WO2019169625A1 - 一种人工改良的杀虫蛋白质及其编码基因与应用 - Google Patents

一种人工改良的杀虫蛋白质及其编码基因与应用 Download PDF

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WO2019169625A1
WO2019169625A1 PCT/CN2018/078572 CN2018078572W WO2019169625A1 WO 2019169625 A1 WO2019169625 A1 WO 2019169625A1 CN 2018078572 W CN2018078572 W CN 2018078572W WO 2019169625 A1 WO2019169625 A1 WO 2019169625A1
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plant
mutant
insecticidal
gene
insecticidal protein
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PCT/CN2018/078572
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English (en)
French (fr)
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崔洪志
王建胜
黄达锋
王君丹
张楠
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创世纪种业有限公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/127—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or 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
    • 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

Definitions

  • the invention belongs to the field of plant molecular biology, in particular to the field of transgenic crop breeding in agricultural biotechnology research, in particular to insect-resistant transgenic crop research, in particular to an artificially improved insecticidal protein and its coding gene and application.
  • Bacillus thurigiensis is a Gram-positive bacterium, and the companion crystals produced during sporulation are called delta-endotoxin, or insecticidal crystal protein.
  • the Bt insecticidal proteins found so far can poison insects such as Lepidoptera, Diptera, Coleoptera, etc. (Aronson, Microbiol. Rev. 50: 1-14, 1968).
  • There are currently 74 classes of 793 Bt insecticidal crystal protein (Cry) genes reported (Crickmore, 2017, http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ Index.html).
  • Bt insecticidal protein controls the specificity and high selectivity of pests, it is not toxic to plants and animals including humans, and is environmentally friendly. Therefore, it has been widely used and used as a biological insecticide. Agent.
  • GM technology provides a more ideal solution for the worldwide problem of pests causing serious losses to agricultural production. People can control pests by planting Bt transgenic insect-resistant crops. In 2016, the global GM crop planting area was 185.1 million hectares, of which 53% of Bt insect-resistant crops were planted, including compound or single insect-resistant GM crops, ie, the planting area of Bt insect-resistant GM crops in 2016 was about 98.1 million hectares. (Clive J., ISAAA Briefs, 2016).
  • Cry2Aa Bt insecticidal protein is resistant to lepidopteran pests and is a kind of Bt insecticidal protein which is deeply researched in the field of insect-resistant transgenic plants.
  • Cui Hongzhi et al. engineered the CmCry2Aa and Cm1Cry2Aa insecticidal genes and performed functional verification in model plant tobacco (ZL200810065477.2).
  • new Bt insect-resistant genes with improved insecticidal activity and improved effects are still needed in the art to improve the insect-resistant efficiency of insect-resistant plants and delay the evolution rate of pest-resistant plants.
  • the present invention carried out a series of mutation design, and used the site-directed mutagenesis technique to mutate and identify the cmCry2Aa insecticidal gene (ZL200810065477.2).
  • a single point mutation with enhanced insecticidal activity of W78F, L144A and R213E, combined with the N-terminal 28 amino acid deletion mutation, can enhance the insecticidal effect of Cry2Aa insecticidal protein.
  • the present invention provides a mutant Cry2Aa insecticidal protein, the amino acid sequence of the mutant Cry2Aa insecticidal protein having the following one or two based on the amino acid sequence shown in SEQ ID NO. Or three mutations:
  • the amino acid sequence has an N-terminal 28 amino acid deletion.
  • the invention provides a gene encoding a mutant Cry2Aa insecticidal protein of the first aspect of the invention.
  • the invention provides a recombinant expression vector comprising the coding gene of the second aspect of the invention and the nucleotide sequence of the coding gene is operably linked to an expression control sequence of the expression vector.
  • the present invention provides a method for improving the insect resistance of a plant, comprising: introducing the coding gene of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention into a plant or plant tissue and encoding the same Gene expression; preferably, the plant is tobacco.
  • the present invention provides a method of producing a transgenic plant, comprising: cultivating a plant or plant comprising the coding gene of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention under conditions effective to produce a plant organization.
  • the present invention provides the use of the coding gene of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention for improving the insect resistance of a plant and for use in plant breeding.
