WO2009109123A1 - 人工改造合成的杀虫基因及其编码的蛋白质与应用 - Google Patents

人工改造合成的杀虫基因及其编码的蛋白质与应用 Download PDF

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WO2009109123A1
WO2009109123A1 PCT/CN2009/070560 CN2009070560W WO2009109123A1 WO 2009109123 A1 WO2009109123 A1 WO 2009109123A1 CN 2009070560 W CN2009070560 W CN 2009070560W WO 2009109123 A1 WO2009109123 A1 WO 2009109123A1
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insecticidal
gene
cmcry2aa
plant
cotton
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PCT/CN2009/070560
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English (en)
French (fr)
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崔洪志
陈文华
杨年松
王君丹
江辉松
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创世纪转基因技术有限公司
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Publication of WO2009109123A1 publication Critical patent/WO2009109123A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to the field of plant genetic engineering, in particular to an artificially modified synthetic insecticidal gene and a plant expression vector constructed thereby and the application thereof in the development of insect-resistant transgenic plants. Background technique
  • Plants are susceptible to pests during their growth, and the annual loss of crops caused by pests is enormous. In order to prevent the occurrence of plant pests, it is necessary to spray a large number of agricultural pesticides. The long-term use of chemical pesticides can lead to increased resistance of pests, and will kill a large number of natural enemies, ecological imbalances, and enter a vicious circle.
  • bud package thuringiensis ⁇ dried bacteria (Bacillus thuringiensis, Bt) insecticidal crystal protein Insecticidal crystal proteins (ICPs) genes: such as Cry 1 Ac, CrylF, CrylAb, etc.; (2) proteinase inhibitors (PSS) genes: Vigna sinensis trypsin inhibitor 7 (cowpea trysin inhibitor, CpTI) Gene CpTI ⁇ , (3) Amylase inhibitor (AI) gene: Rue Phaseolus book
  • v /gn « -amylase inhibitor gene a - ai et al; (4) plant lectin gene: such as Galanthus nivalis aggulutinin (GNA) gene g; ) An insect-specific neurotoxin gene: such as the scorpion toxin gene Bank I ⁇ 4 and the like.
  • the most successful genes used in production are: the insecticidal crystal protein gene and the CpTI gene. The most widely used is the insecticidal crystal protein gene, the Bacillus thuringiensis (Bt) endotoxin crystal protein gene, which was cloned into Bacillus thuringiensis.
  • Bacillus thuringiensis is a Gram-positive soil Bacillus found in 1901 and is known to produce against a variety of insects, such as Lepidopterans, Coleopterans, and Diptera ( Dipterans) are a variety of parasporal crystalline proteins that are toxic to crop pests. This insecticidal protein has been found in more than 50 categories, more than 300 species. Bt insecticidal protein is hydrolyzed by digestive enzymes in the digestive tract of insects, releasing about 60 ⁇ 70kDa of active toxin molecules against protease. The active toxin molecule binds to a specific receptor on the limbal membrane of the intestinal epithelial cells and acts to perforate the cell membrane.
  • Oy2A ⁇ 3 ⁇ 4 has insecticidal effects on cotton bollworm, red bollworm, corn borer, tobacco budworm, cotton budworm, larvae, rice leaf roller, soybean worm, tropical mosquito and other pests. .
  • An insecticidal gene having the nucleotide sequence shown in SEQ ID NO: 1.
  • An insecticidal gene having the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 2 being 5' compared to the nucleotide sequence of SEQ ID NO: The end is 84 bases less after the start codon.
  • the nucleotide sequence uses a plant preference codon.
  • the nucleotide sequence uses a cotton preference codon.
  • a second object of the present invention is to provide a pesticidal protein comprising the above insecticidal gene and which can express the insecticidal gene in a plant.
  • a pesticidal gene-encoded protein CmCry2Aa insecticidal protein which is encoded by the insecticidal gene of SEQ ID NO: 1, has the amino acid sequence of SEQ ID NO: 3 and consists of 633 amino acid residues.
  • a pesticidal gene-encoded protein CmlCry2Aa insecticidal protein, encoded by the insecticidal gene of SEQ ID NO: 2, having the amino acid sequence of SEQ ID NO: 4, consisting of 605 amino acid residues, CmlCry2Aa insecticidal protein Compared with CmCry2Aa insecticidal protein, 28 amino acids are deleted at the N-terminus, both of which have insecticidal activity.
  • a plant expression vector comprising the above insecticidal gene.
  • An insecticidal plant cell transformed with a plant expression vector containing the above insecticidal gene.
  • Plant tissues, organs and plants transformed with the plant expression vector containing the above insecticidal gene Plant tissues, organs and plants transformed with the plant expression vector containing the above insecticidal gene.
  • a third object of the present invention is to provide an application of a pesticidal gene for the cultivation of insect-resistant plant varieties, particularly for the cultivation of insect-resistant cotton.
  • the invention utilizes cotton optimized codon to synthesize CmCry2Aa and Cml Cry2Aa insecticidal protein genes, and constructs plant expression vectors respectively.
  • the results of transgenic tobacco showed that the designed CmCry2Aa Cml CrylAa insecticidal gene had extremely significant resistance to cotton bollworm.
  • the N-terminal amino acid lacking the insecticidal protein can simplify the activation of insecticidal protein while maintaining its insecticidal activity. Expand its insecticidal spectrum.
  • Figure 1 is a CmCry2AaDl-pGEM plasmid map
  • Figure 2 is a CmC / 2Afl ) 2-p [/ C57 plasmid map
  • Figure 4 is ⁇ 2 ⁇ )2. -p[/C57 plasmid map;
  • Figure 5 is a CmC / 2Afl-p [/ C57 plasmid map
  • Figure 6 is a flow chart showing the construction of the ⁇ 2 ⁇ insecticidal gene plant expression vector ⁇ - ⁇ ?5 ⁇ 2 ⁇ ;
  • Figure 7 is a PCR amplification and identification diagram of the CmCry2Aa insecticidal gene plant expression vector Ml-BAR-35S-CmCry2Aa;
  • Figure 8 is a CmCry2Aa insecticidal gene plant expression vector Enzyme digestion identification map
  • Figure 9 is the identification map of plasmid MJ-MR-S-CmC ⁇ la transformed tobacco resistance
  • Figure 10 is a flow chart showing the construction of the CmCry2Aa insecticidal gene plant expression vector Ml-Pnos-NPTII-35S-CmCry2Aa;
  • Figure 11 is a restriction diagram of the CmC / 2Aa insecticidal gene plant expression vector M Pnos-NPTII-35S-CmCry2Aa;
  • Figure 12 is a CmJCry2Aa - ⁇ ⁇ ⁇ plasmid map
  • Figure 13a, b is a flow chart for constructing a CmlCry2Aa plant expression vector Ml-Pnos-NPTII-35S-Cml CrylAa;
  • Figure 14 is a diagram showing the identification of the insect resistance of the plasmid Ml-Pnos-NPTII-35S-Cml CrylAa transformed tobacco;
  • Figure 15 is a flow chart for constructing the prokaryotic expression vector CmCry2A a -pET30a of the CmCry2Aa gene;
  • Figure 16 is the prokaryotic expression vector CmCry2Aa-pET30a of the CmCry2Aa gene
  • Figure 17 is a flow chart showing the construction of the prokaryotic expression vector CmlCry2Aa-pET30a of the CmJCry2Aa gene;
  • Figure 18 is a restriction map of the CmlCry2Aa gene prokaryotic expression vector CmlCry2Aa-pET30a;
  • Figure 19 is the prokaryotic expression of the CmCry2Aa and CmlCry2Aa insecticidal proteins. Protein electrophoresis map;
  • LB medium Tryptone lOg/1, yeast powder 5g/l, sodium chloride lOg/1, PH: 7.0 ⁇ 7.2, solid medium plus 1.5% agar powder
  • Kan kanamycin 50mg/ml
  • Chi chloramphenicol 34mg/ml
  • Tris-Hcl 121.2g Tris is dissolved in 1L of water, adjusted to pH 6.8 with concentrated hydrochloric acid, high pressure sterilization
  • Tris-Hcl ( PH8.8 ): 181.7g Tris is dissolved in 1L, adjusted to pH 8.8 with concentrated hydrochloric acid, high pressure sterilization
  • IPTG 0.25g of IPTG is dissolved in 10mL of ddH20
  • Tris-Glycine Buffer Tris 15. lg, Glycine 94g SDS 5.0g to 1L water
  • Coomassie brilliant blue staining solution 0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% water acetic acid to volume 1L water
  • Coomassie Brilliant Blue Decolorizing Solution 100ml of acetic acid, 50ml of ethanol to 1L of water.
