Apple laccase LAC3 gene and application thereof in improving aphid resistance of plants

Abstract

The invention provides an apple laccase LAC3 gene and application thereof in improving aphid resistance of plants, the apple laccase LAC3 gene is cloned, a nucleotide sequence and a protein structure of the apple laccase LAC3 gene are clarified, a primer is designed on the basis, an overexpression vector of the apple laccase LAC3 gene is constructed, and the overexpression vector is transferred into tobacco through tobacco tissue culture seedling culture and agrobacterium mediation to obtain a transgenic plant. Homozygote strains are obtained through screening, aphid feeding behavioral observation is carried out through an EPG technology, aphid inoculation research is carried out, and the aphid resistance function of the aphid strains is clarified. Compared with a wild plant, the growth vigor density of the overground part of the transgenic plant is larger than that of the wild plant, stalks are thick and strong, lignin is increased, a good resistance effect on aphids is achieved, and a theoretical basis is provided for aphid-resistant molecule improved breeding.

Description

Technical Field

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

[0002] Aphids are important agricultural pests, and the woolly apple aphid is a major pest of fruit trees. Apple trees infected with the woolly apple aphid will have wooly white substances attached to the infected parts. The affected fruit trees will suffer a 10%-30% reduction in yield, weaken the tree vigor, and cause poor fruit quality. In severe cases, the fruit trees will 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 3R problems; in addition, chemical control has brought considerable economic pressure to fruit farmers, ultimately leading to a decline in the overall economic benefits of fruit farmers. Therefore, alternative methods for killing aphids to chemical control are urgently needed.

[0004] Plant insect-resistant breeding and improving the plant's own resistance to pests are sustainable development strategies for preventing and controlling agricultural pests. Clarifying the insect-resistant characteristics of plants and exploring the insect-resistant genes of plants are key issues in insect-resistant breeding. However, apples are woody plants, and it is difficult to screen insect-resistant genes in woody plants. The development of genetically modified technology to verify gene function is relatively slow, and woody plants are difficult to cultivate, so the screening and utilization of apple insect-resistant genes has been slow, which has limited the sustainable management of apple orchard pests. There is an urgent need to explore insect-resistant genes and use apples' own insect resistance 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 resistance to aphids. The present invention clones the apple laccase LAC3 gene, clarifies its nucleic acid sequence and protein structure, designs primers on this basis, constructs its overexpression vector, and transfers it into tobacco through tobacco tissue culture seedling culture and Agrobacterium-mediated, obtains transgenic plants, and screens to obtain homozygous strains, observes aphid feeding behavior through EPG technology, inoculates aphids for research and clarifies its anti-aphid function. Compared with the wild type, the growth density of the aboveground part of the transgenic plant is greater than that of the wild type, the stem is thicker, the lignin is increased, and it has a good resistance to aphids, providing a theoretical basis for the molecular improvement and breeding of apple resistance to aphids.

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

[0007] 1) Protease having the amino acid sequence of SEQ ID NO: 1

[0008] MEALNAIFVNKIRFTLCLFGLCLLVASSTMSLAEPQIHKHEFVVQATPVKRLCKTQNSITVNGQLPGPTLEVNNNGDTLVVKVTNRAQYNVTI

[0009] HWHGIRQMRTGWADGPEFVTQCPIRPGGSYTYRFTIQGQEGTLWWHAHSSWLRATVYGALIIHPKQGDSYPFTKPKSETTLLLGEWWNANPI

[0010] DVLRQATMTGAAPNVSDAYTINAQPGDLYNCSSQDTVIVPIDSGETNLLRVINAALNQPLFFSVANHKLTVVSADASYTKPFTTTVLMLGPG

[0011] QTTDVLITGDQSPARYYLAASAYFSAQNAAFDNTTTTAILEYKSAPCSPNCKNSSAAKPIMAQLPAFNDTNTASAFTKSFRSPRKVEVPTEI

[0012] DENLFFTIGLGLNNCPKNFGSQRCQGPNGTRFTASMNNVSFVLPNNISILQAYLQNIPGVFTADFPANPPTKFDYTGNVSRSLWQPMSGTRG

[0013] YKLKYGSRVQVVLQDTSIVTPENHPIHLHGYDFYILAEGFGNFNAQTDTKKFNLIDPPLRNTVAVPANGWAVIRFVADNPGAWIMHCHLDVHINWGLAMVFLVDNGVGALQSVEQPPVDLPLC;

