CN105085643A - Varroa destructor toxic protein and coding gene thereof, and application thereof - Google Patents

Abstract

The invention discloses a Varroa destructor toxic protein and a coding gene thereof, and application thereof. According to the invention, the novel coding gene VMP of the Varroa destructor toxic protein VMP is cloned from the genome of Varroa destructor; an expression product of the coding gene, i.e., the Varroa destructor toxic protein VMP, has toxic effect on honeybees; thus, the Varroa destructor toxic protein VMP and the coding gene thereof can be applied to lethal drugs for honeybees. Moreover, conditions are created for basic scientific research on the mechanism of harm to honeybees by the Varroa destructor.

Description

A kind of varroa mite toxic protein and its coding gene and application

Technical field:

The invention belongs to the field of biochemistry and molecular biology, and specifically relates to a toxic protein of Varroa dissimilar mite, its coding gene and its application.

Background technique:

Varroa mite has always been one of the most important hazards to beekeeping in the world, and it has not been found in Australia only in the world (Zhou Ting, 2005; Rosenkranze et al., 2010). Varroa dissimilar sucks the body fluids of adult bees or larvae, causing the decline of bee physique, and at the same time reducing the cognitive ability and sense of direction of bees (Kralje et al., 2007), reducing the return rate of busy bees, and shortening the lifespan of bees (Dainate et al., 2012), the bee colony is weak. The most important thing is that Varroa disis also spreads bacterial and viral diseases of bees (Tentcheva et al., 2004; Yan et al., 2009; Prisco et al., 2011, Aie et al., 2012), and activates these latent viruses by inhibiting the host's immunosuppressive response (Yang&Cox -foster, 2005; Santillán-Galicia et al., 2008). The bee colony collapse disorder (CCD) that appeared in the United States, Greece and other countries caused panic in the world and attracted great attention from scientists. After research, its cause is related to the harm of bee mites (Cox-Foster et al., 2007). Every year, the direct loss caused by bee mites alone in my country’s beekeeping industry reaches hundreds of millions of RMB, and bee products are polluted by the use of drugs to control bee mites (Wang Qiang et al., 2006; Wu Jie et al., 2007; Zhou Ting et al., 2007) and Disva et al. The impact of mite resistance and drug residues on the environment is immeasurable (Hu Fuliang et al., 2004; Huang Wencheng, 2005; Satta et al., 2005; Zeng Zhijiang et al., 2007; Adamczyketal., 2010; Damianie et al., 2011; González-Gómezetal ., 2012).

With the spread of Varroa mite and the mutual adaptation with the host, some Italian bees and Africanized Apismellifera also showed tolerance to Varroa mite (Aumeier et al., 2000; Navajas et al. 2008 ; LeConte et al., 2011). Therefore, researchers once again focused on the tolerance of bees to Varroa mite, and believed that effective cleaning behavior is the most important tolerance factor (Moretto et al., 2006; Calderónetal., 2009; LeConteetal., 2011). But how is cleaning behavior controlled? Is it smell? Because the saliva secreted by the Varroa mite when it stings food may contain something that allows the bees to sense the presence of the mite. In fact, Chinese bees only react strongly when Varroa dissecta mite first enters the hive or moves, and bees cannot perceive it when Varroa dissecta mite is stationary (Rath, 1999). The transcriptome of cleaner bees also does not support that cleaner bees have stronger olfactory sensitivity (LeConte et al., 2011), and pointed out that it may be the herd immunity of honey bees that makes bee colonies resistant to Varroa diss. etal. (2012) believed that the cleaning behavior of Italian bees is based on the damaged larvae (remnant wings caused by Varroa mite and DWV virus), which belongs to the immune response of bees. As the original host, Apis chinensis and Varroa dissimilar have developed tolerance in the process of long-term mutual adaptation, which has attracted widespread attention.