  • the present invention provides a method of producing a mutant gene encoding the mutant Cry2Aa insecticidal protein of the first aspect of the invention
  • the method comprises sequentially mutating a DNA sample of the sequence set forth in SEQ ID NO. 2 with one, two or three of the following three sets of primers:
  • Figures 1A-C show the W78F, L144A and R213E mutant cmCry2Aa insecticidal genes.
  • A W78F mutant cmCry2Aa insecticidal gene showing nucleotide variation of W78F mutation
  • B L144A mutant cmCry2Aa insecticidal gene showing nucleotide change of L144A mutation
  • C R213E mutation cmCry2Aa insecticidal gene, Nucleotide changes in the R213E mutation are shown.
  • Figure 2 shows the PCR and restriction enzyme digestion of the mutant cmCry2Aa-pET30a prokaryotic expression vector: (A) PCR identification: 1-11: mutant cmCry2Aa-pET30a clone sample, +: cmCry2Aa-pET30a; (B) digestion identification: 1 , 5, 8: mutation cmCry2Aa-pET30a/NcoI+SacI, M: ⁇ DNA marker.
  • Figure 3 shows SDS-PAGE electrophoresis analysis of prokaryotic expression soluble protein of mutant cmCry2Aa insecticidal gene such as W78F, L144A and R213E.
  • A 1:A460L;2:W78F;3:L144A;4:cmCry2Aa;5:T615TFNP;6:ATNL353AVF;7:Loop3;8:G324V;9:pET30a (empty vector control);
  • 10 Molecular weight protein Marker
  • B 1:1 mCry2Aa; 2: 2 mCry2Aa; 3: R213A; 4: R213E; 5: R213K; 6: T439R; 7: cmCry2Aa; 8: pET30a; 9: BSA (2 ⁇ g); 10: medium molecular weight protein Marker.
  • Figure 4 shows the results of bioassay of the cotton bollworm by the cmCry2Aa partial mutant insecticidal protein.
  • Figure 5 shows the PCR and restriction enzyme digestion during the construction of some plant expression vectors:
  • A PCR identification: 1-10: p2300 vector self-ligated clone; 11-16: L144A clone; 17-22: D108L clone 23-28: QFQ163LFR clone; 29-34: MH183LR clone, 35-40: ATNL353AVF clone; 41: L144A-pET30a positive control, 42: p2300 vector plasmid negative control, 43: blank negative control;
  • B Identification by digestion: 1, 2: MH183LR; 3, 4 ATNL353AVF; 5, 6 SQF163LFR; 7, 8 D108L (3, 5); 9, 10 L144A.
  • Figure 6 shows a 35S-cmCry2Aa-Pnos-NPTII-p2300 plasmid map.
  • Figure 7 shows the Cry2Aa Elisa protein standard curve.
  • Figure 8 shows the amount of insecticidal leaf protein expression in transgenic tobacco.
  • Figure 9 shows the comparison of the R213E mutation cmCry2Aa with the cmCry2Aa tobacco bollworm (B. sinensis for 4 days).
  • Figure 10 shows the construction of the plant expression vector 35S-Cry2Aa-Pnos-NPTII-2300.
  • WL, WLR and WLRN represent different polymerizations of W78F, L144A, R213E and NS336LR, respectively.
  • the Cry2Aa insecticidal protein has the amino acid sequence shown in SEQ ID NO. 1, and according to the amino acid sequence of the Cry2Aa insecticidal protein, according to the cotton codon usage, the subdomain is designed and synthesized to construct the cmCry2Aa insecticidal gene.
  • the gene fragments were artificially synthesized and cloned into the vectors pGEM and pUC57, respectively, and further spliced the complete cmCry2Aa Bt insecticidal gene.
  • the gene fragment was synthesized and cloned according to the functional domain of Cry2Aa insecticidal protein, which is considered to facilitate further study on the structure and function of the gene.
  • a 29-amino acid Coli structural fragment is present at the N-terminus, which is located at the front end of the domain 1 ⁇ -helix 1.
  • the protoxin is activated by the trypsin hydrolysis of the insect digestive tract.