[0014] 2) a protease having no less than 90% homology with the protease in 1);

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

[0016] The apple laccase LAC3 gene provided by the present invention has a specific nucleotide sequence 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, the present invention further provides a plant expression vector, wherein the expression vector carries the nucleic acid fragment of the apple laccase LAC3 gene.

[0040] The present invention also provides a recombinant Agrobacterium, wherein the recombinant Agrobacterium carries the above-mentioned plant expression vector;

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

[0042] Another aspect of the present invention provides a method for improving tobacco resistance to aphids, wherein the method comprises increasing 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 resistance to aphids, and the screening is performed by screening the expression level of the apple laccase LAC3 gene.

[0044] The invention uses apple as material, obtains LAC3 gene by cloning, constructs overexpression vector pBI121-CutGUS-LAC3 on this basis, transfers it into Nicotiana benthamiana to obtain transgenic plants, and performs iterative screening to obtain plants that can stably inherit the transferred genes for phenotypic observation and biological index determination. The results of gene function identification show that compared with non-transgenic plants, the transgenic tobacco plants grow faster and denser, and have anti-aphid function. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0046] Figure 2 The nucleic acid sequence diagram of MdLac3 in the present invention;

[0047] Figure 3 is a graph showing the expression level of MdLac3 in the transgenic tobacco plants of the present invention;

[0048] Figure 4 This is a graph showing the duration ratio of the EPG waveform when aphids feed on tobacco for 6 hours in the present invention;

[0049] Figure 5 This is a comparative diagram of the survival of tobacco aphids on two different tobacco varieties in the present invention;

[0050] Figure 6 The maximum and average aphid yields of tobacco aphids on different tobacco varieties in the present invention are shown in the figure;

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

[0052] Figure 8 : Photos of the growth of the control group and the Lac3 gene-transfected tobacco in January. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific embodiments. In the following embodiments, the operations not described in detail are all routine biological experimental operations, which can be performed with reference to the molecular biology experiment manual and existing published journals, or according to the kit and product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

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

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

[0056] The Escherichia coli strain used in the present invention is DH5α, purchased from Novozyme Biotech Co., Ltd.;

[0057] The Agrobacterium used in the present invention is EHA105, which was purchased from Ang Yu Biotechnology Co., Ltd.

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

[0059] 1. Primer Design

[0060] The LAC3 gene sequence was screened out by blast comparison of 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 target fragment recovery

[0064] ①The total RNA of the new red star apple branches was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Novagen). The specific method was referred to the instruction manual.

[0065] ② Prepare reverse transcription reaction solution 1: Total RNA 1μg, RNase-Free ddH2O-xμL, with a total volume of 8μL; heat reverse transcription reaction solution 1 at 65℃ for 5min, and then quickly put it in an ice bath for 2min.

[0066] Prepare reverse transcription reaction solution 2: 8 μL of the previous mixture, 2 μL of 5×g DNA wiper mix, total volume 10 μL. After mixing, heat at 42°C for 2 min.

[0067] Prepare reverse transcription reaction solution 3: 10 μL of the previous mixture, 2 μL of 10×RT Mix, 2 μL of HiScriptⅢEnzymeMix, 1 μL of Oligo(dT)20VN, 5 μL of RNase-Free ddH2O, and mix well to a total volume of 20 μL. Perform reverse transcription reaction using a PCR instrument. The reaction procedure is: 25℃5min; 37℃45min; 85℃5s; store at -20℃.

