Application of PtoJAZ6 gene in regulating plant resistance to pests

Abstract

The application provides application of a PtoJAZ6 gene in regulation of plant insect resistance, and belongs to the technical field of molecular biology. The application takes a poplar endogenous insect resistance related gene PtoJAZ6 as a research object, deeply analyzes the function of the gene in poplar insect resistance response, and provides a new endogenous gene resource for poplar insect resistance breeding. This not only breaks through the limitations of traditional insect resistance breeding, but also lays a theoretical foundation for cultivating a new transgenic insect-resistant poplar variety which is efficient and safe.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of the PtoJAZ6 gene in regulating plant insect resistance. Background Technology

[0002] The relationship between plants and herbivorous insects has existed for hundreds of millions of years. In poplar plantations, methods such as spraying pesticides, light traps, or introducing natural enemies are often used to control pest populations. While these methods are effective to some extent, they also have negative environmental impacts. In contrast, strategies based on biological control and transgenic technology have greater sustainability advantages. The transgenic application of Bacillus thuringiensis (Bt) is particularly prominent. This strain can produce a toxic crystal protein that induces insects to feed on plants containing the Bt gene. The toxin enters the insect's gut, destroying intestinal epithelial cells and causing death. Because Bt protein is a toxic exogenous substance, its impact on the ecological environment, soil microorganisms, and beneficial insects requires strict management and estimation. Therefore, exploring endogenous insect-resistant gene resources in poplar has become a green and efficient new approach. With the continuous development of molecular biology, several endogenous stress-resistance genes in poplar have been successfully cloned, and their functions of insect resistance, drought resistance, and salt tolerance have been successfully verified.

[0003] Chinese patent CN117511898A discloses a plant insect resistance-related protein UGT198 and its applications, specifically disclosing the nucleic acid and protein sequences of the UGT198 gene. It also discloses the application of UGT198, specifically that transgenic plants overexpressing the UGT198 gene can improve resistance to fall webworm larvae and delay larval development. This invention focuses on the poplar endogenous insect resistance-related glycosyltransferase UGT198 gene to explore its resistance to fall webworm larvae. The results show that transgenic plants overexpressing this gene can improve resistance to fall webworm larvae and delay larval development.

[0004] However, existing technologies lack more genetic resources related to transgenic breeding of insect-resistant poplar trees. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the PtoJAZ6 gene in regulating plant insect resistance, which is of great significance for transgenic breeding of poplar trees with insect resistance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a plant quality-related gene, PtoJAZ6, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a protein encoded by the above-mentioned plant quality-related gene PtoJAZ6, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides the application of the above-mentioned plant quality-related gene PtoJAZ6, characterized in that the insect resistance of crops is improved by overexpressing PtoJAZ6.

[0010] Preferably, the method for overexpressing PtoJAZ6 includes the following steps:

[0011] The cloned PtoJAZ6 was constructed into a gene overexpression vector to obtain a recombinant vector;

[0012] The resulting recombinant vector was transformed into Agrobacterium;

[0013] Agrobacterium is inoculated into crops to achieve gene overexpression.

[0014] Preferably, the insect resistance of the crop targets pests including the fall webworm larvae.

[0015] Preferably, the plant is a poplar.

[0016] The present invention also provides an amplification primer set for detecting the above-mentioned plant quality-related gene PtoJAZ6, the primer set comprising an upstream primer and a downstream primer;

[0017] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3;

[0018] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0019] The present invention also provides an identification primer set for detecting the above-mentioned plant quality-related gene PtoJAZ6, the primer set comprising an upstream primer and a downstream primer;

[0020] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.5;

[0021] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.6.

[0022] The present invention also provides an identification primer set for detecting the above-mentioned plant quality-related gene PtoJAZ6, the primer set comprising an upstream primer and a downstream primer;

[0023] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.9;

[0024] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.10.

[0025] This invention provides the application of the above primer set in crop quality-related research, for insect resistance-assisted breeding of crops.

