Application of gene 15-PGDH in regulating the weight and length of honey bee workers

Abstract

The present invention relates to the field of biotechnology, and discloses the application of gene 15-PGDH in regulating the weight and body length of honey bees. The present invention reduces the expression of 15-PGDH gene in worker bee larvae by feeding 15-PGDH gene siRNA reagent during the honey bee worker bee larvae. After the worker bees are grown and leave the room, the initial weight and body length of the worker bees are measured, and it is found that compared with the normal worker bees leaving the room, the initial weight and body length of the worker bees treated with 15-PGDH gene siRNA are significantly increased. The regulation of 15-PGDH gene in regulating the weight and body length of honey bees is negative regulation, that is, the expression amount of 15-PGDH gene is reduced, and the weight and body length of honey bees are increased. The present invention has important application and promotion value for improving the weight and body length traits of honey bees and for honey bee breeding.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to the application of gene 15-PGDH in regulating the weight and body length of honey bee workers. Background Art

[0002] Bees are social insects and important pollinators. Most crops in the world require bee pollination. According to data from the Food and Agriculture Organization of the United Nations, about 85% of flowering plants and 90% of fruit trees in the world require bee pollination. The value of bee pollination contributed by bees to China's major production crops exceeds 300 billion yuan, accounting for more than 10% of China's total agricultural output value. Honey, royal jelly, bee pollen and other bee products produced by bee colonies can improve human immunity and have high nutritional and health value.

[0003] There are three types of bee colonies: queen bee, worker bee and drone bee. Both queen bee and worker bee are female bees developed from fertilized eggs, but the queen bee has a complete reproductive system. There is only one queen bee in a colony. It is large and mainly responsible for laying eggs. The worker bee has an incomplete reproductive system and is mainly responsible for the labor of the colony. There are tens of thousands of worker bees in a colony. They are smaller than the queen bee and are mainly responsible for cleaning the nest cells, preparing honey and pollen, feeding larvae, collecting nectar, pollen and gum, building honeycombs, guarding the hive, etc. The drone bee is responsible for mating with the queen bee and does not undertake any labor work. As can be seen from the division of labor, worker bees are crucial to the survival and development of the colony.

[0004] The size of worker bees is closely related to their collecting, pollinating and defensive abilities. The honey sac of a worker bee is located in the abdomen of the worker bee. When the abdomen of the worker bee is longer, the honey sac has more space to store honey, and the worker bee has a stronger ability to collect honey. Larger bees have an advantage in pollination efficiency because they have larger hind legs, and the pollen baskets on the tibia of the hind legs can carry more pollen, thereby improving pollination efficiency. In addition, larger bees can resist more effectively when faced with attacks from natural enemies such as wasps, thereby improving the defense capabilities of the bee colony. In beekeeping production, breeders prefer to raise larger bees because such bees produce more honey, are healthier, and have high economic benefits. Therefore, breeding larger worker bees is an important goal of bee breeding.

[0005] At present, in beekeeping production, traditional breeding methods are usually used to perform phenotypic selection on large bee colonies, in order to gradually increase the body length of worker bees through generation-by-generation selection. The main method is to select a bee colony with larger worker bees in the bee colony, use the queen bee of the bee colony as the seed queen bee to breed the next generation, and do this for each generation, breeding larger bees generation by generation. However, traditional breeding has slow genetic progress and a long breeding cycle. It takes multiple generations to significantly increase the body length of worker bees, and the breeding cost is high. Moreover, the larger bees obtained by the existing methods are easily affected by the variety and regional environment. For example, when large bees bred in the north are raised in the south, their size advantage may not be obvious.

[0006] Molecular breeding has been applied to livestock and poultry to accelerate genetic progress, but molecular markers have not been applied to bees to carry out breeding work to increase the length or weight of bees. Studies have shown that the body size of bees is affected and regulated by hormones, genes, etc. In order to create an ideal large-bodied bee variety and obtain a simpler and more direct method to regulate the body size of bee workers, it is necessary to study the genes that regulate the body size of bee workers. Summary of the invention

[0007] The purpose of the present invention is to provide application of gene 15-PGDH in regulating the weight and body length of honey bee workers.