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

[0069] The PCR reaction system was: cDNA 2 μL, Primer-F (10 μM) 2 μL, Primer-R (10 μM) 2 μL, 2×PhantaMax Master Mix 25 μL, ddH2 O 31 μL, and the total volume was 50 μL.

[0070] The PCR reaction program was: 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; and stored at -20°C.

[0071] After the reaction was completed, the amplified products were separated by electrophoresis using 1% agarose gel; the band of the correct size was cut out and recovered and purified.

[0072] The nucleotide sequence of the amplified apple laccase LAC3 gene CDS region was determined to be SEQ ID NO:2, and the amino acid sequence of the encoded protein was determined to be 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 Novozyme single fragment cloning website, and the primer sequences were as follows: PBI121-LAC3-F: gagaacacgggggactctagaATGGAGGCACTCAACGCTATTT;

[0076] PBI121-LAC3-R:cgatcggggaaattcgagctcTTAACAAAGAGGCAGATCCACCG.

[0077] The plant expression vector PBI121 used in the present invention was purchased from Shandong Miaoling Plasmid Platform. Glycerol bacteria containing the PBI121 plasmid were taken for shaking (Kan resistance), and the plasmid was extracted using a plasmid purification kit (Takara).

[0078] According to the instructions, the PBI121 vector was double-digested with the fast-cutting enzymes XbaI and SacI to remove the Gus region on the vector. After gel electrophoresis, the PBI121 (without GUS) vector was recovered and purified using a gel recovery kit. The target gene was constructed into the vector pBI121 using the Novogene recombination kit to obtain the recombinant plant expression vector pBI121-Lac.

[0079] The recombination reaction system is as follows: linearized vector XμL, inserted fragment YμL, 5×CEII Buffer 4μL, ExnaseⅡ2μL, supplemented with ddH2O to a total volume of 20μL, where the volume of linearized vector and target fragment depends on the plasmid concentration. The reaction procedure is: 37℃ 0.5h.

[0080] 2. E. coli Transformation

[0081] The reaction product was transformed into competent E. coli cells, spread on LB solid medium containing Kan, cultured overnight at 37°C, picked up single clones on the plate for expansion and bacterial liquid PCR detection, gel electrophoresis, and selected single clones with correct bands for sequencing. The optimal strain was saved according to the sequencing results and subsequently transformed into Agrobacterium.

[0082] 3. Agrobacterium Transformation

[0083] Take 1 μg of the plasmid to be transformed, add 100 μl EHA105 Agrobacterium competent cells, mix well, and then place on ice for 5 minutes, freeze in liquid nitrogen for 5 minutes, heat shock at 28°C for 5 minutes, and quickly ice bath for 5 minutes. Add 700 μL LB liquid culture medium, shake the bacteria at 28°C 200rpm for 3 hours, spread on the plate, and culture at 28°C for 48 hours. On the third day, select a single clone and perform bacterial liquid PCR detection. Use glycerol bacteria to store the correct bacteria in a -80°C refrigerator.

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

[0085] 1. Cultivation of sterile Nicotiana benthamiana seedlings

[0086] The seeds of Nicotiana benthamiana were sterilized with 75% ethanol and 2.5% NaClO for 30 seconds, washed with sterile water for 3 to 5 times, inoculated on MS medium, and cultured at 25°C for 4 weeks. The leaves of tissue culture seedlings were used for transformation experiments.

[0087] 2. Agrobacterium-mediated transformation into tobacco

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

[0089] ② Agrobacterium-mediated tobacco transformation: Refer to Horsch et al. (1985) to transform tobacco using the leaf disc method. Prepare Nicotiana benthamiana leaves into leaf discs with a radius of 5 mm, place them on a 1mg / L 6-BA MS pre-culture medium, and pre-culture for two days; soak the leaf discs in the prepared Agrobacterium infection solution, dry the soaked leaf discs with sterile filter paper, and place them on a 1mg / L 6-BA MS pre-culture medium for four days in the dark until there are obvious bacterial stains at the bottom of the leaf disc; place the leaf discs in a culture medium containing 1mg / L6-BA+200mgL Cef+50mg / L Kan for differentiation and screening culture; until the regenerated buds grow, transfer the buds to a rooting medium of 200mgL Cef+50mg / L Kan, and after the regenerated buds take root and the root system is well developed, transfer the seedlings to nutrient soil for culture.

