Application of eno gene in improving resistance to brown planthopper and rice with resistance to brown planthopper
By introducing the ENO gene into rice and leveraging its importance in the glycolysis process, the problem of pest resistance caused by chemical control was solved, achieving highly efficient control of brown planthoppers without pesticide residues, improving rice resistance and reducing environmental impact.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHOUKOU NORMAL UNIV
- Filing Date
- 2026-01-27
- Publication Date
- 2026-06-09
AI Technical Summary
In existing technologies, chemical control of brown planthoppers leads to increased pest resistance, high control costs, and negative environmental impacts. There is an urgent need to develop pesticide-free and safe pest control technologies.
By introducing the ENO gene into rice and leveraging its importance in glycolysis, the resistance of rice to brown planthopper was improved. The specific steps included extracting total RNA, reverse transcribing to obtain cDNA, amplifying the coding region of the ENO gene, ligating it with a vector, and transforming it into rice recipient materials for overexpression.
It significantly improved rice's resistance to brown planthoppers, reduced the pest's dependence on pesticides, lowered control costs, and had no negative impact on the environment.
Smart Images
Figure CN122168663A_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of biotechnology, and in particular to the application of an ENO gene in improving resistance to brown planthoppers and rice with resistance to brown planthoppers. Background Technology
[0002] Rice, as a major global food crop, plays a crucial role in food security. Among the many challenges facing rice production, the brown planthopper is particularly prominent. As a major rice pest in my country and many other Asian countries, the brown planthopper causes widespread damage to rice fields in my country every year, resulting in significant rice losses. This piercing-sucking insect feeds on the phloem sap of rice plants using its stylet, causing nutrient loss and hindering the transport of water and nutrients with its saliva. Furthermore, the brown planthopper is a vector for toothed dwarf disease and grassy dwarf disease, indirectly harming rice. In severe cases, brown planthoppers can cause large-scale rice mortality, leading to significant yield reductions or even total crop failure. Currently, chemical control remains the primary method for controlling brown planthoppers, but long-term use has led to resistance to various pesticides, increasing control costs and making the situation increasingly challenging. Simultaneously, pesticide residues have a significant negative impact on food safety and the ecological environment. Therefore, there is an urgent need to develop pesticide-free, safe, and environmentally friendly pest control technologies.
[0003] To address the problems of existing technologies, this disclosure provides an application of the ENO gene in enhancing brown planthopper resistance and rice exhibiting brown planthopper resistance. The technical solution is as follows: On the one hand, this disclosure provides an application of the ENO gene in improving the resistance of brown planthoppers, the application including: using the ENO gene to improve the resistance of brown planthoppers.
[0004] Specifically, the applications include: Total RNA was extracted from rice materials; The total RNA was reverse transcribed to obtain cDNA; Using the cDNA as a template, amplification was performed using forward and reverse primers to obtain the amplification product, which is the coding region of the ENO gene. The sequence of the amplification product is shown in SEQ ID NO: 1 in the sequence listing, the sequence of the forward primer is shown in SEQ ID NO: 2 in the sequence listing, and the reverse primer is shown in SEQ ID NO: 3 in the sequence listing.
[0005] Specifically, the application further includes: adding an A tail to the blunt end of the amplification product and ligating it with an enzyme-digested pCXUN vector to obtain a ligation product; and converting the ligation product into a receptor material through infection.
[0006] Furthermore, the rice material is Nipponbare.
[0007] Specifically, each 50 μL amplification system includes: 5 μL of 10× buffer; 5 μL of 2 mM dNTP; 1.5 μL of the forward primer at a concentration of 10 μM; 1.5 μL of the reverse primer at a concentration of 10 μM; 1 μL of cDNA; 1 μL of KOD-Plus-Neo polymerase at a concentration of 1 U / μL; and 35 μL of ddH2O.
[0008] Specifically, the amplification program includes: pre-denaturation at 98°C for 3 min; followed by 30 cycles, each cycle consisting of: denaturation at 98°C for 15 sec, annealing at 55°C for 20 sec, and extension at 68°C for 50 sec.
