Target gene for preventing and controlling cotton aphid on salt-stressed cotton and application of target gene

Abstract

The invention discloses a target gene for controlling cotton aphid on salt-stressed cotton, the gene is UDP-glucuronyl transferase (UGT2) gene, and the gene expression and metabolite change of cotton aphid on 0, 75 and 150 mM NaCl stressed cotton are detected by transcriptomics and metabonomics technologies. The key gene of the cotton aphid responding to the salt stress cotton is verified through difference analysis screening and RNAi (Ribonucleic Acid Interference) technology. The gene is silenced to reduce the fitness of the cotton aphid on the salt-stressed cotton, and the reproductive capacity, the survival rate and the fitness of the cotton aphid UGT2 gene on the salt-stressed cotton are remarkably reduced after RNAi silents the cotton aphid UGT2 gene on the salt-stressed cotton. Therefore, aiming at silencing of the gene, the technical effect of effectively controlling the cotton aphid can be achieved, and the gene has relatively good popularization and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic prevention and control of pests, and specifically relates to a new technical field of cotton aphid salt stress resistance gene. Background Art

[0002] Salinized soil refers to soil with excessive salt content, causing the salt concentration to exceed the normal range required for plant growth, thus negatively impacting plant growth and development. According to the Food and Agriculture Organization of the United Nations, approximately 833 million hectares of land worldwide are affected by salinization, accounting for approximately 7% of the global land area and 33% of the global arable land area. Furthermore, due to factors such as excessive fertilizer use, inappropriate irrigation methods, industrial pollution, the gradual depletion of marine resources, and mineral weathering, saline-alkali land is expanding globally at a rate of approximately 10% per year. It is estimated that by 2050, approximately 50% of the world's arable land will be affected by salinization. As a result, the cultivated area of non-salinity-tolerant crops such as corn and wheat is gradually decreasing, while more salt-tolerant crops such as cotton are rapidly shifting to saline-alkali land.

[0003] Cotton, a globally important textile and cash crop, is moderately salt-tolerant, but its growth, development, and metabolism are still affected by salt stress. Numerous studies have shown that salt stress can affect the growth and development of herbivorous pests by affecting host plant metabolism. In recent years, the widespread cultivation of Bt transgenic cotton has effectively controlled the occurrence of cotton bollworms, but cotton aphids have gradually become a major pest. Previous studies have found that the developmental period, adult lifespan, fecundity, survival rate, and fitness of cotton aphids on salt-stressed cotton are significantly reduced. Metabolomics analysis of salt-stressed cotton metabolomes revealed that salt stress primarily affects amino acid metabolism, secondary metabolite biosynthesis, and carbohydrate metabolism. Specifically, polyphenols such as gossypol, flavonoids such as dihydromyricetin and neoastilbin, and terpenes such as chamolide lactone E and inosanol increased consistently with increasing salt stress concentration and were significantly correlated with cotton aphid growth, development, and fecundity. Summary of the Invention

[0004] Aiming at the problems of increasing soil salinization in Xinjiang and environmental pollution caused by chemical pesticide control of cotton aphids. The technical problem to be solved by the present invention is to screen out some key genes of cotton aphids in responding to salt stress cotton, verify their functions in responding to salt stress cotton through RNAi technology, and screen out a target gene that can reduce the fitness of cotton aphids on salt stress cotton after silencing. Based on the phenomenon of increasing soil salinization and reduced fitness of cotton aphids on salt stress cotton, the present invention screens out differential detoxification enzyme genes of cotton aphids in response to salt stress cotton through combined transcriptome and metabolome analysis, and verifies the role of this gene in cotton aphids responding to salt stress cotton through RNAi technology, revealing a genetic control method for cotton aphids on salt stress cotton. The silencing of this target gene significantly reduces the fitness of cotton aphids on salt stress cotton. Therefore, the control strategy for cotton aphids targeting this target gene is in line with the green control concept of "protecting benefits and controlling pests" in current plant protection.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions: The present application provides a target gene for controlling cotton aphids under salt stress. UGT2 Gene, the primer sequence used to amplify the specific interference fragment of the gene coding region includes or consists of the following sequence: 1) the nucleotide sequence shown in SEQ ID No. 1 to SEQ ID No. 2; or 2) The nucleotide sequences shown in SEQ ID No. 3 to SEQ ID No. 4.