[0090] 3. Screening and identification of transgenic tobacco

[0091] The leaves of transgenic and common tobacco plants that had been hardened for one month were collected, and the DNA of tobacco plants was extracted using the FastPure Plant DNA Isolation Mini Kit of Novazon Biotech Co., Ltd. The method was referred to the instructions for DNA level identification. PCR detection was performed using M13 universal upstream primer (M13–F: caggaaacagctatgac) and M13 universal downstream primer (M13-R: gtaaaacgacggccagt). The PCR reaction system: template 1μL, 2xTaq Plus Master MixⅡ12.5μL, M13-F (10μM) 1μL, M13-R (10μM) 1μL, ddH2O 9.5μL, total system 25μL. The PCR reaction program was: 95℃3min; 95℃15s, 56℃20s, 72℃3min, a total of 35 cycles; 72℃10min; 4℃ storage. Finally, 1% agarose gel electrophoresis was performed.

[0092] The FastPure Universal Plant Total RNA Isolation Kit of Novagen Biotech Co., Ltd. was used to extract RNA from tobacco plants. The method was referred to the instructions for RNA level identification. Fluorescence quantitative detection was performed using q-Lac3 upstream primers (q-Lac3–F: tacttggcggcgagtgctta) and q-Lac3 downstream primers (q-Lac3-R: tggctgctgaactgtttttgc). Quantitative reaction system: sample 1μL, 2xRealStar FastSYBR qPCR Mix 5μL, q-Lac3–F (10μM) 0.25μL, q-Lac3-R (10μM) 0.25μL, ddH2O 3.5μL, total system 10μL. The qPCR reaction program was: 95℃30s; 95℃5s, 60℃30s, 39 cycles in total; Melt Curve 65℃to95℃, increment 0.5℃, 5S. Based on the data processing results, plants with high expression multiples were selected for subsequent anti-aphid function determination experiments.

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

[0094] 1. EPG analysis of feeding behavior of tobacco aphid

[0095] To observe the feeding behavior of tobacco aphids on different tobacco varieties, adult aphids were selected for EPG experiments on two tobacco varieties. Before the experiment, individual aphids were examined under a microscope to ensure that their mouthparts were not damaged. The insects' probe potential spectra were recorded using a Giga-8dd DC-EPG (input resistance: 1GΩ, digital-to-analog converter: 100Hz / 14-bit resolution) system. In order to shield the electromagnetic interference of the external environment, the feeding experiments were carried out in Faraday cages (80cm×60cm×100cm). The test insects were placed on an ice box, and then a gold wire with a diameter of 18μm and a length of 3-4cm was glued to the pronotum of the test insects with conductive silver glue, and the other end of the gold wire was glued to the end of the copper wire with conductive silver glue (the copper wire and the copper nail were soldered by tin). The glued insects were starved for 1 hour, and then the aphids were placed on the back of the tobacco leaves at the same position of different plants. When the aphid's stylet pierces the plant's tissue, the circuit is closed and amplified, then converted to a digital signal and amplified by the Giga-8d control unit to be stored in a computer. The digital signal is converted into a waveform and displayed on the screen using Stylet+d software, which is also used to interpret the waveform and convert it into a digital file that can be used for pattern recognition. The data for each insect test was recorded 15 times, and each test was recorded for 6 hours. Insects that did not show a feeding wave or no waveform within the 6 hours of recording 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 saliva secretion waves (e1), and phloem feeding waves (e2) using Stylet+a software according to Tjallingii's criteria.

[0096] The EPGs of tobacco aphids feeding on different tobacco varieties were compared, and the experimental data were subjected to independent sample T test using SPSS25.0 software.

