Use of a ce1 gene in improving resistance to brown planthopper

Abstract

The present disclosure provides an application of a CE1 gene in improving resistance to brown planthopper, belonging to the technical field of biotechnology. The application comprises: using the CE1 gene to improve the resistance to brown planthopper. The present disclosure provides an application of a CE1 gene in improving resistance to brown planthopper, which comprises using the CE1 gene to improve the resistance to brown planthopper of rice, and the resistance to brown planthopper of the recipient plant can be significantly improved, and the effect is remarkable.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to the application of a CE1 gene in enhancing resistance in brown planthoppers. Background Technology

[0002] Rice is a major food crop worldwide, with nearly half the world's population relying on it as their staple food. With the rapid growth of the global population, the demand for food is continuously increasing. Therefore, increasing rice yield is an urgent issue to be addressed in the near future. Insect pests are a major cause of reduced rice yields. Statistics show that over 100 types of pests can reduce rice yields, with the brown planthopper being the most serious. The brown planthopper is a typical piercing-sucking insect, using its stylet to pierce the phloem of rice leaf sheaths and suck sap, causing significant loss of nutrients and water, affecting rice growth and development, and in severe cases, leading to plant death, a phenomenon known as "planthopper fire," resulting in no harvest. Furthermore, the brown planthopper can act as a vector for rice dwarf virus and rice toothed leaf dwarf virus, further threatening rice production.

[0003] Currently, the main methods for controlling brown planthoppers are the use of chemical pesticides and the cultivation of resistant rice varieties. Chemical pesticides are convenient to use and fast-acting, killing large numbers of brown planthoppers in a short time. However, this method causes serious environmental damage and also has certain impacts on humans and livestock. Furthermore, the large-scale and frequent use of chemical pesticides can lead to pesticide resistance in brown planthoppers, poisoning non-target organisms and increasing the difficulty of controlling them. Cultivating resistant rice varieties is an economical, effective, and environmentally friendly method for controlling brown planthoppers. Since the 1970s, the International Rice Research Institute has successively bred resistant rice varieties containing the brown planthopper resistance genes Bph1 and Bph2, respectively, and applied them in production. However, due to the emergence of new biotypes of brown planthoppers, the resistance of these early resistance genes has been gradually overcome. Therefore, identifying and discovering new resistance genes is of great significance for the breeding of new brown planthopper-resistant rice varieties.

[0004] Public content

[0005] To address the problems of existing technologies, this disclosure provides an application of the CE1 gene in enhancing resistance in brown planthoppers. The technical solution is as follows:

[0006] This disclosure provides an application of the CE1 gene in improving the resistance of brown planthoppers, the method comprising: using the CE1 gene to improve the resistance of brown planthoppers.

[0007] Specifically, the applications include:

[0008] Total RNA was extracted from rice materials;

[0009] The total RNA was reverse transcribed to obtain cDNA;

[0010] Using the cDNA as a template, amplification is performed using forward and reverse primers to obtain the amplification product, which is the coding region of the 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.

[0011] Furthermore, 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.

[0012] Furthermore, the rice material is Nipponbare.

[0013] Furthermore, 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-Neopolymerase; and 35 μL of ddH2O.

[0014] Furthermore, the amplification program includes: pre-denaturation at 98°C for 3 min; followed by 32 amplification 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.

[0015] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides an application of the CE1 gene in improving the resistance of brown planthoppers. The application includes using the CE1 gene to improve the resistance of brown planthoppers in rice. The resistance of brown planthoppers in recipient plants can be significantly improved, and the effect is significant. Attached Figure Description

[0016] 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.

[0017] Figure 1 This is a comparison diagram of brown planthopper resistance of control group Nip, experimental group CE1-28 and experimental group CE1-29 provided in Embodiment 2 of this disclosure. In the figure, Nip represents the control group Nipponbare, and CE1-28 and CE1-29 represent the two experimental group materials, respectively.

[0018] Figure 2 This is a comparative graph of the statistical results of the resistance readings of brown planthoppers in the control group Nip, the experimental group CE1-28, and the experimental group CE1-29 provided in Embodiment 2 of this disclosure. In the graph, Nip represents the control group Nipponbare, and CE1-28 and CE1-29 represent the two experimental groups, respectively. The data in the graph represent the mean (at least 15 seedlings) ± standard error. The asterisks on the error line indicate that there is a significant difference compared with the control group (the p-value is calculated by one-way ANOVA, **p<0.01).

