies have shown that with the increase of age, the intestinal digestion ability of Drosophila decreases, and the intestinal environment changes from weakly acidic to weakly alkaline, suggesting that the intestinal Acidity may be related to organ aging. The RagA knockout fruit flies involved in the present invention exhibit a phenotype of abnormal intestinal acidity similar to aging fruit flies, and replenishing RagA can inhibit the phenotype of reduced intestinal acidity caused by RagA knockdown.

Contents of the invention

The purpose of the present invention is to provide a method for constructing and identifying a transgenic Drosophila model with abnormal intestinal acidity in view of the above-mentioned deficiencies in the prior art.

To achieve the above object, the present invention provides the following specific technical solutions:

A method for constructing a transgenic fruit fly model with abnormal intestinal acidity. The invention designs an RNA interference target sequence based on the fruit fly RagA gene and inserts it into a transgenic vector for the fruit fly RNA interference. Construction of RNA interference Drosophila targeting RagA expression sequence. The concrete steps of this method are:

a. Based on the Drosophila RagA gene, design the target CTCGGAGGCTACGCTAGTCAA of the RNA interference sequence, and prepare the pValium20-RagA RNAi vector;

b: The Drosophila transgenic vector obtained in step a is used to construct RagA RNAi transgenic Drosophila by means of microinjection combined with genetic engineering techniques.

The specific operation method of the above-mentioned step a is:

a-1: Synthesize primers based on the target sequence of the RNA interference sequence:

aattcgcCTCGGAGGCTACGCTAGTCAAtatgcttgaatataactaTTGACTAGCGTAGCCTCCGAGactg

and

ctagcagtCTCGGAGGCTACGCTAGTCAAtagttatattcaagcataTTGACTAGCGTAGCCTCCGAGgcg;

a-2: After annealing and renaturation, the primers were inserted into the pvalium20 vector after double digestion with NheI and EcoRI.

a-3: screening on the agar plate containing ampicillin to obtain the pValium20-RagA RNAi vector for transgene.

The concrete steps of above-mentioned step b are:

b-1: Inject the transgenic vector pValium20-RagA RNAi into the tail of fertilized eggs of y,v;attP2 Drosophila, and culture them in a humid box at 16°C;

b-2: The larvae hatched from the fertilized eggs obtained in step b-1 can be selfed after they emerge into adults, and the fruit flies with red eye color in the offspring are the transgenic fruit flies with UAS-RagA RNAi ;

b-3: crossing the transgenic fruit flies obtained in step b-2 with the double-balanced fruit fly SP/SM6; MKRS/TM6 for two generations, and screening to obtain UAS-RagA RNAi fruit flies that can be stably inherited;

b-4: Cross the transgenic fruit fly obtained in b-3 with the genotype NP1-GAL4 fruit fly to obtain a transgenic fruit fly NP1-GAL4;UAS-RagA RNAi with abnormal gastrointestinal acidity.

A method for identification of a transgenic fruit fly model with abnormal intestinal acidity. The transgenic fruit flies are identified by bromophenol blue feeding assay and immunofluorescence.

The present invention uses microinjection technology and fruit fly hybridization technology to construct a model of transgenic fruit flies with abnormal intestinal acidity, which provides materials and ideas for screening intestinal disease-related drugs faster and more conveniently, and has good application prospect.

The invention has the advantages of simple operation and strong repeatability. The transgenic fruit fly model of abnormal intestinal acidity can be used to explore the mechanism of abnormal intestinal acidity, and can also be used to screen targeted drugs for the treatment of intestinal diseases. It has a good application prospect.

Description of drawings

Figure 1 shows the anterior midgut ( ) width chart.

Figure 2 shows the intestinal tract of control, RagA knockdown Drosophila ( NP1-GAL4; UAS-RagA RNAi ), RagA knockdown + overexpression Drosophila ( NP1-GAL4/UAS-RagA; UAS-RagA RNAi ) fed bromophenol blue Copper cell area color statistics map.

Figure 3 shows the intestinal tract of control, RagA knockdown Drosophila ( NP1-GAL4; UAS-RagA RNAi ), RagA knockdown + overexpression Drosophila ( NP1-GAL4/UAS-RagA; UAS-RagA RNAi ) fed cresyl violet Copper cell area color statistics map.