[0009] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides an application of the ENO gene in improving the resistance of brown planthoppers. Enolase (ENO) is ubiquitous and highly conserved in prokaryotes and eukaryotes. In organisms, ENO catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate, participating in glycolysis. Glycolysis, as a core primary metabolic pathway common to almost all organisms, not only provides ATP and various biosynthetic precursors to organisms, but its functional integrity is also crucial for growth and development. This disclosure utilizes the importance of ENO in plants, applying the ENO gene to improve the resistance of brown planthoppers, and the recipient plants can effectively improve the resistance of rice to brown planthoppers, with significant results. Attached Figure Description
[0010] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0011] Figure 1 This is a comparison chart of the relative expression levels of real-time fluorescence quantitative PCR amplification provided in Embodiment 1 of this disclosure. In the chart, Nip is the control group, and OsENO-OE-6 and OsENO-OE-9 are the experimental groups, respectively. Figure 2 This is a comparison diagram of brown planthopper resistance in the control group Nip, the experimental group OsENO-OE-6, and the experimental group OsENO-OE-9 provided in Embodiment 1 of this disclosure; Figure 3This is a comparative graph of the statistical results of the resistance readings of brown planthoppers in the control group Nip, the experimental group OsENO-OE-6 and the experimental group OsENO-OE-9 provided in Embodiment 1 of this disclosure. The data in the graph represent the mean (at least 10 seedlings) ± standard error. The asterisks on the error line indicate that there is a significant difference compared with the control group (p value calculated by one-way ANOVA, **p<0.01). Figure 4 This embodiment of the present disclosure provides a comparative chart of the honeydew secretion results of brown planthoppers in the control group Nip, experimental group OsENO-OE-6, and experimental group OsENO-OE-9 48 hours after feeding. The data in the chart represent the mean (honeydew secretion of 30 brown planthoppers) ± standard error. The asterisks on the error lines indicate significant differences compared with the control group (p-values were calculated by one-way ANOVA, *p<0.05, **p<0.01). Figure 5 This is a comparison chart of the weight gain of brown planthoppers 48 hours after feeding, provided in Embodiment 1 of this disclosure, for the control group Nip, the experimental group OsENO-OE-6, and the experimental group OsENO-OE-9. The data in the chart represent the average (weight gain of 30 brown planthoppers) ± standard error. The asterisks on the error lines indicate significant differences compared to the control group (p-values were calculated using one-way ANOVA, *p<0.05, **p<0.01). Detailed Implementation
[0012] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings. Example
[0013] This embodiment provides an application of the ENO gene in improving the resistance of brown planthoppers, the application including: using the ENO gene to improve the resistance of brown planthoppers.
[0014] Specifically, the application includes: Total RNA was extracted from rice materials; Total RNA was reverse transcribed to obtain cDNA; Using cDNA as a template, amplification was performed using forward and reverse primers to obtain the amplification product, which is the coding region of the ENO gene. The sequence of the amplification product is shown in SEQ ID NO: 1 in the sequence listing. The sequence of the forward primer is shown in SEQ ID NO: 2 in the sequence listing, specifically: ATGTCTCGCATCCATCGAAACCC. The sequence of the reverse primer is shown in SEQ ID NO: 3 in the sequence listing, specifically: GTAGGGCTCCACGGGTGCGC.
[0015] Specifically, total RNA was extracted from the rice material Nipponbare: In this embodiment, a whole rice plant of Nipponbare that has grown for 20 days was used. In other embodiments, other rice varieties can also be used, and the process can be the same as in this embodiment. Total RNA was extracted from the rice plant using the RNAsimple Total RNA Extraction Kit (TIANGEN Code: DP419), and the specific process is as follows: 1. Grind fresh rice material thoroughly in a mortar with liquid nitrogen, and add less than 150 μL of lysis buffer RZ to 50-100 mg of tissue to obtain a homogenized sample; 2. Place the homogenized sample at 15~30℃ for 5 minutes.
[0016] 3. Centrifuge at 4°C and 12,000 rpm for 5 minutes to obtain the supernatant. Transfer the supernatant to a new RNase-free centrifuge tube.
[0017] 4. Add 200 μL of chloroform to the supernatant, cap the tube, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.
[0018] 5. Centrifuge at 4°C and 12,000 rpm for 10 minutes to obtain the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-free centrifuge tube.
[0019] 6. Slowly add 0.5 times the volume of anhydrous ethanol to the upper aqueous phase, mix well, and immediately transfer the entire mixture into the CR3 adsorption column. Centrifuge at 12,000 rpm for 30 seconds at 4°C and discard the waste liquid.
[0020] 7. Add 500 μL of protein removal solution RD to the adsorption column CR3, centrifuge at 12,000 rpm for 30 seconds at 4°C, discard the waste liquid, and put the adsorption column CR3 back into the collection tube.
[0021] 8. Add 500 μL of washing buffer RW to the adsorption column CR3, let stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 30 seconds at 4°C, and discard the waste liquid.
[0022] Repeat step 8 once.
[0023] 9. Place the adsorption column CR3 into a new 2 mL collection tube, centrifuge at 12,000 rpm for 2 minutes, and then let the adsorption column CR3 air dry at room temperature for a while.