  • BIR2 cmCry2Aa
  • BIR2-1 1 mCry2Aa (N-terminal deletion mutation, N-terminal deletion of 28 a.a.)
  • BIR2-2 2mCr2Aa (N-terminal deletion mutation, N-terminal deletion of 43 a.a.).
  • the BIR2 (cmCry2Aa) sequence is shown in SEQ ID NO.
  • the amino acid sequence has a deletion of 28 amino acids at the N-terminus, and the corresponding gene mutation is a deletion of nucleotides 4 to 87 of the 5' end of SEQ ID NO. 2; the N-terminal 43 amino acids of the amino acid sequence are deleted, corresponding to SEQ ID NO The 5' end of the .2 is deleted at nucleotides -132.
  • mutant primers PCR was used for site-directed mutagenesis. According to the design, the mutation primers designed by three single-point mutations are respectively shown in SEQ ID NO. 3 to 8 as follows:
  • the PCR reaction system using the mutant CmCry2Aa-pUC57 as a template and using the mutant primers for site-directed mutagenesis is as follows:
  • the CmCry2Aa-pUC57 plasmid carrying the BIR2 insecticidal gene was used as a template, and the cmCry2Aa gene was subjected to site-directed mutagenesis by PCR single point mutation experiment using the designed mutant primers.
  • the PCR reaction system is as follows:
  • Reaction procedure pre-denaturation at 95 ° C for 4 min, denaturation at 94 ° C for 30 sec, annealing at 54 ° C for 30 sec, extension at 68 ° C for 15 min, 16 cycles.
  • a prokaryotic expression vector comprising BIR2-pET30a (control 1), BIR2-1-pET30a, L144A-pET30a, R213E-pET30a, and W78F-pET30a was further constructed using a plasmid carrying the mutant cmCry2Aa insecticidal gene.
  • the construction implementation steps are as follows:
  • the pET30a expression plasmid was digested with restriction endonuclease (the same enzyme cleavage site NdeI+NotI), and the digested product was subjected to agarose electrophoresis, and the vector DNA fragment was recovered by magnetic beads method;
  • PCR amplification and digestion of mutant insecticidal genes using the cloned mutant insecticidal gene plasmid as a template to design a NIRI at the 5' end, and a BIR2 insecticidal gene extension primer for the SacI at the 3' end, and the amplified PCR product NcoI+SacI was digested and electrophoresed.
  • Primer sequences are as follows SEQ ID NO. 9 and 10
  • the recovered fragment was mixed with the vector in an appropriate ratio, and ligated at 16 ° C for 3 hours to transform E. coli.Rosetta competent cells and plated, and the plasmid DNA was extracted and identified by restriction enzyme digestion to obtain the gene of interest. , a genetically engineered strain capable of prokaryotic expression.
  • Each plasmid was identified by PCR and double enzyme digestion, as shown in Figure 2, to ensure that each strain was correct.
  • the prokaryotic expression of the mutant insecticidal gene is carried out according to the following method steps:
  • the cells were cultured to an OD 600 ⁇ 0.6; IPTG was added to a final concentration of 0.6 mM, and cultured at 28 ° C for 4 hrs; the induced bacteria were centrifuged at 12000 r/m for 5 min to collect the cells; the medium supernatant was discarded.
  • the electrophoresis results of prokaryotic expression samples can be seen in Figures 3A and B, and there is a target band at a size of about 66KD. Based on the results of the electrophoresis, the amount of insecticidal protein per strip was estimated to be about 1 ⁇ g by reference to the standard BSA protein.
  • some single-point mutant BIR2 insecticidal proteins were tested for cotton bollworm, including: W78FCry2Aa, L144ACry2Aa, A460LCry2Aa, T615TFNPCry2Aa, ATNL353AVFCry2Aa, Loop3Cry2Aa, G324VCry2Aa, MH183LRCry2Aa, P616QCry2Aa, D578RCry2Aa, NS336LACry2Aa, QFQ163LFRCry2Aa BIR2 insecticidal protein and four control samples of Cry1Ab/Cry1Ac, Cry2Aa, 1mCry2Aa and pET30a. Where pET30a is a negative control for empty vector expression.