[0006] Furthermore, the present application also provides the use of the target gene in at least one of the following: 1) Reduce the fitness, fecundity, and survival rate of cotton aphids under salt stress; 2) Cultivate crop varieties with disease and pest resistance.

[0007] The preferred application of the invention in reducing the fitness, fecundity and survival rate of cotton aphids under salt stress is to use a target gene UGT2 The ds was artificially synthesized using the T7 RiboMAXTM Exp-ress RNAi System synthesis kit. UGT2 , diluted to 250-750ng / µL, and sprayed evenly onto the aphids using a spray method.

[0008] Preferably, ds UGT2 Dilute to 750ng / µL and spray evenly onto the aphids using a spray method.

[0009] Furthermore, the present application also provides a method for reducing the fitness of cotton aphids under salt stress, wherein the target gene in cotton aphids is UGT2 Knockout or silencing.

[0010] Preferably, RNAi technology, CRISPR-Cas technology, T-DNA and promoter insertion, chemical mutagenesis or physical mutagenesis are used to modify the target gene in cotton aphids. UGT2 Knockout or silencing.

[0011] Furthermore, the present application also provides a method for reducing the fitness of cotton aphids under salt stress, wherein ds UGT2 Overexpression.

[0012] Preferably, CRISPR-Cas technology, T-DNA and promoter insertion, chemical mutagenesis or physical mutagenesis are used to modify the ds UGT2 Overexpression. Through the above technical solutions, this application achieves the following technical effects: The present invention utilizes RNAi technology to silence cotton aphid UGT2 Gene, found that compared with the control group, cotton aphid ds UGT2 The number of cotton aphids produced in the treatment group decreased significantly to 15.41. GFP and ds UGT2 The longest survival time of cotton aphids in the treatment groups were 22.33, 21.67 and 18.33 days, respectively, and the survival rates on the 8th day were 72.22%, 65.55% and 44.11%, respectively, and the fitness decreased significantly by 30.25%. Based on this, UGT2 This gene is an important gene for cotton aphids to cope with salt stress in cotton and can become a target gene for cotton aphid resistance. This also provides a theoretical reference for the development of RNAi pesticides or the cultivation of transgenic aphid-resistant cotton. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure Figure 1 shows the volcano plot of detoxification enzyme-related genes.

[0014] Figure 2 Shown is the agarose gel electrophoresis detection result.

[0015] Where M is 2000 bp DNA marker; 1 is ds before nuclease digestion GFP ; 2 is ds after nuclease digestion GFP ; 3 is ds before nuclease digestion UGT2 ; 4 is ds after nuclease digestion UGT2 Agarose gel electrophoresis Figure 3 Shown are the results of different concentration treatments of cotton aphids for 36h, 48h, 72h and 96h. UGT2 Relative gene expression levels.

[0016] Panel A shows the 250 ng / µL treatment group; Panel B shows the 500 ng / µL treatment group; and Panel C shows the 750 ng / µL treatment group.

[0017] Figure 4 Shown is spraying ddH2O, 750ng / µL ds GFP and ds UGT2 The results of the fecundity and survival rate of cotton aphids are shown in the figure.

[0018] Figure A shows the results of the fecundity test; Figure B shows the results of the survival rate test. DETAILED DESCRIPTION

[0019] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0020] The reagents used in the present invention are: LB (Luria-Bertani) medium; T7 RiboMAX TM Express RNAi System was purchased from Promega; E.Z.N.A. ® Gel Extraction Kit was purchased from OMEGA; pET1 expression vector, Escherichia coli competent cells, EasyPure Plasmid MiniPerp Kit, TransZol Up Plus RNA Kit TransScript All-in-One First-Strand cDNA Synthesis, SuperMix for qPCR, and PerfectStart Green qPCR SuperMix Kit were purchased from Quanshijin Biotechnology Co., Ltd.

