CN115181745B - siRNA for inhibiting honeybee Rho1 gene and application thereof - Google Patents

Abstract

The invention provides siRNA capable of obviously inhibiting Rho1 gene transcription of bees and application thereof, and the siRNA can be injected into the abdomen of a queen bee to stably obtain small-volume fertilized eggs of the bees, thereby solving the problem that the small-volume fertilized eggs of the bees are difficult to obtain in large quantity in the existing scientific research activities.

Description

siRNA for inhibiting honeybee Rho1 gene and application thereof

Technical Field

The invention relates to biotechnology, in particular to siRNA for inhibiting honeybee Rho1 gene and application thereof.

Background

The bee has important economic value and ecological value and is an important model organism for biological research. As a true social insect, bees have a fine social structure and rich social behaviors, and particularly, a reproductive division exists, namely a female queen specializes in spawning, while the feeding work of offspring is shared by other females. Reproduction is an important part of life history, and is the "investment" action for reproducing offspring, and natural selection always favors species and individuals that can optimize the balance between "cost" and "benefit" and improve their adaptability in the niche. Therefore, the reproductive investment requires that the mother can dynamically adjust the resources allocated to the offspring according to the external environmental conditions and the physiological state of the mother so as to maximize the adaptability of the offspring and the mother. In the bee colony, the queen bee and the worker bee are mutually matched and mutually influenced in the reproduction behavior, so that the survival and development of the bee colony are jointly ensured, and a good model is provided for the reproduction regulation and control research of social organisms.

The fertilized eggs laid by queen bees in the cells with larger volume are obviously larger than the eggs laid in the smaller cells, and the bees developed by the large eggs under the same feeding conditions are heavier and have more egg tubes. The method has practical guiding significance for the healthy breeding of the bees by detecting the difference of the large eggs and the small eggs on the molecular level and the difference of the physiological functions of the developed bees. However, the problem of obtaining a large amount of bee zygotes with significant size difference stably to provide biological materials for subsequent research has not been solved.

Disclosure of Invention

The invention aims to provide siRNA for inhibiting honeybee Rho1 gene, which is used for stably obtaining a small-volume honeybee fertilized egg.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

an siRNA for inhibiting the honeybee Rho1 gene, said siRNA comprising the following four sequences:

s1 siRNA, wherein the sense strand is: 5 'GGCAGCCAUUAAGAAAGAAATT-3', wherein the antisense strand is 5 'UUCUUCUUCUAAUGGCUGCCTT-3';

s2 siRNA, wherein the sense strand is 5;

s3 siRNA, wherein the sense strand is 5;

s4 siRNA, wherein the sense strand is 5.

The invention also provides application of the siRNA for inhibiting the honeybee Rho1 gene in regulating and controlling the size of a honeybee fertilized egg.

Preferably, the use is for reducing bee zygote volume.

The invention also provides a method for obtaining the small-volume bee fertilized eggs, which comprises the following steps: injecting the mixture of 4 kinds of siRNA in claim 1 into the 4 th-5 th abdominal section of the egg-laying queen bee, and returning the queen bee to the original bee colony after the queen bee is recovered to continue laying eggs to obtain the low-volume bee fertilized egg.

Preferably, the method comprises the following steps: taking out the spawning queen bees from the bee colony, anesthetizing the spawning queen bees with carbon dioxide, injecting the mixture of 4 kinds of siRNA in claim 1 into the 4 th-5 th abdominal section of the spawning queen bees, returning to the original bee colony after the spawning queen bees wake up, and collecting the produced new eggs after the spawning queen bees lay eggs to obtain the low-volume fertilized eggs of the bees.

Preferably, the injection amount of the mixture of 4 siRNAs is 1-3. Mu.l/mouse.

Preferably, the mixture is an equal mixture of 4 siRNAs, and the final concentration of the equal mixture of 4 siRNAs is 1. Mu.g/. Mu.l.

Compared with the prior art, the invention has the beneficial technical effects that:

the invention provides siRNA capable of obviously inhibiting Rho1 gene transcription, and the siRNA can be injected into the abdomen of a queen bee to stably obtain a small-volume bee fertilized egg, thereby solving the problem that the small-volume bee fertilized egg is difficult to obtain in large quantity in the existing scientific research activities.

Drawings

FIG. 1 is a graph comparing the transcript levels of the Rho1 gene in the queen bee ovaries of each group in example 1 of the present invention;

FIG. 2 is a comparison graph of fertilized eggs of a queen bee produced before and after injection in example 2 of the present invention;

FIG. 3 is a graph showing the comparison of the statistical results of the sizes of fertilized eggs laid by the queen bees before and after injection in example 2 of the present invention;

FIG. 4 is a graph showing a comparison of the transcript levels of the Rho1 gene in ovaries before and after injection in the egg-laying queen bee in example 2 of the present invention.

The specific implementation mode is as follows:

the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the following embodiments, the experimental methods not specifying the specific conditions are generally carried out by the conventional conditions, for example, the methods described in molecular cloning, A laboratory Manual (J. SammBruk, D.W. Lassel, huang Peyer, wang Jia Rong, zhu Jia Foundation, et al, 3 rd edition, beijing: scientific Press, 2002).

