Application of PagWRKY40 gene in regulation and control of insect resistance of poplar

Abstract

The invention discloses application of a PagWRKY40 gene in regulation and control of insect resistance of poplars, relates to the field of biotechnology and genetic engineering, and particularly relates to application of the PagWRKY40 gene or a protein coded by the PagWRKY40 gene in regulation and control of insect resistance of poplars, and a nucleotide sequence of the PagWRKY40 gene is shown as SEQ ID No.1. According to the invention, the PagWRKY40 gene of the 84K poplar is cloned, and an over-expression transgenic plant of the gene is obtained by utilizing an agrobacterium tumefaciens-mediated genetic transformation mode. Research results provide a basis for improvement of insect-resistant varieties of poplar trees and have important scientific significance for revealing excellent genes for forest tree genetic engineering breeding, and the 84K poplar PagWRKY40 gene has obvious insect-resistant ability and does not influence plant growth, so that the gene has very important application prospects in growth of transgenic plants, especially transgenic forest trees.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic engineering, specifically relating to the PagWRKY40 gene and the amino acids it encodes and its application in insect resistance. Background Technology

[0002] The main methods for controlling poplar pests are physical control, chemical control, and biological control. Physical control mainly utilizes various mechanical equipment or simple tools to control pests; however, this method is inefficient, time-consuming, labor-intensive, and easily limited by environmental conditions. Chemical control, while effective quickly, can lead to pesticide resistance in pests, pollute the environment, kill beneficial organisms, and threaten human and animal health. Biological control, while environmentally friendly, is typically costly, slow-acting, and has a narrow range of applications.

[0003] Facing the challenges of forestry pests and diseases, research on transgenic insect-resistant poplar has made significant progress. Several Bt-transgenic insect-resistant poplars have been obtained, contributing to green pest control and productivity improvement in forestry. Besides the Bt gene, genes for common insect neurotoxins, exogenous lectins, and scorpion venom have also been applied. However, the application of exogenous genes has certain limitations: their insertion location, copy number, and regulatory sequences may affect their expression levels; exogenous genes may be lost or recombine during poplar propagation; transgenic poplars may indirectly affect non-target insects (such as beneficial insects and pollinators) or soil microorganisms; and long-term reliance on Bt protein may accelerate the evolution of pest resistance. With the development of molecular biology techniques and the expansion of the scope of insect-resistant gene research, in addition to the Bt gene, more endogenous plant insect-resistant genes should be developed. The application of endogenous genes can better avoid the above limitations and provide more diverse options for insect-resistant poplar breeding.

[0004] The WRKY transcription factor family is one of the largest transcription factor families in plants. Its main functions include participation in plant defense and responses to abiotic stresses. It also plays a role in plant senescence, seed germination, and pollen, embryo, and seed development. Under stress conditions, WRKY transcription factors can specifically recognize target promoters, bind to W-box cis-elements (TGACCA / T), and regulate multiple signaling pathways, such as jasmonic acid, abscisic acid, and mitogen-activated protein kinase. Summary of the Invention

[0005] In this invention, the use of the WRKY gene to improve the insect resistance of poplar trees is a more efficient method for improving the insect resistance of poplar trees.

[0006] The application of the PagWRKY40 gene in regulating the insect resistance of poplar trees, wherein the nucleotide sequence of the PagWRKY40 gene is shown in SEQ ID No. 1.

[0007] Furthermore, the insect in question is the American white moth.

[0008] Furthermore, the poplar tree mentioned is an 84K poplar.

[0009] Furthermore, the application of the PagWRKY40 gene in regulating poplar insect resistance improves the poplar's insect resistance without affecting its growth.

[0010] Furthermore, the PagWRKY40 gene enhances the insect resistance of poplar trees through overexpression.

[0011] Furthermore, the PagWRKY40 gene enhances the insect resistance of poplar trees under drought stress.

