CN102286509A - DsRNA vector capable of interfering expression of Bx-cpl-1 gene and use thereof in prevention and control of and study on bursaphelenchus xylophilus - Google Patents

Abstract

The "dsRNA carrier for interfering with the expression of Bx-cpl-1 gene and its application in the control and research of pine wood nematode" of the present invention belongs to the plant disease and insect control technology. The dsRNA interference vector is characterized in that a complete or partial sense strand of the Bx-cpl-1 gene and an antisense strand complementary to the sense strand are sequentially inserted into the multiple cloning site of the backbone vector. It can be used for the control and research of pine wood nematode.

Description

A dsRNA carrier that interferes with the expression of Bx-cpl-1 gene and its application in the control and research of pine wood nematode

technical field

The invention belongs to the technical field of plant disease and insect pest control, and in particular relates to a dsRNA carrier that interferes with the expression of Bx-cpl-1 gene and its application in the control and research of pine wood nematode.

Background technique

Pine wood nematode (Bursaphelenchus xylophilu) is a parasitic nematode that is extremely harmful to forests, and it mainly harms coniferous trees represented by pine species. In the twentieth century, pine trees in Japan were devastated by pine xylophilus infection. In my country, pine wood nematode was first discovered in Nanjing in 1982. After that, the epidemic spread rapidly, causing great damage to my country's pine forest resources, and its economic losses are incalculable. Pine wood nematode disease has even directly threatened my country's Zhangjiajie and other world natural and cultural heritage landscapes.

Pine wood nematode disease is known as "the cancer of pine trees", because it spreads extremely fast, and once the disease occurs, the trees will die quickly, so it is very difficult to prevent and control. Since the spread of the disease is closely related to human activities, many countries list it as a category A plant quarantine object. In order to avoid the harm of pine wood nematode to my country's forest resources, the prevention and control of pine wood nematode is imminent.

The spread of pine wood nematode is extremely fast, and once the outbreak occurs, it is difficult to control. The disease has not been effectively controlled so far. Therefore, we must always adhere to the policy of focusing on prevention, and strictly control the spread of pine wood nematode in the affected areas.

At present, there are mainly two methods to treat pine wood nematode disease. Since the pine wood nematode is inside the trunk, insecticides can be injected into the trunk or watered to the roots, and the other is to spray chemicals from an airplane to control longhorn beetles. These two methods have certain curative effect, but their cost performance is relatively low. The cost of trunk injection treatment is high, so it is not suitable for large-scale promotion; and the use of aircraft to spray chemical agents to control longhorn beetles is also expensive and has certain negative effects.

Since chemical control has many drawbacks, more attention has been paid to the biological control of pine wood nematode. Studies have shown that many fungi have inhibitory effects on the survival of nematodes, such as Syntocephalus racemosa and Leucetus japonica, etc. However, due to the astonishing reproduction speed of pine wood nematodes, the effect of biological control is far behind its diffusion speed.

Therefore, existing control measures cannot effectively control pine wood nematode. We can only hope for a breakthrough in the study of the pathogenic mechanism. At present, the most economical and effective way is to use molecular biology techniques to screen the resistance genes of pine trees, and then use transgenic technology to make pine trees resistant to pine xylophilus.

The active site of cysteine proteases contains cysteine residues. The enzyme widely exists in various organisms in nature, such as bacteria, fungi, plants, mammals and humans. Therefore, there are many studies on this enzyme. Studies have shown that cysteine proteases play a crucial role in nematode invasion of tissues or cells, providing nutrients required for embryonic development, moulting, digesting host proteins, regulating immune responses, and evading clearance from host immune responses .

Cysteine proteases belong to the class of proteolytic enzymes. The enzyme is a precursor substance when it is first synthesized in the organism, and the substance includes two regions: the precursor region and the catalytic region. In the cysteine protease family, there is a highly conserved typical triplet catalytic active center Cys-His-Asn. Nematode cysteine protease is the main "digestive enzyme" of nematodes, usually expressed in the esophageal glands and intestinal cells of nematodes, exerting proteolytic activity. The functions of nematodes ingesting food, invading host cells, and immunity are closely related to cysteine protease. Therefore, the research on this enzyme has become more extensive and in-depth in recent years.

Studies have found that cysteine protease exists in a variety of nematodes. Different nematodes secrete this enzyme in different parts, and the distribution parts of the enzyme are also different. Some nematode oocytes contain L-type cysteine proteases, such as Caenorhabditis elegans; B-type and L-type cysteine proteases are found in the intestinal lumen of many nematodes at different developmental stages. Examples include Haemonchus controtus and Meloidyne incognita.

Lu Chunhua (Lv Chunhua. Cloning and sequence analysis of the S-type cysteine protease gene of the similar perforator nematode [J]. Plant Protection, 2008, 34(5): 17-22.) and others isolated the cysteine from B. xylophilus Two genes (Bx-cpl-1, Bx-cpl-2) of the amino acid protease family were initially expressed in E. coli, but the functions of these two genes in the nematode life cycle were not revealed and proved.

RNA interference (RNAi) refers to the mechanism of post-transcriptional gene silencing induced by double-stranded RNA. With the development of technology, RNA interference technology has attracted more and more attention from researchers. At present, RNA interference technology has been widely used in nematode gene analysis. Ford et al. analyzed the role of histone-like cysteine protease genes in adult worms of B. malayi by RNA interference. Treatment of male and female adults of B. malayi with Bm-cpl-1 and Bm-cpl-5 of the filarial CPL gene family, or with Bm-cpz-1 dsRNA, resulted in a decrease in the number of microfilariae. Treatment of female uteri with Bm-cpl-5 or Bm-cpl dsRNA and siRNA showed that embryo development was significantly affected, resulting in malformed embryos and different embryo stages. Functional analysis of Caenorhabditis elegans L-type cathepsin (Ce-cpl-1) and Z-type cathepsin (Ce-cpz-1) identifies both genes controlling them as essential in early embryonic development , Ce-cpl-1 plays a regulatory role during vitellin breakdown, and Ce-cpz-1 plays an important role in molting.

For the mediation of RNA interference, at present, bacterial-mediated RNAi technology plays an important role in the study of model nematode gene function, and has become the main method for the study of model nematode gene function. At present, the means of RNAi has also become an important method for the gene function of pine wood nematode, but the soaking method is used at present, and the dsRNA synthesized in vitro is soaked in pine wood nematode to analyze the function of the gene. This method has great shortcomings, mainly It is shown that the interference cannot be sustained, and it is extremely unstable. How to realize the stable interference of RNA is also a further goal of the present invention.

Contents of the invention

According to the gaps in the above fields and the needs of pine xylophilus control, the present invention provides a dsRNA carrier that interferes with the expression of Bx-cpl-1 gene and its application in the control and research of pine xylophilus.