[0019] Figure 3 This is a comparative graph showing the statistical results of honeydew secretion by brown planthoppers in the control group Nip, experimental group CE1-28, and experimental group CE1-29 48 hours after feeding, as provided in Embodiment 2 of this disclosure. In the graph, Nip represents the control group Nipponbare, and CE1-28 and CE1-29 represent the two experimental groups, respectively. The data in the graph represent the mean (honeydew secretion of 30 brown planthoppers) ± standard error. The asterisks on the error line indicate significant differences compared with the control group (p-value calculated by one-way ANOVA, **p<0.01).

[0020] Figure 4 This is a comparative graph showing the weight gain of brown planthoppers 48 hours after feeding, according to Embodiment 2 of this disclosure, in the control group Nip, experimental group CE1-28, and experimental group CE1-29. In the graph, Nip represents the control group Nipponbare, and CE1-28 and CE1-29 represent the two experimental groups, respectively. The data in the graph represent the mean (weight gain of 30 brown planthoppers) ± standard error. The asterisks on the error lines indicate significant differences compared to the control group (p-value calculated by one-way ANOVA, **p<0.01). Detailed Implementation

[0021] 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.

[0022] Example 1

[0023] This disclosure provides an application of the CE1 gene in improving the resistance of brown planthoppers, the method comprising: using the CE1 gene to improve the resistance of brown planthoppers.

[0024] Specifically, the application includes linking the coding region of the CE1 gene to an overexpression promoter to improve resistance in brown planthoppers.

[0025] Specifically, the application includes:

[0026] Total RNA was extracted from the rice material Nipponbare. In this embodiment, a whole Nipponbare rice plant grown for 15 days was used. In other embodiments, other rice varieties can be used, and the process can be the same as in this embodiment. Total RNA was extracted from the rice material using the RNAsimple Total RNA Extraction Kit (TIANGEN Code: DP419). The specific process is as follows:

[0027] 1. Grind the tissues of fresh rice material into powder in liquid nitrogen.

[0028] 2. Take 50-100 mg of powder and put it into a 1.5 mL centrifuge tube free of RNase contamination. Add 1 mL of lysis buffer to the centrifuge tube, shake vigorously, and homogenize using a vortex mixer. Then let it stand at room temperature for 5 minutes to allow the protein to be completely separated.

[0029] 3. Centrifuge at 13,200 rpm for 5 min at 4℃ to obtain the supernatant. Transfer the supernatant to a new 1.5 mL centrifuge tube free of RNase contamination.

[0030] 4. Add 200 μL of chloroform to the new centrifuge tube, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0031] 5. Centrifuge at 13,200 rpm for 10 min at 4℃ to obtain the supernatant again. Transfer the supernatant to a new 1.5 ml centrifuge tube free of RNase contamination.

[0032] 6. Add 0.5 times the volume of anhydrous ethanol to a new centrifuge tube, mix well, transfer the solution to the CR3 adsorption column, and centrifuge briefly at 13,200 rpm at 4℃. Discard the waste liquid in the collection tube.

[0033] 7. Add 500 μL of RD protein removal solution to the adsorption column CR3, centrifuge briefly at 13,200 rpm at 4℃, and discard the waste liquid in the collection tube.

[0034] 8. Add 500 μL of RW washing solution to the adsorption column CR3, let it stand at room temperature for 2 min, centrifuge briefly at 13,200 rpm at 4℃, and discard the waste liquid in the collection tube.

[0035] 9. Repeat step 8 once.

[0036] 10. Place the CR3 adsorption column into a 2 mL collection tube and centrifuge at 13,200 rpm for 2 min at 4 °C to remove residual liquid. Then let it air dry at room temperature for 2 min.

[0037] 11. Place the adsorption column CR3 into a new 1.5 mL centrifuge tube, add 50 μL of RNase-Free ddH2O, incubate at room temperature for 2 min, and centrifuge at 4℃ and 13,200 rpm for 2 min to obtain the supernatant, which is the total RNA.

[0038] In other embodiments, the rice material can also be other varieties of rice, and the process can be referred to in this embodiment.

[0039] Total RNA was reverse transcribed to obtain cDNA; in this embodiment, PrimeScript from TAKARA was used. TM Reverse transcription was performed using the RT reagent kit with gDNA Eraser (Code: RR047Q). Please refer to the kit's instruction manual for detailed procedures. The specific steps are as follows:

[0040] 1. Take 1.5 μL of total RNA and measure the total RNA concentration using a nucleic acid concentration analyzer (Thermo). Take 1 μL of total RNA and perform agarose gel electrophoresis (concentration of 1%) to check whether the RNA is intact. If the RNA is found to be intact, proceed with the next steps.