However, the Chinese honeybee (A.cerana), which is widely raised in my country, rarely suffers from the devastating damage of Varroa mite. A large number of literatures show that as the original host, Apis chinensis and Varroa dissociates develop tolerance in the process of long-term mutual adaptation, and Apis cerana can maintain the stability of the bee colony by controlling the population of Varroa dissectae within 800 per box (Peng et al., 1987a,b; Delfinado-Baker & Peng, 1995; Rath, 1999). Studies have shown that there are three main factors that affect the population size of Varroa disses in a bee colony: Reproduction, anti-mite behavior of Apis sinensis and natural death of Varroa disses. Factors affecting the fecundity of Varroa dissecta mite include: 1) the fecundity of the female mite; 2) the capping period of the bee pupae; 3) the attractiveness of the bee larvae; 4) the size of the nest. The anti-mite behaviors of Apis cerana include grooming behavior and hygienic behavior (Rath, 1999). The definition of cleaning behavior in early literature (Rothenbuhler, 1964) is relatively narrow, simply refers to the middle bee "opens the house cover and removes the infected pests". But this uncapping and removal behavior is not specific to Varroa mites, but a general cleaning behavior, targeting all foreign substances or larvae endangered by microorganisms. Later, Rath (1999) added to this, defining cleaning behavior as the behavior of "uncovering, removing and burying larvae". The bees can quickly remove the dead larvae in the worker hive, while the dead larvae in the drone hive are difficult to remove. The worker bees in the middle bee keep the dead drone larvae in the hive without opening the cover (drone hive has a thick The cocoon is not easy to open), thus burying the drone larvae infected or killed by Varroa desvara mite.

Invention content:

The object of the present invention is to provide a kind of varroa diss toxic protein VMP and its coding gene VMP which have toxic effects on honeybees.

The amino acid sequence of the varroa mite toxic protein VMP of the present invention is shown in SEQ ID NO.2.

The nucleotide sequence of the coding gene VMP of the varroa mite toxic protein VMP of the present invention is shown in SEQ ID NO.1. It should be understood that, considering the degeneracy of codons, modifying the nucleotide sequence of the above-mentioned coding gene without changing the amino acid sequence also falls within the protection scope of the present invention.

The present invention clones a new coding gene VMP of a new Varroa mite poisonous protein VMP from the genome of the Varroa mite, and its expression product VMP has a toxic effect on honeybees, so the Varroa mite of the present invention can be The varroa mite poisonous protein VMP and its coding gene are applied to the preparation of bee-killing medicine. The invention also creates conditions for the basic scientific research on the mechanism of the Varroa dessica harming honeybees.

Description of drawings:

Figure 1 is the PCR electrophoresis diagram of VMP gene clone, M means WiderangeDNAMarker, from top to bottom are 6kb, 4kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp, the amplified product contains VMP The DNA fragment of the gene reading frame is 405bp (lane 1);

Figure 2 is the SDS-PAGE electrophoresis diagram of the purification of expressed protein VMP, M indicates the pre-stained protein marker, from top to bottom are 170kDa, 130kDa, 95kDa, 72kDa, 55kDa, 43kDa, 34kDa, 26kDa, 17kDa, 10kDa, Line1 indicates purification The expressed protein VMP, Line2 is the eluted protein when purifying VMP.

Figure 3 is an analysis diagram of the toxicity assay of the purified and expressed Varroa mite toxic protein VMP to honeybees. The purified VMP is diluted to the following concentrations: 10.08ng/μL, 2ng/μL, 1ng/μL, 0.1ng/μL, 0.01ng/μL Inject Apis chinensis, Apis mellifera worker bee prepupa and 5th instar wax moth respectively. Inject approximately 0.2 μL per worm. The following treatments were used as controls (1): purified saliva protein in isolated saliva, (2) desalted background expression protein (background expression product) (Flowthrough), (3) PBS, (4) no injection of any Substance larvae (CK). Three replicates were set up for each treatment, and each replicate had 8 worms. Culture at 34°C, 80% RH. Mortality was calculated after 11 days. Data processing: SPSS16.0, ANOVA, one-way analysis of variance (Duncan). Figure 4 is a morphological diagram of the toxicity determination of purified and expressed Varroa mite toxic protein VMP to honeybees. The purified VMP is diluted to the following concentrations: 10.08ng/μL, 2ng/μL, 1ng/μL, 0.1ng/μL, 0.01ng/μL Inject Apis chinensis, Apis mellifera worker bee prepupa and 5th instar wax moth respectively. Inject approximately 0.2 μL per worm. The following treatments were used as controls: (1) purified saliva protein in isolated saliva, (2) background expression protein (background expression product) of VMP expression strain (Flowthrough), (3) PBS, (4) no Larvae (CK) injected with any substance. After 11 days, the typical morphology of Apis cerana and Apis mellifera treated with different treatments.

