Apple laccase LAC18 gene and aphid-resistant application of apple laccase LAC18 gene in catalytic synthesis of plant lignin

Abstract

The invention provides a laccase gene LAC18 screened from apples and aphid resistance application of the laccase gene LAC18 in catalytic synthesis of plant lignin, the nucleotide sequence of the gene is SEQ ID NO: 1, and the amino acid sequence of encoded protein of the gene is SEQ ID NO: 2. According to the method, the aphid resistance of the transgenic tobacco is comprehensively evaluated by inoculating aphids on leaves of the transgenic laccase gene tobacco and observing indexes such as the survival rate and the aphid yield. The result shows that the laccase activity and the lignin content of the transgenic laccase tobacco are remarkably improved, the lignin of cell walls is thickened, and the lignification degree is remarkably improved. The translaccase gene tobacco not only can effectively reduce the survival rate of myzus persicae, but also has a remarkable inhibition effect on the average reproductive capacity of the myzus persicae, and the aphid resistance of plants is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically relating to an apple laccase LAC18 gene and its catalytic synthesis of plant lignin for aphid resistance. Background Technology

[0002] Aphids are a serious type of piercing-sucking pest in agricultural production. Among them, the woolly apple aphid and the tobacco aphid pose significant threats to economic crops such as apples and tobacco, respectively. China, as the core area of ​​global apple production, contributes over 80% to the world's apple production increase. However, the woolly apple aphid, an invasive pest, sucks sap from fruit trees, causing wart-like protrusions at the affected sites, covering branches and leaves with a white, cottony substance, significantly hindering photosynthesis and causing economic losses to the apple industry of up to 30%. The tobacco aphid has a wider host range, parasitizing not only tobacco but also vegetables, fruit trees, and many other crops. It not only affects crop growth by robbing nutrients but also transmits various plant viruses. In the tobacco planting sector alone, the annual yield loss and control costs due to tobacco aphids exceed hundreds of millions of yuan, becoming a key bottleneck restricting high-quality and high-yield tobacco production.

[0003] Currently, chemical control remains the primary method for controlling apple woolly aphids and tobacco aphids in agricultural production, achieved through the application of insecticides. However, chemical control has several unavoidable drawbacks: long-term use of chemical pesticides easily leads to aphid resistance, causing aphids to become less effective year by year, forcing farmers to increase the dosage and frequency of pesticide use; pesticide residues pollute the soil, water sources, and other ecological environments, while also harming human health; furthermore, broad-spectrum insecticides, while killing aphids, also accidentally kill aphids' natural enemies, disrupting the ecological balance of farmland and further exacerbating the risk of pest outbreaks. Yellow sticky traps, as a common physical control measure, have the advantage of not polluting the environment, but this method lacks species specificity, attracting a large number of aphid natural enemies such as parasitic wasps while trapping aphids, which greatly limits its practical application in production.

[0004] Given the significant shortcomings of existing control measures, finding efficient, environmentally friendly, and sustainable new aphid control technologies has become a critical research bottleneck in the field of agricultural pest and disease management. Against this backdrop, developing biological control technologies utilizing plants' own insect resistance mechanisms has become a highly promising research direction. Plant insect resistance mechanisms often exhibit cross-species universality, providing a theoretical basis for the cross-crop application of insect-resistant genes. Discovering aphid-resistant functional genes from highly resistant apple varieties holds promise for providing a novel approach to tobacco aphid control in other crops such as tobacco. Furthermore, there are significant differences in aphid resistance among different apple varieties. Among the main cultivated varieties, the New Red Star shows relatively strong resistance to the woolly apple aphid, while Red Fuji is a highly susceptible variety. This difference in resistance provides an ideal material basis for screening aphid-resistant genes. Summary of the Invention

[0005] The purpose of this invention is to provide an apple laccase LAC18 gene and its catalytic synthesis of plant lignin for aphid resistance, thereby overcoming the shortcomings of the prior art.

[0006] The LAC18 gene screened from apples provided by this invention encodes a protein with the following amino acid sequence: (SEQ ID NO: 2); The gene in question has the following specific nucleotide sequence: The present invention also provides a recombinant expression vector that can express exogenous genes in plant cells and plants, wherein nucleotide fragments of the above-mentioned genes are inserted therein; In another aspect, the present invention provides an Agrobacterium, wherein the above-described recombinant expression vector is carried.