The dsRNA interference carrier that interferes with the expression of the Bx-cpl-1 gene of the pine wood nematode is characterized in that the complete or partial positive-sense strand of the Bx-cpl-1 gene is inserted forward in sequence on the multiple cloning site of the backbone vector and is compatible with all The sense strand is complementary to the antisense strand.

The backbone vector is an expression vector mediated by Agrobacterium.

The nucleotide sequence of the sense strand is shown in Seq ID No.2, the nucleotide sequence of the sense strand is shown in Seq ID No.3, and the carrier mediated by the Agrobacterium is PHDT-RH, The sense strand is inserted into the 5' end multiple cloning site between LB and RB of PHDT-RH, and the antisense strand is inserted into the 3' end multiple cloning site between LB and RB of PHDT-RH to obtain The dsRNA interference vector used is PHD-RH-CPL1.

Transform into the bacterial strain with the dsRNA interference vector mentioned above.

Said strains refer to filamentous fungi.

The fungus refers to Botrytis cinerea that has been transformed into PHD-RH-CPL1.

Application of the aforementioned dsRNA interference carrier in the control of pine wood nematode.

A new method for studying the function of the Bx-cpl-1 gene, characterized in that the above-mentioned dsRNA interference carrier is transformed into filamentous fungi, and then the mycelium of the filamentous fungal transformants is fed to pine wood nematodes, and the observation of pine wood Phenotypes of nematodes.

The filamentous fungus refers to Botrytis cinerea.

The inventors of the present invention studied the role of the cysteine protease gene Bx-cpl-1 obtained by the predecessors in the life cycle of nematodes, and used a partial fragment of the gene to construct an RNA interference vector, which was mediated by Agrobacterium. Into Botrytis cinerea, the transgenic gray mold hyphae fed pine wood nematodes to conduct interference experiments on the CPL of pine wood nematodes, and fluorescence quantitative PCR was used to detect the cysteine protease gene Bx-cpl-1 of the disturbed pine wood nematodes. Post-transcriptional expression, the results showed that with the increase of the interference time, the transcriptional level of the cysteine protease gene gradually decreased, and the infection effect had a significant difference compared with the control. The measurement of cysteine protease activity showed that the expression of Bx-cpl-1 gene was continuously disturbed for 10 generations of B. xylophilus, the activity of cysteine protease was reduced by 20.9%, and the reproduction number within 5 days was reduced by 40%. The body length of nematodes was also significantly shortened. These experimental data indicate that the expression of Bx-cpl-1 gene plays a key role in the embryonic development of B. xylophilus, and thus interfering with the expression of this gene can achieve the purpose of biological control of B. xylophilus.

Based on these experimental results, the present invention provides a dsRNA interference vector that interferes with the expression of the Bx-cpl-1 gene of pine wood nematode, which is characterized in that the positive-sense strand of the complete or partial fragment of the Bx-cpl-1 gene is inserted into the backbone vector and the antisense strand that is completely complementary to the sense strand. As for the backbone carrier, those skilled in the art may select many conventional backbone vectors for the construction of RNA interference vectors. The construction method is also to sequentially construct the sense strand and anti Sense chain. It can be the complete Bx-cpl-1 gene, or a partial fragment thereof. The complete fragment of the Bx-cpl-1 gene or which part of the fragment is selected is related to the restriction site of the selected backbone vector. Such selection work is a routine skill possessed by those skilled in the art.

In the present invention, the preferred backbone vector is the expression vector mediated by Agrobacterium, which can carry the target gene and transfer it into various organisms, including yeast, filamentous fungi, and human cells in addition to natural plants. Compared with traditional transformation methods, AMT technology has the advantages of high transformation efficiency and good genetic stability. Therefore, constructing an Agrobacterium-mediated RNA interference vector is beneficial to transfer the interference fragment into more types of hosts for expression dsRNA for interfering with pine xylophilus.

In a preferred embodiment of the present invention, with PHDT-RH as the backbone carrier, dsRNA is constructed with the fragment shown in Seq ID No.2 and its antisense strand in the Bx-cpl-1 gene as the core fragment of the dsRNA interference carrier Interference vector PHD-RH-CPL1.

The dsRNA interference carrier claimed in the present invention is transferred into fungi, and these transformed fungi are used to feed pine wood nematodes, which express dsRNA and interact with Bx-cpl-1 of pine wood nematodes to make the expression of Bx-cpl-1 gene Disturbed to affect the growth and development of pine wood nematode, to achieve the purpose of prevention and control.

In the embodiment of the present invention, the constructed dsRNA interference vector PHD-RH-CPL1 is transferred into Botrytis cinerea (Botrytis cinerea), and the transformation of Botrytis cinerea that can be used to interfere with the expression of Bx-cpl-1 gene of pine wood nematode is obtained son.

Another contribution of the present invention is to provide a method for studying the function of Bx-cpl-1 in the growth and development of B. xylophilus, that is, to transfer the above-mentioned dsRNA interference vector into the filamentous fungus favored by B. xylophilus , and then fed the mycelium of the filamentous fungal transformant to the pine xylophilus at various stages to observe the growth and development of the pine xylophilus.

Description of drawings

Figure 1. Schematic diagram of the structure of Agrobacterium-mediated filamentous fungal transformation vector PHDT;

Figure 2. Schematic diagram of the structure of protoplast-transformed filamentous fungal RNAi vector Psilent-1;

Figure 3. Schematic diagram of the structure of the carrier PHD-RH;

Fig. 4. The electrophoresis diagram of the cloned product of the partial fragment of the CPL gene of pine xylophilus,

M: DNAMarker 2000; 2: Amplification result of the sense strand of Bx-cpl-1 gene; 4: Amplification result of the antisense strand of Bx-cpl-1 gene; 6: Amplification result of the sense strand of Bx-cpl-2 gene; 8: Amplification results of the antisense strand of Bx-cpl-2 gene; the rest are controls.

Figure 5. PCR electrophoresis of bacterial solution:

M: DNA Marker 2000; 2-6, amplification result of positive sense strand of Bx-cpl-1 gene; 8-12: amplification result of antisense strand of Bx-cpl-1 gene; 14-18: positive sense strand of Bx-cpl-2 gene Chain amplification results; the rest are controls.

Figure 6. Hind III and Xba I double digested fragment

M: DNAMarker 2000; 1-6: The digested product of the vector pGM-T containing the insert.

Figure 7. HindIII and BglII double enzyme digestion detection sense strand

Figure 8. HindIII and BglII double enzyme digestion detection antisense strand

Figure 9. Hygromycin PCR electrophoresis

M: DNA Marker 2000; 1-3: PCR with hygromycin primers; 4: control.

Figure 10. PCR electrophoresis of the target fragment

M: DNAMarker 2000; 1-2: PCR of target fragment primers; 3: control.