[0041] 2. Prepare the first reaction mixture on ice. The first reaction mixture consists of 2 μg total RNA, 2.0 μL of 5×gDNAEraserBuffer and 1.0 μL of gDNAEraser. Add the volume of the first reaction mixture to 10 μL using RNase-free dH2O. The reaction conditions are: 42℃ for 2 min, and storage at 4℃ to obtain the first reaction product with genomic DNA removed.

[0042] 3. Prepare the second reaction mixture on ice for cDNA synthesis. The second reaction mixture consists of: 10.0 μL of the first reaction product, 1.0 μL of PrimeScript RT Enzyme Mix I, 4.0 μL of RT PrimerMix, 4.0 μL of 5×PrimeScript Buffer 2, and 1.0 μL of N-ase Free dH2O, with a total volume of 20 μL. The reaction conditions are: 37℃ for 15 min, 85℃ for 5 sec, to obtain cDNA.

[0043] Amplification of the CE1 coding sequence

[0044] Using cDNA as a template, amplification was performed using forward and reverse primers to obtain the amplified product, which is the coding region of the gene, used to improve rice resistance to brown planthopper. The sequence of the amplified product is shown in SEQ ID NO: 1 of the sequence listing, the sequence of the forward primer is shown in SEQ ID NO: 2 of the sequence listing, and the reverse primer is shown in SEQ ID NO: 3 of the sequence listing. In this embodiment, the coding sequence of CE1 was amplified using KOD-Plus-Neo high-fidelity DNA polymerase (Takara Code: KOD-401). Specific operating procedures are detailed in the kit's instructions. The specific operating procedures are as follows:

[0045] The amplification system was 50 μL, and the specific amplification system is shown in Table 1.

[0046] Table 1 shows the amplification system.

[0047] reagents Usage 10×buffer 5.0μL 2mMdNTP 5.0μL Forward primer (10 μm) 1.5μL Reverse primer (10 μm) 1.5μL cDNA 1.0μL KOD-Plus-Neopolymerase 1.0μL <![CDATA[ddH2O]]> 35μL

[0048] The amplification procedure is shown in Table 2.

[0049] Table 2 shows the amplification procedure.

[0050]

[0051] After purification and recovery of the amplification product, the CE1 coding sequence fragment was obtained, which was 1008 bp in length.

[0052] Example 2

[0053] This invention provides an application of the CE1 gene in improving resistance to brown planthoppers. The application includes: adding an A tail to the blunt end of the amplified product and ligating it with an enzyme-digested pCXUN vector to obtain a ligation product.

[0054] The linker product is transferred into the acceptor material through infection.

[0055] Construction of CE1 overexpression vector

[0056] The purified and recovered CE1 coding sequence fragment obtained in Example 1 was ligated with an A tail added to the end and then ligated with the pCXUN vector linearized by XcmI restriction enzyme digestion to obtain the ligation product. After correct sequencing, the CE1 overexpression transgenic vector driven by the UBI promoter was obtained.

[0057] Construction of rice materials with enhanced resistance to brown planthopper

[0058] First, the CE1 overexpression transgenic vector was transformed into Agrobacterium EHA105, which was purchased from Shanghai Weidi Biotechnology Co., Ltd. Then, the CE1 overexpression transgenic vector was transformed into the recipient material Nipponbare using the Agrobacterium infection method. The specific steps are as follows:

[0059] 1. Inducing callus tissue:

[0060] Select 14g of mature, plump, hulled seeds from Nipponbare rice and place them in a 50mL centrifuge tube. Rinse 5 times with tap water, then 3 times with 30mL deionized water, then 5 minutes with 70% ethanol solution, then 3 times with distilled water. Finally, sterilize with 0.15% HgCl2 at 100rpm on a shaking incubator for 18 minutes. After sterilization, pour out the HgCl2 on a sterile table, wash the seeds 4 times with sterile water, spread the seeds on sterile filter paper, and place them in a laminar flow hood for 1 hour until they are dry. Place the dried seeds on N6 solid culture medium and culture at 28℃ in the dark for 1 month to obtain pale yellow and dense embryogenic callus tissue.

[0061] 2. Culture and suspension of Agrobacterium:

[0062] An appropriate amount of Agrobacterium containing the CE1 overexpression vector was spread onto LB agar medium containing kanamycin and rifampin antibiotics and incubated overnight at 28°C. The cultured Agrobacterium was scraped into 10 mL of 1 / 2 N6 liquid culture medium and thoroughly mixed by pipetting to obtain a suspension. The suspension concentration was adjusted to OD using 1 / 2 N6 liquid culture medium. 600 =0.8, and add AS cell culture medium with a final concentration of 150 μM to obtain Agrobacterium suspension.