[0021] Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0022] Example 1: A target gene for controlling cotton aphids under salt stress A target gene for controlling cotton aphids under salt stress, wherein the target gene is UGT2 Gene, the primer sequence used to amplify the specific interference fragment of the gene coding region includes or consists of the following sequence: 1) the nucleotide sequence shown in SEQ ID No. 1 to SEQ ID No. 2; or 2) The nucleotide sequences shown in SEQ ID No. 3 to SEQ ID No. 4.

[0023] Furthermore, the present application also provides the use of the target gene in at least one of the following: 1) Reduce the fitness, fecundity, and survival rate of cotton aphids under salt stress; 2) Cultivate crop varieties with disease and pest resistance.

[0024] The preferred application of the invention in reducing the fitness, fecundity and survival rate of cotton aphids under salt stress is to use a target gene UGT2 The ds was artificially synthesized using the T7 RiboMAXTM Exp-ress RNAi System synthesis kit. UGT2 , diluted to 250-750ng / µL, and sprayed evenly onto the aphids using a spray method.

[0025] Preferably, ds UGT2 Dilute to 750ng / µL and spray evenly onto the aphids using a spray method.

[0026] Furthermore, the present application also provides a method for reducing the fitness of cotton aphids under salt stress, wherein the target gene in cotton aphids is UGT2 Knockout or silencing.

[0027] Preferably, RNAi technology, CRISPR-Cas technology, T-DNA and promoter insertion, chemical mutagenesis or physical mutagenesis are used to modify the target gene in cotton aphids. UGT2 Knockout or silencing.

[0028] Furthermore, the present application also provides a method for reducing the fitness of cotton aphids under salt stress, wherein ds UGT2 Overexpression.

[0029] Preferably, CRISPR-Cas technology, T-DNA and promoter insertion, chemical mutagenesis or physical mutagenesis are used to modify the ds UGT2 Overexpression. Example: Functional identification of a target gene that significantly reduces the fitness of cotton aphids under salt stress This example provides a method for functional identification of a target gene that significantly reduces the fitness of cotton aphids under salt stress, which specifically includes the following steps: (1) Salt stress cotton The cotton variety used in the experiment was Zhongmianso 49, a conventional early-mid-maturing upland cotton. Five to six seeds were sown in pots filled with nutrient soil and placed in blue plastic boxes to grow the seedlings until they had two true leaves. Healthy seedlings of equal size were removed from their pots, their roots rinsed with running water, and then transplanted into black culture boxes filled with 1L of 1 / 2 Hoagland's nutrient solution. Each box contained one cotton seedling secured in a planting basket and sponge. The seedlings were hydroponically cultivated for six days to allow them to acclimate to the nutrient solution. The solution was then replaced with full Hoagland's nutrient solution and the seedlings were hydroponically cultivated until they had five true leaves. The Hoagland's nutrient solution was changed every six days. At the fifth true leaf stage, NaCl stress treatment began. Three NaCl concentrations (0, 75, and 150 mM) were used, with 30 seedlings treated at each concentration. To avoid salt shock, the concentration was gradually increased to reach the desired treatment concentration. For the 75 mM NaCl stress treatment, the cotton seedlings of each treatment were first cultured in a 25 mM NaCl nutrient solution for 2 days. After the cotton seedlings adapted to the salt concentration, the nutrient solution was replaced with 50 mM NaCl and continued to be cultured for 2 days. Subsequently, the nutrient solution was replaced with 75 mM NaCl and continued to be cultured. For the 150 mM NaCl stress treatment, the cotton seedlings of each treatment were first cultured in a 25 mM NaCl nutrient solution for 2 days. After the cotton seedlings adapted to the salt concentration, the nutrient solution was replaced with 50 mM NaCl and continued to be cultured for 2 days. Subsequently, the nutrient solution was replaced with 75 mM NaCl and continued to be cultured for 2 days. Finally, the nutrient solution was replaced with 150 mM NaCl and continued to be cultured. The culture medium of the cotton seedlings under each treatment was replaced every 6 days. Cotton seedlings were cultivated in an artificial climate chamber at the Korla Experimental Base of the Institute of Plant Protection, Xinjiang Uygur Academy of Agricultural Sciences under the following environmental conditions: temperature (26 ± 1) °C, relative humidity (RH) 50% ± 5%, and photoperiod of 16 h:8 h (L:D).