[0007] The present invention also provides an application of the gene in increasing the lignin content of plants.

[0008] In another aspect, the present invention also provides an use of the said gene in improving tobacco resistance to tobacco aphids; In another aspect, the present invention provides a method for improving tobacco antagonism against tobacco aphids, wherein the method involves increasing the expression level of the gene in tobacco. Furthermore, the method involves using the aforementioned Agrobacterium to infect the leaves of tobacco tissue culture seedlings, and after a series of cultivation processes, obtaining tobacco plants with the laccase gene.

[0009] This invention involves inoculating tobacco leaves with a laccase gene and observing indicators such as survival rate and aphid production to comprehensively evaluate the aphid resistance of transgenic tobacco. Results showed that the laccase activity and lignin content of the transgenic tobacco were significantly increased, as were the lignin thickening of the cell walls and the degree of lignification. The transgenic tobacco not only effectively reduced the survival rate of tobacco aphids but also significantly inhibited their average reproductive capacity, greatly improving the plant's aphid resistance. Attached Figure Description

[0010] Figure 1 Cloning of the new red star laccase gene and EGFP gene; where in the figure: A. Laccase gene clone; M. DL2000 Marker; 1. A. Laccase gene. B. Predicted tertiary structure of the laccase gene; C. EGFP gene cloning; M. DL2000 Marker; Figure 2 pBI121-LAC18 recombinant vector map; Figure 3 : Screening of positive clones of recombinant vectors; where in the figure: A. positive screening verification of pBI121-Lac18; M. DL2000 Marker; 1. pBI121-LAC18; B. positive screening verification of pBI121-GFP; M. DL2000 Marker; 1-2. pBI121-GFP; Figure 4 Microscopic observation of leaf sections from tobacco plants transgenic with the EGFP gene; Figure 5DNA level identification diagram of LAC18 transgenic plants; where: M.DL5000 Marker; 1. Negative control; 2-9. Identification results of pBI121-LAC18 transgenic tobacco plants; Figure 6 : Schematic diagram of the tobacco seedling hardening process in Benedict's tobacco; Figure 7 ; RNA level identification diagram of transgenic plant LAC18; Figure 8 Image showing the growth of LAC18 genetically modified tobacco mother plants after one month of hardening-off; Figure 9 : Growth dynamics of LAC18 transgenic tobacco and conventional tobacco T1 generation; where: A. Growth at 30 days B. Growth at 45 days C. Growth at 60 days D. Plant height measurement within 90 days, t-test analysis for significance of differences; *: Significant difference at p<0.05 level; Figure 10 A comparative graph showing the EPG levels of tobacco aphids feeding on the phloem of transgenic tobacco plants; where a represents common tobacco and b represents transgenic tobacco. Figure 11 Figure: Survival rate and aphid production of tobacco aphids in LAC18 transgenic tobacco plants; Figure 12 : Graph showing the detection of laccase activity in transgenic tobacco; where: t-test analysis was used to determine the significance of differences; *: significant difference at the p<0.05 level; Figure 13 Figure: Determination of lignin content in genetically modified tobacco; where: t-test analysis shows significant differences; *: significant differences at the p<0.05 level; Figure 14 Photograph of a stained section of stem from a genetically modified tobacco plant. Detailed Implementation

[0011] Using apple varieties resistant to the woolly apple aphid as research material, the inventor's team combined transcriptomic and proteomic analysis and other techniques to screen a key laccase gene from the lignin synthesis pathway. To clarify the aphid-resistant function and application value of this gene, the inventor introduced it into tobacco through transgenic technology. The study confirmed that this gene can effectively promote the synthesis of lignin in tobacco and significantly improve the tobacco's resistance to tobacco aphids by enhancing the physical barrier function of the cell wall.

[0012] The apple laccase gene and its aphid-resistant application method screened in this study break through the limitations of existing aphid control technologies and provide a new, efficient, and environmentally friendly technical solution for the sustainable management of plant aphids. This has important theoretical and practical significance for promoting the breeding of new aphid-resistant crop varieties and ensuring safe agricultural production.