[0063] 3. Agrobacterium infection:

[0064] Agrobacterium suspension was placed in a glass bottle, and embryogenic callus was transferred into the bottle. The mixture was inoculated on a shaker at a low speed of 80 rpm for 18 min. The Agrobacterium suspension was then poured out, and the embryogenic callus was transferred to sterile filter paper and allowed to stand for 2 h. The callus was then transferred to 1 / 2 N6 solid medium containing 50 μM NAS and incubated in the dark at 20°C for 2 days.

[0065] 4. Removal of Agrobacterium

[0066] After 2 days of culture, the callus was transferred to a 200mL glass bottle and washed three times with sterile water. Then, N6 liquid culture medium containing 500mg / L cephalosporin was added, and the mixture was shaken at low speed for 20 minutes. The culture medium was then discarded, and this process was repeated three times. Finally, the callus tissue was transferred to sterile filter paper and placed on a laminar flow hood for 3 hours to allow the callus tissue to dry completely.

[0067] 5. Screening of callus tissue

[0068] Dried callus tissue was transferred to N6 solid medium containing 250 mg / L cephalosporin and 50 mg / L hygromycin, and incubated in the dark at 28°C for 30 days. Callus tissue free from Agrobacterium contamination and in good growth condition was transferred to fresh N6 solid medium containing 250 mg / L cephalosporin and 50 mg / L hygromycin, and incubated in the dark at 28°C for approximately 15 days.

[0069] 6. Differentiation and Transplantation

[0070] The selected callus tissue was transferred to MS medium and cultured in the dark at 28°C for about 10 days. Then, it was cultured in the light at 28°C for about 20 days. When the green shoots that sprouted from the callus reached a length of about 1-2 cm, they were transferred into test tubes containing 1 / 2 MS rooting medium. When the callus developed roots and the seedlings reached the height of the test tubes, the T0 generation plants were obtained.

[0071] After two generations of self-pollination, T0 generation plants were used to obtain T2 generation lines for subsequent experiments. The self-pollination was carried out using existing conventional self-pollination methods.

[0072] The T2 generation (CE1-28 and CE1-29) and the rice material Nipponbare (Nip) were used as experimental and control groups, respectively. Seeds from both groups were soaked and germinated. The seeds were then sown in 10cm diameter circular plastic cups, 20 seeds per cup, with three cups sown per plant line as three replicates. When the seedlings reached the two-leaf stage, they were covered with a mesh net and inoculated with 8 2nd-3rd instar brown planthopper nymphs per seedling. Photos were taken after all the susceptible controls had died. Results are as follows: Figure 1 As shown, by Figure 1 It can be seen that the resistance of brown planthoppers in the experimental groups (CE1-28 and CE1-29) was significantly improved compared with that of the control group, Nip.

[0073] When all the control group Nip plants died (resistance value of 9), the degree of damage to each seedling in the experimental material was examined to determine the resistance value of the transgenic line. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the resistance readings of the experimental groups (CE1-28 and CE1-29) were significantly lower than those of the control group (Nipponbare). The lower the reading, the stronger the resistance.

[0074] Meanwhile, the amount of honeydew secretion and weight gain of brown planthoppers were measured 48 hours after feeding on the control group (Nip) and the experimental groups (CE1-28 and CE1-29).

[0075] The specific method is as follows:

[0076] The weight of newly emerged female brown planthoppers was measured using a 1 / 100000 electronic balance (Shimadzu; Type: AUW120D), 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) at the base of Nip, CE1-28, and CE1-29 plants. After 48 hours of free 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 3 and Figure 4 As shown. By Figure 3 and Figure 4 It was found that the honeydew secretion and weight gain of the brown planthopper feeding groups (CE1-28 and CE1-29) were significantly lower than those of the control group (Nip) 48 hours later. Higher honeydew secretion and weight gain indicate weaker resistance.

[0077] 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 a CE1 gene in improving resistance to brown planthopper in rice, characterized in that, The application includes: transforming a CE1 overexpression transgenic vector into rice recipient material, wherein the coding region sequence of the CE1 gene is shown in SEQ ID NO:

1.

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 is performed using forward and reverse primers to obtain the amplification product, which is the coding region of the 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 recipient material through infection.

4. The application according to claim 2, 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 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 polymerase; and 35 μL of ddH2O.

6. The application according to claim 2, characterized in that, The amplification program included: pre-denaturation at 98°C for 3 min; followed by 32 amplification cycles, each consisting of denaturation at 98°C for 15 sec, annealing at 55°C for 20 sec, and extension at 68°C for 50 sec.

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