Detailed ways:

The following examples are to further illustrate the present invention, rather than limit the present invention. The specific experimental conditions and methods are not indicated in the following examples, and the technical means used are generally conventional means well known to those skilled in the art.

Example 1:

1. Cloning of toxin protein gene

Get about 60 Varroa mites, use TriZolReagent (Invitrogen Company, its article number: 15596026) to extract total RNA, use agarose gel electrophoresis and ultraviolet spectrophotometer to detect the purity and amount of total RNA, and take 1 μg of total RNA to make To start the reverse transcription reaction, the reverse transcription kit used was SMARTer ^TM RACE cDNA Amplification Kit (Clontech, catalog number: 634923). For the steps of the reverse transcription reaction, refer to the instructions of the kit to obtain the reverse transcription product.

Using the reverse transcription product as a template, design specific primers for the VMP gene:

VMPF1 (5'ATGTTCAAACTTCTCGTTATCG3')

VMPR1 (5'TTAGGAGGCGAGCGCCTGCTGGA3')

Use high-fidelity Taq enzyme for PCR. The PCR reaction system is: reverse transcription product 1 μL, 10xBuffer 5 μL, dNTP (each 2.5 mM) 4 μL, VMPF1 (10 μM) 1 μL, VMPR1 (10 μM) 1 μL, Taq enzyme (5U/μL) 1 μL, ddH ₂ O 37 μL. Mix well after adding the sample on ice. The PCR reaction conditions were: 94°C for 5 min; 30 cycles of 94°C for 30 sec, 44.5°C for 30 sec, and 72°C for 45 sec; 72°C for 5 min. A 405bp fragment was obtained by PCR amplification. The results of electrophoresis are shown in Figure 1. After the fragment was recovered by agarose gel electrophoresis, it was connected to the pEASY ^TM -T1Simple vector (Beijing Quanshijin Biotechnology Co., Ltd., its product number: CT111-01). For specific steps, refer to the instruction manual of the vector. Then the connection vector was sequenced, and the analysis showed that the sequence contained an open reading frame, which was 405 bases, and its sequence was shown in SEQ ID NO.1. No similar sequence was found through BLAST comparison analysis, and the gene was named Di Varroa mite virulence gene VMP. The encoded protein has 134 amino acid residues, and its sequence is shown in SEQ ID NO.2. According to BLASTx analysis, the highest homology is 44% (XM_003740358.1).

Two, embodiment 2: transform Transetta, express VMP

Using the pEASY ^™ -T1Simple recombinant plasmid containing the virulence gene VMP gene cDNA of Varroa mite in Example 1 as a template, design primers F1 (5' GAATTC ATGTTCAAACTTCTCGTTATCG3') (the underline indicates the EcoRI restriction site) and R1 (5' AAGCTTTTAGGAGGCGAGCGCCTGCTGGA3 ') (the underline indicates the HindIII restriction site). The reaction system is: pEASY ^TM -T1Simple recombinant plasmid (50ng/μL) containing cDNA of Varroa mite virulence gene VMP gene (50ng/μL), 10xBuffer 5μL, dNTP (each2.5mM) 4μL, F1 (10μM) 1μL, R1 (10μM) 1μL, Taq enzyme (5U/μL) 1 μL, ddH ₂ O 37 μL. Mix well after adding the sample on ice. The reaction conditions are: 94°C for 5 min; 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec; 72°C for 5 min. A DNA fragment of 405 bp was obtained after the PCR reaction, and the fragment was purified and inserted forward into the EcoRI restriction site of the expression vector pET-32a(+).