[0008] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides the use of gene 15-PGDH in regulating the weight and length of honey bee workers, wherein the regulation is negative regulation.

[0009] The gene 15-PGDH encodes 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which comes from the honey bee gene 15-PGDH in NCBI's Gene ID: 409847.

[0010] In a second aspect, the present invention provides siRNA for gene 15-PGDH, the sequences of its sense strand and antisense strand are as follows:

[0011] Sense chain: 5'-GCUCUCACCUCGGUAUGUATT-3', wherein the sequence of GCUCUCACCUCGGUAUGUA is shown in SEQ ID NO: 1, and the base TT at the 3' end is DNA, which is overhanging and has a stabilizing effect, and can also form a sticky end to facilitate unzipping.

[0012] Antisense strand: 5'-UACAUACCGAGGUGAGAGCTT-3', wherein the sequence of UACAUACCGAGGUGAGAGC is shown in SEQ ID NO: 2, and the base TT at the 3' end is DNA, which is overhanging and has a stabilizing effect, and can also form a sticky end to facilitate unzipping.

[0013] In a third aspect, the present invention provides biological materials containing the siRNA or a nucleotide sequence encoding the siRNA, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0014] In a fourth aspect, the present invention provides the use of the siRNA or a biological material containing the siRNA or a nucleotide sequence encoding the siRNA in increasing the body weight and body length of honey bee workers.

[0015] In a fifth aspect, the present invention provides a method for increasing the weight and body length of honey bee worker bees, the method comprising: utilizing genetic engineering means to knock down or silence the gene 15-PGDH in honey bee worker bees; wherein.

[0016] Furthermore, siRNA of the gene 15-PGDH (the sequences of the sense chain and the antisense chain are as described above), is designed and synthesized, and the siRNA or the nucleotide sequence encoding the siRNA is introduced into honey bees by feeding. For example, it is added to the food of worker bee larvae and enters the body of the worker bees by feeding, thereby knocking down the mRNA expression of 15-PGDH in the worker bees.

[0017] Furthermore, the siRNA is fed to honey bee worker bee larvae.

[0018] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0019] (I) The present invention provides the application of 15-hydroxyprostaglandindehydrogenase (15-PGDH) gene (gene ID: 409847) in regulating the body length and weight of honey bee workers. The present invention reduces the expression of 15-PGDH in the worker bees by feeding them with 15-PGDH siRNA reagent during the larval stage; after the worker bees leave the room, the birth weight, the length of the third dorsal plate, the length of the fourth dorsal plate, and the length of the hind tibia are measured. It is found that compared with normal worker bees, the birth weight of the worker bees in the 15-PGDH interference group increased by 10% (p<0.05), the length of the third dorsal plate increased by 9% (p<0.05), the length of the fourth dorsal plate increased by 5% (p<0.05), and the length of the hind tibia increased by 8% (p<0.05). By using the present invention, the purpose of regulating the birth weight and body length of worker bees can be achieved. The method is simple and easy to operate, and has important application and promotion value for honey bee breeding.

[0020] (2) The breeding cycle is short, and the body length of worker bees can be quickly increased within one generation, saving breeding costs.

[0021] (3) This method is less affected by the environment and region and is equally applicable in the south and the north. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of qRT-PCR detection of 15-PGDH interference in worker bee larvae in a preferred embodiment of the present invention.

[0023] Figure 2 This is the result of the effect of 15-PGDH interference on the initial weight of worker bees in a preferred embodiment of the present invention.

[0024] Figure 3 This is the result of the effect of 15-PGDH interference on the body length index of worker bees in a preferred embodiment of the present invention.