[0013] In the examples provided in the instruction manual, sterile *Nicotiana benthamiana* were cultured in a tissue culture room at 24±2℃, relative humidity of 50±10%, and photoperiod (light:dark) of 16:8; experimental tobacco aphids were raised in an intelligent artificial climate chamber at 25±2℃, relative humidity of 60±10%, and photoperiod (light:dark) of 16:8; and soil-grown tobacco seedlings obtained from the sowing and growth of *Nicotiana benthamiana* seeds were used for propagation.

[0014] The following information is provided for the instruments and equipment used: FastPure Plant DNA Isolation Mini Kit, QuickCut™ Xba |, and QuickCut™ Sac | were all purchased from Takara Bio Engineering (Dalian) Co., Ltd.; 2×RealStar Fast SYBRqPCR Mix was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.; EHA105 Agrobacterium competent cells G6046 were purchased from Shanghai Angyu Biotechnology Co., Ltd.; Kanamycin (50 mg / mL), Cephalosporin (200 mg / mL), Rifampin (50 mg / mL), MS medium, 6-BA, and other tissue culture reagents were purchased from Beijing TransGen Biotech Co., Ltd.; Silver colloid was purchased from Professor Yan Fengming of Henan Agricultural University; Laccase activity assay kit was purchased from Beijing Solarbio Biotechnology Co., Ltd.; Lignin content assay kit was purchased from Suzhou Greens Biotechnology Co., Ltd.; Lignin staining solution (phloroglucinol method) was purchased from Beijing Regen Biotechnology Co., Ltd.

[0015] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0016] Example 1: Screening and Cloning of the Apple LAC18 Gene Three *Acer rubrum* 'New Red Star' apple trees with uniform growth were selected. The second leaf, extending downwards from the top of each shoot, was aseptically cut and stored at 4℃ for later use. Total RNA was extracted from the leaves using an RNA kit, and cDNA was synthesized via reverse transcription. Homologous recombination primers (Table 1) were designed using the Novizuma single-fragment cloning website, as referenced to the target gene and vector sequences, and synthesized by Sangon Biotech, with EGFP as a technical control. Using cDNA as a template, PCR amplification was performed using 2×Phanta Max Master Mix (Dye Plus) with the primers in Table 1. The reaction system contained 25 µL of Mix, 2 µL each of forward and reverse primers, 1 µL of cDNA, and 25 µL of ddH2O. The program was: 95℃ pre-denaturation for 3 min, followed by 35 cycles of 95℃ for 15 s, 55℃ for 15 s, and 72℃ for 1 min / kb, with a final extension at 72℃ for 5 min, and then storage at 4℃. The target gel block was weighed, purified from the gel, and the PCR product was used for downstream experiments.

[0017] Table 1: Primer list for gene cloning containing homologous arms

[0018] Figure 1 Figure A indicates that the laccase gene was successfully cloned at approximately 2000 bp. Sequencing revealed its full length to be 1752 bp (SEQ ID NO:1), encoding 583 amino acids (SEQ ID NO:2), with a predicted molecular weight of 64418.28 Da. Amino acid sequence analysis showed the presence of a signal peptide structure, with the predicted cleavage site located at amino acids 31-32 (predicted probability 0.867). CD-search was used to predict the functional domains of the SM-LAC18 gene, revealing three functional domains: CuRO_1_LCC_plant (positions 37-153), CuRO_2_LCC_plant (positions 168-316), and CuRO_3_LCC_plant (positions 429-566). Tertiary structure prediction is shown below. Figure 1 B, Figure 1 C cloned the EGFP gene, with the size matching the preset value, serving as a technical control.

[0019] Example 2: Construction of plant overexpression vectors Use QuickCut™ Xba | QuickCut™ Sac | The pBI121 plasmid was linearized by double enzyme digestion. The reaction products were purified and recovered using a gel extraction kit, and the purified linearized DNA was identified by agarose gel electrophoresis. The linearized products were used directly for subsequent ligation reactions or stored at -20°C for later use.

[0020] The ligation reaction was performed using the purified PCR product of the target gene and the linearized vector product, following the instructions in the ClonExpress® Ⅱ OneStep Cloning kit C112. The vector pattern is shown below. Figure 2 As shown. The product was transformed into DH5α competent cells using a heat shock method. PCR reaction system was prepared according to 2×Taq Plus Master Mix II (Dye Plus), and PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis.