[0012] Furthermore, the amino acid sequence of the protein encoded by the PagWRKY40 gene is shown in SEQ ID No. 2.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] This invention cloned the PagWRKY40 gene from 84K poplar and obtained transgenic plants overexpressing this gene using Agrobacterium tumefaciens-mediated genetic transformation. The leaves of transgenic and wild-type poplar trees were fed to fall webworm larvae, and the extent of insect damage was compared between the two types. Physiological indicators were also measured to investigate the effect of the PagWRKY40 gene on poplar insect resistance. The results showed that PagWRKY40, as a positively regulatory gene in response to insect stress, can positively regulate poplar insect resistance, and therefore can serve as an excellent gene for insect-resistant poplar breeding. These findings provide a basis for improving insect-resistant poplar varieties and have significant scientific value in revealing superior genes for forest tree genetic engineering breeding. The 84K poplar PagWRKY40 gene exhibits significant insect resistance without affecting plant growth; therefore, its application in the growth of transgenic plants, especially transgenic forests, has very important prospects. Attached Figure Description

[0015] Figure 1 This is a graph showing the expression level of the PagWRKY40 gene in the transgenic line.

[0016] Figure 2 Photographs showing the feeding preferences of the American white moth on different genetically modified poplar leaves;

[0017] Figure 3 Figure showing the weight gain of the American white moth in insect feeding experiments with different transgenic strains;

[0018] Figure 4 Figure showing leaf feeding amount in insect feeding experiments for different transgenic strains;

[0019] Figure 5A graph showing the CAT activity analysis of leaves from different transgenic lines under feeding conditions of American white moth larvae;

[0020] Figure 6 A graph showing the PPO activity analysis of leaves from different transgenic lines after feeding by American white moth larvae;

[0021] Figure 7 A graph showing the LOX activity analysis of leaves from different transgenic lines fed on by American white moth larvae. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0023] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0024] Example 1: Construction of PagWRKY40 gene overexpression vector

[0025] The nucleotide sequence of the PagWRKY40 gene is shown in SEQ ID No. 1, as follows:

[0026] ATGGATTATTCATCGTGGGTTGATACTTCTTTGGATCTTAATATTAATCCTTTAAGAGTGAAAAGTGAAGTTCCGGTAGATGCAGAAAGATTCGGGATGGCAAGAGAATTGAAACCCACTTTCATGGATTTTCTGACCACGCCTTCAGCCAAAGAAGAGACTGGAGCTTTGGTAGTGGAAATGAACCGAGTGAGCGAAGAAAACAGGAAGCTAACTGAAATGCTAACTGTGATGTGTGAGAGCTACAATGCTTTAAGAAGCCAGTTGATGGATTACATGAGCAAGAATGGAGAAAAGGAGCTTCTTGCCCCATCAAAGAAAAGAAAGTCTGAAAGCAGCAACAACAACGATAATAACATTGCAAAGAACGGGCACTCTGAGAGCAGCTCAACTGATGAAGAATCATCCAAGAAACCAAGGGAAGAAGTCATTAAAGATAAGATTTCGAAGGCTTATGTCAGGACTGAAGCTGGTGATACAAGCCTTATTGTGAAAGATGGATATCAATGGAGGAAATATGGCCAAAAAGTCACAAGAGATAACCCTTGTCCAAGAGCTTACTTCAAGTGCTCTTTTGCTCCAAGCTGCCCTGTCAAAAAGAAGGTTCAAAGGAGCATCGATGACCAATCTGTTCTAGTGGCAACTTATGAAGGAGAGCACAACCATCCACATCCTTCAATGGAGGCAACATCAGGTTCAAACCGTAGTCTAACACGCGGTCCAGTACCCTGCATAGCCTCCCTAGCCTCATCTGGGCCAACCGTTACTCTTGATCTCGCAAAATCTAAGTCAAGCAATGATGACAGGAGCTCAGAACCAAGAGCGGATACGCCTGAAGTCCGGAAATTCTTGGTGGAGCAGATGGCCTCTTCGCTGACGAAAGATCCCAATTTCACAGCAGCACTGGCCGCAGCAATCTCAGGAAGAATGCTTCAGCAAAGTCACAGCGAGAAGTGGTGA

[0027] The amino acid sequence encoded by the PagWRKY40 gene is shown in SEQ ID No. 2, as follows:

[0028] MDYSSWVDTSLDLNINPLRVKSEVPVDAERFGMARELKPTFMDFLTTPSAKEETGALVVEMNRVSEENRKLTEMLTVMCESYNALRSQLMDYMSKNGEKELLAPSKKRKSESSNNNDNNIAKNGHSESSSTDEESSKKPREEVIKDKISKAYVRTEAGD TSLIVKDGYQWRKYGQKVTRDNPCPRAYFKCSFAPSCPVKKKVQRSIDDQSVLVATYEGEHNHPHPSMEATSGSNRSLTRGPVPCIASLASSGPTVTLDLAKSSKSNDDRSSEPRADTPEVRKFLVEQMASSLTKDPNFTAALAAAISGRMLQQSHSEKW

[0029] Primers for the PagWRKY40 gene were designed based on its sequence. Sac I and BamH I restriction sites were introduced into the upstream and downstream primers, respectively. Using cDNA from 84K poplar leaves as a template, PCR amplification was performed using a high-fidelity enzyme. The specific upstream and downstream primers are as follows:

[0030] PagWRKY40-F (5'-3'): ggatccATGGATTATTCATCGTGGGTTGA

[0031] PagWRKY40-R (5'-3'): aagcttTCACCACTTCTCGCTGTGAC

[0032] The PCR reaction system is as follows:

[0033] Ex Taq mix10ul

[0034] PagWRKY40-F1ul

[0035] PagWRKY40-R1ul

[0036] cDNA2ul

[0037] ddH2O up to 20ul

[0038] The PCR reaction procedure is as follows:

[0039]

[0040] The target band was obtained by gel extraction and recovery. Subsequently, the target fragment was purified using a purification kit. The purified target fragment was ligated into the pEASY-T1 vector. All ligation products were transformed into *E. coli* and plated on LB agar plates containing KAN resistance for overnight incubation. The specific method for transforming *E. coli* is as follows:

[0041] ① Take 100 μl of competent cells (DH5α) stored at -80°C, thaw them on ice, and then transform them. Add all the ligation products (5 μl) and gently mix them by pipetting.

[0042] ②Incubate the mixed competent cells on ice for 30 minutes.

[0043] ③ Quickly place the centrifuge tubes in a 42°C water bath for 30 seconds.

[0044] ④ Quickly transfer the centrifuge tubes to ice and let them stand for 2 minutes. Be careful not to shake the centrifuge tubes too much.

[0045] Add 250 μl of LB medium without any antibiotics to the centrifuge tube and incubate at 37°C with shaking for 1 hour.

[0046] Place the shaken bacterial culture in a centrifuge and centrifuge at 4000 rpm for 2 minutes to precipitate the bacterial cells.

[0047] In a clean bench, discard the supernatant from the centrifuge tubes, gently agitate the precipitated bacterial cells with a pipette tip, mix thoroughly, and then spread the bacterial solution onto LB agar plates containing the appropriate antibiotics. Seal the plates with sealing film and incubate overnight in a shaker at 37°C. Invert the plates.

[0048] Single-clone bacterial plaques were selected as templates, and PCR detection was performed using universal vector primers. The detected bacterial culture was sequenced, and after correct sequence alignment, the culture was expanded. Plasmids were extracted using a plasmid extraction kit. The pCMBIA1300 vector plasmid was digested with Sac I and BamH I restriction endonucleases, purified, and ligated with the target fragment. The recombinant product was transformed into *E. coli* and plated on kanamycin selection medium for preliminary screening. Positive clones were detected by PCR using universal vector primers. Bacterial cultures corresponding to a single band of the expected size were sent to the company for sequencing, ultimately obtaining the pCMBIA1300-PagWRKY40 recombinant plasmid. The extracted plasmid was transformed into *Agrobacterium* strain GV3101, completing the construction of the plant overexpression vector. The specific method for transforming *Agrobacterium* is as follows:

[0049] ① Take 100 μl of Agrobacterium GV3101, add 0.4 μl of plasmid, gently mix with a pipette, and incubate on ice for 5 minutes.

[0050] ② Freeze in liquid nitrogen for 5 minutes, then immediately place in a 37°C water bath for 5 minutes.

[0051] ③ Add 700 μl of liquid LB medium to the centrifuge tube, incubate at 28°C with shaking for 2.5 hours, centrifuge at 6000 rpm for 2 minutes, and collect the bacterial cells.