  • Example 1 Synthesis of ⁇ 2 ⁇ insecticidal gene
  • the codon usage of the synthesized ⁇ 2 ⁇ insecticidal gene is as follows: UCAG
  • Domain I Domain I DI gene fragment: BamHI-ATG-DI-Apal, the specific sequence thereof is shown as: SEQ NO ID: 5.
  • Domain II ( Domainll ) DII gene fragment Apal-DII-SspI, the specific sequence thereof is shown as: SEQ NO ID: 6.
  • Domain III ( Domainlll ) Dili gene fragment Xbal - Hpal-DIII-SacI, the specific sequence thereof is shown as: SEQ NO ID: 7.
  • CmCry2AaDl-pGEM The three domain coding genes of Cm0 2Aa insecticidal gene, D1, D2 and D3, were synthesized and cloned into pGEM and /?[/57, respectively, and the plasmids were named: CmCry2AaDl-pGEM, and the plasmid map is shown in Figure 1.
  • the Hpal _ D3 _ Sad fragment in CmCry2AaD3- P UC57 was cloned between the Sspl and Sacl sites in CmCry2AaD2-pUC57, and a blunt-end-adhesive link was ligated to construct the vector plasmid CmCry2AaD2.D3 - /?[/ 57, The plasmid map is shown in Figure 4.
  • the Sphl-Apal D1 fragment in CmCry2AaDl-pGEM was cloned into the vector to obtain the plasmid CmCry2Aa-pUC57.
  • the plasmid map is shown in Figure 5, and the CFM was completed.
  • PCR identification results and the enzyme digestion identification results in the screening process of Ml-BAR-35S-CmCry2Aa are shown in Fig. 7 and Fig. 8, respectively.
  • lanes 1, 2 and 3 represent clones 1, 2 and 3, respectively.
  • the target band was amplified for the template, and " + " represents the plasmid
  • a target band was amplified for the template (positive control), " - " indicates that the target band was not amplified with water as a template (negative control);
  • the endonuclease Xba l + Sacl cut plasmid MJ-MR-rwo ⁇ G s, cut GUS, about 1.8Kbp, 2, 3, 4, 5 and 6 lanes are restriction endonucleases Xbal + Sacl cut 1 , 2, 3, 5, 6 clones, cut down 35S + CmCrylAa, about 2.7Kbp. The results showed that clones 1, 2 and 3 were all correctly cloned.
  • Example 3 Conversion of ⁇ 2 ⁇ insecticidal gene tobacco
  • Nptll is a construction of a CmC/ 2Afl insecticidal gene plant expression vector Ml-Pnos-NPTII-35S-CmCry2Aa
  • the Agrobacterium transformation of cotton was further carried out. Due to the original CmC / 2Aa plant expression vector
  • the selection marker gene ⁇ is not conducive to the Agrobacterium transformation system of cotton (glyphosate inhibits the regeneration and differentiation of cotton cells), and the cloning route was designed to obtain the CmCry2A i insecticidal gene plant expression vector M Pnos with Nptll as a selectable marker gene.
  • -NPTII-35S-CmCry2Aa ⁇ k ⁇ 21 The Np // gene obtained by PCR was cloned and verified by sequencing, and it was confirmed that the amplified Np // gene nucleotide sequence was correct. The entire cloning process is shown in Figure 10.
  • the lanes 1 and 2 are EcoRI and the clones of Ml-Pnos-NPTII-35S-CmCry2Aa are cloned 7 and 22, and the band of 1.7Kb can be cut and positive; 3 lanes For EcoR I, Pnos-NPTII-Tnos-PBS was used as a positive control, and a band of about 1.7 Kbp was cut; in the 4, 5 lane, Ncol + Kpnl was used to cut the clones of Ml-Pnos-NPTII-35S-CmCry2Aa.
  • a 29-amino acid Coli structural fragment is present at the N-terminus of the insecticidal gene, which is located at the front end of the domain 1 ⁇ -helix 1 .
  • the amino acid sequence needs to be removed by the trypsin hydrolysis of the insect digestive tract when the protoxin is activated. Analysis by DNAman software revealed a trypsin recognition cleavage site between amino acids 29 and 30.
  • the mutant primer was designed to remove the 28 amino acids at the 5' end of CmCry2Aa, so that the expressed protein is directly active protein, which is beneficial for expanding the insecticidal spectrum (when the insect can not activate the original toxin correctly) and improving the insecticidal efficiency (activation efficiency) Not 100%).
  • CmJC/ 2Aa The CmlCrylAa 5-terminal primer was added with a BamHI site, and the 3-terminal primer was added to the Sacl site for subsequent cloning.
  • the designed primers are as follows:
  • Primer 1 2Aa5 5'-G GGATCC ATGTCTTTGGACACTATCCA-3'
  • Primer 2 2Aa3 5'-GAGCTCTTAGTACAAGGGT-3'
  • the target band amplified was 1820 bp. Is the amplified CmJC ⁇ Aa cloned to ⁇ ⁇ ? On S- ⁇ , it is named: CmlCry2Aa -pMD18, and its plasmid map is shown in Figure 12.
  • the Agrobacterium tumefaciens-mediated leaf disc method was used to transform tobacco (a method known to the scientific researcher in the field), and the resistant plants were obtained by kanamycin rooting, and 25 resistant plants were obtained, and the total DNA of the resistant tobacco leaves was extracted and identified by PCR. 22 positive plants were tested for resistance to cotton bollworm, and 5 hatching larvae were taken from each leaf. After three days, some results are shown in Figure 14. 1, 2 is not transferred. Insecticidal tobacco leaves (control), 3 and 4 are transformed CFM ⁇ 2 ⁇ insecticidal tobacco leaves, respectively, and the results indicate that the synthesis The insecticidal gene resistance to cotton bollworm is basically equivalent to the ⁇ 2 ⁇ insecticidal gene.