The procedure for recombining the PCR fragment into the EcoRI restriction site of pET-32a(+) is as follows: ①Take 5 μg of the pET-32a(+) plasmid, use EcoRI and HindIII restriction endonucleases for restriction endonuclease digestion and linearization, and Purify the linearized plasmid and dissolve it in ddH ₂ O to make the final concentration 50ng/μL; ②Purify the PCR fragment and dissolve it in ddH ₂ O to adjust the final concentration to 50ng/μL; ③Refer to T4DNALigase (TAKARA, Product No. D2011A) for the recombination ligation reaction. The specific steps are: prepare the following ligation reaction system in a centrifuge tube (total volume is 20 μL), 8 μL of PCR fragment, 1 μL of EcoRI and HindIII linearized pET-32a(+) vector, 2 μL of 10×T4 DNA Ligase Buffer, 1 μL of T4 DNA Ligase, and 7 μL of ddH ₂ O. Mix well and connect overnight at 16°C. ④ Take 10 μl of the ligation product and add it to 50 μl of thawed expression host Escherichia coli competent cell TransettaDE3, and spread it on a resistant LB plate containing carbenicillin (Car, 100 μg/mL) for culture. The positive colony is identified by conventional colony PCR method. The primers are still F1 and R1. The positive clones were sent to the company for sequencing to reconfirm that the virulence gene VMP of Varroa mite was inserted into the expression vector pET-32a(+), and transformed into the expression host Escherichia coli competent cell TransettaDE3.

In this way, the expression strain TransettaDE3-pET-32a(+)-VMP transformed with the expression vector pET-32a(+) containing the Varroa mite virulence gene VMP gene was obtained.

3. Expression and purification of toxic protein VMP

The expression strain TransettaDE3-pET-32a(+)-VMP transferred with the expression vector pET-32a(+) containing the VMP gene was picked and inoculated in 50mL LB (containing Car100μg/μL) liquid medium, and cultured overnight at 37°C with shaking. Transfer 9mL of seed solution to a 500mL Erlenmeyer flask of fresh 300mL (containing Car100μg/μL) LB liquid medium, cultivate at 37°C and 180rpm until the OD value is 0.4-0.6, add a certain amount of IPTG (final concentration 0.2mM) in Induced at 16°C for 6h. 4°C, 8000g, centrifuge for 10min to collect the bacteria; wash the precipitate with PBS in a pre-weighed centrifuge tube, remove the supernatant after centrifugation and weigh, add 10mL protein purification solution per gram of wet weight of the bacteria, and ultrasonically lyse in an ice bath Bacteria: 300W, act for 4s, rest for 6s, repeat 100 times as a cycle, a total of two cycles. Centrifuge at 12000×g for 10 min at 4°C and collect the supernatant. The supernatant was passed through a 0.22 μm filter membrane to remove impurities and air bubbles, passed through a Ni-column (IMAC, BIO-RAD, catalog number: 7324612), and the purification steps were carried out according to the instructions. PAGE electrophoresis analysis (Figure 2), the concentration was determined by the Bradford method, and the kit used was Bradford Protein Assay Kit (Shanghai Sangong, product number: SK3041). The concentration of the obtained purified and expressed VMP, a toxic protein of Varroa dissociates, was 10.08 ng/μL.

4. Determination of the toxicity of VMP to bees

Dilute the above-mentioned purified and expressed varroa mite toxic protein VMP to the following concentrations: 10.08ng/μL, 2ng/μL, 1ng/μL, 0.1ng/μL, 0.01ng/μL, and inject them into Apis cerana and Apis mellifera worker bee prepupa and the 5th instar wax moth. Inject approximately 0.2 μL per worm. The following treatments were used as controls: (1) purified saliva protein in isolated saliva, (2) desalted background expression protein (background expression product) (Flowthrough), (3) PBS, (4) no injection of any Substance larvae (CK). Three replicates were set up for each treatment, and each replicate had 8 worms. Culture at 34°C, 80% RH. Mortality was calculated after 12 days. Data processing: SPSS16.0, ANOVA, one-way analysis of variance (Duncan), it was found that the purified expression of the toxic protein VMP of Varroa mite at a concentration>1.01ng/μL has obvious lethal toxicity to Chinese honeybees (Figure 3, 4).

Claims (3)

1. Yi Zhong Di Siwa mite toxic protein VMP, it is characterized in that, its aminoacid sequence is as shown in SEQIDNO.2.

2. an encoding gene VMP of Di Siwa mite toxic protein VMP according to claim 1, it is characterized in that, its nucleotide sequence is as shown in SEQIDNO.1.

3. the application of Di Siwa mite toxic protein VMP according to claim 1 in preparation honeybee lethal drug.