  • the mutant insecticidal proteins obtained by prokaryotic expression are incorporated into the feed at different doses for insect resistance test, and the specific embodiments are as follows:
  • each protein sample is diluted with 6 ml of 6 ml, 2 ml, 660 ⁇ l, 220 ⁇ l, 73 ⁇ l, 24 ⁇ l), and then mix well, then Pour into the tray and try to smooth it up. After cooling, use a scalpel to make a strip of 1cm wide. Then cut the strip into 1cm cubes and use the scalpel tip to pick the cut feed block into 24 holes.
  • BT-pET30a, Cry2Aa-pET30a was used as a positive control, and pET30a was used as a negative control.
  • the larvae feeding status and larval death status were examined on the 4th to 7th day according to the test conditions. The number of larvae dead insects and live insects were recorded, and the larvae feeding status was visually observed.
  • the corrected mortality rate is calculated according to formula (2):
  • Xt larva corrected mortality, in %
  • X1 treated mortality, in %
  • X0 control mortality, in %.
  • the W78F mutant insecticidal activity has not changed, but it is pre-designed, that is, the mutation can improve the tolerance of the insecticidal protein to ultraviolet radiation in the natural environment without affecting the insecticidal activity, and further testing of the insect resistance in the transgenic plant is required. effect.
  • mutant insecticidal genes in the first example the following 15 mutant insecticidal gene plant expression vectors were constructed: 35S-cmCry2Aa-Pnos-NPTII-p2300, 35S-SG324Cry2Aa-Pnos-NPTII-p2300, 35S-R213ACry2Aa -Pnos-NPTII-p2300, 35S-R213ECry2Aa-Pnos-NPTII-p2300, 35S-R213KCry2Aa-Pnos-NPTII-p2300, 35S-SQF163Cry2Aa-Pnos-NPTII-p2300, 35S-Loop3Cry2Aa-Pnos-NPTII-p2300, 35S-L148Cry2Aa -Pnos-NPTII-p2300, 35S-SW78Cry2Aa-Pnos-NPTII-p2300, 35S-SATN353Cry2Aa-
  • Vector digestion The 35S-cmCry2Aa-Pnos-NPTII-p2300 plasmid was digested with a restriction enzyme (the same primer cleavage sites PstI and KpnI), and the digested product was subjected to agarose electrophoresis using magnetic beads. The vector fragment 35S-Pnos-NPTII-p2300 vector fragment is recovered by the method;
  • cmCry2Aa-pstI 5'-ATATCTGCAGGAATAACGTTCTTAATTCTGG-3’
  • cmCry2Aa-KnpI 5'-AAGGGGTACCTGAGTTGAGAGTCAC-3’
  • the amplified PCR product is recovered, and then the PstI and KpnI are digested;
  • the constructed vector was identified by PCR or restriction enzyme digestion as shown in FIG. Figure 6 is a plasmid map of 35S-cmCry2Aa-Pnos-NPTII-p2300, and other mutant insecticidal gene plant expression vectors are constructed in the same manner. Identification of the correct plant expression vector is ensured by sequencing.
  • the constructed mutant cmCry2Aa insecticidal gene plant expression vector was transferred into the model plant tobacco Xanthi to obtain transgenic tobacco plants.
  • the specific method is as follows:
  • Agrobacterium competent preparation A single colony of LBA4404 was picked and inoculated into 5 ml of YEB (containing streptomycin 100 ⁇ g/ml), and cultured at 28 ° C, 250 rpm overnight. 2 ml of the colony was pipetted into 50 ml of YEB medium, and the cultivation was continued until the OD value was about 0.6. The bacterial solution was transferred to a sterile centrifuge tube, ice-bathed for 30 minutes, centrifuged at 5000 rpm for 5 minutes, and the cells were resuspended in 2 ml of 20 mM CaCl 2 and dispensed in a sterile small centrifuge tube at 200 ⁇ l per tube.