[0021] Based on the gel electrophoresis results, the correct positive clones were selected and sent to Qingdao Qingke Biotechnology Co., Ltd. for sequencing to further confirm that the constructed vector was accurate.

[0022] LAC18 and pBI121 were ligated using the Novavitt recombinant kit, and the correct recombinant expression vectors pBI121-Lac18 and pBI121-EGFP were obtained after PCR verification and sequencing. Figure 3pBI121-Lac18 and pBI121-EGFP were transformed into EHA105 cells using a freeze-thaw method. PCR confirmed the successful construction of EHA105-pBI121-Lac and EHA105-pBI121-EGFP, and the bacterial cultures were preserved for subsequent experiments.

[0023] Example 3: Agrobacterium-mediated genetic transformation of tobacco leaf discs Positive clones with correct sequencing were cultured in a shaker and plasmids were extracted. Transformation was performed according to the EHA105 Agrobacterium competent cell instructions. The transformation solution was plated on plates containing Kan and Rif, and incubated upside down at 28°C for 48 h. The next day, single clones were selected for colony PCR identification. Successful Agrobacterium clones were used directly in experiments or stored at -80°C with 25% glycerol.

[0024] The seeds of Nicotiana benthamiana were disinfected with 75% ethanol and 2.5% NaClO for 30 seconds, rinsed three times with sterile water, and then air-dried. They were inoculated into MS medium and cultured aseptically under light at 25°C for 4 weeks. Leaves were then used for transformation.

[0025] First, activate Agrobacterium to OD. 600 =0.8~1.2. The leaf disc method of Horsch et al. (1985) was used for transformation. The leaves were made into leaf discs with a radius of 5 mm and pre-cultured in MS medium containing 1 mg / L 6-BA for 2 days. They were then infected with Agrobacterium tumefaciens for 3-4 min, blotted dry with sterile filter paper, and cultured in the dark for 4 days. They were then transferred to differentiation medium containing 1 mg / L 6-BA, 200 mg / L Cef and 50 mg / L Kan for selection culture. After the regenerated shoots grew, they were transferred to rooting medium containing 200 mg / L Cef and 50 mg / L Kan. After the root system was well developed, they were transplanted to nutrient soil for hardening.

[0026] Example 4: Identification and Screening of Genetically Modified Tobacco Leaves were collected from transgenic and control tobacco plants that had been hardened off for one month. DNA was extracted from the tobacco plants using the FastPure Plant DNA Isolation Mini Kit, and PCR detection was performed using the M13 universal primers (Table 2). The PCR results were then detected by 1% agarose gel electrophoresis to identify positive transgenic tobacco plants.

[0027] Table 2: Primer list for identifying transgenic tobacco

[0028] Based on the DNA level identification results, RNA was extracted from positive plants and cDNA was synthesized by reverse transcription. The expression level of the target gene in transgenic tobacco plants was detected using qPCR. Primers used for qPCR are listed in Table 2. The quantitative reaction system consisted of 1 μL sample, 5 μL of 2×RealStar Fast SYBR qPCR Mix, 0.25 μL of q-Lac18–F (10 μM), 0.25 μL of q-Lac18 R (10 μM), and 3.5 μL of ddH2O, for a total volume of 10 μL. The qPCR reaction program was: 95℃ for 30 s, 95℃ for 5 s, and 60℃ for 30 s, for a total of 39 cycles; Melt Curve: 65℃ to 95℃, increment 0.5℃, 5 s. Based on the data processing results, plants with high expression levels and good growth were selected for subsequent aphid resistance assays.

[0029] The results showed that, under a fluorescence microscope, obvious green fluorescence signals were visible in the villi region of EGFP-transgenic tobacco leaves. Figure 4 (Right), while the control group (non-standard tobacco eye patches) showed no green fluorescence signal ( Figure 4 (Left). The results show that the experimental technique is reliable. After 45 days of culture, sterile tobacco seedlings underwent pre-culture, Agrobacterium infection, dark culture, and differentiation screening to obtain regenerated shoots and transgenic plants. DNA identification yielded 8 LAC18-positive plants (…). Figure 5 ).