[0025] Figure 4 This is a comparison diagram of adult worker bees in the 15-PGDH siRNA experimental group and the normal control group in a preferred embodiment of the present invention.

[0026] Figure 5 This is the qRT-PCR detection result of 15-PGDH overexpression in queen larvae in a preferred embodiment of the present invention.

[0027] Figure 6 This is the result of the effect of 15-PGDH overexpression on the queen birth weight in a preferred embodiment of the present invention.

[0028] Figure 7 This is the result of the effect of 15-PGDH overexpression on the queen bee body length index in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention relates to the regulatory effect of 15-PGDH gene on the body weight and body length of honey bees.

[0030] The larval development period is a critical period that determines the body size of honey bees. Queen bees and worker bees develop from the same genome, but due to different food in the larval stage, they develop into two castes with significantly different body sizes: the queen bee is large, has complete ovaries, and can reproduce normally; the worker bees are small, have incompletely developed ovaries, and cannot reproduce normally. The inventor's previous research found that when the larvae are 5 days old, the 15-PGDH gene in the queen bee is lowly expressed, while the 15-PGDH gene in the worker bee is highly expressed. It is speculated that the expression of the 15-PGDH gene in the larval stage may be involved in regulating the development of honey bee larvae, thereby affecting the size of adult honey bees.

[0031] The present invention reduces the expression of 15-PGDH gene in worker bee larvae by feeding 15-PGDH gene siRNA reagent during the larval stage of honey bees. After the worker bees are grown and leave the room, the initial weight and body length of the worker bees are measured, and it is found that compared with the normal worker bees leaving the room, the initial weight and body length of the worker bees treated with 15-PGDH gene siRNA are significantly increased. The regulation of 15-PGDH gene in regulating the weight and body length of honey bees is negative regulation, that is, the expression amount of 15-PGDH gene is reduced, and the weight and body length of honey bees are increased. The present invention has important application and promotion value for improving the weight and body length traits of honey bees and honey bee breeding.

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0033] Explanation of terms in this invention:

[0034] Length of the third dorsal plate: The length of the third dorsal plate along the body axis is an indicator of the body length of the bee.

[0035] Length of the fourth dorsal plate: The length of the fourth dorsal plate along the body axis, which is an indicator of the body length of the bee.

[0036] Length of the hind tibia of worker bees: The length of the hind tibia of worker bees is an indicator of the worker bees' ability to carry pollen.

[0037] Example 1

[0038] 1. Experimental Methods

[0039] 1.1 Experimental bee colony and group settings for the 15-PGDH gene interference group

[0040] All samples were obtained from the Nankou pilot base bee farm of the Chinese Academy of Agricultural Sciences. In June 2024, Italian honey bees with the same genetic background ( Apis mellifera ligustica ) bee colony, the experimental bee colony was subjected to queen removal at 10 am, and the queen was released 24 hours later to obtain bee eggs of the same age. After 72 hours, the eggs on the experimental honeycomb turned into larvae, which was 0 hours in the honeybee larval stage. The worker bee larvae were continuously fed with siRNA of the 15-PGDH gene at 48 hours, 72 hours, and 96 hours. The reagent concentration was 500 ng / μL, and each bee was fed 2 μL each time, with a total of 1 μg of reagent each time. Samples of worker bee larvae were collected 24 hours after the last feeding of 15-PGDH siRNA and stored in liquid nitrogen. Three groups were set up: experimental group, nonsense sequence group, and blank control group. There were 40 bees in each group, of which 20 in each group were used to collect samples of worker bee larvae 24 hours after the last feeding, and the other 20 in each group were left in the honeycomb to be capped and released normally for subsequent adult bee morphology determination.

