An apple laccase LAC3 gene and its application in improving plant resistance to aphids

Abstract

The present invention provides an apple laccase LAC3 gene and its application in improving plant aphid resistance. The apple laccase LAC3 gene was cloned in the present invention, its nucleic acid sequence and protein structure were clarified. On this basis, primers were designed, an overexpression vector was constructed, and through tobacco tissue culture seedling cultivation and Agrobacterium-mediated transformation, it was transferred into tobacco to obtain transgenic plants, and homozygous lines were obtained through screening. The feeding behavior of aphids was observed by EPG technology, and aphids were inoculated to study and clarify its aphid resistance function. Compared with the wild type, the growth density of the above-ground part of the transgenic plants is greater than that of the wild type, the stems are thick, the lignin content increases, and it plays a good role in resisting aphids, providing a theoretical basis for molecular improvement breeding of aphid resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant pest control, and particularly relates to an apple laccase LAC3 gene and its application in improving plant aphid resistance. Background Art

[0002] Aphids are important agricultural pests, and the woolly apple aphid is a major fruit tree pest. For apple trees infected with the woolly apple aphid, white wool-like substances will adhere to the infected parts. The fruit trees are damaged and the yield is reduced by 10%-30%. It can weaken the tree vigor, the fruit quality is poor, and in severe cases, the fruit trees will wither and die, causing huge losses every year.

[0003] Chemical control is an effective method for controlling aphid pests such as the woolly apple aphid, but it is prone to the 3R problem; in addition, chemical control brings a certain economic pressure to fruit farmers, ultimately leading to a decline in the overall economic benefits of fruit farmers. Therefore, alternative aphid control methods to chemical control need to be developed urgently.

[0004] Plant insect-resistant breeding and improving the plant's own resistance to pests are sustainable development strategies for controlling agricultural pests. Clarifying the insect-resistant characteristics of plants and excavating the insect-resistant genes of plants are the key issues in insect-resistant breeding. However, apples are woody plants, and it is difficult to screen for insect-resistant genes in woody plants. The development of gene function verification technology using transgenic technology is relatively slow, and coupled with the difficulty in cultivating woody plants, the progress in screening and utilization of apple insect-resistant genes is slow, which restricts the sustainable management and development of orchard pests in apple orchards. There is an urgent need to excavate insect-resistant genes and utilize the natural insect resistance of apples to sustainably manage orchard pests. Summary of the Invention

[0005] The purpose of the present invention is to provide an apple laccase LAC3 gene and its application in improving plant aphid resistance. The present invention cloned the apple laccase LAC3 gene, clarified its nucleic acid sequence and protein structure, designed primers on this basis, constructed its overexpression vector, transferred it into tobacco through tobacco tissue culture seedlings and Agrobacterium-mediated transformation, obtained transgenic plants, and screened to obtain a homozygous strain system. The feeding behavior of aphids was observed by EPG technology, and aphids were inoculated to study and clarify its aphid resistance function. Compared with the wild type, the growth density of the above-ground part of the transgenic plant is greater than that of the wild type, the stem is thick, the lignin content increases, and it plays a good role in aphid resistance, providing a theoretical basis for molecular improvement breeding of apple aphid resistance.

[0006] The present invention first provides an apple laccase LAC3 gene, and the gene encodes:

[0007] 1) A protease with an amino acid sequence of SEQ ID NO:1

[0008] MEALNAIFVNKIRFTLCLFGLCLLVASSTMSLAEPQIHKHEFVVQATPVKRLCKTQNSITVNGQLPGPTLEVNNGDTLVVKVTNRAQYNVTI

[0009] HWHGIRQMRTGWADGPEFVTQCPIRPGGSYTYRFTIQGQEGTLWWHAHSSWLRATVYGALIIHPKQGDSYPFTKPKSETTLLLGEWWNANPI

[0010] DVLRQATMTGAAPNVSDAYTINAQPGDLYNCSSQDTVIVPIDSGETNLLRVINAALNQPLFFSVANHKLTVVSADASYTKPFTTTVLMLGPG

[0011] QTTDVLITGDQSPARYYLAASAYFSAQNAAFDNTTTTAILEYKSAPCSPNCKNSSAAKPIMAQLPAFNDTNTASAFTKSFRSPRKVEVPTEI

[0012] DENLFFTIGLGLNNCPKNFGSQRCQGPNGTRFTASMNNVSFVLPNNISILQAYLQNIPGVFTADFPANPPTKFDYTGNVSRSLWQPMSGTRG

[0013] YKLKYGSRVQVVLQDTSIVTPENHPIHLHGYDFYILAEGFGNFNAQTDTKKFNLIDPPLRNTVAVPANGWAVIRFVADNPGAWIMHCHLDVHINWGLAMVFLVDNGVGALQSVEQPPVDLPLC;

[0014] 2) A protease having a homology of not less than 90% with the protease in 1);

[0015] Furthermore, the homology is not less than 95%;

[0016] For the apple laccase LAC3 gene provided by the present invention, a specific nucleotide sequence thereof is as follows:

[0017] ATGGAGGCACTCAACGCTATTTTCGTCAACAAGATTCGCTTCACGTTATGCCTTTTTGGTCTTTGCCTTCTCGTGGCGTC

[0018] ATCGACAATGTCCTTGGCAGAACCCCAAATTCACAAGCATGAGTTTGTTGTTCAAGCAACACCAGTGAAGAGGCTGTGCA

[0019] AAACCCAAAACTCCATCACAGTGAATGGACAGCTCCCTGGACCAACCTTGGAAGTAAACAATGGTGACACTCTTGTCGTC

[0020] AAAGTCACCAACAGAGCTCAATACAACGTCACCATCCACTGGCATGGGATTAGGCAAATGAGAACTGGATGGGCAGATGG

[0021] GCCAGAATTTGTGACTCAGTGCCCGATTAGGCCAGGAGGGAGCTACACCTACCGCTTTACAATTCAAGGGCAAGAGGGTA

[0022] CTCTGTGGTGGCATGCTCACAGCTCATGGCTTAGAGCCACCGTTTACGGAGCACTCATCATTCATCCTAAACAAGGAGAC

[0023] TCCTACCCCTTCACTAAACCGAAAAGTGAAACAACCCTTCTTCTCGGTGAATGGTGGAACGCTAACCCTATCGATGTCTT

[0024] GAGGCAGGCGACTATGACAGGAGCAGCTCCAAATGTTTCTGATGCATACACCATCAATGCTCAACCTGGTGATCTTTACA

[0025] ACTGCTCAAGCCAAGACACTGTCATAGTTCCTATAGACTCCGGCGAGACCAACCTTCTTAGAGTCATCAACGCTGCACTC

[0026] AACCAACCTCTTTTCTTCTCCGTGGCCAACCACAAGCTCACCGTTGTTAGCGCTGATGCCTCCTACACCAAACCTTTCAC

[0027] TACCACGGTTCTCATGCTAGGGCCTGGGCAGACCACTGATGTTTTAATCACCGGTGACCAGTCACCAGCCCGGTACTACT

[0028] TGGCGGCAAGTGCTTATTTCAGCGCGCAAAATGCAGCATTCGACAACACCACCACCACCGCCATTCTTGAATACAAGTCT

[0029] GCCCCTTGCAGCCCCAATTGCAAAAACAGTTCAGCAGCCAAACCAATTATGGCACAACTCCCTGCTTTCAATGACACAAA

[0030] CACTGCTTCTGCTTTCACCAAGAGTTTCAGAAGTCCAAGAAAAGTTGAAGTCCCAACTGAAATCGATGAAAATCTCTTCT

[0031] TCACAATTGGTCTTGGACTCAACAACTGCCCGAAAAATTTTGGATCCCAAAGGTGCCAAGGCCCAAATGGGACACGCTTC

[0032] ACAGCCAGCATGAACAATGTGTCCTTCGTGCTTCCAAACAACATTTCAATCCTTCAGGCATACCTACAAAACATCCCTGG

[0033] AGTTTTCACTGCTGATTTTCCAGCAAACCCTCCGACGAAATTCGACTACACTGGCAATGTGAGTCGCTCTCTCTGGCAAC

[0034] CAATGTCAGGCACTAGAGGATACAAGTTGAAGTATGGATCAAGGGTGCAGGTTGTGCTGCAGGACACAAGCATCGTCACA

[0035] CCAGAAAACCATCCTATCCATCTTCACGGATACGATTTTTACATCCTTGCTGAGGGTTTTGGAAATTTCAATGCCCAGAC

[0036] TGATACCAAAAAGTTCAACCTTATTGATCCACCTCTGAGGAACACAGTGGCAGTGCCTGCGAATGGATGGGCAGTCATTC

[0037] GATTTGTCGCTGACAATCCAGGTGCATGGATAATGCATTGTCACTTGGATGTCCACATCAACTGGGGTTTGGCCATGGTG

[0038] TTCTTGGTGGACAATGGAGTTGGGGCACTGCAGTCAGTCGAGCAACCACCGGTGGATCTGCCTCTTTGTTAA(SEQ ID NO:2)。

[0039] In another aspect of the present invention, there is also provided a plant expression vector, and the nucleic acid fragment of the apple laccase LAC3 gene is carried in the expression vector.

[0040] The present invention also provides a recombinant Agrobacterium, and the above-mentioned plant expression vector is carried in the recombinant Agrobacterium;

[0041] The present invention also provides a use of the apple laccase LAC3 gene, which is an application in increasing the biosynthesis amount of lignin in tobacco;

[0042] In another aspect of the present invention, there is also provided a method for improving the aphid resistance of tobacco, and the method is to increase the expression level of the apple laccase LAC3 gene in tobacco plants;

[0043] The present invention also provides a method for screening tobacco plants with better aphid resistance, which is to screen by screening the expression level of the apple laccase LAC3 gene.

[0044] Taking apple as the material, the LAC3 gene was cloned by the present invention. On this basis, an overexpression vector pBI121-CutGUS-LAC3 was constructed and transferred into Nicotiana benthamiana to obtain transgenic plants, and iterative screening was carried out to obtain plants that could stably inherit the transferred gene for phenotypic observation and determination of biological indicators. The results of gene function identification showed that compared with non-transgenic plants, transgenic tobacco plants grew faster, were more lush, and had aphid-resistant functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the electrophoresis gel result diagram of MdLac3 gene cloning in the present invention;

[0046] Figure 2 It is the MdLac3 nucleic acid sequence diagram in the present invention;

[0047] Figure 3 This is the expression level diagram of MdLac3 in transgenic tobacco plants in the present invention;

[0048] Figure 4 This is the proportion diagram of the duration of the EPG waveform when aphids feed on tobacco for 6 h in the present invention;

[0049] Figure 5 This is the comparison diagram of the survival of Myzus persicae on two different varieties of tobacco in the present invention;

[0050] Figure 6 This is the diagram of the maximum and average aphid production of Myzus persicae on different varieties of tobacco in the present invention;

[0051] Figure 7 This is the diagram of the total lignin content in the leaves of different varieties of tobacco in the present invention.

[0052] Figure 8 : Photograph of the growth of the control group and transgenic Lac3 tobacco in January. Detailed implementation mode

[0053] The present invention will be further described below in conjunction with specific embodiments. In the following embodiments, the operations not described in detail are all conventional biological experimental operations, which can be referred to the molecular biology experimental manual and existing published journal literatures, etc., or carried out according to the kit and product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0054] The plant materials used in the embodiments of the present invention are apple (Malus domestica) and Nicotiana benthamiana, and their culture conditions are 25°C and 16 h of light.