[0026] The beneficial effects of this invention are:

[0027] Poplar is an important economic tree species in my country, but its plantations are susceptible to insect infestation due to their simple stand structure, which seriously restricts sustainable development. Traditional breeding methods are difficult to develop new insect-resistant varieties, while the development of transgenic insect-resistant poplars often relies on exogenous genes, which has problems such as narrow insect resistance spectrum or uncertain safety, limiting their widespread application. This invention focuses on the endogenous insect-resistant gene PtoJAZ6 in poplar, deeply analyzing its function in the poplar's insect resistance response, providing a new endogenous gene resource for poplar insect resistance breeding. This not only breaks through the limitations of traditional insect resistance breeding, but also lays a theoretical foundation for the development of efficient and safe transgenic insect-resistant poplar varieties. Attached Figure Description

[0028] Figure 1 Obtaining the target gene, as shown in the figure: (A) is an RNA electrophoresis diagram; (b) is the cloning of the PtoJAZ6 gene; M: DL2000 DNA Marker (from top to bottom 2000, 1000, 750, 500, 250, 100bp); M1: 5000 DNA Marker.

[0029] Figure 2 Construction of the CAM-FLAG-GFP-PtoJAZ6 overexpression vector. Figure shows: (A) amplification of the target gene; (B) E. coli colony PCR identification; (C) Agrobacterium colony PCR identification. P: Positive control (plasmid); CK: Negative control (water); M: Maker 2000;

[0030] Figure 3 Transgenic plants were obtained. In the diagram: A: sterile seedlings; B: callus tissue; C: resistant shoots; D: rooting culture.

[0031] Figure 4 PCR detection of transgenic 84K. In the figure: M: DL2000; WT: wild type; CK: water; P: Agrobacterium tumefaciens culture.

[0032] Figure 5 The relative expression levels of the PtoJAZ6 gene in the transgenic line are shown in the figure. Different marker letters indicate significant differences (P<0.05).

[0033] Figure 6 Changes in leaf loss during forced feeding by American white moth larvae;

[0034] Figure 7 Changes in leaf loss during the selective feeding process of fall webworm larvae;

[0035] Figure 8 Enzyme activity was detected. The significance of the differences between the control group and the treatment group was analyzed separately. Different labels indicate significant differences (P<0.05). Detailed Implementation

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] The PtoJAZ6 nucleic acid sequence is shown in SEQ ID NO.1:

[0038] ATGTCTGGCTCGACGGAATTCGTTGAGAAGATGGGAAAGATGTGCGAGAAGCCGAGCTTCTCGCAAACTTGTAGTCTGTTGAGTCAATACTTGAAGGAGAGAGGTAGTTTCGGAGATCTTAATCTTGGCATGGCATCCAACAGCGAATCAACCCCGAATAAAAACGGTCCGTCTGAGATGCTGCGTCGCTCCCCGTCCACCATGAATCCGTTTCCAGTGAGCGAGAAACCAGGTCACATTTCGTGCCAAAGCATGGGGGCTCCTCGGAATTTCACGTCAATGGATTTGTTCCCTCAACAAGCTGGATTTGCTCCCAAGGAAGATGTCCCAAAGAAACTTGACTCAAGTGTTAGCAAGTCTGCCACCGCAGAACCCCAAACTGCGCAAATGACTATATTCTATGCCGGAAGAGTTATTGTCTTCAACGATTTCCCGGCTGACAAGGCTAAGGAAGTGATGCTCTTAGCCAGCAAGGGAAGCTCCCAGATCCAGAATGCTTTTCCTTCTATTCCAGCCAACAGTCACCCTGCCCTCGCTCCTAATATATCAAAAACTCCAATTGAGTCCACCATTTCAATTCCGTCTAGCTCAAATGCTCCTCCTAATTTTGGCAATAACTTGATTCAAGAGTGCATGCAACCAGCCCCTCAACCTATAGCTAATGATCTTCCAATTGCAAGGAGAGCATCCCTCCACCGTTTTTTGGAGAAGAGAAAAGACAGGATCATCGCAAAGGCTCCATACCAAATAAATCCTGCAGCAACTACGTCTAATAAGCCAGTCGAAGGCGAGTTCTCGTGGCTCGGCTTGGCTGCTCCATCTACACACTAG

[0039] >PtoJAZ6 protein sequence, as shown in SEQ ID NO.2:

[0040] MSGSTEFVEKMGKMCEKPSFSQTCSLLSQYLKERGSFGDLNLGMASNSESTPNKNGPSEMLRRSPSTMNPFPVSEKPGHISCQSMGAPRNFTSMDLFPQQAGFAPKEDVPKKLDSSVSKSATAEPQTAQMTIFYAGRV IVFNDFPADKAKEVMLLASKGSSQIQNAFPSIPANSHPALAPNISKTPIESTISIPSSSNAPPNFGNNLIQECMQPAPQPIANDLPIARRASLHRFLEKRKDRIIAKAPYQINPAATTSNKPVEGEFSWLGLAAPSTH

[0041] Example

[0042] Cloning and overexpression vector construction of the PtoJAZ6 gene

[0043] RNA was extracted from 741 Yang and reverse transcribed into cDNA. Primers for amplifying the PtoJAZ6 gene (P.x_tomentosa59633) were designed (upstream primer: TCATTTGGAGAGGACACGCATGTCTGGCTCGACGGAA, as shown in SEQ ID NO.3; downstream primer: TCGTCATCCTTGTAGTCGAAGTGTGTAGATGGAGCAGCCA, as shown in SEQ ID NO.4). The amplified product was ligated into the overexpression vector CAM-FLAG-GFP and transformed into E. coli DH5α competent cells. Positive single colonies were picked and sent to the company for sequencing. Single colonies with correct sequencing were used for propagation and plasmid extraction, and transformed into Agrobacterium GV3101 competent cells.

[0044] 84K Poplar Genetic Transformation

[0045] Agrobacterium colonies containing the PtoJAZ6 gene overexpression vector were inoculated into LB broth for propagation. The bacterial suspension was then transferred to LB broth, cultured with shaking, and colonies were collected. The bacterial cells were collected by centrifugation and resuspended in a suspension for later use. Leaves from 84K poplar seedlings were pre-cultured for 2-3 days, resulting in visibly thickened and enlarged leaves. Genetic transformation was performed using the Agrobacterium-mediated leaf disc method in a clean bench. Two to three uniform, flat incisions were made on the leaves using a sterile scalpel. The leaves were then placed in the resuspension, and AS (antibiotic oxidase) was added to promote transformation. After 10 minutes of immersion in the resuspension, the leaves were removed, blotted dry with sterile paper, and placed in a co-culture dish. The leaves were incubated in the dark at 28°C for 2-3 days until Agrobacterium was visible around the leaves. After co-culture, the leaves were transferred to a selection medium, which was changed every 7 days to prevent antibiotic inactivation. The selected resistant shoots were transferred to a rooting medium containing antibiotics. After 7 days, adventitious roots were induced to form complete plants, which were preliminarily identified as positive lines.

[0046] PCR identification of resistant strains and qRT-PCR detection of positive strains

[0047] When the plantlets in the bottle reached 10-15 cm in length, fresh, tender leaves were cut and placed in a mortar. Liquid nitrogen was added, and the mixture was ground into powder. DNA was extracted from each plantlet. Wild-type 84K poplar was used as a negative control, water as a blank control, and Agrobacterium tumefaciens solution as a positive control. PCR identification was performed on the resistant plantlets (upstream primer: AGAAGACGTTCCAACCACG, as shown in SEQ ID NO.5; downstream primer: AATCCAGCTTTGTTGAGGGAAC, as shown in SEQ ID NO.6) to confirm the presence of the PtoJAZ6 target gene, thus identifying PtoJAZ6 overexpressing transgenic positive plants. Using wild-type 84K poplar as a control, qRT-PCR was performed on the PtoJAZ6 overexpressing plantlets. The third fully expanded leaf of each plant was cut, and total RNA was extracted from both the PtoJAZ6 overexpressing plantlets and wild-type 84K poplar. This RNA was reverse transcribed into cDNA, and the relative expression level of the PtoJAZ6 gene was detected using qRT-PCR. qRT-PCR primer information is as follows:

[0048] Internal reference primers

[0049] ActinF: GAAGTCCTCTTCCAGCCTTCTC, as shown in SEQ ID NO.7

[0050] ActinR: CTTGATCTTCATGCTGCTTGGG, as shown in SEQ ID NO.8

[0051] Detection primers

[0052] J6qPCR-F: TGACAAGGCTAAGGAAGTG, as shown in SEQ ID NO.9

[0053] J6qPCR-R: TTGTTGGCTGGAATAGAAGG, as shown in SEQ ID NO.10.