  • E. coli Rosetta (DE3) strain was activated on LB medium plates, single colonies were picked and inoculated into 5 ml of LB liquid medium, and shaken at 37 °C overnight. 500 ⁇ l of the bacteria solution was added to 100 ml LB. In liquid medium, 37°.
  • the prokaryotic expression vector pET30a (+ X CmCry2Aa-pET30a and CmlCry2Aa-pET30a prokaryotic expression vector were transferred into the competent E. coli Rosetta (DE3) competent cells, respectively.
  • the specific method was as follows: E. from the -70 °C water tank. Coli Rosetta (DE3) competent cells, thawed on water, added ⁇ ligation product to competent cells on a clean bench, mix gently, bath for 30 min, then heat for 90 sec in a 42 °C water bath.
  • PAGE analysis refers to "Molecular Cloning"
  • concentration of concentrated gel is 5%
  • concentration of separation gel is 10%
  • electrophoresis with 80v voltage
  • the bromophenol blue indicator enters the separation gel
  • increase the voltage to ⁇ about lhr30min
  • the electrophoresis was terminated, stained with Coomassie blue staining solution overnight, and decolorized with a decolorizing solution until the protein bands were clear.
  • FIG 19 shows the results of SDS-PAGE electrophoresis of CmCry2Aa and CmlCry2Aa genes, 1 and
  • the prokaryotic expression vectors of CmCry2Aa-pET30a, CmlCry2Aa-pET30a and CrylAc-pET30a were transferred into the strain E. coli Rosetta (DE3), and induced respectively, and the concentration of the target insecticidal protein was adjusted to near level by SDS electrophoresis.
  • Three concentration gradients were prepared: protein extracts 73 ⁇ 1, 660 ⁇ 1, 2000 ⁇ 1 were added to each 20g artificial diet, and the feed was prepared by mixing.
  • Helicoverpa armigera and Ostrinia nubilalis were separately raised on the prepared tanning materials for toxicity determination.
  • test results are as follows:
  • Example 8 Obtaining Transgenic Cotton Using CmC/2Aa and Cm:iCry2Aa Insecticidal Plant Expression Vectors
  • the CmCry2Aa or CmlCry2Aa insecticidal gene plant expression vector was transformed into cotton by Agrobacterium-mediated transformation to obtain transgenic cotton.
  • Agrobacterium-mediated transformation is a plant genetic transformation method well known to researchers in the field. The specific operating procedures are:
  • the constructed insecticidal gene plant expression vector was electroporated into Agrobacterium strain LBA4404, and Agrobacterium single colony was inoculated with kanamycin (km) 50 mg/L, rifampicin (rif) 25 Mg/L in LB or YEB in liquid medium. Shake dark culture at 28 °C overnight until the logarithm of bacterial growth Period. The bacterial solution was diluted with LB or YEB liquid medium, and shaken for 4-6 hours, and the bacterial solution was diluted to an OD600 value of 0.3 0.35.
  • Cotton seeds are separated from linters with sulfuric acid (H 2 S0 4 ), tap water is washed away from the surface of the seeds, dried, and the seeds are surface-sterilized with 70% ethanol for 1 min, then 10% -15 % peroxidized. Hydrogen (H 2 0 2 ) treatment for 2 ⁇ 4 h, rinse with sterile water for 2 ⁇ 3 times;
  • the co-cultured hypocotyl segments were placed in callus induction medium (MS + 2,4-D 0.1 mg/L + KT 0.1 mg/L + MgC12 0.91 g/L + Gelrite 2.0 g/L + Km) 50400 mg/L + Cef 500 mg/L + glucose 30 g/L, pH 5.8), cultured under normal conditions (25 °C) for 2 months (the same medium was changed once a month).
  • callus induction medium MS + 2,4-D 0.1 mg/L + KT 0.1 mg/L + MgC12 0.91 g/L + Gelrite 2.0 g/L + Km
  • the callus was picked a little for the ELISA test of the selectable marker gene nptll or the reporter gene was detected.
  • the callus with positive test results continued to be subcultured, and the non-positive callus was eliminated.
  • the frequency of cotton resistant callus was 50% to 76%.
  • the induced resistant callus was introduced into a proliferation medium (MS medium + MgC12 0.91 g/L + Gelrite 2.0 g/L + glucose 30 g/L, pH 5.8), and cultured under normal conditions (25 ° C). , subculture every other month until the callus differentiates. After the first and second transfer to the proliferation medium, some of the callus browning died, and the normal callus did not proliferate rapidly. After the second passage, the callus proliferation rate increased.
  • the planted regenerated cotton seedlings were placed in an artificial incubator with a temperature control of 22 °C and a humidity control of 80-85% for 5-7 days, and then transplanted in a greenhouse for 10-20 days, and then transplanted into a soil pot or a field.
  • Example 9 Analysis of insect resistance of CmCry2Aa and CmlCry2Aa transgenic cotton to cotton bollworm Using the newly hatched larvae of Helicoverpa armigera, 45 strains of CmCry2Aa and 22 strains of CmlCry2Aa were transplanted in the greenhouse, and the T. transgenic cotton plants were identified for their insecticidal activity against cotton bollworm. Take the 2nd fruit branch of the transgenic cotton.
  • the damage of cotton leaves is divided into 5 levels: 0: slight, insects do not penetrate to form holes; Grade 1: lighter, forming 3-5 needle-eye holes; : Light, forming 10 or less holes less than 1 mm 2 ; Class 3: Medium, forming more than 10 small holes less than 1 mm 2 ; Class 4: Heavier, forming more than 10 small holes or forming 1-3 larger than 1 Square mm hole; Grade 5: Heavy, forming more than 3 holes larger than 1 mm 2 .
  • the test results are shown in the table below.