Figure 4 is the immunofluorescence staining images of the copper cell area of the control, RagA knockdown Drosophila ( NP1-GAL4; UAS-RagA RNAi ), and RagA knockdown + overexpression Drosophila.

Detailed ways

The present invention will be further described below in combination with specific embodiments.

Embodiment 1: Construction and verification of Drosophila transgenic vector;

1. To obtain the Drosophila transgenic vector pValium20-RagA RNAi, design and synthesize primers according to the target sequence of the RNA interference sequence:

aattcgcCTCGGAGGCTACGCTAGTCAAtatgcttgaatataactaTTGACTAGCGTAGCCTCCGAGactg

and

ctagcagtCTCGGAGGCTACGCTAGTCAAtagttatattcaagcataTTGACTAGCGTAGCCTCCGAGgcg;

Plasmid pValium20 was double-digested with NheI and EcoRI, and the gel was recovered for later use.

The pValium20-RagA RNAi vector for transgene was obtained by screening on the agar plate containing ampicillin.

with primers

aattcgcCTCGGAGGCTACGCTAGTCAAtatgcttgaatataactaTTGACTAGCGTAGCCTCCGAGactg

and

ctagcagtCTCGGAGGCTACGCTAGTCAAtagttattcaagcataTTGACTAGCGTAGCCTCCGAGgcg

The constructed Drosophila transgenic vector pValium20 was identified by PCR.

Embodiment 2: Microinjection experiment and verification of transgenic fruit flies;

1. In order to obtain the transgenic fruit flies with abnormal intestinal acidity, the genetic engineering technology of microinjection was used to construct the transgenic fruit flies.

2. Put the transgenic carrier pValium20-RagA RNAi at a certain concentration ratio into a glass microelectrode with an elongated opening, and inject this mixture into the newly produced Drosophila within 1 hour to remove the egg shell with the help of the pneumatic mechanical injection arm The tails of the fertilized eggs were cultured in a humid box at 16°C after injection.

3. After the injected fertilized eggs hatch into larvae, they can self-fertilize after they emerge into adults, and the fruit flies with red eye color in the offspring are selected as transgenic fruit flies; the resulting transgenic fruit flies and double-balanced fruit flies SP/SM6; MKRS/TM6 were crossed for two generations to obtain the stable hereditary SP/SM6; UAS-RagA RNAi/TM6 fruit flies; the UAS- RagA RNAi transgenic fruit flies were crossed with the genotype NP1-GAL4 fruit flies to obtain Transgenic fruit flies expressing RagA interfering sequences; UAS-RagA /SM6; RagA RNAi/TM6 Drosophila Drosophila is crossed with genotype NP1-GAL4 Drosophila to obtain transgenic fruit that simultaneously expresses RagA interfering sequences and RagA coding region sequences fly.

4. After dissecting 3-day-old control fruit flies, transgenic fruit flies expressing RagA interference sequences, and transgenic fruit flies expressing RagA interference sequences and RagA coding sequences, the intestines were fixed, mounted, and photographed under a microscope. Count the widths of the anterior midgut, copper cell area, and posterior midgut of the Drosophila gut. The results are shown in Figure 1. Knocking down RagA makes the Drosophila intestine thicker; on the basis of knocking down RagA, supplementing RagA can inhibit the phenotype of gut thickening.

5. Mix the pH indicator-bromophenol blue with the fruit fly food (final concentration 0.2%), feed the transgenic fruit flies expressing the RagA interference sequence of the control fruit flies, and the transgenic fruit flies expressing the RagA interference sequence and the RagA coding sequence, after 24 hours The fruit flies were dissected, and the proportion of fruit flies with a pH value of less than 4.0 in the copper cell area of the gastrointestinal tract was counted. The results are shown in Figure 2. RagA knockdown Drosophila presents a phenotype of abnormal intestinal acidity, and supplementation of RagA can inhibit the phenotype of decreased intestinal acidity caused by RagA knockdown.