Figure 11. Transcript level of Bx-cpl-1 gene after 2 generations of interference (Bar value is standard error)

Figure 12. Transcript level of Bx-cpl-1 gene after interference for 30 generations (Bar value is standard error)

Figure 13. Transcript level of Bx-cpl-2 gene after 2 generations of interference (Bar value is standard error)

Figure 14. Transcript level of Bx-cpl-2 gene after 30 generations of interference (Bar value is standard error)

Figure 15. Determination of enzyme activity using GLUpNA as substrate after interference for 10 generations

◆: Control; ■: Interference with CPL1; △: Interference with CPL2

Figure 16. Determination of enzyme activity using GLUpNA as substrate after 35 generations of interference

◆: Control; ■: Interference with CPL1; △: Interference with CPL2

Figure 17. RNA interference affects the reproductive ability of B. xylophilus adults (Bar value is standard error)

Figure 18. Body length change of B. xylophilus after disturbance (Bar value is standard error).

Detailed ways

The implementation of the present invention and its beneficial effects will be described in detail below through experiments and data.

biomaterials:

The pine wood nematode (Bursaphelenchus xylophilus) (US1) comes from the United States and has been subcultured in our laboratory for many years.

Botrytis cinerea was provided by the Institute of Microbiology, Chinese Academy of Sciences.

Agrobacterium tumefaciens strain EHA105, commercially available.

Agrobacterium-mediated filamentous fungal transformation vector PHD-T (see Figure 1), a known vector, was kindly donated by Researcher Liu Xingzhong, Institute of Microbiology, Chinese Academy of Sciences.

Psilent-1, an existing known carrier, was donated by Liu Xingzhong, a researcher at the Institute of Microbiology, Chinese Academy of Sciences.

The applicant declares that the above biological materials are kept in the applicant's laboratory and can be released to the public for verification tests.

Main instruments and reagents

Instruments: Olympus solid microscope, American Polyscience refrigerated centrifuge (9500), constant temperature incubator, refrigerator, electronic balance, autoclave, PCR instrument, electrophoresis instrument, gel imaging system, water bath, pipette, homogenizer , centrifuge tubes, pipette tips, beakers and pH meters, etc.

Reagent:

The Trizol Reagent used for RNA extraction, RT-PCR reverse transcription kit was purchased from Invitrogen;

SanPrep column DNA mini-extraction kit and SanPrep column DNA gel recovery kit were purchased from Sangon Bioengineering Co., Ltd.;

TA cloning kit, Tag DNA polymerase, DNA Marker 2000 and Escherichia coli competent cells used for cloning were all purchased from Tiangen Biological Company;

Restriction endonucleases HindIII, BglII, XbaI and SpeI were all purchased from Takara Biological Company.

TAE buffer solution (50×): Tris base 242g, glacial acetic acid 57.1ml, 0.5mol/L EDTA (pH8.0) 100ml.

Potato medium (PDA), used to cultivate Botrytis cinerea: Weigh 100g of peeled potatoes, chop them, add 500ml of distilled water, and boil for 30min. Filter the potatoes with gauze, make up 500ml and put them into a conical flask, and adjust the pH value to 6.0. Then weigh 10g of glucose and 7.5g of agar powder, add to the flask and shake well. 121°C, 20min high-pressure steam sterilization and poured into plates.

LB medium, used for Escherichia coli blue-white screening. Yeast powder 5g/L, peptone 10g/L, NaCl 10g/L.

YEM medium, divided into solid and liquid, used for the activation of Agrobacterium: yeast powder 0.2g, mannitol 5g, MgSO4 7H2O 0.1g, K2HPO40.25g, NaCl 0.05g, agar 6g, dissolved in water , to 500ml (liquid medium without agar).

MM medium is the basic medium used for expanding the cultivation of Agrobacterium tumefaciens. The formula of 500ml is as follows: 5ml K-buffer (pH 7.0): KH2PO4 145g/L, K2HPO4200g/L; 10ml M-N: NaCl 7.5g/L, MgSO4. 7H2O15g/L; 5ml glucose 20g/L (stored in a refrigerator at 4°C after filter sterilization); 5ml FeSO40.01g/L (stored in a refrigerator at 4°C after filter sterilization); 0.5ml CaCl2.2H2O 1g/L; 2.5ml Spore Elements (CuSO4.5H2O0.1g/L, ZnSO4.7H2O 0.1g/L, Na2MoO4.2H2O0.1g/L, MnSO4.H2O 0.1g/L, H3BO30.1g/L); 1.25ml, NH4NO320g/L ; 470.75ml sterile water.

IM medium, used to induce Agrobacterium tumefaciens, 1L formula: 0.8ml 1.25M K-buffer, (adjust the pH to 4.9); 20ml MN: NaCl 15g/L, MgSO4.7H2O 30g/L; 1ml CaCl _{2.2H2O 2g} /L; 10ml 50% glycerol; 2.5ml NH4NO3 _20g /L; 5ml Spore Elements; 40ml MES 1M (adjust _pH to 5.5), adjust pH with NaOH (filter sterilized, 4 ℃ storage); 10ml FeSO4 0.01g/L (filter sterilized, stored at 4 ℃); 10ml glucose 20g/L (filter sterilized); 2ml AS 100mM (dissolved in absolute ethanol, placed at -20 ℃); 898.7ml water.

Co-cultivation medium (Co-IM) for co-cultivation of Botrytis cinerea and Agrobacterium tumefaciens. The amount of glucose in the induction medium IM was halved when preparing.

Kanamycin: dissolved in water, prepared at a concentration of 10mg/ml, stored at -20°C, and diluted to 10μg/ml when used;

Rifampicin: dissolved in methanol, prepared at a concentration of 10mg/ml, stored at -20°C, and diluted to 34μg/ml when used;

Hygromycin: 50mg/ml, store at 4°C away from light, and dilute to 200μg/ml when used;

Cephalosporin: dissolve in water, 200mg/ml, store at -20°C, and dilute to 400μg/ml when used.

MES buffer (pH5.5): MES 0.15M, DTT 4Mm, EDTA 4mM

Substrate GLUpNA (0.5 mg/mL): 1.5 mg of substrate was dissolved in 1 ml of DMSO and diluted to 0.5 mg/ml with MES buffer.

SYBR Premix Ex Taq was purchased from TaKaRa Company.

Mix SYBRMix with dye: Mix 1ml, DyeII 200μl.

Example 1: Construction of Agrobacterium-mediated filamentous fungus expression dsRNA vector-PHD-RH

Agrobacterium-mediated filamentous fungal transformation vector PHD-T, protoplast transformation filamentous fungal RNAi vector Psilent-1 (Figure 2) is a known vector, donated by researcher Liu Xingzhong, Institute of Microbiology, Chinese Academy of Sciences.