[0030] After obtaining the transgenic plants, they were hardened off and cultured in an indoor natural environment for 7 days. Figure 6 The tobacco plants were removed from MS medium, cleaned, and transplanted into potting soil to continue growing under natural conditions.

[0031] One month after hardening off, RNA was extracted from the plants, and the expression of the target gene was detected using quantitative real-time PCR. The results showed that the expression levels in the four LAC18 transgenic tobacco plants were significantly upregulated, increasing by 18604-fold, 52711-fold, 21373-fold, and 62523-fold compared to the WT plants. Figure 7 Through qualitative and quantitative analysis, the optimal LAC18 transgenic tobacco plants were selected for subsequent experiments.

[0032] A 30-day observation of transgenic tobacco seedlings revealed that the laccase gene-transgenic tobacco plants exhibited significantly better growth than the control group. Figure 8The effects were observed in tobacco plants transgenic with the laccase gene: faster growth rate, more vigorous above-ground growth, larger and greener leaves, and increased branching. After seed production, seeds of LAC18 tobacco (Nicotiana benthamiana) were collected to obtain the T1 generation. Positive transgenic lines were screened by PCR after sowing, and the growth of transgenic SM-LAC18 plants and ordinary tobacco plants was observed at 30, 45, and 60 days. Figure 9 The study found that the LAC18 gene-transformed lines exhibited significant growth advantages throughout the entire growth period, with more robust plant type and faster leaf development. After 45 days of cultivation, the laccase gene-transformed tobacco reached a height of 9.97 cm (p<0.05), an increase of 65.3% compared to the control. This result indicates that the introduction of the LAC18 gene significantly promoted the growth and development of Nicotiana benthamiana, providing a basis for further research on laccase function.

[0033] Example 5: Determination of Aphid Resistance in Transgenic Tobacco 1. Comparison of EPG indices of tobacco aphids feeding on tobacco plants with translaccase gene. To investigate the feeding behavior of tobacco aphids on transgenic tobacco, adult aphids were used in an EPG experiment. The Giga-8dd DC-EPG system was used to record the potential changes during aphid probing and feeding on plants in Faraday cages. Each sample was tested 15 times, with one aphid per plant, and each test lasted 6 hours. Valid data were identified and labeled using the Stylet+a system according to Tjallingii (1988) criteria: non-probing wave (np), intercellular pathway wave (C), mechanical damage wave (pd), phloem salivary secretion wave (E1), phloem continuous feeding wave (E2), and xylem feeding wave (G). Independent samples t-tests were performed on the EPG data of tobacco aphid feeding on different tobacco varieties using SPSS 25.0 software.

[0034] From the non-phloem stage of tobacco feeding by tobacco aphids, when feeding on tobacco plants transgenic with the SM-LAC18 gene, the number of probing attempts, the time of the first probing attempt, the total time of the np wave, the total time of the G wave, and the number of pd waves were all significantly higher than those of ordinary tobacco. There was no significant difference in the total time of the C wave between the two types of tobacco (Table 3).

[0035] Table 3: Comparison of EPG indices from tobacco aphids feeding on the non-phloem of laccase-transgenic tobacco plants.

[0036] The data in the table are mean ± standard error. * after each other indicates that the differences are statistically significant at the p < 0.05 level according to the independent samples t-test.

[0037] From the perspective of the phloem stage of tobacco feeding by tobacco aphids, when tobacco aphids feed on LAC18 gene-transformed tobacco plants, the total time of E1 wave, the proportion of E1 in the total waveform, the time taken to first reach the phloem, and the time of the first appearance of E2 wave are all significantly higher than those of ordinary tobacco; the total time of E2 wave, the proportion of E2 in the total waveform, and the proportion of E2 > 10 min are all significantly lower than those of ordinary tobacco (Table 4).

[0038] Table 4: Comparison of EPG levels in tobacco aphids feeding on the phloem of transgenic tobacco plants.

[0039] The data in the table are mean ± standard error. * after each other indicates that the differences are statistically significant at the p < 0.05 level according to the independent samples t-test.

