CN118755728B - OfJHAMT gene of Asian corn borer and its application - Google Patents
OfJHAMT gene of Asian corn borer and its application Download PDFInfo
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Abstract
本发明公开了一种亚洲玉米螟OfJHAMT基因及其应用。亚洲玉米螟OfJHAMT基因的RNA干扰序列片段合成的dsRNA在亚洲玉米螟防治中的应用，采用了一种亚洲玉米螟OfJHAMT基因，核苷酸序列如SEQ ID NO:1所示；亚洲玉米螟OfJHAMT基因的RNA干扰序列片段，核苷酸序列如SEQ ID NO:2所示；将亚洲玉米螟OfJHAMT基因的RNA干扰序列片段合成dsRNA，通过dsRNA特异性地沉默OfJHAMT基因mRNA表达，干扰亚洲玉米螟5龄幼虫滞育，使亚洲玉米螟5龄幼虫解除滞育，化蛹致残，难以蜕变为成虫，直至死亡。本发明可有效减少化学杀虫剂的使用，解决环境污染和害虫抗药性问题。
The invention discloses an OfJHAMT gene of an Asian corn borer and its application. The application of dsRNA synthesized from RNA interference sequence fragments of the OfJHAMT gene of an Asian corn borer in the prevention and treatment of an Asian corn borer comprises the following steps: using an OfJHAMT gene of an Asian corn borer, the nucleotide sequence of which is shown in SEQ ID NO:1; an RNA interference sequence fragment of the OfJHAMT gene of an Asian corn borer, the nucleotide sequence of which is shown in SEQ ID NO:2; synthesizing the RNA interference sequence fragment of the OfJHAMT gene of an Asian corn borer into dsRNA, specifically silencing the mRNA expression of the OfJHAMT gene through dsRNA, interfering with the diapause of the fifth instar larvae of the Asian corn borer, causing the fifth instar larvae of the Asian corn borer to terminate the diapause, pupate and become disabled, and have difficulty in metamorphosing into adults, until death. The invention can effectively reduce the use of chemical pesticides and solve the problems of environmental pollution and pest resistance.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an Asiatic corn borer OfJHAMT gene and application thereof.
Background
Asian corn borer (Ostrinia fucalis) is one of main pests of corn in inner Mongolia, recently is increased as a national crop pest, and has wide distribution, serious hazard and strong environmental adaptability. The larvae are mainly used for damaging all parts on the ground of the corn plant, so that the corn yield is reduced and the quality is reduced. Diapause is a state of growth, development and reproduction arrest caused by insects resisting adverse environmental conditions, and is a seasonal adaptive strategy regulated by genes to cope with environmental changes. Insects often utilize diapause to circumvent extreme ecological environments, thereby preserving individual survival. Asian corn borers belong to facultative diapause insects and enter diapause with 5-instar larvae. Because chemical control is still mainly performed on Asiatic corn borers at present, the traditional chemical control method has the problems of environmental pollution and pest resistance, and therefore, a new biological control strategy is needed.
RNA interference (RNAi) refers to the phenomenon that when dsRNA homologous to an endogenous mRNA coding region is introduced, the mRNA is degraded to silence the expression of a target gene, and the target gene is silenced to weaken or delete the normal biological function of the target gene to achieve the aim of the biological function of a specific gene. The premise of pest control based on RNAi is that the target sequence is screened to obtain dsRNA with high lethal effect on insects.
Juvenile Hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acid or inactive precursors of JH to active JH in the last step of insect JH biosynthesis and is therefore an excellent target for the development of insect growth regulators. In addition, as the human, land animals and most aquatic animals do not contain downstream paths for synthesizing juvenile hormone, the novel pesticide molecule designed by taking JHAMT as a target is harmless to human and livestock and is more environment-friendly. Through the expression Of the gene OfJHAMT Of dsRNA (Of which Of is abbreviated as Asian corn borer), the diapause Of the Asian corn borer is disturbed, so that the 5-instar larva Of the Asian corn borer is released from diapause, pupated and disabled and is difficult to be molted into adults until death, thereby achieving the aim Of biological control, providing an environment-friendly borer control technology and being a research subject which is urgently needed at present.