[0024] 10. Transfer the adsorption column CR3 into an RNase-Free centrifuge tube, add 30 μL of RNase-Free ddH2O to the center of the adsorption membrane, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes at 4°C to obtain total RNA.
[0025] Further, total RNA was reverse transcribed to obtain cDNA: In this embodiment, reverse transcription was performed using the PrimeScript RT reagent Kit with gDNAEraser (Code: RR047Q) from TAKARA. For specific operating procedures, please refer to the kit's instruction manual.
[0026] 1. Genomic DNA removal reaction: Prepare the reaction mixture on ice according to the following composition: First, prepare the premixed solution for the genomic DNA removal reaction by the amount of "reaction number + 2", dispense it into each reaction tube, then add 1.5 μg of total RNA, and finally make up the total volume to 10 μL with RNase-free deionized water. Incubate the reaction solution at 42°C for 2 minutes, and then place it on ice.
[0027] 2. Preparation of reverse transcription reaction system: On ice, add 4 μL of 5×PrimeScript Buffer, 2.4 μL of RNase-Free ddH2O, 1 μL of RT Primer Mix and 1 μL of PrimeScriptRT Enzyme Mix I to the above 10 μL reaction solution in sequence to make the total volume 20 μL. Gently mix the reaction mixture.
[0028] 3. Perform reverse transcription reaction: Place the prepared reaction mixture in a PCR instrument and run the following program: react at 37℃ for 15 minutes; react at 85℃ for 5 seconds to inactivate reverse transcriptase; finally cool to 4℃ and store to obtain cDNA.
[0029] In this embodiment, the coding sequence of the ENO gene was amplified using KOD-Plus-Neo high-fidelity DNA polymerase (Takara Code: KOD-401).
[0030] Specifically, each 50 μL amplification system includes: 5 μL of 10× buffer; 5 μL of 2 mM dNTP; 1.5 μL of 10 μM forward primer; 1.5 μL of 10 μM reverse primer; 1 μL of cDNA; 1 μL of 1 U / μL KOD-Plus-Neo high-fidelity DNA polymerase; and 35 μL of ddH2O.
[0031] Specifically, the amplification program includes: pre-denaturation at 98°C for 3 min; followed by 30 cycles, each cycle consisting of: denaturation at 98°C for 15 sec, annealing at 55°C for 20 sec, and extension at 68°C for 50 sec.
[0032] Construction of ENO overexpression vector The purified and recovered coding sequence fragment of the ENO gene was amplified by adding an A tail to the blunt end of the product and then ligating it into the pCXUN vector, which had been linearized by XcmI restriction enzyme digestion. The ligation product was then sequenced to obtain the ENO overexpression transgenic vector driven by the UBI promoter.
[0033] Construction of ENO-overexpressing transgenic rice materials First, the ENO overexpression transgenic vector was transformed into Agrobacterium tumefaciens EHA105, and then the ENO overexpression transgenic vector was transformed into the rice recipient material Nipponbare through Agrobacterium infection. After two generations of self-pollination of the T0 generation plants, the T2 generation lines were obtained and identified as homozygous lines.
[0034] RNA was extracted from the stems of transgenic plants and reverse transcribed into cDNA. The expression level was detected using real-time quantitative PCR. The results are as follows: Figure 1 As shown. By Figure 1 It can be seen that, compared with the control group rice material Nipponbare (Nip), the expression of the ENO gene in the two different transgenic lines OsENO-OE-6 and OsENO-OE-9 was significantly upregulated.
[0035] Resistance identification of transgenic plants The T2 generation transgenic lines OsENO-OE-6 and OsENO-OE-9 were used as the experimental group, and the rice material Nipponbare (Nip) was used as the control group. Seeds from both groups were soaked and germinated separately. The seeds were then sown in 10cm diameter circular plastic cups, with 12 seeds per cup. Three cups were sown per line as three replicates. When the seedlings reached the two-leaf stage, they were covered with a mesh net and inoculated with 10 2nd-3rd instar brown planthopper nymphs per seedling. After all control group seedlings had died, photographs were taken. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that, compared with the control group Nip, the experimental groups OsENO-OE-6 and OsENO-OE-9 grew well, indicating that both experimental groups OsENO-OE-6 and OsENO-OE-9 have good resistance to brown planthoppers.