[0041] 1.2 Experimental bee colony and group settings for the 15-PGDH gene overexpression group

[0042] All samples were from the bee farm of the Nankou pilot base of the Chinese Academy of Agricultural Sciences. In June 2024, Italian honey bee colonies with the same genetic background were raised using standard beekeeping techniques. The experimental colonies were deprived of the queen at 10 a.m., and the queen was released 24 hours later to obtain the same 0-day-old bee eggs. After 72 hours, the eggs on the experimental honeycombs became larvae, which was 0 hours in the larval stage of the honey bees. At this time, the honey bee larvae were transferred to the royal jelly strips by using a worm transfer needle. The queen larvae were continuously fed with the overexpression solution of the 15-PGDH gene at 48 hours, 72 hours, and 96 hours. The reagent concentration was 500 ng / μL, and each was fed 2 μL each time, for a total of 1 μg of reagent each time. Queen larvae samples were collected 24 hours after the last feeding of 15-PGDH siRNA and stored in liquid nitrogen. Three groups, experimental group, empty vector group and blank control group, were set up, with 40 bees in each group, of which 20 bees in each group were used to collect queen larvae samples 24 hours after the last feeding, and the other 20 bees in each group remained in the bee colony and capped normally for subsequent adult bee morphology determination.

[0043] 1.3 15-PGDH siRNA sequence

[0044] Shanghai GenePharma Co., Ltd. was commissioned to design and synthesize 15-PGDH siRNA and 15-PGDH overexpression vector. The sequence of 15-PGDH siRNA is: sense: 5'-GCUCUCACCUCGGUAUGUATT-3'; antisense: 5'-UACAUACCGAGGUGAGAGCTT-3'. The nonsense sequence is: sense: 5'-UUCUCCGAACGUGUCACGUTT-3'; antisense: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0045] 1.4 Construction of 15-PGDH overexpression vector

[0046] The mRNA fragment (768 bp, XM_006560114.3: 232-999) of synthetic 15-PGDH (the 15-PGDH gene sequence is shown in SEQ ID NO: 3) was amplified using oligo mix, and the 15-PGDH mRNA fragment was cloned into the pCDNA3.1(+) vector using BamHI and EcoRI endonucleases.

[0047] 1.5 RNA extraction

[0048] Extract high-quality RNA from samples using an RNA extraction kit (TranaZol Up Plus RNA Kit, Beijing Quanshijin Biotechnology Co., Ltd., catalog number: ER501-01-V2) according to the kit instructions. The specific steps are as follows:

[0049] ① Add two zirconium beads to the sample and use a high-throughput tissue homogenizer to thoroughly homogenize the sample.

[0050] ② Add 1 mL TransZol and 0.2 mL RNA Extraction Agent for every 50-100 mg sample and vortex at room temperature for 5 minutes.

[0051] ③ Use a centrifuge at 4℃ 10000rpm for 15 minutes.

[0052] ④ Take the colorless aqueous phase at the top of the tube and put it into a new centrifuge tube. Add anhydrous ethanol with an equal volume to the colorless aqueous phase and gently invert to mix.

[0053] ⑤ Add all the mixed solution into the centrifuge column, centrifuge at 12000rpm at 4℃ for 30s and discard the effluent.

[0054] ⑥ Add 500 μL of Clean Buffer 9 to the centrifuge column, centrifuge at 12000 rpm at 4°C for 30 seconds, and discard the effluent.

[0055] ⑦Repeat the steps once.

[0056] ⑧ Add 500 μL Wash Buffer 9 to the centrifuge column (96 mL of anhydrous ethanol must be added before use), centrifuge at 4°C, 12,000 rpm, for 30 seconds, and discard the effluent.

[0057] ⑨Repeat the steps once.

[0058] ⑩ Centrifuge at 4℃ 12000rpm for 2min to completely remove residual ethanol.

[0059] Place the centrifuge column in an RNase-free centrifuge tube, add 150 μL of RNase-free double-distilled water in the center of the centrifuge column, let it stand at room temperature for 1 min, then add 150 μL of RNase-free double-distilled water in the center of the centrifuge column and let it stand at room temperature for 1 min.

[0060] Centrifuge at 4°C 12000rpm for 1 min to extract high-quality RNA.