[0052] ④ In a clean bench, discard some of the supernatant, gently blow away the precipitated bacterial cells with a pipette tip, mix them evenly, spread them on an LB agar plate containing rifampin and kanamycin, and incubate at 28°C for 2 days.

[0053] Select a single colony for PCR testing; if the band is correct, the bacteria can be stored for genetic transformation.

[0054] 2. Acquisition and identification of genetically modified poplar trees

[0055] Obtaining transgenic plants: Transgenic 84K poplar plants were obtained using the Agrobacterium-mediated leaf disc method.

[0056] Identification of overexpressing plants: Overexpressing plants were detected using PCR. The primer sequences were 35SF (5'-3'): AAAGTCGACATGGTGCAAGGGCGAGGAGCTGA; PagWRKY40-R (5'-3'): aagcttTCACCACTTCTCGCTGTGAC. PCR results showed that 11 overexpressing plants were obtained, indicating that the target gene had been successfully inserted into the plant genome.

[0057] Identification of PagWRKY40 gene expression in overexpressing plants: The expression level of PagWRKY40 gene in all overexpressing lines was detected using qRT-PCR. RNA was extracted from leaves of all transgenic PagWRKY40 gene (OE1 to OE11) and non-transgenic PagWRKY40 gene (wild-type WT) poplar trees, and cDNA was reverse transcribed from each. Quantitative primers qRT-PagWRKY40-F and qRT-PagWRKY40-R were designed based on the non-conserved domain sequences of the PagWRKY40 gene, with Ptaction used as an internal control primer. The primer sequences are shown below. Real-time quantitative PCR was performed using an ABI 7500. The algorithm calculated the relative expression level of the PagWRKY40 gene. The results showed that the expression levels of the OE4, OE7, and OE10 lines were significantly higher than those of the non-transgenic lines. Figure 1 ).

[0058] Ptactin-F (5'-3'): TCATCGGAATGGAAGCTGCTGGTA

[0059] Ptactin-R (5'-3'): TAGTGGAACCACCACTGAGCACAA

[0060] qPagWRKY40-F (5'-3'): AGAGCACAACCATCCACATC

[0061] qPagWRKY40-R (5'-3'):TTGCGAGATCAAGAGTAACGG

[0062] 3. Insect resistance analysis of transgenic poplar

[0063] (1) Selective feeding of the fall webworm

[0064] To observe the resistance of PagWRKY40 transgenic 84K poplar material to the fall webworm, the feeding preferences of the fall webworm were tested. Ten healthy second-instar fall webworm larvae of similar growth, development, and weight were collected. Leaves of similar size from the OE4, OE7, OE10, and WT lines were placed in transparent petri dishes to observe the feeding preferences of the fall webworms and to photograph and record the leaf area loss of each genotype line. After 4 hours of feeding, it was found that the insects mainly concentrated on the leaves of the wild-type line, which had the most severe leaf consumption. Leaf loss was also observed in all transgenic lines. After 8 hours, it was found that almost no leaves remained in the wild-type line. Figure 2 Meanwhile, it was found that the leaves of the OE10 strain were the most intact and showed the least amount of damage. This result indicates that the fall webworm larvae have a certain deterrent effect on the PagWRKY40 transgenic poplar.

[0065] (2) Analysis of leaf feeding by the American white moth

[0066] Wild-type (WT) and PagWRKY40 gene-transgenic 84K poplar lines (OE4, OE7, OE10) with consistent growth were selected for a feeding experiment on the fall webworm to assess the insect resistance of the overexpressing lines. Healthy second-instar fall webworms were starved for 12 hours before the feeding experiment. Five to six leaves of similar size were taken from both the transgenic and wild-type lines and weighed, then placed in plastic petri dishes. Simultaneously, three healthy second-instar fall webworm larvae were taken, weighed, and placed on the leaves of each line for feeding. The amount of leaf food consumed and the weight gain of the fall webworm larvae were recorded at 2, 4, and 8 hours after feeding. The larval weight gain rate was calculated as follows: (weight of larvae after feeding - weight of larvae before feeding) / weight of larvae before feeding. The leaf food consumption rate was calculated as follows: (weight of leaf before feeding - weight of leaf after feeding) / weight of leaf before feeding. The results showed that the weight gain rate of American white moth larvae feeding on wild-type leaves was significantly higher than that of the transgenic strain; among them, after 8 hours of feeding, the weight gain rate of larvae feeding on wild-type leaves was more than twice that of the transgenic strain group. Figure 3 After being fed on by the fall webworm, the wild-type strain consumed significantly more leaves than the transgenic strain. Figure 4 This further demonstrates that the larvae of the fall webworm have a certain anti-feeding effect on poplar trees transgenic with the PagWRKY4 gene.