[0040] In the EPG waveform, the E waveform is divided into E1 and E2 waveforms. E1 waveform indicates that the aphid is secreting saliva from its stylet in preparation for feeding; E2 waveform indicates that the aphid is actually feeding. Analysis of the time proportions of each waveform when aphids feed on SM-LAC18 transgenic tobacco and regular tobacco revealed that when aphids feed on SM-LAC18 transgenic tobacco, the total time of the G wave was 3.76%, lower than that of regular tobacco (8.35%); the total time of the E1 wave (saliva secretion in the phloem) was 1.30%, higher than that of regular tobacco (0.54%); the total time of the E2 wave (feeding in the phloem) was 0.23%, lower than that of regular tobacco (4.34%); and the time proportion of the E wave (E1+E2) was 1.53%, lower than that of regular tobacco (4.88%). Figure 10 The above data indicate that on genetically modified tobacco, tobacco aphids need to spend more time finding suitable feeding sites. After finding a suitable site, they secrete saliva for a longer time and need to secrete more saliva to digest food, but the feeding time is shorter, suggesting that genetically modified laccase tobacco is not conducive to tobacco aphid feeding.

[0041] 2. The effect of tobacco laccase gene transfection on the survival and reproduction of tobacco aphids. Tobacco leaves containing the laccase gene were harvested, and the petioles were inserted into petri dishes containing 25 mL of agar medium. Six second-instar female tobacco aphids were inoculated onto each leaf, with 15 replicates. Common tobacco was used as a control. From inoculation, the number of surviving aphids and the amount of aphids laid were observed and recorded daily for 7 days. After the experiment, the survival rate of tobacco aphids was analyzed using a log-rank test using GraphPad Prism 8 software, and the amount of aphids laid was analyzed using an independent samples t-test to investigate the effects of different tobacco varieties on the survival and reproduction of tobacco aphids.

[0042] result: Figure 11The results showed that the survival rate of tobacco aphids on day 8 in transgenic SM-LAC18 tobacco (18.33%) was significantly lower than that in conventional tobacco (32.73%). Regarding reproduction, the average aphid production on transgenic SM-LAC18 tobacco plants (16.57 aphids) was significantly lower than that in the control group (36.93 aphids); however, the maximum aphid production (51 aphids) did not show a significant difference compared to the control group (72.67 aphids). This indicates that transgenic LAC18 significantly inhibits the average reproductive capacity of tobacco aphids while reducing their survival rate.

[0043] Example 6: Detection of laccase activity in transgenic tobacco To quantitatively determine laccase activity in tobacco leaves, this experiment used the Solarbio laccase activity assay kit and employed the microplate method. 0.1 g of transgenic laccase and ordinary tobacco leaves were accurately weighed and placed separately in an ice bath. 1 mL of laccase extraction solution was added, and the samples were thoroughly homogenized using a homogenizer. Subsequently, the homogenized samples were centrifuged at 10000 g for 10 min at 4°C. The supernatant was carefully collected and placed on ice for subsequent analysis.

[0044] Laccase reacts specifically with the ABTS substrate in the kit to generate ABTS radicals. The rate of ABTS radical generation is dynamically monitored at 420 nm using a microplate reader. Enzyme activity is defined as the amount of enzyme required to oxidize 1 nmol of ABTS substrate per minute per gram of sample. The formula for calculating laccase activity (U / g) is as follows: ΔA ÷ (ε × d) × V1 × 10 9 ÷ (V2 × W ÷ V3) ÷ T = 61.7 × ΔA ÷ W, where ΔA is the absorbance difference, ε is the ABTS molar extinction coefficient, d is the cuvette path length, V1 is the total reaction volume, V2 is the sample volume in the reaction, V3 is the volume of extract added, W is the sample mass, and T is the reaction time. A t-test was performed on the data using GraphPad Prism 8 to analyze the significance of the differences.

[0045] result: Figure 12 The results showed that the average laccase activity of common Nicotiana benthamiana was approximately 53.70 u / g. After the LAC18 gene was introduced, the average laccase activity of tobacco increased to approximately 85.71 u / g, representing an overall increase of approximately 59.61% in laccase activity compared to the wild type.