Therefore, in the prior art, there is a need for the application of dsRNA synthesized by Asiatic corn borer OfJHAMT gene, RNA interference sequence fragment of Asiatic corn borer OfJHAMT gene and RNA interference sequence fragment of Asiatic corn borer OfJHAMT gene in preventing and controlling Asiatic corn borer.
Disclosure of Invention
The invention aims to provide an application of dsRNA synthesized by an Asiatic corn borer OfJHAMT gene, an Asiatic corn borer OfJHAMT gene RNA interference sequence fragment and an Asiatic corn borer OfJHAMT gene RNA interference sequence fragment in Asiatic corn borer prevention and treatment.
The invention aims at realizing the following technical scheme:
the nucleotide sequence of the Asiatic corn borer OfJHAMT gene is shown as SEQ ID NO. 1. After designing the corresponding degenerate primer, the conserved region is obtained by PCR amplification, and the product has a fragment length of 961 bp, designated OfJHAMT, an open reading frame of 948 bp and codes 315 amino acids by agarose gel electrophoresis detection.
The nucleotide sequence of the RNA interference sequence fragment of the Asiatic corn borer OfJHAMT gene is shown as SEQ ID NO. 2. Designing upstream and downstream primers according to SEQ ID NO. 1, and obtaining the primer through PCR amplification.
And further synthesizing the dsRNA according to the corresponding interference sequence fragments by using a related kit. The invention provides dsRNA synthesized by RNA interference sequence fragments of Asiatic corn borer OfJHAMT genes.
The dsRNA is applied to the prevention and treatment of Asiatic corn borer, and is injected into the abdomen of 5-year-old Asiatic corn borer diapause larva, and the dsRNA can specifically silence OfJHAMT gene mRNA expression, interfere the diapause of the 5-year-old Asiatic corn borer larva, so that the 5-year-old Asiatic corn borer larva is unhaused, pupated and disabled, and is difficult to be molted into an adult until death.
The beneficial effects of the invention are as follows: the invention obtains Asian corn borer OfJHAMT gene, asian corn borer OfJHAMT gene RNA interference sequence fragment and dsRNA synthesized by Asian corn borer OfJHAMT gene RNA interference sequence fragment, provides a biological control method for controlling the Asian corn borer by using the dsRNA, specifically silences OfJHAMT gene mRNA expression through the dsRNA, and interferes Asian corn borer 5 larva diapause through RNA interference, so that the Asian corn borer 5 larva is diapaused, pupated residues are difficult to be decomposed into adults until death. The application of the invention can effectively reduce the use of chemical pesticides and solve the problems of environmental pollution and pest drug resistance.
Drawings
FIG. 1 shows agarose gel detection of the open reading frame sequence length of the transcriptome of Asian corn borer OfJHAMT (M is DL2000 DNA MARKER, the bands are 100, 250, 500, 750, 1000, 2000 bp in sequence from small to large, and 1 is the band size of the Asian corn borer JHAMT gene);
FIG. 2 shows the gene expression level of Asian corn borer after dsOfJHAMT (i.e., dsRNA) injections (dsOfJHAMT is the experimental group injected with dsRNA, dsGFP is the control group);
FIG. 3 is a phenotype of diapause 5-instar larvae remaining in diapause 5-instar after injection dsGFP;
FIG. 4 is a phenotype of diapause-relieving abnormal pupation of diapause larvae after dsOfJHAMT injections;
FIG. 5 is a comparison of larval phenotypes injected dsOfJHAMT and dsGFP (dsOfJHAMT is the experimental group injected with dsRNA, dsGFP is the control group);
FIG. 6 is a statistical analysis of diapause rate of Asian corn borer after injection dsOfJHAMT and dsGFP (dsOfJHAMT is experimental group injected with dsRNA, dsGFP is control group);
Fig. 7 is a statistical analysis of asian corn borer survival after injections dsOfJHAMT and dsGF (dsOfJHAMT is the experimental group injected with dsRNA, dsGFP is the control group).