[0061] The RNA purity was checked by spectrophotometer and then stored at -80°C.

[0062] 1.6 cDNA synthesis

[0063] Use a reverse transcription kit for quantitative PCR (PrimeScript™ RT reagent Kit with gDNAEraser (Perfect Real Time, Catalog No.: TaKaRa, RR047A) to reverse transcribe RNA into cDNA; the specific steps are as follows:

[0064] ① Add 1μL of gDNA Eraser and 2μL of 5×gDNA Eraser Buffer to a 0.2mL PCR tube, and then normalize the RNA purity (1000 divided by RNA concentration) and add the corresponding RNA solution. Make up the rest with ddH2O, for a total of 10μL.

[0065] ②Process with PCR instrument, 42℃ for 2min, and store at 4℃.

[0066] ③ Add Primescript RT Enzyme Mix Ⅰ 1μL, 5× PrimeScript Buffer2 4μL, RT Primer Mix 1μL, and RNase-free dH2O 4μL to the PCR product, a total of 20μL.

[0067] ④ Place in PCR instrument and use the following program: 37℃ 15min; 85℃ 5s; store at 4℃. Then obtain 20μL of cDNA product and store at -20℃.

[0068] 1.7 Real-time quantitative PCR

[0069] The TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) kit (Cat. No.: TaKaRa, RR820A) was used to detect the expression of 15-PGDH in worker bee larvae samples by qRT-PCR to verify whether the feeding reagent was effective. The specific steps are as follows:

[0070] ① Shanghai Shenggong Biotechnology Co., Ltd. was commissioned to synthesize primers (primer sequences are shown in Table 1).

[0071] ② Mix the sample cDNA stock solution, 10 -1 , 10 -2 , 10 -3 , 10 -4 The cycle conditions were 95°C 30s, 95°C 5s, and 60°C 30s for 45 cycles to obtain fluorescence, obtain amplification curves and melting curves, and thus determine the optimal dilution multiple of the sample and verify that the designed primers are usable.

[0072] ③ Prepare a qRT-PCR reaction with a total volume of 20 μL, take 1 μL cDNA (200 ng), 0.8 μL upstream primer (10 μM), 0.8 μL downstream primer (10 μM), 10 μL TB Green Premix Ex Taq Ⅱ, 7.4 μL h2 O. The cycle conditions are 95℃ 30s, 95℃ 5s, 60℃ 30s, and obtain fluorescence in the 45th cycle.

[0073] Table 1 Primer sequences

[0074]

[0075] 1.8 Morphological determination of adult honey bees

[0076] Worker bees and queen bees in the bee colony were sampled immediately after they emerged from the hive. The birth weight of the worker bees and queen bees was measured with an electronic balance. A LEICA DMS 300 digital microscope was used to take photos of the hind tibia, third dorsal plate, fourth dorsal plate of worker bees and the third dorsal plate and fourth dorsal plate of queen bees at an appropriate and fixed magnification. After obtaining the photos, the photos were measured using the software Adobe Photoshop2023 to obtain the actual lengths of the third dorsal plate, fourth dorsal plate and hind tibia.

[0077] 2. Results

[0078] 2.1 Results of worker bee 15-PGDH interference

[0079] qRT-PCR results showed that compared with the control group, the expression level of 15-PGDH mRNA in worker bee larvae decreased by 64% after 15-PGDH siRNA was continuously fed for 3 days (p<0.01). Figure 1 The results suggested that feeding 15-PGDH siRNA to worker bee larvae could significantly inhibit the expression of 15-PGDH mRNA.

[0080] 2.2 Effects of 15-PGDH interference on the morphological development of worker bees

[0081] The birth weight, length of the third dorsal plate, length of the fourth dorsal plate and length of the hind tibia of the worker bees after leaving the hive were tested. It was found that compared with the control group, the birth weight of the worker bees in the 15-PGDH interference group increased by 10% (p<0.05), the length of the third dorsal plate increased by 9% (p<0.05), the length of the fourth dorsal plate increased by 5% (p<0.05), and the length of the hind tibia increased by 8% (p<0.05). Figure 2~Figure 4 .