[0046] Example 7: Determination of Lignin Content in Transgenic Tobacco This experiment used the Suzhou Greens Lignin Content Assay Kit to determine the total lignin content in tobacco leaves containing the laccase gene. The acetylation method was employed for detection, with non-transgenic tobacco serving as a control. Tobacco leaves with synchronized growth cycles and at the same location on the plant were selected. Reagent 1, Reagent 2, and acetic acid were added sequentially to the treated leaf samples. After thorough mixing, the samples were placed in a microplate reader, and the absorbance at 280 nm was analyzed. The total lignin content of the tobacco leaves was calculated according to the standard curve in the kit instructions: Lignin (mg / g) = [(ΔA + 0.003) ÷ 10.615] × V1 ÷ W × 2, where ΔA is the absorbance difference, V1 is the total volume after finalization, and W is the sample weight. A t-test was performed using GraphPad Prism 8 to analyze the significance of differences.

[0047] Results: Data as follows Figure 13 The results showed that the average lignin content in wild-type Nicotiana benthamiana leaves was approximately 54.92 mg / g. After transfection with the SM-LAC18 gene, the average lignin content in the leaves increased to approximately 80.95 mg / g, representing an overall increase of approximately 47.40% compared to the wild type. Based on these results, it is inferred that overexpression of the SM-LAC18 gene significantly promotes lignin biosynthesis in Nicotiana benthamiana leaves.

[0048] Example 8: Tissue structure changes of lignin in the stems of transgenic tobacco plants To observe the changes in the lignin structure of the cell walls of tobacco plants that have undergone laccase gene modification, this study used a lignin staining kit based on the phloroglucinol method. Translaccase-modified tobacco plants and non-transgenic tobacco plants with similar physiological conditions and approximately 8 weeks of growth were selected. Stems from the same location were cut and slides were prepared. Reagents A and B were added sequentially, allowing the reagents to fully react with the samples. After standing for 3 minutes, a coverslip was placed on top. The samples were then observed under a microscope.

[0049] Result: As Figure 14 The results showed that the lignin in non-GMO tobacco stained lightly, appearing reddish-purple; while the lignin in SM-LAC18 transgenic tobacco stained much darker, with a significantly larger stained area, especially prominent in the vascular tissue. The changes in the lignin structure of the stems indicated that, compared to non-GMO tobacco, GMO tobacco had a significantly higher lignin content and more developed vascular tissue.

[0050] Tobacco plants transgenic with the apple laccase gene (LAC18) were successfully obtained using Agrobacterium-mediated transformation, and their genetic transformation, aphid resistance function, and mechanism were systematically analyzed. At the DNA level, PCR detection showed a 100% positive transformation rate for the LAC18 gene. This is partly due to the high efficiency of exogenous gene integration in *Nicotiana benthamiana* as a genetic transformation recipient material. Furthermore, parameters such as Agrobacterium activity, OD value (optical density value) of the infection solution, pre-culture state of the leaf disc, and infection time significantly influenced the transformation efficiency during the transformation process. The study introduced the EGFP gene as a visual reporter system; its fluorescence characteristics are not limited by the host species and do not require substrate assistance, providing an efficient and non-destructive detection method for transgenic identification.

[0051] This invention uses aphid inoculation on leaves to observe indicators such as survival rate and aphid production, comprehensively evaluating the aphid resistance of transgenic tobacco. The results show that LAC18 transgenic tobacco not only effectively reduces the survival rate of tobacco aphids but also significantly inhibits their average reproductive capacity.

Claims

1. A gene screened from apples, characterized in that, The amino acid sequence of the protein encoded by the gene is SEQ ID NO:

2.

2. The gene as described in claim 1, characterized in that, The gene in question has the nucleotide sequence SEQ ID NO:

1.

3. A recombinant expression vector capable of expressing exogenous genes in plant cells and plants, characterized in that, The recombinant expression vector contains an inserted nucleotide fragment of the gene described in claim 1.

4. An Agrobacterium, characterized in that, The Agrobacterium carries the recombinant expression vector as described in claim 3.

5. The application of the gene described in claim 1 in increasing the lignin content of plants.

6. The application of the gene of claim 1 in improving tobacco resistance to tobacco aphids.

7. A method for improving tobacco's resistance to tobacco aphids, characterized in that, The method is to increase the expression level of the gene of claim 1 in tobacco.

8. The method as described in claim 7, characterized in that, The method involves using Agrobacterium as described in claim 4 to infect tobacco leaves.

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