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Description

人工改造合成的杀虫基因及其编码的蛋白质与应用 技术领域
本发明涉及植物基因工程领域， 尤其涉及一种人工改造合成的杀虫基因以及其 构建的植物表达载体及其在抗虫转基因植物研制方面的应用。 背景技术
植物生长过程中易遭受虫害， 每年因虫害造成的农作物损失量十分巨大。 为防 治植物虫害发生， 需大量喷施农说药， 长期大量使用化学农药， 可导致害虫抗药性增 强， 并且会大量杀伤其天敌， 生态失衡， 步入恶性循环。 利用基因工程手段使植物 获得抗虫性， 目前已被广泛釆用， 用于植物抗虫基因工程的基因主要包括： （ 1 ) 苏 云金芽 包^ :干菌 ( Bacillus thuringiensis, Bt )杀虫晶体蛋白 ( insecticidal crystal proteins, ICPs )基因： 如 Cry 1 Ac, CrylF, CrylAb等；（ 2 )蛋白酶抑制剂（ proteinase inhibitors, PIS )基因： 如 豆 ( Vigna sinensis )胰蛋白酶^7制剂 ( cowpea trysin inhibitor, CpTI ) 基因 CpTI ψ, ( 3 )淀粉酶抑制剂 ( amylase inhibitor, AI )基因: 如莱 Phaseolus 书
v /g n ) « -淀粉酶抑制剂基因 a - ai 等； （4 )植物外源凝集素 （lectin )类基因： 如雪花莲外源凝集素（ Galanthus nivalis aggulutinin, GNA )基因 g 等； ( 5 )昆虫特 异性神经毒素（ neurotoxin )基因： 如蝎毒素基因 Bank I Γ4等。 在生产上利用最成功 的基因主要有： 杀虫晶体蛋白基因和 CpTI基因。 其中应用最广泛的是 杀虫晶 体蛋白基因， 即苏云金芽孢杆菌 ( Bt ) 内毒素晶体蛋白基因， 克隆于苏云金芽孢杆 菌。 苏云金芽孢杆菌 （β )是 1901年发现的一种革兰氏阳性土壤芽孢杆菌， 已知其 是可产生对多种昆虫， 如鳞翅目（Lepidopterans)、 鞘翅目（Coleopterans)和双翅目 (Dipterans)等作物害虫有毒性的多种伴孢结晶蛋白质。这种杀虫蛋白质已经发现了五 十多大类， 三百多种。 Bt杀虫蛋白在昆虫消化道内消化酶的作用下， 蛋白被水解， 释放出约 60 ~ 70kDa抗蛋白酶的活性毒素分子。 活性毒素分子可与肠道上皮细胞紋 缘膜上的特异性受体结合， 并发生作用而使细胞膜穿孔。 消化道上皮细胞的离子、 渗透压平衡遭到破坏， 最终导致昆虫死亡。 由于其杀虫的专一性和高度选择性， 所 以对植物和包括人在内的动物没有毒害， 而且是环境可以接受的。 自 80年代后期以 来， 许多实验室已将 Bt杀虫基因导入到不同植物组织的细胞中， 并已在被转化的细 胞和植物中表达了这种来源与微生物的杀虫基因， 使转基因植物具有抗虫性。
不同种类 Bt杀虫蛋白杀虫谱可能不同，目前人们已经发现了对鳞翅目、鞘翅目、 双翅目据有毒性的杀晶体蛋白的大小及性状也不同。 在 1995年无脊推病理学会 (Society for Invertebrate Pathology, SIP)年会上专门成立了由 Crickmore等人组成的 Bt 杀虫晶体蛋白基因命名委员会， 提出了以杀虫蛋白氨基酸序列同源性为唯一标准的 分类命名体系， 将杀虫基因分为 17类， 36亚类 (Crickmore et al. 1995) , 2008年增补为 54类 , 101亚类 ( http：〃 www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ )。其中 Oy2A<¾ 对棉铃虫、 红铃虫、 玉米螟、 烟芽夜蛾、 粉紋夜蛾、 黎豆夜蛾、 稻纵卷叶螟、 大豆 夜蛾、 热带家蚊等害虫均具有杀虫效果。
由于遗传密码有 64种， 但是绝大多数生物倾向于利用这些密码子中的一部分， 如果直接将昆虫的基因直接转化至植物中， 在一定程度上会影响其蛋白的表达， 利 用偏爱密码子 (preferred codons)并避免利用率低的或稀有的密码子合成基因，可以增 加蛋白表达量。 另外 杀虫晶体蛋白的 Ν端小段氨基酸残基在毒蛋白活化过程中被切除是杀虫 活性所必须的。 发明内容
为提高转基因植物的抗虫性， 本发明的目的在于提供一种在植物中能高效表达 的杀虫基因。
一种杀虫基因， 具有 SEQ ID NO: 1所示的核苷酸序列。
一种杀虫基因， 具有 SEQ ID NO: 2所示的核苷酸序列， SEQ ID NO: 2所示的 核苷酸序列与 SEQ ID NO: 1所示的核苷酸序列相比， 5' 端在起始密码子后少 84 个碱基。
所述核苷酸序列釆用了植物偏爱性密码子。
所述核苷酸序列釆用了棉花偏爱性密码子。
本发明的第二个目的在于提供一种含有上述杀虫基因并可在植物中表达该杀虫 基因编码的杀虫蛋白质。
一种杀虫基因编码的蛋白质 CmCry2Aa杀虫蛋白， 由 SEQ ID NO: 1所示杀虫 基因编码， 具有 SEQ ID NO: 3所示的氨基酸序列， 由 633个氨基酸残基组成。
一种杀虫基因编码的蛋白质 CmlCry2Aa杀虫蛋白， 由 SEQ ID NO: 2所示杀虫 基因编码， 具有 SEQ ID NO: 4所示的氨基酸序列， 由 605个氨基酸残基组成， CmlCry2Aa杀虫蛋白与 CmCry2Aa杀虫蛋白相比 N端缺失 28个氨基酸， 二者都具 有杀虫活性。
一种含有上述杀虫基因的植物表达载体。
用含有上述杀虫基因植物表达载体转化的具有杀虫能力的植物细胞。
用含有上述杀虫基因植物表达载体转化的植物组织、 器官及植株。
本发明的第三个目的在于提供杀虫基因在培育抗虫植物品种中的应用， 尤其是 在培育抗虫棉花中的应用。
本发明利用棉花优化密码子合成了 CmCry2Aa以及 Cml Cry2Aa 杀虫蛋白基因， 并分别构建了植物表达载体。 转基因烟草鉴定结果表明所设计合成的 CmCry2Aa Cml CrylAa杀虫基因对棉铃虫具有极显著的抗性， 缺失 杀虫蛋白的 N端氨基酸 可简化杀虫蛋白质的活化， 在保持其杀虫活性的同时， 扩大其杀虫谱。 附图说明
图 1是 CmCry2AaDl-pGEM质粒图谱；
图 2是 CmC/ 2Afl )2-p[/C57质粒图谱；
图 3是
Figure imgf000003_0001
谱；
图 4是 Οη 2Αα )2. ？ -p[/C57质粒图谱；
图 5是 CmC/ 2Afl-p[/C57质粒图谱；
图 6是 Οη 2Αβ杀虫基因植物表达载体 Μ^-βΑ ·?5 Οηθ 2Αβ的构建流程 图；
图 7是 CmCry2Aa杀虫基因植物表达载体 Ml-BAR-35S-CmCry2Aa PCR扩增鉴定 图；
图 8是 CmCry2Aa杀虫基因植物表达载体
Figure imgf000003_0002