The construction of the vector PHD-RH is mainly based on the Agrobacterium-mediated filamentous fungal transformation vector PHDT as the backbone, and its T-DNA region is modified. A DNA sequence (Seq ID No.1) was synthesized by Shanghai Bioengineering Company. Its structure KpnI restriction site, Aspergillus nidulans tryptophan promoter, 5' multiple cloning site, intron, 3' multiple cloning site, Aspergillus nidulans tryptophan terminator, BstxI restriction site ( 5'------3'). The synthesized sequence was inserted between the Kpn I and Bstx I sites of the vector PHDT, and the positive clone was identified and verified by sequencing, and the obtained vector was named PHD-R. Its 5' multiple cloning site contains Xho I, Snab I, HindIII, and Xba I restriction sites in sequence, and the 3' multiple cloning site contains Stu I, Spe I, BglII, and ApaI restriction sites. A hygromycin resistance gene was added on the vector PHD-R as a selection marker. Primers HYG-F and HYG-R (with KPN I restriction sites added at both ends) were designed to amplify the hygromycin resistance gene HPH using the vector PHDT as a template. The amplified product was connected to the carrier PHD-R, and after sequencing confirmation, it was inserted into the Kpn I restriction site of the carrier PHD-R to obtain the carrier PHD-RH (Figure 3). The positive clones were identified by enzyme digestion and then confirmed by sequencing.

HYG-F: 5’-TGGTA CCTA GA CGTTA A CTGATATTGA A-3’

HYG-R: 5'-TGGTACCTAAACCCAGGGCTGGTGACGGA-3'

The cultivation and separation of embodiment 2 pine xylophilus

1. Cultivation of B. xylophilus

B. xylophilus was cultured with Botrytis cinerea grown on PDA medium.

Sterilize the PDA medium at 121°C for 20 minutes by high-pressure steam, and pour it into a petri dish at about 60°C; culture

After the base is cooled, insert Botrytis cinerea into the ultra-clean bench and place it in a constant temperature incubator at 25°C for cultivation; after 3 to 4 days, sterilize the pine wood nematode with hydrogen peroxide, put it into a petri dish, and place it in a constant temperature incubator at 25°C After 6 to 7 days, remove the culture dish from the incubator and store it at 4°C.

2. Isolation of B. xylophilus

Wrap the medium containing pine xylophilus with four layers of gauze, suspend it in a beaker filled with sterilized distilled water; place the beaker in a constant temperature incubator at 25°C for 24 hours; remove the gauze and medium, pour Supernatant; Pour the remaining liquid into a sterilized Petri dish, use a pipette to pick out B. xylophilus under a microscope, and then proceed to subsequent experiments.

3. Extraction of total RNA

Trizol Reagent (purchased from Invitrogen) was used for extraction, and the operation was referred to the instructions.

4. Synthesis of first-strand cDNA

RT-PCR kit (purchased from Invitrogen) was used to synthesize cDNA, and the operation was referred to the instruction manual, and the synthesized cDNA was stored for future use.

5. Primer Design

The full-length cDNA of Bx-cpl-1 is 1220bp, and its GenBank ID is EU651860.

The full-length cDNA of Bx-cpl-2 is 1161bp, and its GenBank ID is EU659123.

Both genes consist of 2 introns and 3 exons.

DNAMAN software was used to analyze the target fragment and the restriction site on the carrier PHDT-RH, and screen out the restriction site that is not in the target gene but on the carrier.

A Bx-cpl-1 sense strand primer and a Bx-cpl-1 antisense strand primer were designed at 481 bp. The sense strand primer was inserted into the HindIII and Xba I restriction sites, and the antisense strand primer was inserted into the Spe I and BglII restriction sites.

The Bx-cpl-2 sense strand primer and the Bx-cpl-2 antisense strand primer were designed at 830bp. The sense strand primer was inserted into the HindIII and Xba I restriction sites, and the antisense strand primer was inserted into the Spe I and BglII restriction sites.

Table 1. Primers for introducing restriction sites for Bx-cpl-1 and Bx-cpl-2 genes

Embodiment 2. Cloning of cysteine protease gene partial fragment

2.1. Amplify the target fragment

Amplify the pine xylophilus cDNA with the primers in Table 1 of Example 1 to obtain the target fragments for the construction of RNA interference vectors such as Seq ID No. 3, 4 and Seq ID No. 5, 18 with four restriction sites :

The PCR reaction system is as follows:

Pine xylophilus cDNA 1μl 10×Buffer(+Mg ²⁺ ) 2μl dNTPs(10mM) 1.6μl Upstream primer (10mM) 0.5μl Downstream primer (10mM) 0.5μl Taq enzyme (5U/μl) 0.3μl ddH ₂ O 14.1μl Final V 20μl

PCR reaction program: 94°C 4min; 94°C 30sec, Tm 30sec, 72°C 1min, 35 cycles; 72°C 10min.

1% agarose gel electrophoresis to detect PCR results. Four specific fragments of about 350 bp were obtained respectively ( FIG. 4 ).

2.2 Recycling target fragments

Use 50 μl of the system to recover the target fragment, use the SanPrep Column DNA Gel Recovery Kit, and store the recovered fragment at -20°C.

2.3. The target fragment is connected to the pGM-T vector

The target fragment was ligated with the pGM-T vector, and ligated at 16°C for 12h. The reaction system is as follows:

target fragment 7.5μl pGM-T vector 0.5μl DNA ligase (3U/μl) 1μl 10×Ligase Buffer 1μl Final V 10μl

The ligation product was transformed into Escherichia coli competent cells, and positive clones were screened by blue and white spots. The positive clones were cultured in LB liquid medium containing ampicillin in a water bath at 37°C and shaken at 220 rpm for 3 hours.

2.4 Bacteria liquid PCR

The above tubes of bacterial liquid were subjected to bacterial liquid PCR, and the system was as follows:

Escherichia coli liquid 1μl 10×Buffer(Mg ²⁺ ) 2μl dNTP (10mM) 1.6μl Upstream primer (10mM) 0.5μl Downstream primer (10mM) 0.5μl Taq enzyme (5U/μl) 0.3μl ddH ₂ O 14.1μl Final V 20μl

The PCR amplification procedure of the bacterial solution is the same as that in 2.1, and the primers are listed in Table 1.

1% agarose gel electrophoresis was used to detect the results of bacterial liquid PCR. After connecting the sense strand or antisense strand with the pGM-T vector, transform Escherichia coli, perform blue-white screening, and pick positive clones for bacterial liquid PCR. The PCR results are shown in Figure 5, and the conversion efficiency is very high, close to 100%.

Embodiment 3. Construction of RNA interference vector

The pGM-T loaded with the sense strands of Bx-cpl-1 and Bx-cpl-2 genes and the PHDT-RH vector were double-digested with Hind III and Xba I enzymes respectively, and Bx-cpl-1 and Bx - The sense chain pGM-T vector of cpl-2 gene was digested to obtain fragments of about 350 bp, and the fragment size was the same as expected. Recover the target fragment and the digested carrier PHDT-RH for ligation, transformation, and screening of positive transformants. In the positive transformants, the downstream of the sense strand of the target gene has introduced IT (Intron) sequence. Sent to Beijing Sangong for sequencing. The PHDT-RH vector inserted into the sense strand and the PHDT-RH vector of the antisense strand of the target gene after correct sequencing were double digested with Spe I and BglII enzymes. The target fragment and the digested vector are recovered, ligated, transformed, screened, and sequenced. The constructed vector contains the sense strand and antisense strand of the target gene. After shaking the bacteria, extract the plasmid with a plasmid extraction kit. details as follows:

3.1 Double digestion of target fragment and PHDT-RH plasmid

The sense strands of Bx-cpl-1 gene and Bx-cpl-2 gene and the PHDT-RH vector were digested with HindIII and Xba I respectively, and the effect of enzyme digestion was detected by 1% agarose gel electrophoresis.