[0055] The vector used in the present invention is pBI121, purchased from Shandong Miaoling Plasmid Platform;

[0056] The Escherichia coli strain used in the present invention is DH5α, purchased from Novoprotein Scientific Inc.;

[0057] The Agrobacterium used in the present invention is EHA105, purchased from Angyu Biotechnology Co., Ltd.;

[0058] Example 1: Cloning of apple laccase LAC3 gene

[0059] 1. Primer design

[0060] The LAC3 gene sequence was screened by blast alignment in the NCBI database, and primers were designed using Primer Premier5 software. The primer sequences are as follows:

[0061] LAC3-F: ATGGAGGCACTCAACGCTATTT;

[0062] LAC3-R: TTAACAAAGAGGCAGATCCACCG.

[0063] 2. Gene cloning and recovery of target fragment

[0064] ① Use the FastPure Universal Plant Total RNA Isolation Kit (Vazyme) to extract the total RNA from the branches of Red Delicious apples. The specific method refers to the instruction manual.

[0065] ② Prepare reverse transcription reaction solution 1: 1 μg of total RNA, RNase-Free ddH2O - x μL, with a total volume of 8 μL; Heat reverse transcription reaction solution 1 at 65 °C for 5 min, then quickly ice-bath for 2 min.

[0066] Prepare reverse transcription reaction solution 2: 8 μL of the above mixture, 2 μL of 5×gDNA wiper Mix, with a total volume of 10 μL. After mixing, heat at 42 °C for 2 min.

[0067] Prepare reverse transcription reaction solution 3: Mix 10 μL of the above mixture, 2 μL of 10×RT Mix, 2 μL of HiScriptⅢ EnzymeMix, 1 μL of Oligo(dT)20VN, and 5 μL of RNase-Free ddH2O, with a total volume of 20 μL. Use a PCR instrument for reverse transcription reaction. The reaction program is: 25 °C for 5 min; 37 °C for 45 min; 85 °C for 5 s; Store at -20 °C.

[0068] ③ Use the P525 high-fidelity enzyme for gene cloning with the cDNA synthesized by reverse transcription as the template.

[0069] The PCR reaction system is: 2 μL of cDNA, 2 μL of Primer-F (10 μM), 2 μL of Primer-R (10 μM), 25 μL of 2×PhantaMax Master Mix, 31 μL of ddH2O, with a total volume of 50 μL.

[0070] The PCR reaction program is: 95 °C for 3 min; 95 °C for 15 s, 55 °C for 15 s, 72 °C for 2 min, for a total of 35 cycles; 72 °C for 5 min; Store at -20 °C.

[0071] After the reaction is completed, the amplified product is separated by electrophoresis on a 1% agarose gel; Cut out the band with the correct size and recover and purify it.

[0072] The nucleotides of the CDS region of the amplified apple laccase LAC3 gene are SEQ ID NO:2, and the amino acid sequence of the encoded protein is SEQ ID NO:1.

[0073] Example 2: Construction of expression vector and transformation of Escherichia coli and Agrobacterium

[0074] 1. Construction of expression vector

[0075] Homologous recombination primers were designed using the Novoprotein single-fragment cloning website, and the primer sequences are as follows: PBI121-LAC3-F: gagaacacgggggactctagaATGGAGGCACTCAACGCTATTT;

[0076] PBI121-LAC3-R: cgatcggggaaattcgagctcTTAACAAAGAGGCAGATCCACCG.

[0077] The plant expression vector PBI121 used in this invention was purchased from the Shandong Miaoling plasmid platform. Take the glycerol bacteria containing the PBI121 plasmid for shaking culture (Kan resistance), and extract the plasmid using a plasmid purification kit (Takara).

[0078] According to the instructions, use the fast-cutting enzyme XbaI and SacI to perform double digestion on the PBI121 vector to remove the Gus region on the vector. After gel electrophoresis, use a gel recovery kit to recover and purify the PBI121 (without GUS) vector. Use the Novoprotein homologous recombination kit to construct the target gene into the vector pBI121 to obtain the recombinant plant expression vector pBI121-Lac.