Example 1

The siRNA of the bee Rho1 gene comprises 4 siRNA (S1, S2, S3 and S4) which are respectively as follows: the sense strand sequence is a nucleotide sequence shown by S1-F or the antisense strand sequence is a nucleotide sequence shown by S1-R; the sense strand sequence is a nucleotide sequence shown by S2-F or the antisense strand sequence is a nucleotide sequence shown by S2-R; the sense strand sequence is a nucleotide sequence shown by S3-F or the antisense strand sequence is a nucleotide sequence shown by S3-R; the sense strand sequence is a nucleotide sequence shown by S4-F or the antisense strand sequence is a nucleotide sequence shown by S4-R.

1. Preparation of siRNA molecule and Rho1 gene real-time quantitative PCR primer

The sequence of bee Rho1 gene (LOC 409910, SEQ ID NO. 1) was found in NCBI net (http:// www.ncbi.nlm.nih.gov /), and four pairs of siRNA were designed and synthesized. A pair of irrelevant control siRNAs (scramblesiRNA) were synthesized simultaneously.

S1 siRNA:

Sense strand (SEQ ID NO. 2): 5 'GGCAGCCAUUAAGAAAGAAATT-doped 3' (S1-F)

Antisense strand (SEQ ID NO. 3): 5 'UUCUUCUAAUGGCUGCCTT-doped 3' (S1-R)

S2 siRNA:

Sense strand (SEQ ID NO. 4): 5 'GCCCAAAUGUGCCCAUUAUTT-3' (S2-F)

Antisense strand (SEQ ID NO. 5): 5 'AUAAUGGGCACALUAUUUGGGGCTT-3' (S2-R)

S3 siRNA:

Sense strand (SEQ ID NO. 6): 5 'GGAAGGUAUUAUUAAGGGAAGUATT-doped 3' (S3-F)

Antisense strand (SEQ ID NO. 7): 5 'UACUUCCCUAAUACCUCTT-3' (S3-R)

S4 siRNA:

Sense strand (SEQ ID NO. 8): 5 'GAUGUUGCUCCUAAUUTT-containing 3' (S4-F)

Antisense strand (SEQ ID NO. 9): 5 'AAUUAAGGAGCCCAACAUUCTT-3' (S4-R)

scramble siRNA:

Sense strand (SEQ ID NO. 10): 5' UUCCCGAACGUCACGUTT-

Antisense strand (SEQ ID NO. 11): 5 'ACGUGACACGUUCGGAGAATT-3' (SS-R)

Real-time quantitative PCR primers were designed and synthesized based on the gene sequence of CAX 72463.

Sense strand (SEQ ID NO. 12): 5 'AAACAAGTGGAACTGGCTCT-doped 3' (Rho 1-F)

Antisense strand (SEQ ID NO. 13): 5 'CAGGGAATCTGGATCTATCA-3' (Rho 1-R)

2. Injection of siRNA

Selecting healthy queen bees with strong spawning ability as female parents, artificially culturing a batch of queen bees, and putting the queen bees into a mating box. After the natural mating and normal spawning, the spawning queen bee is transferred to a normal bee colony to spawn. The egg-laying queen bees were divided into three groups, namely a blank group (PBS), a negative control group (NC) and an siRNA treatment group, and each group had three queen bees.

Transferring the oviposition queens of each group into a laboratory, and anesthetizing with carbon dioxide.

siRNA treatment group: an equal mixture of 4 siRNAs was injected using a microinjector into the queen bee 4 th-5 th abdominal node, the final concentration of the 4 siRNA mixtures was 1. Mu.g/. Mu.l (solvent was ultrapure water) and the injection amount was 1. Mu.l/body.

Negative control group (NC group): scarmble siRNA was injected into the queen bee at 4 th to 5 th abdominal joints using a microinjector (final concentration: 1. Mu.g/. Mu.l, injection amount: 1. Mu.l/individual).

Blank group: no treatment is done.

And after the queens are revived, putting all groups of queens back to the original bee colony. After 72h, cutting and killing the queen bee, quickly freezing the queen bee ovary by using liquid nitrogen, and then putting the queen bee ovary into a refrigerator at the temperature of minus 80 ℃ for later use.

Real-time quantitative PCR validation of RNA interference effects

Extracting total RNA of queen bee ovary by Trizol method, reverse transcribing into cDNA, and performing real-time quantitative PCR analysis by using the cDNA as a template. As shown in FIG. 1, siRNA significantly reduced the transcript level of Rho1 gene.

Example 2

1. Selecting healthy queen bees with strong spawning ability as female parents, artificially culturing a batch of queen bees, and putting the queen bees into a mating box. After the queen bees naturally copulate and normally lay eggs, the queen bees are transferred to normal bee colonies to lay eggs. The egg-laying queen bees are divided into two groups, namely a negative control group (NC group) and an siRNA treatment group, and each group comprises three queen bees. Queen bees were restricted to lay eggs on an empty comb for 6 hours by a queen bee controller, randomly collected eggs (50) on the comb, photographed under a microscope (fig. 2, left), and the size of each group of eggs was measured (fig. 3).