The enzyme digestion system is as follows:

Enzyme 1 2.5μl Enzyme 2 2.5μl BSA 5μl 10×Buffer(T) 5μl DNA 35μl Final V 50μl

The sense strand fragment and PHDT-RH plasmid were recovered.

3.2 Sense strand connected to PHDT-RH plasmid

Ligate the recovered sense strand to the PHDT-RH carrier, and ligate overnight at 16°C. The connection system is as follows:

chain of justice 13μl PHDT-RH 3μl DNA ligase (3U/μl) 1μl 10×Ligase Buffer 3μl Final V 20μl

The recombinant plasmid was transformed into Escherichia coli, and positive colonies were screened by blue and white spots.

3.3. Positive colonies were shaken, bacterial liquid PCR, sequencing, plasmid extraction to obtain PHDT-RH plasmid with positive sense strand.

Sense strand sequencing primer IT-F: AAATGGAAGGGACTGACAAG

Antisense strand sequencing primer IT-R: GAGAGGCAAGAAGAAAGGCT

3.4 Digestion of antisense strand and PHDT-RH plasmid

The antisense strands of Bx-cpl-1 gene and Bx-cpl-2 gene and the PHDT-RH vector connected with the sense strand were double-digested with Spe I and Bgl II respectively, and the effect of enzyme digestion was detected by 1% agarose gel electrophoresis.

Ligate the recovered antisense strand to the PHDT-RH vector containing the sense strand, and ligate overnight at 16°C. The recombinant plasmid was transformed into Escherichia coli, and the PHDT-RH plasmid connected to the sense strand and the antisense strand was screened by blue and white spots.

The positive colonies were shaken, and PCR was performed on the bacterial liquid, sequenced, and plasmids were extracted.

The result of enzyme digestion showed: the positive recombinant plasmid after the sense strand of Bx-cpl-1 or Bx-cpl-2 gene was connected with PHDT-RH was detected by double digestion with HindIII and Bgl II, and HindIII was located at the beginning of the sense strand. The sense strand is located upstream of the intro (148bp), and Bgl II is located downstream of the intro, so the excised fragments should have the sense strand and intro sequence and the intermediate restriction site. The gel electrophoresis image obtained after enzyme digestion showed that a fragment of about 550 bp was obtained, which was in line with expectations ( FIG. 7 ). Therefore, recombinant plasmids with sense strands can be used for further experiments.

The positive recombinant plasmid after the ligation of the antisense strand and PHDT-RH connected with the sense strand was detected by double digestion with HindIII and Bgl II. Bgl II was located downstream of the antisense strand, and a fragment of about 900bp was obtained after digestion (Fig. 8) It shows that the excised fragment has sense strand, intro sequence and antisense strand in order. That is, the sense and antisense strands of Bx-cpl-1 or Bx-cpl-2 are connected to the PHDT-RH plasmid, so far the RNA interference vectors of the two genes are obtained, named PHD-RH-CPL1 and PHD-RH- CPL2.

Sequencing verification results showed:

As can be seen from Seq ID No.6,

Restriction site HindIII (AAGCTT), Xba I (TCTAGA) and intro primer (AGAGAGGCAAGAAGAAAGGCT) can be found in the positive sense strand sequencing results of Bx-cpl-1 gene. It shows that the sense strand of Bx-cpl-1 gene is successfully connected with PHDT-RH plasmid.

It can be seen from Seq ID No.7:

Restriction site Spe I (ACTAGT), BglII (AGATCT) and intro primer (AAATGGAAGGGACTGACAAG) can be found in the antisense strand sequencing results of Bx-cpl-1 gene. It shows that the antisense strand of Bx-cpl-1 gene is successfully connected with PHDT-RH plasmid.

Example 4. Establishment of Agrobacterium tumefaciens-mediated Botrytis cinerea genetic transformation system

In this experiment, Agrobacterium tumefaciens was used as the mediator to transform the T-DNA fragment (with hygromycin resistance) capable of expressing double-stranded dsRNA into Botrytis cinerea. By optimizing the transformation conditions, the Botrytis cinerea transformants were successfully constructed.

4.1 Cultivation of Agrobacterium tumefaciens

1. Streak-inoculate Agrobacterium tumefaciens on YEB medium and culture at a constant temperature of 28°C for 2-3 days;

2. Pick a single colony and inoculate it in the basic medium, shake and culture at 28°C and 200rpm for 24h;

3. Collect the bacteria in a centrifuge tube, add liquid induction medium, adjust the OD600 to about 0.20, 28 ° C, 200rpm shaking culture for 6 hours, stop when the OD600 rises to about 0.4.

4.2 Preparation of Competent Agrobacterium:

(1) Pick a single colony of Agrobacterium EHA105, inoculate it in 20ml liquid YEB medium (containing 50mg/L rifampicin), and cultivate it at 28°C and 200rpm for 48h;

(2) Transfer 500 μl shake-cultured EHA105 to 50 ml liquid YEB (containing 50 mg/L rifampicin) medium, culture at 28°C and 200 rpm until the OD600 value is about 0.6;

(3) Place the bacterial solution in an ice bath for 30 minutes, centrifuge at 5000 rpm for 10 minutes at 4°C, and discard the supernatant;

(4) Suspend the cells with 50 ml pre-cooled 10% glycerol, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, and collect the cells;

(5) Suspend the cells with 25ml pre-cooled 10% glycerol, centrifuge at 4°C, 5000rpm for 10min, discard the supernatant, and collect the cells;

(6) Suspend the cells with 10 ml of pre-cooled 10% glycerol, centrifuge at 5000 rpm for 10 min at 4°C, discard the supernatant, and collect the cells;

(7) Suspend the bacterial cells with 1-2ml of pre-cooled 10% glycerol, aliquot 50 μl/tube, freeze in liquid nitrogen and store at -80°C. 4.3 Transformation of Agrobacterium by electroporation:

1. Add 1 μl of PHD-RH-CPL1 and PHD-RH-CPL2 plasmids to 50 μl of Agrobacterium competent cells, mix well in a centrifuge tube, transfer to an electric shock cup, and ice-bath for 30 sec;

2. Put the electric shock cup in an appropriate position, press and hold the electric shock button to start electric shock;

3. Add 500 μl of YEB liquid medium without any antibiotics, shake at 28°C, 220 rpm for 3 hours, and activate the bacteria;

4. Mix the bacterial solution evenly with a sterilized pipette tip, draw 100 μl and evenly spread it on the YEB solid medium (Kana 50 mg/L and Rifampicin 50 mg/L), and incubate at 28°C for 2 days.