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
Example 1: asian corn borer OfJHAMT gene open reading frame sequence acquisition and amino acid sequence analysis
1. CDNA fragment of Asiatic corn borer OfJHAMT gene
Based on the existing Asian corn borer transcriptome database, searching the Unigene, and determining to obtain the Asian corn borer OfJHAMT gene fragment after NCBI Blastx analysis.
2. Obtaining the open reading frame sequence of Asiatic corn borer OfJHAMT gene
Splicing the gene fragments by GeneDoc software, and designing an upstream primer and a downstream primer by PRIMER PREMIER 5.0.0 software:
OfJHAMT: an upstream primer: ofJHAMT A
5’-ACATGAACAACGCCGAACTCTACCA-3’
A downstream primer: ofJHAMT A
5’-AGTCACATTTGCTCCCGTACACCAC-3’
The primers used in the present invention were synthesized by the division of biological engineering (Shanghai).
Larvae of 5 years old which are healthy to grow and have the same size are selected and frozen in liquid nitrogen. Each Asian corn borer 5-instar larva was treated as described above and RNA was extracted according to the Freezol Reagent kit of Vazyme. The extracted RNA is reversely transcribed into first strand cDNA by HiScript ® III RT SuperMix for qPCR (+ GDNA WIPER) (Vazyme), the first strand cDNA is used as a template, the upstream primer and the downstream primer are combined, PCR amplification and detection are carried out, an open reading frame fragment of Asian corn borer JHAMT gene is obtained, PCR product recovery and purification are carried out by Eastep ® GEL AND PCR Clenup Kit (Promega), the PCR product is cloned into pMD19-T vector (TaKaRa), the mixture is transferred into E.coli competent cells E.coli DH5 alpha (Tiangen), plasmid is extracted by GeneJET PLASMID MINIPREP KIT Kit (Thermo Scientific) after bacterial liquid is cultured, and after detection, bacterial liquid is sent to Shenzhihua large gene technology Co for sequencing, and nucleotide sequence of SEQ ID NO: 1 is obtained after sequencing.
As shown in FIG. 1, agarose gel detection of Asian corn borer OfJHAMT transcriptome cDNA sequence length was 961 bp, open reading frame was 948 bp, encoding 315 amino acids.
3. Amino acid sequence analysis of Asian corn borer OfJHAMT gene
The obtained Asian corn borer OfJHAMT gene nucleotide is translated by ExPaSy on-line software, the open reading frame of the Asian corn borer OfJHAMT is predicted to encode 315 amino acids, the molecular weight is 36.39KD, and the isoelectric point is 7.86.
Example 2: dsRNA synthesis of Asiatic corn borer OfJHAMT
1. Primer design of Asian corn borer OfJHAMT gene dsRNA
Based on Asian corn borer OfJHAMT gene sequence, PRIMER PREMIER 5.0.0 software was used to design dsRNA primer (T7 promoter: TAATACGACTCACTATAGGG) with interference sequence as SEQ ID NO. 2:
OfJHAMT: an upstream primer: ofJHAMT-RNAiF
5’-TAATACGACTCACTATAGGGATCACAAGGACGAGCGGACT-3’
A downstream primer: ofJHAMT-RNAiR
5’-TAATACGACTCACTATAGGGGGGGTTCACTGCCTGAACTG -3’
All primers of the present invention were synthesized by the division of biological engineering (Shanghai).