2. Transferring the oviposition queens of each group into a laboratory, and anesthetizing with carbon dioxide.

siRNA treatment group: an equal mixture of 4 siRNAs (final concentration 1. Mu.g/. Mu.l, injection amount 1. Mu.l/individual) was injected in the queen bee 4-5 abdominal segments using a microinjector.

Negative control group (NC group): scramble siRNA was injected into queen bee at 4 th-5 th abdominal segment using a microinjector (final concentration 1. Mu.g/. Mu.l, injection amount 1. Mu.l/individual).

3. And after the queens are revived, putting all groups of queens back to the original bee colony.

4. 72h after injection, queen bees are restricted to lay eggs on an empty honeycomb for 6 hours by a queen bee controller, and the eggs (50) on the honeycomb are randomly collected, and a photograph of the eggs is taken under a microscope (fig. 2, right), and the size of each group of eggs is measured.

The experimental results are shown in fig. 2 and fig. 3, and the injection of siRNA significantly reduced the volume of fertilized eggs laid by queen bees.

5. Real-time quantitative PCR verification of RNA interference effect

Cutting and killing two groups of queen bees, quickly freezing queen bee ovary with liquid nitrogen, and placing into a refrigerator at-80 deg.C for use. Extracting total RNA of queen bee ovary by Trizol method, reverse transcribing into cDNA, and performing real-time quantitative PCR analysis by using the cDNA as a template. The experimental results are shown in fig. 4, and the transcript level of Rho1 gene in queen bee ovary of laying ova was significantly reduced after siRNA injection.

Sequence listing

<110> bee institute of Chinese academy of agricultural sciences

<120> siRNA for inhibiting honeybee Rho1 gene and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 621

<212> DNA

<213> honeybee Rho1 gene (Apis mellifera ras-like GTP-binding protein Rho 1)

<400> 1

atggcagcca ttagaaagaa attggttatt gtgggtgatg gtgcttgtgg taaaacatgc 60

ttacttatag tcttcagcaa agaccaattt ccagaagttt atgtacctac agtgtttgaa 120

aattatgttg cagatattga agttgatggg aaacaagtgg aactggctct ttgggacaca 180

gctggacaag aagattatga taggttgcgt ccactatcat atcctgacac agatgtaatc 240

ttaatgtgtt tctctattga tagtccagat tccctggaaa acattcctga gaaatggaca 300

ccagaggtga agcacttttg cccaaatgtg cccattatcc ttgttggaaa caaaaaagac 360

ttacgcaatg atcctaatac aatcaaagaa cttagtaaga tgaaacaaga accagttaag 420

ccagaagaag gaagagctat ggctgaaaaa atcaatgcct ttgcttatct tgaatgttct 480

gctaaaagta aggaaggtat tagggaagta tttgaaacag ccactcgggc agcattacaa 540

ttcgtaggta aaaaagaaga agaagggaag atgttggctc ctataattct tgaattatca 600

ccgataacga gacaacacta a 621

<210> 2

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggcagccauu agaaagaaat t 21

<210> 3

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

uuucuuucua auggcugcct t 21

<210> 4

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcccaaaugu gcccauuaut t 21

<210> 5

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

auaaugggca cauuugggct t 21

<210> 6

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggaagguauu agggaaguat t 21

<210> 7

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

uacuucccua auaccuucct t 21

<210> 8

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gauguuggcu ccuauaauut t 21

<210> 9

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aauuauagga gccaacauct t 21

<210> 10

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

uucuccgaac gugucacgut t 21

<210> 11

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acgugacacg uucggagaat t 21

<210> 12

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaacaagtgg aactggctct 20

<210> 13

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cagggaatct ggactatcaa 20

Claims (1)

1. The application of siRNA for inhibiting honeybee Rho1 gene in regulation and control of honeybee fertilized egg size is characterized in that the application is reduction of honeybee fertilized egg volume, the application does not belong to a disease treatment method, the siRNA is a mixture of 4 siRNAs, and the sequences of the 4 siRNAs are as follows:

s1 siRNA, wherein the sense strand is: 5 'GGCAGCCAUUAAGAAAGAAATT-3', wherein the antisense strand is 5 'UUCUUCUUCUAAUGGCUGCCTT-3';

s2 siRNA, wherein the sense strand is: 5 'GCCCAAAUGUGCCCAUUAUTT-3', and the antisense strand is 5 'AUAAUGGGCACACAAUUUGGGGCTT-3';

s3 siRNA, wherein the sense strand of the siRNA is: 5' and 5' are UACUUCCCUAAUACCUUCTT-3 ';

s4 siRNA, wherein the sense strand is: 5 'of GAUGUUGGCUCCUUAAUUTT-3', and the antisense chain of the 5 'of AAUUAAUAGGAGCCAACAUUCTT-3'.

Images