5. Extract the Agrobacterium plasmid for enzyme digestion identification or detection by bacterial liquid PCR.

4.4 Preparation of infection bacteria solution:

1. Streak inoculate the transformed Agrobacterium on YEB solid medium, culture at 28°C for 40 hours, until a single colony appears;

2. Pick a single colony and inoculate it in YEB liquid medium, 28°C, 220rpm, shaking culture, stop when OD600 reaches about 0.6;

3. Centrifuge at 5000rpm for 10min, discard the supernatant, and the precipitate is the collected bacteria;

4. Add 0.5 times the volume of MS liquid, and blow with the tip of the pipette to suspend the bacteria.

4.5 Obtaining Botrytis cinerea mycelium for Agrobacterium-mediated transformation

Pick the Botrytis cinerea on the PDA solid medium with an inoculation loop, and inoculate it in 200ml of PDA liquid medium. Culture at 25° C. with shaking at 220 rpm for 2 days. The hyphae were collected by filtration with sterile gauze. Add liquid nitrogen and mycelia to a sterilized mortar for grinding, and add appropriate amount of sterile water after fully grinding.

4.6 Co-cultivation and screening process

1. After the co-cultivation medium is sterilized by high pressure steam, MES, AS, glucose, and FeSO 4 must be cooled to about 50°C before adding MES, AS, glucose, and FeSO ₄ into the mix, otherwise these four components are easily decomposed or oxidized at high temperature . After the culture medium is solidified, spread a sterilized filter paper strip of the same size as the petri dish in the ultra-clean bench to drive away the air bubbles between the filter paper and the culture medium;

2. Mix the mycelial fragments of Botrytis cinerea and the induced Agrobacterium in a ratio of 4:1, and apply 400 μl evenly on the solid co-cultivation medium;

3. Protect from light, culture at 23°C for 3 days;

4. Peel off the filter paper strip on the medium, spread the reverse side on the PDA medium (cephalosporin 400 μg/mL, hygromycin 200 μg/mL), and culture at 18°C for 2 days;

5. Remove the filter paper strip from the culture medium, put the culture dish at 18°C for 1 to 3 days, and the colonies grown are the colonies resistant to cephalosporin;

6. Pick out the cephalosporin-resistant single colony and carry out further screening on PDA medium (containing cephalosporin). In order to ensure the effectiveness of cephalosporin, negative and positive controls are required for each plate.

4.7 Identification of Botrytis cinerea transformants

Extraction of Botrytis cinerea DNA:

1. Add liquid nitrogen and Botrytis cinerea mycelium into the mortar and grind thoroughly;

2. Take 10ml of the ground sample and add it to a 50ml centrifuge tube, then add 15ml Trizol to the tube, shake vigorously to mix, put in a water bath at 65°C for 40min, and shake from time to time;

3. Add 15ml of phenol: chloroform (1:1) solution, mix upside down; centrifuge at 10000rpm at room temperature for 10min;

4. Take the supernatant to a new 50ml centrifuge tube, add 15ml of phenol:chloroform (1:1) solution; centrifuge at 10000rpm for 10min at room temperature;

5. Take the supernatant to a new 50ml centrifuge tube, add 15ml chloroform; centrifuge at 10000rpm for 10min at room temperature;

6. Discard the supernatant, add 900μl 70% ethanol to rinse, and centrifuge at 12000rpm for 5min at room temperature;

7. Redissolve the DNA in 20μl TE and store at -20℃;

8.1% agarose gel electrophoresis was used to detect the content and quality of DNA.

4.8 Hygromycin primer PCR, target gene primer PCR:

The primers of the cysteine protease gene used in the construction of the vector were used for PCR verification, and those that amplified specific bands could be identified as positive Botrytis cinerea transformants.

Hygromycin primers are as follows: HPH-F: ACTGGTACCTAGACGTTAACT,\

PH-R CAGGTACCTAAACCCAGGGCT.

The target gene primers are as follows:

Primer name Nucleic acid sequence (5'-3') CPL-1uF CCAG AAGCTT TTGACTGGAGAGAAA CPL-1uR AGAGC TCTAGA CTCATCTCTGGCAA CPL-1dF AGTC ACTAGT CTCATCTCTGGCAAT CPL-1dR TCTTC AGATCT TTGACTGGAGAGAA

The PCR reaction system is as follows:

DNA 1μl 10×PCR Buffer 2μl Mg ²⁺ (25mM) 2μl dNTP (10mM) 0.4μl Upstream primer (10mM) 1μl Downstream primer (10mM) 1μl Taq enzyme (5U/μl) 0.4μl ddH ₂ O 12.2μl Final V 20μl

PCR reaction program: PCR reaction program: 94°C 4min 94°C 30sec 56°C 30sec 72°C 1min, 35cycles; 72°C 10min

The results showed that: the DNA of the Botrytis cinerea transformant was detected with hygromycin primers, and a fragment of about 2000 bp was obtained (Fig. 9). Since there is no hygromycin gene in the Botrytis cinerea genome, it can be determined that the hygromycin gene comes from integration into Botrytis cinerea. PHD-RH-CPL1 and PHD-RH-CPL2 plasmids on the genome. Both LB and RB of the plasmid PHDT-RH can be integrated into the host genome, so it is preliminarily shown that the sense strand, antisense strand and intro are also integrated into the Botrytis cinerea genome.

Further use the primers of the sense strand and the antisense strand of the Bx-cpl-1 gene and the Bx-cpl-2 gene (as shown in Table 1) to carry out PCR amplification to the DNA of the Botrytis cinerea transformant, and all obtain a fragment of about 350bp (see Figure 10). It indicated that the sense strand and antisense strand were successfully integrated into the Botrytis cinerea genome along with the region between LB and RB of PHDT-RH. So far, Botrytis cinerea transformants that interfere with Bx-cpl-1 gene and Bx-cpl-2 gene have been successfully constructed.