[0054] PtoJAZ6 overexpression strains for insect resistance testing

[0055] Forced insect feeding: Healthy, green, and uniformly sized leaves from the transgenic strain and control (CK) were picked and placed in different dishes. Moistened filter paper was placed at the bottom of the dishes to prevent the leaves from losing water and affecting the experimental results. About 30 American white moth larvae were placed on the leaves. The growth environment was 25±1℃ and the relative humidity was 70±5%. The loss rate of poplar leaves was photographed and recorded after 0h, 12h, 24h, 36h and 48h. Each treatment was performed in 3 biological replicates.

[0056] Selective insect feeding: Young leaves of transgenic lines and CK poplars were picked and placed in the same dish. Moistened filter paper was placed at the bottom of the dish to prevent the leaves from losing water. About 30-40 American white moth larvae were placed on the leaves. The growth environment was 25±1℃ and the relative humidity was 70±5%. The loss rate of poplar leaves was photographed and recorded after 0h, 12h, 24h, 36h and 48h. Each treatment was performed in 3 biological replicates.

[0057] Assay of defense-related enzyme activity

[0058] Take leaves from wild-type 84K and transgenic lines before and after feeding insects, grind them thoroughly in a mortar, add the detection reagent, and place them in an ELISA reader for detection. For specific methods, refer to the instructions of the polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) kits from Suzhou Mingen Biotechnology Co., Ltd. Each experiment was repeated three times.

[0059] The results are as follows:

[0060] (1) Cloning of the PtoJAZ6 gene

[0061] RNA was extracted from 741 Yang and subjected to 1% gel electrophoresis for quality assessment. The electrophoresis results showed clear 28S, 18S, and 5S rRNA bands. Figure 1 A) RNA OD 260 / OD 280 The ratio is 1.9, ensuring that it is not contaminated by proteins, salt ions, organic solvents, etc., and can be used for subsequent experiments. Figure 1 B shows that the product obtained by primer PCR amplification matches the expected fragment, which is 831bp.

[0062] (2) Construction of overexpression vector

[0063] The PtoJAZ6 gene fragment was successfully amplified using PCR technology. Agarose gel electrophoresis analysis showed that the size of the target band was consistent with the expectation. Figure 2 A). After T-vector ligation and sequencing verification, the correct PtoJAZ6 gene fragment was ligated into the linearized CAM-FLAG-GFP vector and transformed into E. coli competent cells. Colony PCR was used to identify eight single colonies, and the results showed that all colonies amplified the target band in the correct position. Figure 2 B). After extracting plasmids from positive clones and transforming them into competent Agrobacterium cells, colony PCR was used for further verification. The experiment was set up with the recombinant plasmid as a positive control (P) and sterile water as a negative control (CK). The results showed that the positive control and each transformed single clone could specifically amplify the PtoJAZ6 gene fragment, with a single band and the size as expected, while the negative control showed no amplification product. Figure 2 C). The above results indicate that the recombinant expression vector CAM-FLAG-GFP-PtoJAZ6 containing the PtoJAZ6 gene has been successfully transformed into Agrobacterium, laying the foundation for subsequent genetic transformation experiments.

[0064] (3) Obtaining and detecting transgenic lines

[0065] Select well-grown 84K poplar rooted seedlings ( Figure 3 A) Infecting injured leaves with Agrobacterium and applying resistance selection pressure resulted in the formation of callus tissue at the wound site. Figure 3 B). Plants transformed with the overexpression vector grew normally. Figure 3 C), while the lines without the vector turned white and died during antibiotic selection. After the resistant shoots were transferred to a rooting medium containing antibiotics, the successfully transformed lines grew adventitious roots, and six transgenic lines were initially obtained ( Figure 3 D).

[0066] DNA was extracted from six transgenic 84K lines, and PCR amplification was performed using specific primers (upstream primer AGAAGACGTTCCAACCACG, as shown in SEQ ID NO.11; AATCCAGCTTTGTTGAGGGAAC, as shown in SEQ ID NO.12), detecting the 450bp target band. Gel electrophoresis results showed that all lines exhibited a clear band at 450bp (P is the positive control of PtoJAZ6 Agrobacterium tumefaciens culture), confirming that the PtoJAZ6 gene had been successfully integrated into the 84K poplar genome. These six transgenic lines were named JAZ6-3, JAZ6-6, JAZ6-8, JAZ6-11, JAZ6-12, and JAZ6-13.