  • Insecticidal plant expression vector transformed into Agrobacterium 2 ug of plant expression vector plasmid DNA was added to 200 ⁇ l of LBA4404 competent cells, placed in an ice bath for 5 minutes, then transferred to liquid nitrogen for 8 minutes, and rapidly incubated in a 37 ° C water bath for 5 minutes. Thereafter, 800 ⁇ l of YEB medium was added, and pre-expressed at 28 ° C, 250 rpm for 4 to 5 hours, and then coated with a YAB solid plate containing kanamycin, and cultured at 28 ° C for 24 to 48 hours.
  • the Elisa test was first carried out to identify the insect resistance, and the transgenic tobacco transplanting greenhouse was selected to harvest the seeds.
  • the summary of the transformation of transgenic tobacco is shown in Table 6.
  • Elisa detection of insecticidal protein expression was performed using the Elisa detection kit from Amar, India.
  • the specific experimental steps are as follows:
  • the OD value was read with a microplate reader at 450 nm absorbance.
  • the protein expression levels of different materials were compared based on the OD values of the samples.
  • a standard curve of the OD value and the protein concentration can be prepared using the standard protein, and the expression level of the insecticidal protein can be calculated based on the detected OD value.
  • the standard curve was prepared by using different concentrations of Cry2Aa protein standard dilution as the sample, and the concentration was serially diluted from 156.252 ng/mL to 0.153 ng/mL, and each dilution was added in parallel to the ELISA plate. The antibodies were tested for optimal dilution of the antibody and were run as described in the Elisa protocol above.
  • the standard concentration of the recombinant Cry2A protein was plotted on the ordinate and the OD value was plotted on the abscissa to make the double antibody sandwich Elisa standard curve (Fig. 7).
  • the coefficient of determination R2 1.
  • genotypes corresponding to the letters in the material numbers in the table are: “D”-R213A; “F”-D108L; “G”-BIR2-1; “H”-BIR2-2; “I”-BIR1+ BIR2-1.
  • the transgenic tobacco with positive insecticidal protein expression was successively tested for insect resistance bioassay.
  • the first batch of insect resistance identification materials included 8 single-point mutant cmCry2Aa transgenic tobaccos such as TPGGA456 ⁇ 460SSSSV (Loop3), with unmutated cmCry2Aa (BIR2) and 2mCry2Aa (BIR2-2, N-terminal deletion of 43 a.a.) as controls.
  • the specific materials and identification results are shown in Table 8.
  • Table 8 shows the number of transgenic tobaccos per test gene in the bioassay experiment from 3 to 22, two replicates per plant, and eight insects per experiment.
  • the corrected mortality rate is the data after three days of inoculation. Due to the test system problem, the control test for cotton bollworm mortality was high, and the 5-day mortality rate was mostly close to 100%, which was not analyzed.
  • the test data shows that the standard error is higher, and the data variation range is larger, indicating that the test system has a larger error. The reason may be caused by problems such as heterogeneity of cotton bollworm activity and control of test conditions, and may also be related to the resistance of cotton bollworm in the recipient tobacco variety Xanthi itself, because subsequent trials have found that the mortality rate of the control tobacco Xanthi has been high.
  • the identification materials included four single point mutant cmCry2Aa transgenic tobaccos of W78F, R213E, D578R and MH183LR.
  • the larval survival survey was carried out on the days of insects 2 and 4 days respectively. The results of the identification are shown in Table 9.
  • the data in the table are the multiples of the corrected mortality compared with the control; the “()” in the data indicates the ranking of the mutation in the insect resistance performance of this test.
  • the insecticidal activity of the BIR2-1 deletion mutant was significantly increased; the L144A mutation and the R213E mutation significantly increased the insecticidal activity of cmCry2Aa; meanwhile, studies showed that the W78F mutation also has the effect of increasing the activity of the insecticidal protein.
  • seven polymerase mutant insecticidal gene plant expression vectors were cloned and constructed, together with the control, including the following nine vectors: (1) cmCry2Aa (BIR2, control); (2) 1mCry2Aa (BIR2-1) (control); (3) 1mCry2Aa + W78F (polymerization mutation 1); (4) 1mCry2Aa + L144A (polymerization mutation 2); (5) 1mCry2Aa + R213E (polymerization mutation 3); (6) 1mCry2Aa + NS336LA (polymerization mutation 4 (7) 1mCry2Aa+W78F+L144A (polymerization mutation 5); (8) 1mCry2Aa+W78F+L144A+R213E (polymerization mutation 6); (9) 1mCry2Aa+W78F+L144A+R213E+NS336LA (polymerization mutation 7).