Example 5 RNA interference effect analysis of pine xylophilus CPL gene

Nematodes were inoculated on the positive transgenic Botrytis cinerea obtained in Example 4, and after feeding for a period of time, the RNA of the nematodes was extracted, reverse-transcribed, and the transcription levels of Bx-cpl-1 and Bx-cpl-2 genes were detected by fluorescent quantitative PCR. At the same time, collect a certain amount of nematodes, add substrate after homogenate centrifugation, and measure the activity of cysteine protease. Finally, the changes in phenotype were observed by counting the length of nematodes and the number of reproductions of nematodes. details as follows:

5.1 Real-time quantitative PCR detection of gene expression level Transgenic Botrytis cinerea inoculated nematodes

Select 3 transformants from the positive transformants of each gene (Bx-cpl-1 gene and Bx-cpl-2 gene), inoculate 4 dishes for each transformant, and culture at a constant temperature of 25°C for 3 to 4 days. 1 dish of preserved species was produced. The remaining 3 dishes were inoculated with 200 pine wood nematodes per dish, and cultured at a constant temperature of 25°C for 7-8 days. Gather them as spares. Design qPCR primers

5.2 After analysis by DNAMAN software, 4 pairs of qPCR primers were designed and synthesized for each gene.

The designed primers were subjected to ordinary PCR, and 1% agarose gel electrophoresis was used to detect whether the primers were available. A pair of better primers, that is, primers with less dimer formation, were screened out for each gene. At the same time, a pair of internal reference primers were screened out, the internal reference gene used was actin, and the size of the fragment amplified by the internal reference gene primers was 151bp.

The primers qPCR primers used for the screening of the present invention are shown in Table 2

Primer name Nucleic acid sequence (5'-3') CPL1-478F GACTGGAGAGAAAAGGGCGTT CPL1-573R CCGTGAGCGATTGCGTAC CPL2-177F CCGAGAAAAGCCCAGCAGGAT CPL2-293R ATTGCCTCCAGAAATGCGAC actin-735F ACCGCTGCCTCC TCTTCTTC actin-867R TAGGTGGTCTCGTGGATACC

5.3 Extract nematode RNA, cDNA first-strand synthesis

Botrytis cinerea interfering with Bx-cpl-1 gene, Botrytis cinerea and common gray mold interfering with Bx-cpl-2 gene were all inoculated on PDA medium (containing hygromycin 200 μg/ml), each inoculated in three dishes.

After the nematodes were bred on the transgenic Botrytis cinerea for 2 generations, the nematodes were collected by the method described in Example 2, and after centrifugation, 50 μl was taken to extract RNA. The nematode RNA extraction method was the same as in Example 2. The rest were reserved for subsequent inoculation, and RNA was extracted after 30 generations of continuous interference by the transgenic Botrytis cinerea to compare whether the interference effect changed.

Reverse transcription after extracting RNA, refer to Example 2 for the method of synthesizing the first strand of cDNA.

5.4 Fluorescence quantitative PCR

Dilute the cDNA 10 times, 30 times and 50 times, and do a preliminary experiment to determine the optimal dilution factor. The Ct value has a linear relationship with the logarithm of the cDNA concentration, so that the Ct value is optimal between 18 and 22. If the value is too large or Too small will reduce the credibility of the results.

The fluorescent quantitative PCR system is as follows:

cDNA 1μl Mix (add dye) 10.4μl FP(10mM) 0.4μl RP (10mM) 0.4μl DPEC H ₂ O 7.8μl Final V 20μl

CPL1-478F/CPL1-573R in Table 2 was used to detect the pine wood nematode interfered by the Bx-cpl-1 gene transgenic cinerea, and CPL2-177F/CPL2-293R was used to detect the Bx-cpl-2 gene-transferred cinerea interference pine xylophilus. Four parallel tubes were made for each sample.

The results show that::

After pine xylophilus was bred on the transformed Botrytis cinerea for two generations, the transcription level of the disturbed gene was detected by qPCR, and SPSS statistical analysis was performed. Figure 11 and Figure 13 show that the transcription of the Bx-cpl-1 gene was reduced by 50.1% (F=0.314, t=12.454, df=9.957, P<0.001), and Bx-cpl The transcription of the -2 gene was reduced by 23.9% (F=0.049, t=5.025, df=10, P=0.001). This shows that the dsRNA synthesized in Botrytis cinerea affects the gene transcription of B. xylophilus, and the interference effect is more significant, and the interference effect of Bx-cpl-1 is more obvious than that of Bx-cpl-2.

After 30 generations of continuous interference, the transcription level of the gene after interference was also detected by qPCR, and SPSS statistical analysis was performed. Figure 12 and Figure 14 show that the transcription level of the Bx-cpl-1 gene decreased by 56.4% (F=0.072, t=20.777, df=10, P<0.001) after 30 generations of interference, and the transcription of the Bx-cpl-2 gene Levels were reduced by 31.8% (F=8.979, t=5.153, df=5.751, P=0.002<0.001).

For the Bx-cpl-1 gene, there was no significant difference in the transcription level of the control group between the interference 2 generation and the interference 30 generation (F=5.240, t=1.582, df=7.655, P=0.154>0.05), while the transcription level of the experimental group had Significant differences (F=0.699, t=2.503, df=10, P=0.031<0.05) indicated that the transcription level of Bx-cpl-1 gene decreased gradually with the increase of interference time, and there was a certain cumulative effect. Example 6. Cysteine protease activity assay

Collect disturbed and undisturbed B. xylophilus, prepare homogenate, and use fluorescent substrate GLUpNA to measure the activity of cysteine protease. The substrate can produce p-nitroaniline after hydrolysis catalyzed by cysteine protease. The meter can detect the absorbance of p-nitroaniline at 405nm.

The results showed that: the chromogenic substrate GLUpNA was used to measure the cysteine protease activity of B. xylophilus, and the change of absorbance was calculated (the OD405 measured every 15 minutes was subtracted from the previous measurement value, and the average value was obtained after 6 values were obtained).

The results after 10 generations of continuous interference showed (Table 3): after interfering with the Bx-cpl-1 gene, compared with the control group, the absorbance change at 405nm was reduced from 0.081 to 0.064, and the cysteine protease activity was reduced by 20.9%; After Bx-cpl-2 gene, compared with the control group, the change in absorbance at 405 nm was reduced from 0.081 to 0.074, and the cysteine protease activity was reduced by 8.6% ( FIG. 15 ). The results after 35 generations of continuous interference showed (Table 4): after interfering with the Bx-cpl-1 gene, the change in absorbance at 405 nm was reduced from 0.084 to 0.064, and the cysteine protease activity was reduced by 23.8%; interfering with the Bx-cpl-2 gene Afterwards, compared with the control group, the change in absorbance at 405 nm decreased from 0.084 to 0.067, and the cysteine protease activity decreased by 20.2% ( FIG. 16 ).

Table 3 After 10 generations of interference, the enzyme activity determination with GLUpNA as substrate

Table 4 interferes with GLUpNA after 35 generations and uses GLUpNA as the enzyme activity assay of substrate

Example 7. Effect of RNA interference on nematode reproduction

7.1 Preparation of Botrytis cinerea blocks

1. Botrytis cinerea interfering with the Bx-cpl-1 gene, Botrytis cinerea interfering with the Bx-cpl-2 gene, and common gray mold were all inoculated on PDA medium (hygromycin 200 μg/ml), and each was inoculated in three dishes;

2.25 ℃ constant temperature culture for 3 to 4 days;

3. Use a sterilized toothpick to cut off a 1cm×1cm square for each plate of Botrytis cinerea, put it into a 24-well plate, and mark the edge of the plate.