酶切鉴定图； 图 9是质粒 MJ-MR- S-CmC ^la转化烟草抗虫性鉴定图；
图 10是 CmCry2Aa杀虫基因植物表达载体 Ml-Pnos-NPTII-35S-CmCry2Aa的构 建流程图； 图 11是 CmC/ 2Aa杀虫基因植物表达载体 M Pnos-NPTII-35S-CmCry2Aa的酶 切鉴定图；
图 12是 CmJCry2Aa - ρΜ λ ^质粒图谱；
图 13a、 b是 CmlCry2Aa植物表达载体 Ml -Pnos-NPTII-35S-Cml CrylAa的构建 流程图；
图 14是质粒 Ml -Pnos-NPTII-35S-Cml CrylAa转化烟草抗虫性鉴定图； 图 15是 CmCry2Aa基因原核表达载体 CmCry2Aa-pET30a的构建流程图； 图 16是 CmCry2Aa基因原核表达载体 CmCry2Aa-pET30a酶切鉴定图； 图 17是 CmJCry2Aa基因原核表达载体 CmlCry2Aa-pET30a的构建流程图； 图 18是 CmlCry2Aa基因原核表达载体 CmlCry2Aa-pET30a酶切鉴定图； 图 19是 CmCry2Aa及 CmlCry2Aa杀虫蛋白的原核 达的蛋白电泳图； 具体实施方式
主要试剂配方：
LB培养基： 胰蛋白胨 lOg/1, 酵母粉 5g/l, 氯化钠 lOg/1, PH: 7.0 ~ 7.2, 固体 培养基加 1.5%琼脂粉
抗生素： Kan (卡那霉素） 50mg/ml, Chi (氯霉素） 34mg/ml
1M Tris-Hcl ( PH6.8 ): 121.2gTris溶于 1L水中， 用浓盐酸调节 PH值到 6.8 , 高 压灭菌
1.5M Tris-Hcl ( PH8.8 ): 181.7gTris溶于 1L中， 用浓盐酸调节 PH值到 8.8 , 高 压灭菌
30% Acrylamide: 丙烯酰铵 290g、 N, N-亚甲双丙烯酰铵 10g定容到 1L超纯水 中
0.1mol/l IPTG: 0.25g的 IPTG溶解于 10mL的 ddH20中
1M DTT: 3.09gDTT溶于 20ML的 0.01M醋酸钠 （PH5.2 ) 中， 分装成小份一
20 °C保存
10%过硫酸铵： lg过硫酸铵加水定容至 10ml, 分装成小份一 20 °C保存
1 x SDS-PAGE凝胶上样緩冲液： 50mmol/lTris-HCL ( PH6.8 )、 2%SDS、 0.1%臭 酚蓝、 10%甘油、 100mmol/lDTT (临用时加）
5 X Tris-Glycine Buffer: Tris 15. lg、 Glycine 94g SDS 5.0g定容到 1L水中 考马斯亮蓝染色液： 0.1%考马斯亮蓝 R-250 、 25%异丙醇、 10%水醋酸定容到 1L水中
考马斯亮蓝脱色液： 100ml醋酸、 50ml乙醇定容到 1L水中 实施例 1 Οη 2Αβ杀虫基因的合成
1. 根据 θ 2Αβ杀虫蛋白的氨基酸序列， 根据棉花密码子用法， 分结构域分别 设计合成了构建 Ο 72Αβ 杀虫基因的 3个基因片段。 基因合成委托北京奥科公司 完成。
根据设计， 合成的 Οη 2Αβ杀虫基因的密码子用法如下： U C A G
F 11/17/33 S 25/39/42 Y 12/19/44 C 2/3/50 U
F 22/35/67 S 12/19/20 Y 15/24/56 C 2/3/50 C
u L 0/0/0 S 17/27/28 * 1/2/100 * 0/0/0 A
L 19/30/30 S 0/0/0 * 0/0/0 W 8/13/100 G
L 32/50/51 P 14/22/54 H 8/13/73 R 3/5/8 U
L 10/16/16 P 3/5/12 H 3/5/27 R 0/0/0 c
c L 0/0/0 P 9/14/35 Q 19/30/66 R 3/5/8 A
L 2/3/3 P 0/0/0 Q 10/16/34 R 1/2/3 G
I 25/39/64 T 28/44/49 N 31/49/46 S 1/2/2 U
1 14/22/36 T 14/22/25 N 37/58/54 S 5/8/8 c
A 10/0/0 T 15/24/26 K 2/3/22 R 16/25/44 A
M 11/17/100 T 0/0/0 K 7/11/78 R 13/21/36 G
V 24/38/65 A 16/25/52 D 18/28/75 G 23/36/56 U
V4/6/11 A 7/11/23 D 6/9/25 G 3/5/7 c
G V 0/0/0 A 8/13/26 E 11/17/58 G 8/13/20 A
V 9/ 14/24 A 0/0/0 E 8/13/42 G7/11/17 G 注： 上表中各密码子所编码氨基酸符号后的三个数字分别表示： 密码子数量 /在蛋白中的出 现频率 （％。 ) /密码子用法（％ )
结构域 I ( Domain I ) DI基因片段： BamHI-ATG-DI-Apal, 其具体序列如： SEQ NO ID: 5所示。
结构域 II ( Domainll ) DII基因片段： Apal-DII-SspI,其具体序列如： SEQ NO ID: 6所示。
结构域 III ( Domainlll ) Dili基因片段： Xbal - Hpal-DIII-SacI, 其具体序列如： SEQ NO ID: 7所示。
Cm0 2Aa杀虫基因的三个结构域基因编码片段 Dl、 D2、 D3人工合成后， 分 别克隆到 体 pGEM和 /?[/ 57中， 质粒分别命名为： CmCry2AaDl -pGEM, 质粒 图谱如图 1所示； CmCry2AaD2 -PUC57, 质粒图谱如图 2所示和 CmCry2AaW - pUC57, 质粒图谱如图 3所示。
2. Οη θ 2Αβ杀虫基因的克隆拼接
首先将 CmCry2AaD3-PUC57 中 的 Hpal _ D3 _ Sad 片 段克 隆到 CmCry2AaD2-pUC57中的 Sspl和 Sacl位点之间， 一平端一粘端连接， 构建得到载 体质粒 CmCry2AaD2.D3 - /?[/ 57,质粒图谱如图 4所示，然后将 CmCry2AaDl-pGEM 中的 Sphl - Apal D1片段克隆到该载体中，得到质粒 CmCry2Aa - pUC57 ,质粒图谱 如图 5所示， 完成 CFM
Figure imgf000005_0001
杀虫基因的拼接， Οη θ 2Αβ杀虫基因的核苷酸 序列如 SEQ ID NO: 1 所示。 与天然的 C/ 2Aa ( genebank 登陆号 AY496458 : http://www.ncbi.nlm.nih.gov/entrez/ viewer.fcgi?db =nuccore&id=45685585 )基因对应邵分的核 苷酸序列对比， 其同源性为 75.97%。 其编码蛋白质氨基酸序列的同源性为 99.95%。 实施例 2 CmCry2Aa杀虫基因植物表达载体 Ml-BAR-35S-CmCry2Aa的构建