  • cry2Aa was digested with Sal1+SacI into Arabidopsis transformation vector 35S-Pons-NPTII-2300 to construct cmCry2Aa plant expression vector 35S-Cry2Aa-Pnos-NPTII-2300
  • the plasmid map is shown in Figure 10.
  • the vector construction method using other insecticidal genes is similar.
  • control 1mCry2Aa plant expression vector 1mCry2Aa was amplified by using cry2Aa as a template and the restriction enzyme sites were as follows: SEQ ID NO. 13 and 14: 1m2AAFW: GTCGACATGTCTTTGGACACTATCCAAAAG; 2AARv: GCGAGCTCTTAGTACAAGGGTGGAAGGTTAGT; cloned 1mcry2Aa was cloned into The T vector was subjected to sequencing verification. After verification, the T vector of 1 mCry2Aa was digested with Sal1+SacI, and the target fragment was ligated to the vector of 2300 to construct 35S-BIR2-1-Pnos-NPTII-2300.
  • W78F original TCG-mutation to TTT mutated gene vector has been obtained in the early stage, and the W78F vector plasmid was directly amplified directly with 1m2AAFW and 2AARv primers, and the amplified fragment was cloned into The T vector of W78F was obtained on the T vector, and the vector was digested with Sal1+SacI, and the target fragment was ligated to the vector of 2300 to construct 35S-W78F/BIR2-1-Pnos-NPTII-2300.
  • the plant expression vectors 35S-L144A/BIR2-1-Pnos-NPTII-2300, 35S-R213E/BIR2-1-Pnos-NPTII-2300 were obtained in a similar manner.
  • the constructed plant expression vector was transformed into Agrobacterium strain GV3101, respectively.
  • the specific method steps are as follows:
  • Agrobacterium GV3101 was spotted on a YEB solid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml gentamicin, and cultured at 28 ° C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of YEB liquid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml gentamicin, and cultured overnight (about 12-16 hours) at 28 ° C until the OD600 value was 0.4, forming a seed fungus. liquid.
  • the GV3101 competent cells were thawed on ice, and 1 ⁇ l of the plant DNA of the plant expression vector to be transformed was added to 40 ⁇ l of the competent cells, and the mixture was mixed and ice bathed for about 10 minutes.
  • the mixture of the competent cells after the ice bath and the 35S-BgVP2-2300 plasmid was transferred to an ice-cold 0.1 cm size electric shock cup (purchased from Bio-Rad) using a micropipette, and the suspension was tapped to reach the shock. The bottom of the cup (be careful not to have bubbles).
  • the electric shock cup is placed on the slide of the electric shock chamber, and the slide is pushed to place the electric shock cup to the base electrode of the electric shock chamber.
  • the rock with good water absorption and soft soil was mixed with nutrient soil (1:1) as the soil for Arabidopsis thaliana planting.
  • nutrient soil (1:1) as the soil for Arabidopsis thaliana planting.
  • 20-30 Arabidopsis seeds were seeded per pot (Columbia type, from the Arabidopsis Bioresource Center, Ohio State University).
  • the film is covered with a film to provide a moist environment for plant growth.
  • the temperature was 22 ° C
  • the light intensity was 3500-4000 lx
  • the photoperiod was 12 hours dark / 12 hours light culture
  • 1/2 MS liquid medium was watered every 7 days. After 30 days of culture, 4-5 plants were kept per pot, and the photoperiod was adjusted to 8 hours dark/16 hours light culture.
  • the GV3101 Agrobacterium liquid transformed with the expression vector was inoculated to an LB liquid medium containing 50 ⁇ g/ml rifampicin, 50 ⁇ g/ml gentamicin, 50 ⁇ g/ml kanamycin, and the next morning was pressed. 1:50 inoculation into a new LB medium (1 L) containing 50 ⁇ g/ml rifampicin, 50 ⁇ g/ml gentamicin, 50 ⁇ g/ml kanamycin, and cultured for about 8 hours to Agrobacterium
  • the OD600 is between 1.0 and 1.2.