7.2 Inoculation of pine xylophilus

1. Five juvenile females and five males were inoculated on each piece of Botrytis cinerea, and all nematodes were disturbed for 10 generations by feeding method before inoculation;

2. Wrap the edge of the 24-well plate with parafilm;

3. Culture at a constant temperature of 25°C for 5 days, that is, the time for nematodes to reproduce for one generation.

7.3 Counting the number of offspring

1. Add 1ml of sterile water to each well of the 24-well plate, soak the gray mold block for 60 minutes, and fully transfer the nematodes from the culture medium to the water;

2. Discard the medium, take 50 μl of nematodes and place them on a concave glass slide for counting, take a total of 20 times, and sum the data of 20 times to get the number of nematodes in each well;

3. Replace the tip and count each hole separately;

4. Do 3 parallels and 6 repetitions;

5. Use SPSS software to analyze the difference between the experimental group and the control group, and choose the t test for statistical analysis of the data of the two groups.

The results showed that after 10 generations of continuous interference, the number of offspring reproduced by adults of B. xylophilus was counted.

It can be seen from Figure 17 that compared with the control, interfering with the Bx-cpl-1 gene of adults can reduce the number of offspring from an average of 156 to an average of 91 in 5 days, a reduction of 41.7% (F=1.290, t=10.112, df=34, P<0.001).

Compared with the control, interfering with the Bx-cpl-2 gene of adults can reduce the number of offspring produced within 5 days from 156 to 98, a decrease of 37.2% (F=0.015, t=8.325, df=34, P< 0.001).

It shows that the interference of Bx-cpl-1 gene or Bx-cpl-2 gene can significantly reduce the reproduction of B. xylophilus adults. Therefore, the reproductive ability of B. xylophilus adults can be greatly weakened by RNAi, and the Bx-cpl-1 gene and Bx-cpl-2 gene are related to the embryonic development of B. xylophilus.

Example 8 Effect of RNA interference on nematode body length

8.1 Cultivation of pine xylophilus

1. Botrytis cinerea interfering with the Bx-cpl-1 gene, Botrytis cinerea interfering with the Bx-cpl-2 gene, and common Botrytis cinerea were all inoculated

On the PDA medium (hygromycin 200 μg/ml), inoculate three dishes each,

2. After Botrytis cinerea grows well, 200 pine wood nematodes are inoculated per dish;

3. Take a sterilized 1.5ml centrifuge tube, collect B. xylophilus by the method described in 2.2.1.2, collect one tube per dish, and centrifuge about 50 μl of worms in each tube;

4. Add sterile water to suspend the worms, and the final volume is 2ml;

5.50°C water bath for 5 minutes to kill pine xylophilus.

8.2 Measuring the body length of nematodes

1. Mix pine wood nematodes evenly with a 100 μg pipette tip, randomly take 10 μl of nematodes from each tube, place them on a glass slide, and cover with a cover glass;

2. Take pictures under the microscope and measure the length with a ruler. Since the dead state of nematodes after heat shock does not form a straight line, it needs to be measured according to the following measurement rules: 2nd instar larvae are generally divided into 3 segments, and summed after each measurement, 3rd instar larvae Larvae, 4th instar larvae and females are generally divided into 2 segments, which are summed after measurement respectively, and males are generally divided into 4-5 segments, which are summed after measurement respectively;

3. Measure 60 pieces of 2nd instar larvae, 3rd instar larvae, 4th instar larvae, females and males after 5 generations of interference and undisturbed, and record the data;

8.3 Analyzing data

1. Use SPSS software to analyze whether there is a significant difference between the control group and the experimental group. Since it is necessary to test whether two groups of irrelevant samples come from the population with the same mean value, an independent sample t test is performed on the two groups of data. The test steps are:

Analyze->Compare Means->Independent Samples T test;

2. Observe the results of the independent sample t test, and record the values of F, t, df, P, etc.;

3. Calculate the average value and standard error, and use excel to make a graph. When making a histogram, mark the bar value on the graph, and the bar value is the standard error; when making a scatter graph, give the average value and standard error in the form of a list ;

4. Calculate the percentage change in the average value of the control group and the experimental group.

The results show (table 5, Fig. 18): after interfering with the Bx-cpl-1 gene, the average body length of the 2nd instar larvae of B. xylophilus was shortened from 242.1 μm to 216.9 μm, which was shortened by 10.4% (F=11.234, t=5.576 , df=98.814, P<0.001), the body length of the 3rd instar larvae did not change significantly (F=0.244, t=-1.330, df=118, P=0.186), the average body length of the 4th instar larvae shortened from 663.5μm to 605.5 μm, shortened by 9.7% (F=0.565, t=5.379, df=118, P<0.001), and the average body length of males was shortened from 923.5 μm to 848.1 μm, shortened by 8.2% (F=3.888, t=6.059, df=118, P<0.001), the average body length of females was shortened from 1008.2 μm to 953.3 μm, shortened by 5.4% (F=21.408, t=3.358, df=88.436, P=0.001).

Overall, there was a significant difference in body length between disturbed and undisturbed nematodes, and Bx-cpl-1 and Bx-cpl-2 genes may be involved in the development of nematodes by affecting molting.

Body length change of pine xylophilus after table 5 disturbance

Claims (8)

1. disturb the dsRNA interference carrier of pine wood nematode Bx-cpl-1 genetic expression, it is characterized in that, on the multiple clone site of skeleton carrier in order forward be inserted with the complete of Bx-cpl-1 gene or part positive-sense strand and with described positive-sense strand complementary antisense strand.

2. dsRNA interference carrier according to claim 1, described skeleton carrier are agriculture bacillus mediated expression vector.

3. dsRNA interference carrier according to claim 2, the nucleotide sequence of described positive-sense strand is shown in Seq ID No.2, the nucleotide sequence of described positive-sense strand is as shown shown in the Seq ID No.3, described agriculture bacillus mediated carrier is PHDT-RH, described positive-sense strand is inserted in the LB of PHDT-RH and 5 ' end multiple clone site between the RB, described antisense strand is inserted in the LB of PHDT-RH and 3 ' end multiple clone site between the RB, and the dsRNA interference carrier that obtains is PHD-RH-CPL1.

4. change the bacterial strain of requirement 1～3 arbitrary described dsRNA interference carrier of having the right over to.

5. bacterial strain according to claim 4 refers to change over to the Botrytis cinerea bacterium (Botrytis cinerea) of PHD-RH-CPL1.

6. the application of the arbitrary described dsRNA interference carrier of claim 1～3 in the pine wood nematode control.

7. novel method of studying the Bx-cpl-1 gene function, it is characterized in that, the arbitrary described dsRNA interference carrier of claim 1～3 is transformed in the filamentous fungus,, observes the phenotype of pine wood nematode then by the mycelium of the filamentous fungus transformant pine wood nematode of feeding.

8. novel method according to claim 7, described filamentous fungus refers to the Botrytis cinerea bacterium.

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