[0030] (2) Sampling of cotton aphids on salt-stressed cotton The cotton aphids used in the experiment were collected in 2022 from the cotton fields of the Korla Experimental Base, Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences (Korla Village, Heshilike Township, Korla City, Bayingolin Mongol Autonomous Prefecture, Xinjiang, 41.75ºN, 85.81ºE). No chemical pesticides were applied to the cotton fields before sampling.

[0031] On the sixth day of NaCl stress treatment, 20 adult cotton aphids were placed on the underside of cotton leaves and reared for one day. After nymphs were laid, the adults were brushed off and reared for seven days in an artificial climate chamber at 25°C, 50% humidity, and a 16:8 h light intensity (L:D). Adult cotton aphid samples were collected and placed in 1.5 mL sampling tubes. After rapid liquid nitrogen treatment, they were stored in a -80°C freezer and sent for transcriptomic and metabolomic analysis. Aphids were reared and propagated on cotton in an artificial climate chamber at (26 ± 1)°C, 50% ± 5% relative humidity (RH), and a 16:8 h light intensity (L:D) period.

[0032] (3) Genetic screening The present invention uses salt stress-cotton-aphid as the research system, and uses transcriptomic and metabolomic technologies to analyze the gene expression and metabolite changes of cotton aphids under 0, 75 and 150 mM NaCl stress. Through differential analysis and screening, 12 detoxification enzyme-related differentially expressed genes were found. The expression levels of 4 cytochrome P450 genes and 3 UDP-glycosyltransferase genes of cotton aphids were upregulated under 75 mM NaCl stress; the expression levels of 3 cytochrome P450 genes and 6 UGT genes of cotton aphids were upregulated under 150 mM NaCl stress. Among them, LOC114124293 (UDP-glucuronyltransferase-2, UGT2 ) gene showed the largest fold difference in the 75 and 150 mM NaCl stress treatment groups, see Appendix Figure 1 shown.

[0033] (4) Synthesis of d sGFP and ds UGT2 The online interference site prediction website (https: / / eurofinsgenomics.eu / en / ecom / tools / sirna-design / ) was used to target cotton aphids. UGT2 and GFP Specific interference fragments were designed in the gene coding region, as shown in Table 1. Based on the above interference fragments, two pairs of primers were designed to introduce T7 promoter sequences at the 5' end of the forward and reverse primers, respectively, and PCR amplification and purification were performed (amplification procedure: pre-denaturation: 94°C, 5min; denaturation: 95°C, 30s; annealing: 58°C, 30s; extension: 72°C, 1min; final extension: 72°C, 10min). The purified PCR product was used as a template for dsRNA synthesis, and the dsRNA was artificially synthesized using the T7RiboMAXTM Express RNAi System synthesis kit. The purity was then analyzed by 1% agarose gel electrophoresis and quantified using a NanoDrop-1000 spectrophotometer. And selected GFP The gene was used as a control and ds GFP .

[0034] Table 1: Primer sequences for synthetic dsRNA.

[0035]

[0036] Note: The underlined sequence in the table represents the T7 promoter sequence introduced by the primer 5' (5) ds UGT2 Optimal interference concentration detection Collect newly laid cotton aphid nymphs within 12 hours on two cotyledon stage cotton leaves. UGT2 and ds GFP The solution was diluted to three concentrations (250, 500, and 750 ng / µL) and evenly sprayed onto nymphs using a spray method. ddH2O was used as a blank control. Aphids were collected 36, 48, 72, and 96 hours after the experiment. Three biological replicates were set for each concentration and time point. qPCR was used for detection (qPCR protocol: initial denaturation: 95°C, 5 min; denaturation: 95°C, 15 s; annealing: 58°C, 30 s; extension: 72°C, 30 s) at different concentrations and time points. UGT2 The expression level of the gene, qPCR primers are shown in Table 2, screening ds UGT2 The optimal interference concentration.