[0082] 2.3 Results of Queen 15-PGDH overexpression

[0083] The results of qRT-PCR showed that compared with the control group, the expression level of 15-PGDH mRNA in queen larvae increased by 88% after continuous feeding with the 15-PGDH overexpression vector for 3 days (p<0.01). Figure 5 The results suggested that feeding queen larvae with 15-PGDH overexpression vector could significantly increase the expression level of 15-PGDH mRNA.

[0084] 2.4 Effects of 15-PGDH overexpression on queen morphological development

[0085] The birth weight, length of the third back plate, and length of the fourth back plate of the queen bee after leaving the hive were tested. Compared with the control group, the birth weight of the queen bee in the 15-PGDH overexpression group decreased by 21% (p<0.05), the length of the third back plate decreased by 2%, and the length of the fourth back plate decreased by 2%, but it did not reach a significant level. Figure 6 and Figure 7 .

[0086] The present invention utilizes molecular marker genes to inhibit the expression of 15-PGDH gene, thereby achieving the purpose of increasing the body length of worker bees. The present invention protects the application of 15-PGDH in regulating the birth weight and body length of bees, and the regulation is negative regulation, that is, the expression of 15-PGDH gene is reduced, and the birth weight and body length of bees are increased.

[0087] By using the regulatory effect of 15-PGDH on the birth weight and body length of honey bees in the present invention, molecular breeding can be carried out, which can shorten the generation interval and quickly achieve the breeding goal. The method of the present invention is simple and easy to operate, can be used for improving honey bee traits, and has breeding application value.

[0088] It should be noted that there may be a variety of 15-PGDH siRNA sequences, and any siRNA that can inhibit the expression of the 15-PGDH gene can achieve the purpose of the present invention.

[0089] The time of feeding 15-PGDH siRNA can be different. Feeding worker bee larvae with 15-PGDH siRNA at any time during the larval stage can achieve the purpose of the present invention.

[0090] The amount of 15-PGDH siRNA fed can be different, as long as the expression of 15-PGDH can be inhibited, the purpose of the present invention can be achieved.

[0091] In addition to feeding, injection is also a method of inhibiting the expression of the 15-PGDH gene.

[0092] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. The application of gene 15-PGDH in regulating the weight and length of honey bee workers is characterized in that: The regulation is negative regulation; The gene 15-PGDH from honey bee has Gene ID: 409847 in NCBI.

2. Application of siRNA of gene 15-PGDH or biological materials containing said siRNA or nucleotide sequences encoding said siRNA in increasing the weight and length of honey bee workers; The sequences of the sense and antisense strands of siRNA are as follows: Sense strand: 5′-GCUCUCACCUCGGUAUGUATT-3′; Antisense strand: 5′-UACAUACCGAGGUGAGAGCTT-3′; The gene 15-PGDH from honey bee has Gene ID: 409847 in NCBI; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

3. A method for increasing the weight and body length of honey bees, characterized in that: The method comprises: knocking down or silencing the gene 15-PGDH in honey bee workers by genetic engineering means; The gene 15-PGDH from honey bee has Gene ID: 409847 in NCBI.

4. The method according to claim 3, characterized in that include: The siRNA of the gene 15-PGDH is designed and synthesized, and the siRNA or the nucleotide sequence encoding the siRNA is introduced into honey bee workers through feeding.

5. The method according to claim 4, characterized in that The sequences of the sense strand and antisense strand of the siRNA are as follows: Sense strand: 5′-GCUCUCACCUCGGUAUGUATT-3′; Antisense strand: 5'-UACAUACCGAGGUGAGAGCTT-3'.

6. The method according to claim 5, characterized in that Honey bee worker larvae were fed with the siRNA.

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