[0067] (4) Defensive enzyme activity analysis

[0068] The insect resistance of PagWRKY40 gene-transgenic poplar plants was further evaluated by measuring physiological indicators of wild-type (WT) and PagWRKY40 gene-transgenic 84K poplar lines (OE4, OE7, OE10). Healthy, uniformly growing wild-type and transgenic 84K poplar tissue culture seedlings (one month old) were selected and transplanted into flowerpots. After one month of cultivation in a greenhouse, an insect feeding experiment was conducted. Leaves grafted with second-instar fall webworm larvae were selected, and physiological indicators were measured at 0h, 3h, 6h, 12h, and 24h. Samples were rapidly placed in liquid nitrogen and stored at -80°C. All physiological indicator measurements were performed using a kit from a biotechnology company.

[0069] CAT activity results showed that there was no significant difference in CAT activity among the different strains when the fall webworm had been feeding for 0 h. With increasing treatment time, CAT activity continuously increased. At 12 h and 24 h of feeding, the CAT activities of OE4, OE7, and OE10 were significantly higher than those of WT. At these times, the CAT activities of OE4, OE7, and OE10 were 1.55 times, 1.65 times, and 1.67 times that of WT, respectively. Figure 5 ).

[0070] PPO activity results showed no significant difference in PPO activity among the different lines at 0 h. With increasing treatment time, PPO activity showed an increasing trend, reaching its peak at 24 h. The PPO activity of the transgenic lines was significantly higher than that of the WT line at both 12 h and 24 h. At 24 h after feeding by the fall webworm, the PPO activities of OE4, OE7, and OE10 were 1.06, 1.07, and 1.09 times that of the WT line, respectively. Figure 6 ).

[0071] LOX activity results showed no significant difference in LOX activity among the different lines at 0 h. With increasing treatment time, LOX activity in all lines continuously increased, reaching its peak at 24 h, with transgenic lines showing a greater increase. After 12 h and 24 h of feeding by the fall webworm, the LOX activities of OE4, OE7, and OE10 were significantly higher than those of WT, reaching their peak at 24 h. At this point, the LOX activities of OE4, OE7, and OE10 were 1.53 times, 1.42 times, and 1.53 times that of WT, respectively. Figure 7 ).

[0072] The above results indicate that PagWRKY40 can enhance the insect resistance of transgenic plants by increasing the activity of antioxidant enzymes such as CAT, PPO, and LOX.

Claims

1. The application of the PagWRKY40 gene in regulating insect resistance in poplar trees, characterized by, The nucleotide sequence of the PagWRKY40 gene is shown in SEQ ID No.

1.

2. The application of the PagWRKY40 gene according to claim 1 in regulating poplar insect resistance, characterized in that, The insect in question is the American white moth.

3. The application of the PagWRKY40 gene according to claim 1 in regulating the insect resistance of poplar trees, characterized in that, The poplar tree mentioned is an 84K poplar.

4. The application of the PagWRKY40 gene according to claim 1 in regulating poplar insect resistance, characterized in that, The application of the PagWRKY40 gene in regulating the insect resistance of poplar trees improves their insect resistance without affecting their growth.

5. The application of the PagWRKY40 gene according to any one of claims 1 to 4 in regulating insect resistance in poplar trees, characterized in that, The PagWRKY40 gene enhances the insect resistance of poplar trees through overexpression.

6. The application of the PagWRKY40 gene according to claim 1 in regulating insect resistance in poplar trees, characterized in that, The amino acid sequence of the protein encoded by the PagWRKY40 gene is shown in SEQ ID No. 2.

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