6. Mix the pH indicator-cresyl violet with the fruit fly food (final concentration 0.2%), and feed the transgenic fruit flies expressing the RagA interference sequence, the transgenic fruit flies expressing the RagA interference sequence and the RagA coding sequence of the control fruit flies, after 24 hours The fruit flies were dissected, and the proportion of fruit flies with a pH value of less than 4.0 in the copper cell area of the gastrointestinal tract was counted. The results are shown in Figure 3. RagA knockdown Drosophila exhibits abnormal intestinal acidity phenotype, and supplementation of RagA can inhibit the phenotype of decreased intestinal acidity caused by RagA knockdown.

7. After dissecting 3-day-old control fruit flies, transgenic fruit flies expressing RagA interference sequences, and transgenic fruit flies expressing RagA interference sequences and RagA coding sequences, the intestines were dissected and fixed, and the specific primary antibody of copper cells was used to Cut was immunofluorescent stained and photographed under a fluorescent microscope. The results are shown in Figure 4. The number of copper cells in RagA knockdown Drosophila decreased, and RagA supplementation could inhibit the reduction in the number of intestinal copper cells caused by RagA knockdown.

Therefore, the transgenic Drosophila model of abnormal intestinal acidity of the present invention can be used to analyze the mechanism of abnormal gastrointestinal acidity, and can also be used to quickly screen drugs or compounds for treating abnormal intestinal acidity, and has a good application prospect.

Claims (5)

1. A method for constructing a transgenic fruit fly model with abnormal gastrointestinal acidity, characterized in that, based on the fruit fly RagA gene, an RNA interference target sequence is designed and inserted into a transgenic vector for fruit fly RNA interference; RNA interference expressing sequences in Drosophila. 2. the construction method of the abnormal transgenic fruit fly model of a kind of gastrointestinal acidity according to claim 1, is characterized in that, comprises the following steps: a: Based on the Drosophila RagA gene, design the target CTCGGAGGCTACGCTAGTCAA of the RNA interference sequence, and prepare the pValium20-RagA RNAi vector; b: The Drosophila transgenic vector obtained in step a is used to construct RagARNAi transgenic Drosophila by means of microinjection combined with genetic engineering techniques. 3. the construction method of the abnormal transgenic fruit fly model of a kind of gastrointestinal acidity according to claim 2, is characterized in that, the concrete method of step a is: a-1: Synthesize primers based on the target sequence of the RNA interference sequence: aattcgcCTCGGAGGCTACGCTAGTCAAtatgcttgaatataactaTTGACTAGCGTAGCCTCCGAGactg and ctagcagtCTCGGAGGCTACGCTAGTCAAtagttatattcaagcataTTGACTAGCGTAGCCTCCGAGgcg; a-2: After annealing and renaturation of the primers, insert them into the pvalium20 vector recovered from the gel after double digestion with NheI and EcoRI; a-3: screening on the agar plate containing ampicillin to obtain the pValium20-RagA RNAi vector for transgene. 4. the construction method of the abnormal transgenic fruit fly model of a kind of gastrointestinal acidity according to claim 1, is characterized in that, the specific steps of step b are: b-1: Inject the transgenic vector pValium20-RagA RNAi into the tail of fertilized eggs of yv;attP2 Drosophila, and culture them in a humid box at 16°C; b-2: The larvae hatched from the fertilized eggs obtained in step b-1 can be selfed after they emerge into adults, and the fruit flies with red eye color in the offspring are the transgenic fruit flies with UAS- RagA RNAi ; b-3: Crossing the transgenic fruit flies obtained in step b-2 with the double balancer (Double balancer) fruit flies SP/SM6; MKRS/ TM6 for two generations, and screening to obtain UAS-RagA RNAi fruit flies that can be stably inherited; b-4: Cross the transgenic fruit fly obtained in b-3 with the genotype NP1-GAL4 fruit fly to obtain a transgenic fruit fly NP1-GAL4;UAS-RagA RNAi with abnormal gastrointestinal acidity. 5. An identification method for a transgenic Drosophila model with abnormal gastrointestinal acidity, characterized in that the Drosophila with abnormal gastrointestinal acidity is identified by means of bromophenol blue feeding measurement and immunofluorescence.

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