[0079] The recombinant reaction system is as follows: linearized vector X μL, inserted fragment Y μL, 5×CEII Buffer 4 μL, ExnaseⅡ 2 μL, supplemented with ddH2O to a total volume of 20 μL, where the volumes of the linearized vector and the target fragment depend on their plasmid concentrations. The reaction program is: 37°C for 0.5 h.

[0080] 2. Transformation of Escherichia coli

[0081] Transform the reaction product into Escherichia coli competent cells, coat it on an LB solid medium containing Kan, culture overnight at 37°C, pick the monoclonal colonies on the plate for expansion and perform bacterial liquid PCR detection and gel electrophoresis, and select the monoclonal colonies with the correct bands and send them to the company for sequencing. Save the optimal strain according to the sequencing results for subsequent transformation of Agrobacterium.

[0082] 3. Transformation of Agrobacterium

[0083] Take 1 μg of the plasmid to be transformed, add it to 100 μl of Agrobacterium tumefaciens EHA105 competent cells. After mixing, let it stand on ice for 5 min, quickly freeze it in liquid nitrogen for 5 min, heat-shock it at 28 °C for 5 min, and then quickly ice-bath it for 5 min. Add 700 μL of LB liquid medium, shake the bacteria at 28 °C and 200 rpm for 3 h, then spread it on a plate and culture it at 28 °C for 48 h. On the third day, pick monoclonal colonies and perform colony PCR detection. Preserve the correct bacteria in a -80 °C refrigerator using glycerol bacteria.

[0084] Example 3: Cultivation of tobacco tissue culture seedlings, Agrobacterium-mediated transformation into tobacco, screening and identification of transgenic tobacco

[0085] 1. Cultivation of sterile Nicotiana benthamiana seedlings

[0086] Disinfect Nicotiana benthamiana seeds with 75% ethanol and 2.5% NaClO for 30 s, and rinse them with sterile water 3 - 5 times. Inoculate the seeds on MS medium and culture them under light at 25 °C for 4 weeks. The leaves of the tissue culture seedlings are used for transformation experiments.

[0087] 2. Agrobacterium-mediated transformation into tobacco

[0088] ① Activation of Agrobacterium: Take the verified correct Agrobacterium into LB liquid medium (Rif 20 mg / L, Kan 50 mg / L), and culture it overnight with shaking at 28 °C until OD600 = 0.8 - 1.2 for use.

[0089] ② Agrobacterium-mediated transformation of tobacco: Refer to Horsch et al. (1985) and use the leaf disc method to transform tobacco. Prepare Nicotiana benthamiana leaves into leaf discs with a radius of 5 mm, place them on MS pre-culture medium containing 1 mg / L 6-BA, and pre-culture for two days; soak the leaf discs in the prepared Agrobacterium infection solution, blot the soaked leaf discs dry with sterile filter paper, and place them on MS pre-culture medium containing 1 mg / L 6-BA for dark co-culture for four days until there are obvious bacterial stains at the bottom of the leaf discs; put the leaf discs into a medium containing 1 mg / L 6-BA + 200 mg / L Cef + 50 mg / L Kan for differentiation and screening culture; after the regenerated buds grow, transfer the buds to a rooting medium containing 200 mg / L Cef + 50 mg / L Kan. After the regenerated buds take root and have well-developed roots, harden off the seedlings and transfer them to nutrient soil for cultivation.

[0090] 3. Screening and identification of transgenic tobacco

[0091] Collect the leaves of transgenic and non-transgenic tobacco plants that have been acclimatized and grown for 1 month. Use the FastPure Plant DNA Isolation Mini Kit from Novoprotein Scientific Inc. to extract the DNA of tobacco plants. The method follows the instruction manual for DNA-level identification. Use the M13 universal forward primer (M13–F: caggaaacagctatgac) and the M13 universal reverse primer (M13-R: gtaaaacgacggccagt) for PCR detection. PCR reaction system: 1 μL of template, 12.5 μL of 2x Taq Plus Master MixⅡ, 1 μL of M13-F (10 μM), 1 μL of M13-R (10 μM), 9.5 μL of ddH2O, and the total volume is 25 μL. The PCR reaction program is as follows: 95°C for 3 min; 95°C for 15 s, 56°C for 20 s, 72°C for 3 min, for a total of 35 cycles; 72°C for 10 min; store at 4°C. Finally, perform 1% agarose gel electrophoresis detection.