带有完整 CmCry2Aa杀虫基因的质粒 CmCry2Aa-pUC57 , 进一步按照如图 6所 述程序构建植物表达载体
Figure imgf000005_0002
植物表达载体
Ml -BAR-35S-CmCry2Aa构建过程中筛选鉴定的 PCR鉴定结果及酶切鉴定结果分别 如图 7和图 8所示， 图 7中 1、 2和 3泳道代表以 1、 2和 3号克隆子为模板扩增出 了目标条带， " + " 代表以质粒
Figure imgf000005_0003
为模板扩增出目标条带 （阳 性对照）， " - "代表以水为模板未扩增出目标条带（阴性对照）； 图 8中 1泳道为限 制性内切酶 Xba l + Sacl切质粒 MJ-MR-rwo^G s, 切下 GUS , 大约 1.8Kbp左右， 2, 3 , 4, 5和 6泳道为限制性内切酶 Xbal + Sacl切 1 , 2, 3 , 5 , 6克隆子， 切下来 35S + CmCrylAa , 大约 2.7Kbp左右。 结果表明 1、 2、 3号克隆均为正确克隆。 实施例 3 转 Οη 2Αβ杀虫基因烟草获得
釆用农杆菌介导叶盘法转化烟草（本领域科研人员周知的方法）， 经 PPT生根筛 选， 获得抗性植株 46株， 提取抗性烟草叶片总 DNA, 进行 PCR鉴定， 筛选出 27 株阳性植株， 并进行抗棉铃虫饲虫试验， 每叶接 5头初孵幼虫， 三天后结果如图 9 所示， 1为未转 CmC/ 2Aii杀虫基因烟草叶片， 2、 3和 4分别为转化
Figure imgf000006_0001
杀虫基因烟草叶片， 结果表明所合成 Οη 72Αβ杀虫基因抗棉铃虫效果非常突出。 经调查， 棉铃虫平均校正死亡率 93.3 %。 实施例 4 Nptll 为选择标记的 CmC/ 2Afl 杀虫基因植物表达载体 Ml-Pnos-NPTII-35S-CmCry2Aa的构建
鉴于 Ο 72Αβ杀虫基因具有理想的棉铃虫毒杀效果， 进一步进行了棉花的农 杆菌转化。 由于原 CmC/ 2Aa植物表达载体
Figure imgf000006_0002
中选择标记基 因 ββΓ不利于棉花的农杆菌转化体系 （草丁膦对棉花细胞的再生分化有抑制作用）， 设计了克隆路线， 获得 Nptll为选择标记基因的 CmCry2A i杀虫基因植物表达载体 M Pnos-NPTII-35S-CmCry2Aa^k ρΒΠ21通过 PCR获得的 Np //基因经克隆后经测 序验证， 确信所扩增 Np //基因核苷酸序列正确。 整个克隆流程如图 10。
最终载体
Figure imgf000006_0003
经酶切鉴定， 结果如图 11所示， 1 , 2泳道为 EcoRI切 Ml-Pnos-NPTII-35S-CmCry2Aa的克隆子 7 , 22的结果， 能切下 1.7Kb 左右的带， 阳性； 3泳道为 EcoR I切 Pnos-NPTII-Tnos-PBS作为阳性对照， 切下 1.7Kbp左右的带； 4, 5泳道为 Ncol + Kpnl切 Ml-Pnos-NPTII-35S-CmCry2Aa 的克隆子 7 , 22的结果， 能切下 1.4Kbp左右的带， 为阳性； 6, 7泳道为 Pst I切 Ml-Pnos-NPTII-35S-CmCry2Aa的 i 子 Ί , 22的结果， 能切下 3Kbp左右的带， 阳 性； 证明所构建以 Np //为选择标记基因的 CmCry2Aa植物表达载体完全正确。 实施例 5 CmlCrylAa杀虫基因的克隆及植物表达载体的构建及其功能鉴定
( 1 )
Figure imgf000006_0004
杀虫基因的克隆
根据 CmCry2Aa杀虫蛋白的分子结构， 杀虫基因 N端存在长度为 29个氨基酸的 Coli结构片段， 位于结构域 1 α -螺旋 1的前端。根据杀虫蛋白结构功能关系， 该氨 基酸序列为原毒素活化时需要被昆虫消化道胰蛋白酶水解去除。经 DNAman软件分 析， 在第 29、 30氨基酸之间的确存在胰蛋白酶识别切割位点。
如果杀虫蛋白进入昆虫消化道后如不能切除该段多肽， 杀虫活性降低。 因此， 设 计突变引物， 将 CmCry2Aa的 5' 端 28个氨基酸去除， 使表达出的蛋白直接为活性 蛋白， 对于扩大杀虫谱（昆虫不能正确活化原毒素时）和提高杀虫效率有利 （活化 效率不是 100%时）。 将其命名为 CmJC/ 2Aa。 CmlCrylAa 5端引物加 BamHI位点， 3端引物加 Sacl位点， 以便下一步的克隆。 所设计的引物如下：
引物 1 2Aa5： 5'-G GGATCC ATGTCTTTGGACACTATCCA-3' 引物 2 2Aa3： 5'-GAGCTCTTAGTACAAGGGT-3'
以质粒 CmCry2Aa-pUC57为模板， 扩增出的目的条带为 1820bp。 将扩增出的 CmJC ^Aa克隆到 ρΜ λ？ S-Γ上， 命名为： CmlCry2Aa -pMD18, 其质粒图谱如图 12所示。
( 2 ) CmlCrylAa植物表达载体 Ml -Pnos-NPTII-35S-Cml Cry2Aa的构建 为在植物中验证 Cml Cry2Aa的杀虫功能， 设计构建以 Nptll为选择标记基因的
Figure imgf000007_0001
构建流程如图 13a和 b 所示。
( 3 ) Cml Cry2Aa在烟草中的抗棉铃虫鉴定
釆用农杆菌介导叶盘法转化烟草 （本领域科研人员周知的方法）， 经卡那霉素生 根筛选， 获得抗性植株 25株， 提取抗性烟草叶片总 DNA , 进行 PCR鉴定， 筛选出 22株阳性植株， 并进行抗棉铃虫饲虫试验， 每叶接 5头初孵幼虫， 三天后部分结果 如图 14所示， 1、 2为未转
Figure imgf000007_0002
杀虫基因烟草叶片 （对照试验）， 3和 4分别 为转化 CFM Οηθ 2Αβ杀虫基因烟草叶片， 结果表明所合成
Figure imgf000007_0003
杀虫基因 抗棉铃虫效果与 Οη 2Αβ杀虫基因基本相当。经调查，棉铃虫平均校正死亡率 87.7 %。 统计学分析表明差异不显著， 与设计的预期结果一致。 实施例 6 CmCry2Aa及 CmlCry2Aa杀虫基因原核表达载体的构建及原核表达
( 1 ) CmCrylAa基因原核表达载体 CmCry2Aa-pET30a的构建
CmCrylAa基因原核表达载体 CmCry2Aa-pET30a的构建流程如图 15所示， 其 酶切鉴定结果如图 16所示， "B + S" 表示 BamH I + Sad切 1号 CmCry2Aa-pET30 阳性克隆子质粒， 能切下完整的 Οηθ 2Αβ, 1.9K bp左右， 大小符合； " + " 表示 BamH I + Sad切 CmCry2Aa-pUC57质粒故为正对照， 也能切下完整的 CmCry2Aa, 1.9K bp左右， 大小符合； "B" Bgll单酶双切 CmC/ 2Aa-pE: ?0的阳性克隆子质粒， 能切下 1.3Kbp左右的带， 大小符合， 可见此构建正确。