[0036] When all the control group Nipponbare (Nip) plants died (resistance value of 9), the degree of damage to each seedling in the experimental materials was examined to determine the resistance value of the transgenic lines. In this example, the resistance value of the control group Nipponbare (Nip) was 9, and the resistance values of the experimental groups OsENO-OE-6 and OsENO-OE-9 were 4 and 3.4, respectively. The above resistance values were plotted into a bar chart, and the results are as follows. Figure 3 As shown. By Figure 3It can be seen that the resistance readings of OsENO-OE-6 and OsENO-OE-9 in the experimental group were significantly lower than those of the control group Nipponbare (Nip). The lower the reading, the stronger the resistance. Therefore, it can be seen that the resistance of OsENO-OE-6 and OsENO-OE-9 in the experimental group was significantly higher than that of Nipponbare (Nip) in the control group.
[0037] Simultaneously, the honeydew secretion and weight gain of brown planthoppers feeding on the control group Nip and the experimental groups OsENO-OE-6 and OsENO-OE-9 were measured 48 hours later. The specific methods are as follows: The weight of newly emerged female brown planthoppers was measured using a 1 / 100,000 electronic balance and recorded as their pre-feeding weight. Brown planthoppers of known weight were then fixed into wax bags of known weight (pre-release wax bag weight), and these bags were fixed to the base of plants in the control group (Nipponbare), and the experimental groups (OsENO-OE-6 and OsENO-OE-9), respectively. Forty-eight hours after feeding, the wax bags were removed, and the weight of the brown planthoppers after feeding was measured and recorded as their post-feeding weight. Simultaneously, the weight of the wax bag was also measured and recorded as its post-release weight. The weight gain of the brown planthoppers is equal to the difference in their weight before and after feeding, and the amount of honeydew secretion is equal to the difference in the weight of the wax bag before and after release. The results are as follows: Figure 4 and Figure 5 As shown. By Figure 4 and Figure 5 It was found that the honeydew secretion and weight gain of the brown planthopper-fed experimental groups (OsENO-OE-6 and OsENO-OE-9) were significantly lower than those of the control group (Nip) 48 hours after feeding. Higher honeydew secretion and weight gain indicate weaker resistance. Therefore, the honeydew secretion and weight gain indicate that the resistance of the experimental groups (OsENO-OE-6 and OsENO-OE-9) was significantly higher than that of the control group (Nip). Example
[0038] This disclosure provides a rice variety resistant to brown planthoppers, which contains the sequence shown in SEQ ID NO: 1 in the sequence listing.
[0039] The rice with brown planthopper resistance provided in this embodiment is based on the application of the ENO gene provided in Embodiment 1 in improving brown planthopper resistance. This application includes using the ENO gene to improve brown planthopper resistance. The specific sequence information of the ENO gene can be referred to the above embodiment. Since the rice with brown planthopper resistance adopts part or all of the technical solutions of Embodiment 1, it has at least all the beneficial effects brought about by the technical solutions of Embodiment 1, which will not be repeated here. The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.
Claims
1. The application of an ENO gene in enhancing resistance in brown planthoppers, characterized in that, The application includes using the ENO gene to enhance resistance in brown planthoppers.
2. The application according to claim 1, characterized in that, The applications include: Total RNA was extracted from rice materials; The total RNA was reverse transcribed to obtain cDNA; Using the cDNA as a template, amplification was performed using forward and reverse primers to obtain the amplification product, which is the coding region of the ENO gene. The sequence of the amplification product is shown in SEQ ID NO: 1 in the sequence listing, the sequence of the forward primer is shown in SEQ ID NO: 2 in the sequence listing, and the reverse primer is shown in SEQ ID NO: 3 in the sequence listing.
3. The application according to claim 2, characterized in that, The application also includes: adding an A tail to the blunt end of the amplification product and ligating it with an enzyme-digested pCXUN vector to obtain a ligation product; and converting the ligation product into a receptor material through infection.
4. The application according to claim 3, characterized in that, The rice variety in question is Nipponbare.
5. The application according to claim 2, characterized in that, Each 50 μL amplification system includes: 5 μL of 10× buffer; 5 μL of 2 mM dNTP; 1.5 μL of the forward primer at a concentration of 10 μM; 1.5 μL of the reverse primer at a concentration of 10 μM; 1 μL of cDNA; 1 μL of KOD-Plus-Neo polymerase at a concentration of 1 U / μL; and 35 μL of ddH2O.
6. The application according to claim 2, characterized in that, The amplification program includes: pre-denaturation at 98°C for 3 min; followed by 30 cycles, each cycle consisting of: denaturation at 98°C for 15 sec, annealing at 55°C for 20 sec, and extension at 68°C for 50 sec.
7. A type of rice resistant to brown planthoppers, characterized in that, The rice contains the sequence shown in SEQ ID NO: 1 in the sequence listing.