[0037] Table 2: Reference genes (18S rRNA) and UGT2 qPCR primer sequences of genes

[0038] (6) Silence UGT2 Effects of genes on the growth and development of cotton aphids under salt stress Thirty newly hatched first-instar cotton aphids within 12 hours were placed on 75 mM NaCl-stressed cotton. 750 ng / µL ds UGT2 Spray evenly on cotton aphids, and record the survival and age of nymphs every 12 hours. When they develop into adults, observe the survival of adults and the number of nymphs produced every 24 hours. After each count, brush off the newly produced nymphs until all adults die.

[0039] The results showed that: during the synthesis of dsRNA, samples were taken before and after RNase and DNase digestion, and the purity was detected by 1% agarose gel electrophoresis. UGT2 and ds GFP The lane showed a single band, indicating that ds UGT2 and ds [[ID=⑤4]]GFP Purity qualified see attached Figure 2 250ng / µL ds UGT2 No significant silencing effect; 500ng / µL ds UGT2 There was no significant silencing effect at 36 h, and the highest silencing effect was 48.68% at 72 h; 750 ng / µL ds UGT2 The silencing effect was observed at 36, 48, 72, and 96 hours, with the highest silencing efficiency at 72 hours reaching 62.04%. UGT2 The best silence effect is Figure 3 As shown. Spraying 750 ng / µL dsUGT2 Silencing Aphis gossypii on cotton under salt stress UGT2 After gene addition, compared with 21.89 in the ddH2O-treated group, ds UGT2 The number of cotton aphids produced by the treatment decreased significantly to 15.41, a decrease of 29.59% ( F =25.25, df =2, P <0.05). ddH2O, ds GFP and ds UGT2 The longest survival time of cotton aphids in the treatments were 18.33, 21.67 and 22.33 days, and the survival rates on the 8th day were 72.22%, 65.55% and 44.11%, respectively (X 2 =8.96, df =2, P <0.05), the results are shown in the Appendix Figure 4 The life table parameter results are shown in Table 3, indicating that UGT2 Genes involved in cotton aphid resistance to salt stress.

[0040] Table 3: Silencing of salt-stressed cotton aphids UGT2 Post-genetic life table parameters

[0041] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Its purpose is to enable people who need this technology to understand the content of the present invention and implement it. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A target gene for controlling cotton aphids under salt stress, characterized by: The target gene is cotton aphid UGT2 Gene, the primer sequence used to amplify the specific interference fragment of the gene coding region includes or consists of the following sequence: 1) the nucleotide sequence shown in SEQ ID No. 1 to SEQ ID No. 2; or 2) The nucleotide sequences shown in SEQ ID No. 3 to SEQ ID No.

4.

2. Use of the target gene according to claim 1 in at least one of the following: 1) Reduce the fitness, fecundity, and survival rate of cotton aphids under salt stress; 2) Cultivate crop varieties with disease and pest resistance.

3. The use according to claim 2, characterized in that: The target gene of claim 1 is artificially synthesized using the T7RiboMAXTM Express RNAi System synthesis kit. UGT2 , diluted to 250-750ng / µL, and sprayed evenly onto the aphids using a spray method.

4. The use according to claim 3, characterized in that: ds UGT2 Dilute to 750ng / µL and spray evenly onto the aphids using a spray method.

5. A method for reducing the fitness of cotton aphids on salt-stressed cotton, characterized by: The target gene according to claim 1 is knocked out or silenced in cotton aphid.

6. The method according to claim 5, wherein: RNAi technology, CRISPR-Cas technology, T-DNA and promoter insertion, chemical mutagenesis or physical mutagenesis are used to modify the target gene in the cotton aphid. UGT2 Knockout or silencing.

7. A method for reducing the fitness of cotton aphids on salt-stressed cotton, characterized by: Place cotton in ds UGT2 Overexpression.

8. The method according to claim 7, wherein: The ds in the cotton are modified by using CRISPR-Cas technology, T-DNA and promoter insertion, chemical mutagenesis or physical mutagenesis. UGT2 Overexpression.

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