( 2 ) CmlCry2Aa基因原核表达载体 CmlCry2Aa-pET30a的构建
CmlCry2Aa基因原核表达载体 CmlCry2Aa-pET30a的构建流程如图 17所示， 其酶切鉴定结果如图 18 所示， 1 , DNA Ladder; 2 , BamH I + Sad 切 4 号 CmlCry2Aa-pET30a阳性克隆子质粒， 能切下完整的 CmlCry2Aa, 1.8 K b 左右， 大小符合； 3 , BamH I + Sacl切 1号
Figure imgf000007_0004
阳性克隆子质粒， 能切下 完整的 CmJC/ 2Aa, 1.8 K bp左右， 大小符合（载体酶切不充分）； 4, Kpnl切 4 号 CmlCry2Aa-pET30a阳性克隆子质粒，能切下 1.78 K bp左右基因片段，大小符合； 5 , Kpnl切 1号 CmJCry2Aa-pET30a阳性克隆子质粒， 能切下 1.78 K b 左右的基 因片段， 大小符合。 可见此构建正确。
( 3 ) Οηθ 2Αβ及
Figure imgf000007_0005
基因的原核表达
制备感受态大肠杆菌细胞：在 LB培养基平板上活化 E.coli Rosetta ( DE3 )菌株， 挑取单菌落接种于 5mlLB 液体培养基中， 37 °C摇菌过夜， 取此菌液 500μ1加入到 lOOmlLB液体培养基中， 37°。培养2 ~ 2.51 1", 有大量絮状菌体出现时， 水浴 15min, 4°C、 6000r/m离心 5min, 弃上清， 加入 1/5体积预冷的氯化钙， 重悬菌体， 4°C、 6000r/m离心 5min, 再加 1/3体积的氯化钙， 重悬菌体， 水浴 30min, 4°C、 6000r/m 离心 5min, 弃上清， 吸干， 加入 1/25体积氯化钙重悬菌体， 以 ΙΟΟμΙ菌液分装， - 70°C保存。
分别将空原核表达载体 pET30a (+ X CmCry2Aa-pET30a及 CmlCry2Aa-pET30a 原核表达载体转入菌株 E.coli Rosetta ( DE3 )感受态细胞中， 具体方法为： 从- 70 °C水箱中拿出 E.coli Rosetta ( DE3 )感受态细胞， 置于水上解冻， 在超净工作台上 把 ΙΟμΙ连接产物加入到感受态细胞中， 轻轻混匀， 水浴 30min, 再在 42 °C水浴中热 激 90sec, 迅速再水浴 2min , 再加入 350μ1ίΒ 空白液体培养基， 37°C下摇动培养 40min,再将此管菌体全部加入到含有 5(Vg/ml Kan的固体培养基中，均匀涂布平板， 待液体完全被吸收后， 37 °C倒置培养 12 ~ 16小时。 挑取单菌落接种到含有 5(Vg/ml Kan和 34 g/ml Chi抗生素的 5mL液体培养基 中 37 °C过夜培养， 取 ΙΟΟμΙ接种到含有 5(Vg/ml Kan和 34 g/ml Chi抗生素的 10mL 液体培养基中， 37 °C培养 2hr左右， OD600 « 0.6时加入 IPTG至终浓度 lmmol//l, 37 °C下诱导表达。 诱导表达 4hr后， 各取 lmL菌液， 10000r/m离心 5min, 收集菌 体，加入 ΙΟΟμΙ含有 lOOmmol/lDTT 的 1 x SDS-PAGE凝胶上样緩冲液重悬，取 40μ1 在 100 °C煮 5min, 取 20μ1上样， 进行 SDS-PAGE电泳。 SDS-PAGE分析法参考《分 子克隆》， 浓缩胶浓度为 5% , 分离胶浓度为 10% , 先以 80v电压电泳， 待溴酚蓝指 示剂进入到分离胶后， 加大电压到 ΙΙΟν, 大约 lhr30min待指示剂移动到胶底部时， 结束电泳， 用考马斯亮蓝染色液染色过夜，再用脱色液脱色直至蛋白条带清晰为止。
图 19为 CmCry2Aa及 CmlCry2Aa基因的原核表达 SDS-PAGE电泳结果， 1和
2分别为经大肠杆菌表达的 CmlCry2Aa蛋白及 CmCry2Aa蛋白，可见大小在约 66 kD 附近， CmlCry2Aa目的蛋白较 CmCry2Aa蛋白略小（ pET30a载体上 HIS ' Tag基因 150bp , 编码 50个氨基酸）； 3为转化 pET30a空载体的阴性对照试验； 4为表达的 截断的 CrylAc杀虫蛋白 （专利号 ZL95863.8 )； 5为蛋白质 Marker。 实施例 Ί 利用 CmC/ 2Aa及 CmlCry2Aa杀虫基因原核表达可溶性产物对选择的鳞 翅目昆虫种的杀虫活性分析
CmCry2Aa-pET30a、 CmlCry2Aa-pET30a及 CrylAc-pET30a原核表达载体 转入菌株 E.coli Rosetta ( DE3 )后， 分别进行诱导 达， 利用 SDS电泳结果扫 描定量， 将目的杀虫蛋白浓度调整到接近水平。 制备 3个浓度梯度： 每 20克 人工饲料中分别加入蛋白初提液 73μ1、 660μ1、 2000μ1, 混勾制备饲料。 在制备 的祠料上分别祠养棉铃虫 ( Helicoverpa armigera )和玉米虫冥 ( Ostrinia nubilalis ), 进行毒性测定。
在 48孔板（ Costar )上逐孔放入人工准备的饲料，每个孔中放入一只幼虫， 每种浓度饲料用 24只幼虫， 2次重复， 7天计算校正死亡率。 获得试验结果如 下表:
Figure imgf000008_0001
实施例 8 利用 CmC/ 2Aa及 Cm:iCry2Aa杀虫基因植物表达载体获得转基因棉花
通过农杆菌介导法将 CmCry2Aa或 CmlCry2Aa杀虫基因植物表达载体转 化棉花， 获得转基因棉花。
农杆菌介导法是本领域科研人员熟知的植物遗传转化方法。具体操作程序 为：
1. 菌株培养
将所构建的杀虫基因植物表达载体电激转化到农杆菌菌株 LBA4404中， 农杆菌单菌落接种于含卡那霉素 (kanamycin, km)50 mg/L、 利福平 (rifampicin, rif)25 mg/L的 LB或 YEB液体培养基中。 28 °C振荡暗培养过夜到细菌生长对数 期。 用 LB或 YEB液体培养基稀释菌液， 再振荡培养 4~6 h, 将菌液稀释至 OD600值 0.3 0.35。
2. 无菌苗制备
(1)棉花种子用硫酸 (H2S04)脱去短绒， 自来水洗掉种子表面的硫酸， 晾 干后用 70 %乙醇对种子进行表面消毒 1 min, 再用 10 % -15 %过氧化氢 (H202) 处理 2~4 h , 用无菌水冲洗 2~3次；
(2)在无菌水中浸泡 18~24 h,待种子露白， 再在无菌条件下剥去种皮， 种 入种苗培养基 (1/2 MS + 琼脂 6g/L, pH 6.8)中；
(3) 25 °C -28 °C光培养 3〜5 d 时备用。
3. 棉花外植体与农杆菌的共培养