[0092] Use the FastPure Universal Plant Total RNA Isolation Kit from Novoprotein Scientific Inc. to extract the RNA of tobacco plants. The method follows the instruction manual for RNA-level identification. Use the q-Lac3 forward primer (q-Lac3–F: tacttggcggcgagtgctta) and the q-Lac3 reverse primer (q-Lac3-R: tggctgctgaactgtttttgc) for fluorescence quantitative detection. Quantitative reaction system: 1 μL of sample, 5 μL of 2x RealStar FastSYBR qPCR Mix, 0.25 μL of q-Lac3–F (10 μM), 0.25 μL of q-Lac3-R (10 μM), 3.5 μL of ddH2O, and the total volume is 10 μL. The qPCR reaction program is as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, for a total of 39 cycles; Melt Curve from 65°C to 95°C, increment 0.5°C, 5S. Select the plants with high expression multiples based on the data processing results for subsequent anti-aphid function determination experiments.

[0093] Example 4: Verification of the anti-aphid function of transgenic tobacco

[0094] 1. EPG analysis of the feeding behavior of Myzus persicae

[0095] To observe the feeding behavior of Myzus persicae on different tobacco varieties, adult aphids were selected for EPG experiments on two types of tobacco. Before the experiment began, aphid individuals were examined under a microscope to ensure that their mouthparts were not damaged. A Giga-8dd DC-EPG (input resistance: 1 GΩ, digital-to-analog converter: 100 Hz / 14-bit resolution) system was used to record the electrical penetration graph of the insects. To shield the electromagnetic interference from the external environment, the feeding experiments were carried out separately in a Faraday cage (80 cm × 60 cm × 100 cm). The test insects were placed on an ice box, and then a gold wire with a diameter of 18 μm and a length of 3 - 4 cm was glued to the pronotum of the test insect with conductive silver glue. The other end of the gold wire was glued to the copper wire end (the copper wire and copper nail were soldered with tin). The adhered insects were starved for 1 h, and then the aphids were placed on the back of the tobacco leaves at the same position on different plants. When the stylets of the aphids pierced the plant tissue, the circuit closed and was amplified, then converted into a digital signal, and amplified by the Giga-8d control unit, and stored in a computer. This digital signal was converted into a waveform and displayed on the screen using Stylet+d software, which was also used to interpret the waveform and convert it into a digital file that could be used for pattern recognition. The experimental data of each insect were recorded 15 times, and each experiment was recorded for 6 h. Insects that did not show feeding waves or had no waveforms within the recorded 6 h were not recorded in the data. After the recording was completed, the EPG waveforms of the aphids were divided into non-probing waves (np), path waves (C), non-active intracellular feeding waves (pd), xylem feeding waves (G), phloem salivary secretion waves (E1), and phloem feeding waves (E2) according to the standards of Tjallingii using Stylet+a software.

[0096] For the EPG comparison of Myzus persicae feeding on different varieties of tobacco, independent-samples T-tests were performed on the experimental data using SPSS 25.0 software.

[0097] From the non-phloem stage of Myzus persicae feeding on tobacco, when Myzus persicae fed on the transgenic Hongxing Lac3 tobacco plants, the first probing time, the total time of np waves, the total time of c waves, and the number of pd waves were all significantly higher than those on ordinary tobacco. The number of probing times and the total time of G waves on the two types of tobacco were not significant (Table 1).

[0098] Table 1: Comparison table of EPG indexes of Myzus persicae feeding on different tobacco plants in the non-phloem stage

[0099]

[0100] From the perspective of the phloem stage of Myzus persicae feeding on tobacco, when M. persicae feeds on the tobacco plants transformed with the Lac3 gene of Red Star, the number of E1 times, the total time of E2 waves, and the proportions of E1 and E2 in the total E waves are all significantly lower than those on ordinary tobacco. Among the two tobacco varieties, the total time of E1 waves, the number of E2 times, the time taken to reach the phloem for the first time, and the time when the E2 wave appears for the first time are similar in value and there is no significant difference (Table 2).