[0097] From the perspective of the non-phloem stage of feeding on tobacco, the time of first start of probing, total time of np wave, total time of c wave and number of pd wave of tobacco aphids feeding on the transgenic tobacco plants of New Red Star Lac3 gene were significantly higher than those of ordinary tobacco. The number of probing and total time of G wave on the two tobacco plants were not significant (Table 1).

[0098] Table 1: Comparison of EPG indexes of tobacco aphids feeding on non-phloem of different tobacco plants

[0099]

[0100] From the perspective of the phloem stage of tobacco aphids feeding on tobacco, the number of E1 waves, the total time of E2 waves, and the proportion of E1 and E2 in the total E waves when tobacco aphids fed on tobacco plants with the new red star Lac3 gene were significantly lower than those of ordinary tobacco. In the two tobacco varieties, the total time of E1 waves, the number of E2 waves, the time it took to reach the phloem for the first time, and the time when the E2 wave first appeared were similar, with no significant difference (Table 2).

[0101] Table 2: Comparison of EPG indexes of tobacco aphids 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. The E1 waveform indicates that the aphid's stylet is secreting saliva in preparation for feeding; the E2 waveform indicates that the aphid is feeding. The following conclusions were drawn based on the waveform analysis.

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

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

[0107] 2. Inoculate aphids to clarify its anti-aphid function

[0108] The petioles of tobacco leaves of different varieties were inserted into culture dishes containing agar medium (the amount of agar was 25 ml), and the 2nd instar tobacco aphids were inoculated on the leaves. A total of 15 groups of parallel replicates were conducted, with 6 aphids in each replicate. The survival rate and aphid production were observed every day. The observation period of the entire experiment was 7 days, and the tobacco aphids had completed a life cycle.

[0109] The data results of tobacco aphids surviving on transgenic tobacco and ordinary tobacco are as follows Figure 5 As 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] The total lignin content of leaves of different tobacco varieties was detected using the Greiss lignin content assay kit. The acetylation method was used to acetylate the phenolic hydroxyl groups in lignin, which had a characteristic absorption peak at 280nm. The absorbance value at 280nm was positively correlated with the lignin content.

[0112] Tobacco leaves with the same growth cycle and position were used for sample preparation. After adding reagents 1, 2 and acetic acid, the samples were tested on the machine. The obtained data was 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 follows Figure 7 As shown, the average lignin content of common tobacco is about 47.26 mg / g, and the average lignin content of LAC3 transgenic plants is about 122.18 mg / g. The lignin content of transgenic plants is about 158.53% higher than that of wild-type plants. The above results show that overexpression of LAC3 gene promotes the biosynthesis of lignin.

Claims

1. An apple laccase LAC3 gene, characterized in that: The gene encoding: 1) Protease having the amino acid sequence of SEQ ID NO: 1 2) A protease having not less than 90% homology with the protease in 1).

2. The apple laccase LAC3 gene according to claim 1, characterized in that The homology in 2) is not less than 95%.

3. The apple laccase LAC3 gene according to claim 1, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A plant expression vector, wherein the expression vector carries the nucleic acid fragment of the apple laccase LAC3 gene according to claim 1.

5. A recombinant Agrobacterium, characterized in that: The recombinant Agrobacterium carries the plant expression vector according to claim 4.

6. Use of the apple laccase LAC3 gene according to claim 1 in improving the biosynthesis of lignin in tobacco.

7. A method for improving tobacco's resistance to aphids, characterized in that the method is to increase the expression level of the apple laccase LAC3 gene of claim 1 in tobacco plants.

8. The method according to claim 7, characterized in that The method of increasing the expression level of the apple laccase LAC3 gene of claim 1 is to use the recombinant Agrobacterium of claim 5 to transform tobacco plants.

9. A method for screening tobacco plants with better anti-aphid antibodies, characterized in that: The method is to screen by detecting the expression level of the apple laccase LAC3 gene according to claim 1.

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