取无菌苗的下胚轴， 用解剖刀切成 0.5~0.6 cm小段， 浸入稀释好的菌液中 5-10 min, 然后取出胚轴段， 用灭菌滤纸吸干多余的菌液， 放在共培养培养基 _h(MS + 2.4-D 0.1 mg/L + KT 0.1 mg/L + 葡萄糖 30 g/L + 乙酰丁香酮 200 mg/L + 琼脂 6 g/L, pH5.0 , 表面铺一层灭菌滤纸）， 用封口膜封口。 22°C 45 °C共培养 2天。
4. 诱导愈伤组织及抗性愈伤组织的筛选
(1)愈伤组织的诱导
经共培养后的下胚轴段放入愈伤组织诱导培养基中 （MS + 2,4-D 0.1 mg/L + KT 0.1 mg/L + MgC12 0.91 g/L + Gelrite 2.0 g/L + Km 50400 mg/L + Cef 500 mg/L + 葡萄糖 30 g/L, pH 5.8) , 在常规条件下 （25 °C )培养 2个月 （一个 月换一次相同的培养基)。
(2) 抗性愈伤组织的检测
无菌条件下挑取愈伤组织少许进行选择标记基因 nptll的 ELISA检测或报 告基因 的检测， 检测结果为阳性的愈伤组织继续继代， 非阳性的愈伤组织 淘汰。 通过对《ρ //或 g s基因表达的检测， 获得棉花抗性愈伤组织的频率为 50%~76%。
5. 愈伤组织的增殖继代
诱导出的抗性愈伤组织接入增殖培养基 (MS培养基 + MgC12 0.91 g/L + Gelrite 2.0 g/L + 葡萄糖 30 g/L, pH 5.8)中， 常规条件下 (25 °C )培养, 每隔一个 月继代一次， 直到愈伤组织分化。 在第一次和第二次转入增殖培养基后有部分 愈伤组织褐化死亡， 正常愈伤组织增殖也不快， 第二次继代后， 愈伤组织增殖 速度才加快。
6. 愈伤组织的分化及转基因苗移栽
愈伤组织经继代几次后， 有的愈伤组织转成米粒状颗粒， 将其转入分化培 养基中 （无 NH4+、且 KN03加倍的 MS +谷氨酰胺 1.0 g/L + 天门冬酰胺 0.5 g/L + MgC12 0.91-1.35 g/L + Gelrite 2.0-3.0 g/L + 葡萄糖 20~30 g/L, pH 5.8) , 进一步分化成胚状体， 胚状体长成为小植株后再转入大的三角瓶中， 待根长好 后练苗移栽。 洗去再生棉株根部的培养基， 栽到灭菌蛭石中， 浇足营养液。 栽 好的再生棉苗放入控温 22 °C、 控湿 80~85%的人工培养箱中 5~7 d, 再在温室 中培养 10~20 d后移栽到土盆或大田中。
利用农杆菌介导转化方法，分别将 CmCry2Aa及
Figure imgf000009_0001
杀虫基因植物表达 载体导入棉花中获得了转基因棉花。 实施例 9 CmCry2Aa及 CmlCry2Aa转基因棉花对棉铃虫的抗虫性分析 利用初孵棉铃虫幼虫，对 45株 CmCry2Aa及 22株 CmlCry2Aa已经移栽于温室的 T„代转基因棉花植株进行抗棉铃虫杀虫活性鉴定。 取待测转基因棉花倒数第二果枝 2片展开叶， 叶柄基部用湿润脱脂棉球包裏， 每个叶片置于一个培养亚中（每株设 2 次重复）。 每个培养亚接健康棉铃虫初孵幼虫 5头， 置于室内避光放置， 3天和 5天 进行棉铃虫存活情况以及棉花叶片受危害情况调查。 分别计算平均校正死亡率和确 定叶片受害级别。 死亡率 =死虫数 /10 100%; 校正死亡率 = (死亡率 -对照死亡率） I ( 1-对照死亡率）。 棉花叶片受危害情况分成 5级： 0级： 轻微， 虫取食未穿透形成 孔洞； 1级： 较轻， 形成 3-5个针眼大小孔洞； 2级： 轻， 形成 10个以下小于 1平 方毫米孔洞； 3级： 中等， 形成 10个以上小于 1平方毫米小孔洞； 4级： 较重， 形 成 10个以上小孔洞或者形成 1-3个大于 1平方毫米孔洞； 5级： 重， 形成 3个以上 大于 1平方毫米孔洞。 试验结果如下表所示。
序号 转化事件编号 3天校正死亡率 5天校正死亡率 叶片受害级别
对照 0. 0% 0. 0% 5
1 cm-D10 11. 1% 100. 0% 0
2 cm-Dl l 22. 2% 37. 5% 3
3 cm-D13 22. 2% 50. 0% 5
4 cm-D14 33. 3% 50. 0% 3
5 cm-D15-l 33. 3% 37. 5% 4
6 cm- D 15- 2 22. 2% 50. 0% 3
7 cm-D16-2 0. 0% NA 5
8 cm-D17 33. 3% 62. 5% 4
9 cm-D18 55. 6% 75. 0% 3
10 cm-D20 11. 1% 12. 5% 5
11 cm-D21 88. 9% 100. 0% 1
12 cm-D22-l 100. 0% 100. 0% 1
13 cm-D24 66. 7% 100. 0% 2
14 cm_D3 44. 4% 62. 5% 4
15 cm-D4 44. 4% 75. 0% 3
16 cm_D5 88. 9% 87. 5% 1
17 cm_D6 0. 0% 75. 0% 2
18 cm-D7 33. 3% 50. 0% 4
19 cm-D8 55. 6% 75. 0% 2
20 cm-D9 100. 0% 100. 0% 0
21 cm-D9-2 22. 2% 75. 0% 2
22 cm- G2H1 11. 1% 25. 0% 5
23 cm- HI 88. 9% 87. 5% 1
24 cmHl-2 88. 9% 100. 0% 1
25 cmHlO-1 100. 0% 100. 0% 1
26 cmH10-2 100. 0% 100. 0% 1
27 cmHl l 100. 0% 100. 0% 1
28 cmHl-2 44. 4% 62. 5% 3
29 cmHl-3 11. 1% 37. 5% 4
30 cmH2 77. 8% 87. 5% 1 /:/: O 09so/-o600si>l£AV
Figure imgf000011_0001

Claims

权 利 要 求 书
1、 一种杀虫基因， 具有 SEQ ID NO: 1所示的核苷酸序列。
2、 一种杀虫基因， 具有 SEQ ID NO: 2所示的核苷酸序列。
3、 根据权利要求 1和 2所述的杀虫基因， 其特征在于所述核苷酸序列釆用 了植物偏爱性密码子。
4、 根据权利要求 1和 2所述的杀虫基因， 其特征在于所述核苷酸序列釆用 了棉花偏爱性密码子。
5、 权利要求 1所述的杀虫基因编码的蛋白质， 具有 SEQ ID NO: 3所示的 氛基酸序列。
6、 权利要求 2所述的杀虫基因编码的蛋白质， 具有 SEQ ID NO: 4所示的 氨基酸序列。
7、 含有权利要求 1或 2所述杀虫基因的植物表达载体。
8、 用权利要求 7所述杀虫基因植物表达载体转化的具有杀虫能力的植物细 胞、 组织或植株。
9、 权利要求 8所述的具有杀虫能力的植物细胞、 组织或植株在培育抗虫植 物品种中的应用。
10、根据权利要求 9的具有杀虫能力的植物细胞、组织或植株在培育抗虫植 物品种中的应用， 其特征在于所述植物为棉花。
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