[0101] Table 2: Comparison table of EPG indexes of M. persicae feeding on the phloem of different tobacco plants

[0102]

[0103]

[0104] In the EPG waveform, the E waveform is divided into E1 waveform and E2 waveform. Among them, the E1 waveform indicates that the aphid stylet is secreting saliva to prepare for feeding; the E2 waveform indicates that the aphid is feeding. The following conclusions are drawn based on the waveform analysis.

[0105] When aphids feed on ordinary tobacco, the total time of G waves accounts for 11.5697%, the total time of E1 waves (secreting saliva in the phloem) accounts for 0.4297%, the total time of E2 waves (feeding in the phloem) accounts for 5.4574%, and the time proportion of E waves (E1 + E2) is 5.8871%. The total time of pd waves accounts for 0.3702%. The total time of C waves accounts for 82.1692%, and the total time of np waves accounts for 0.0039% (as Figure 4 a).

[0106] When aphids feed on transgenic tobacco, the total time of G waves accounts for 10.7778%. The total time of E1 waves (secreting saliva in the phloem) accounts for 0.1733%, the total time of E2 waves (feeding in the phloem) accounts for 0.4283%, and the time proportion of E waves (E1 + E2) is 0.6016%, indicating that the effective feeding time of aphids on transgenic tobacco is shorter and that on ordinary tobacco is longer. The total time of pd waves accounts for 0.6172%. The total time of C waves accounts for 85.1567%, and the total time of np waves accounts for 2.8467%, indicating that aphids need to spend more time on transgenic tobacco to find a suitable feeding site (as Figure 4 b).

[0107] 2. Inoculate aphids to clarify their aphid resistance function

[0108] Insert the petioles of tobacco leaves of different varieties into petri dishes filled with agar medium (the amount of agar is 25 ml), inoculate 2nd instar M. persicae on the leaves respectively, with a total of 15 groups of parallel repetitions, and each repetition contains 6 aphids. Observe the survival rate and the number of aphids produced every day. The observation period of the whole experiment is 7 days, and the aphids have completed a life cycle.

[0109] After the green peach aphid survived on transgenic tobacco and ordinary tobacco, the data results are as Figure 5 shown. It can be seen that the survival rate of aphids on transgenic plants is significantly lower than that on ordinary tobacco.

[0110] 3. Determination of total lignin content in transgenic tobacco leaves

[0111] Use the Greiss lignin content determination kit to detect the total lignin content in the leaves of different tobacco varieties. The acetylation method is adopted to acetylate the phenolic hydroxyl groups in lignin, which has a characteristic absorption peak at 280 nm. The absorbance value at 280 nm is positively correlated with the lignin content.

[0112] Take tobacco leaves at the same growth cycle and the same position for sample preparation. After adding reagent 1, 2 and acetic acid, carry out on-machine detection. The obtained data are calculated according to the following formula: lignin (mg / g weight) = [(ΔA + 0.003) ÷ 10.615] × V1 ÷ W × 2 = 0.1413 × (ΔA + 0.003) ÷ W × 2. The results are as Figure 7 shown. The average lignin content of ordinary tobacco is about 47.26 mg / g, the average lignin content of LAC3 transgenic plants is about 122.18 mg / g, and the lignin content of the transgenic type has increased by about 158.53% compared with the wild type. The above results indicate that the overexpression of the LAC3 gene promotes the biosynthesis of lignin.

Claims

1. A method for improving tobacco resistance to aphids, characterized in that: The method is to use recombinant Agrobacterium to transform tobacco plants, thereby increasing the expression level of the apple laccase LAC3 gene with the amino acid sequence of SEQ ID NO: 1; The recombinant Agrobacterium is a plant expression vector containing the apple laccase LAC3 gene with the nucleotide sequence of SEQ ID NO:2.

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