[0067] Total RNA was extracted from wild-type (WT) 84K poplar and transgenic lines, reverse transcribed into cDNA, and then analyzed by qRT-PCR using specific primers. Figure 5 As shown, compared with the wild type, the expression level of PtoJAZ6 was significantly upregulated in all overexpressing transgenic lines. Among them, JAZ6-3, JAZ6-8, and JAZ6-12 had the highest PtoJAZ6 expression levels, approximately 17 times that of the wild type. Therefore, these three high-expression lines were selected for subsequent experimental studies.

[0068] (4) Insect resistance analysis of PtoJAZ6 overexpression lines

[0069] To investigate the insect-resistant function of the PtoJAZ6 gene, leaves from high-expression lines JAZ6-3, JAZ6-8, JAZ6-12, and wild-type (WT) 84K poplar were selected and placed in petri dishes. Each dish was inoculated with 30-40 fall webworm larvae for a 48-hour forced insect-feeding experiment, and leaf damage was recorded every 12 hours. The results showed ( Figure 6 The transgenic lines showed significantly lower leaf loss rates than the wild type, with JAZ6-3 and JAZ6-12 exhibiting the least leaf area loss. Furthermore, the activity of larvae feeding on the transgenic leaves was significantly reduced. These results indicate that overexpression of the PtoJAZ6 gene can enhance the insect resistance of Populus 84K.

[0070] To assess the feeding preference of fall webworm larvae for transgenic plants, leaves from JAZ6-3, JAZ6-8, JAZ6-12, and wild-type (WT) plants with uniform growth were placed in the same petri dish for a 48-hour selective feeding experiment (each group was repeated three times). Leaf damage was recorded every 12 hours during the experiment. Figure 7 The results showed that the larvae significantly preferred to feed on WT leaves, while the leaves of the transgenic line (JAZ6-3 / 8 / 12) were hardly consumed, indicating that overexpression of the PtoJAZ6 gene can significantly reduce the attractiveness of leaves to larvae, further verifying its insect-resistant function.

[0071] (5) Analysis of defensive enzyme activity under insect pest stress

[0072] Polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) play important roles in plant insect resistance. To investigate the insect-resistant mechanism of the PtoJAZ6 gene, the activities of PPO and PAL in transgenic lines JAZ6-3 and JAZ6-12 before and after insect feeding treatment were detected. The results showed that ( Figure 8 Compared with the untreated control group, the activities of PPO and PAL in the leaves of the experimental group fed by larvae were significantly increased, indicating that PtoJAZ6 may enhance the resistance of Populus 84K to insects by activating plant defense-related enzyme systems.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the plant quality-related gene PtoJAZ6, characterized in that, Insect resistance in crops can be enhanced by overexpressing PtoJAZ6; The nucleotide sequence of the plant quality-related gene PtoJAZ6 is shown in SEQ ID NO.1; The insect resistance of the crop is against the fall webworm larvae. The crop in question is poplar.

2. The application of the plant quality-related gene PtoJAZ6 according to claim 1, characterized in that, The method for overexpressing PtoJAZ6 includes the following steps: The cloned PtoJAZ6 was constructed into a gene overexpression vector to obtain a recombinant vector; The resulting recombinant vector was transformed into Agrobacterium; Agrobacterium is inoculated into crops to achieve gene overexpression.

3. The application of any of the following primer sets in crop quality-related research, characterized in that: Assisted breeding for resistance to fall webworm larvae in poplar trees: The amplification primer set for detecting the plant quality-related gene PtoJAZ6 includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.4; The amplification primer set for detecting the plant quality-related gene PtoJAZ6 includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.5 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.6; The amplification primer set for detecting the plant quality-related gene PtoJAZ6 includes an upstream primer with the nucleotide sequence shown in SEQ ID NO.9 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.10; The nucleotide sequence of the plant quality-related gene PtoJAZ6 is shown in SEQ ID NO.1.

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