2. Asian corn borer OfJHAMT-specific dsRNA synthesis
PCR amplification was performed using the Asian corn borer JHAMT gene extraction plasmid as a template and a primer containing the upstream and downstream of the T7 promoter sequence. A gene fragment of length 427 bp (SEQ ID NO: 2) was obtained. The recovery and purification of PCR products using GeneJET PLASMID MINIPREP KIT (Thermo Scientific) kit followed by in vitro transcription of dsRNA was performed according to the T7 RiboMAXTM Express RNAI SYSTEM (Promega) kit. Detecting the concentration of the dsRNA, ensuring that the concentration of the dsRNA reaches at least 300 ng/mu L, and storing in a super low-temperature refrigerator at-80 ℃ for standby.
Example 3: dsRNA interference experiment of Asiatic corn borer OfJHAMT gene
1. Specific dsRNA injection
30 Healthy diapause Asiatic corn borers with 5 ages and consistent sizes are selected for experiments. 1 μl (1000 ng/μl) of the synthesized dsRNA (i.e., dsOfJHAMT) was gently injected into the abdomen of asian corn borer 5-year diapause larvae using a 0.26 mm gauge microinjector (SHIMADZU, japan). Meanwhile, 30 Asian corn borer diapause larvae with the age of 5 are selected as a control group, and dsGFP with the same volume and concentration are injected into the control group. The injected Asian corn borers are placed in a well-ventilated incubator (the conditions of relative humidity of 22+/-1 ℃ is 80+/-5% and the light-dark period is 8 hours: 16 hours), artificial feed is fed every day, and 5-year-old Asian corn borer larvae are rapidly frozen in liquid nitrogen after being selected and treated for 48 hours.
2. Silencing detection of Asiatic corn borer OfJHAMT gene
Collecting 5-year-old Asian corn borer larvae injected for dsOfJHAMT hours, simultaneously selecting dsGFP-year-old Asian corn borer larvae injected with the same head number as a control group, extracting total RNA, reversely transcribing into cDNA, and diluting 10 times as a qPCR template. The relative expression levels of the target gene (OfJHAMT) and the reference gene (beta-actin) are detected by using a Real-time PCR method, so that the expression levels are calculated. Each group was set with 3 biological replicates, each taking 3 first 5-year old asian corn borer larvae.
Referring to fig. 2, the result shows that the expression level of OfJHAMT gene after RNAi is very significantly different from that of the control group (P < 0.01), compared with the control group, the expression level of OfJHAMT gene of the experimental group is inhibited after dsOfJHAMT h injection, and the expression level is only 13.65%. It is shown that injection dsOfJHAMT can cause strong RNAi effect in Asiatic corn borer, resulting in obvious reduction of OfJHAMT gene expression in vivo, and further resulting in diapause release and development of Asiatic corn borer into residual pupa until death.
3. Phenotype observations of 5-year-old Asian corn borers after dsRNA injection
After dsGFP injections, the 5-year old Asian corn borer larvae of the control group were still diapause, see FIG. 3. Diapause is relieved by injecting dsOfJHAMT of 5-year-old diapause Asiatic corn borers of the experimental group, and the diapause is changed into malformed pupae which are difficult to be molted into adults and die, as shown in figure 4; a is Asian corn borer 5-instar larvae of which diapause cannot be relieved in the control group, b is Asian corn borer 5-instar larvae of which diapause cannot be relieved in the experimental group, and the phenotype characteristics of the control group and the experimental group are expressed as that the surfaces of the larvae of the experimental group are changed from normal white to black, and the figure 5 is shown. Referring to fig. 6, diapause rate of asian corn borer treated group was significantly lower than that of dsGFP control group after dsOfJHAMT days of injection; referring to fig. 7, death began on day 3 of injection dsOfJHAMT and the survival rate was 56.67% on day 9, which was evident in the lethal effect compared to the control group injected with dsGFP (survival rate 100%). The pest control research mainly adopts an injection method and a feeding method, and the two methods have the same action principle and effect.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
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