  • the supernatant was discarded, and the Agrobacterium pellet was suspended in the impregnation medium (1/2 MS liquid medium and containing 5% sucrose; adjusted to pH 5.7 with KOH; 0.02% Silwet L-77). Make OD600 around 0.8.
  • the upper part of the Arabidopsis thaliana prepared for transformation prepared in Example 5 was slowly and spirally immersed in the Agrobacterium-containing dyeing medium, and gently shaken clockwise for about 2 minutes, and covered with a transparent plastic cover. Humidity, put in the greenhouse overnight. After 24 hours, the plastic transparent cover was removed and poured through water. After 2-3 weeks, ensure that the plants are hydrated.
  • Seed disinfection first soak for 10 minutes with 70% ethanol, occasionally suspend the seeds; then wash with sterile water four times, and occasionally suspend the seeds. Then, the treated seeds were uniformly coated on the surface of 1/2 MS solid screening medium containing 50 ⁇ g/ml kanamycin (a 150 mm diameter plate was sown up to 1500 seeds), and vernalized at 4 ° C for 2 days, then Incubate for 7-10 days at a constant temperature of 22 ° C, an illumination intensity of 3500-4000 lx, and a photoperiod of 12 hours of darkness/12 hours of light. After germination of the transgenic seeds on the screening medium for 2 weeks, the plants capable of germination and normal growth were transferred to soil for further cultivation.
  • Arabidopsis thaliana seeds transformed by dip-dying were germinated on Kana plates, and green kanamycin-resistant transgenic Arabidopsis plants were screened. Then transgenic Arabidopsis plants were transplanted into nutrient mash, and after 8-10 leaves of the seedlings, the roots were wrapped with absorbent absorbent cotton balls and transferred to a culture flask. Eight larvae of the newly hatched cotton bollworm were incubated with each insect to carry out insect-resistant organisms. Determination.
  • the invention aims to improve the insecticidal activity of cmCry2Aa Bt insecticidal protein against Helicoverpa armigera, and finds four mutations of 1mCry2Aa, W78FCry2Aa, R213ECry2Aa and L144ACry2Aa and their combined mutations can be significantly improved by prokaryotic expression identification and transgenic plant bioassay. Insecticidal activity of cotton bollworm.
  • the invention can be used for the study of insect-resistant transgenic plants.

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Abstract

本发明公开了一种突变Cry2Aa杀虫蛋白质，在SEQ ID NO.1 所示的氨基酸序列的基础上，所述杀虫蛋白质的氨基酸序列具有如下一种、两种或三种突变：A) W78F；B) L144A和C)R213E。优选地，D) 所述氨基酸序列N端28个氨基酸缺失。本发明还公开了突变Cry2Aa杀虫蛋白质的编码基因，包括其的重组表达载体。利用其改善植物抗虫效果的方法。

Description

一种人工改良的杀虫蛋白质及其编码基因与应用 技术领域
本发明属于植物分子生物学领域，尤其是农业生物技术研究中的转基因农作物育种领域，特别是涉及抗虫转基因农作物研究，具体而言涉及一种人工改良的杀虫蛋白质及其编码基因与应用。
背景技术
在世界范围内，虫害给农业生产带来了较大的损失。传统采用化学杀虫剂的防治技术，在传统农业实践中发挥了巨大的作用。但是，这种虫害防治技术存在很大的弊端，一方面，大量有毒化学农药的使用，不仅污染环境，而且易残留，严重威胁人们健康；另一方面，化学农药长期大量使用还能够造成害虫的抗药性，导致虫害的爆发。
苏云金芽孢杆菌(Bacillus thurigiensis，简称Bt)是一种革兰氏阳性菌，在芽孢形成过程中产生的伴胞晶体被称为δ-内毒素，或杀虫晶体蛋白。目前所发现的Bt杀虫蛋白可毒杀鳞翅目、双翅目、鞘翅目等的昆虫(Aronson,Microbiol.Rev.50:1-14,1968)。根据现通用命名规则，目前已经有74类793个Bt杀虫晶体蛋白(Cry类)基因被报导(Crickmore，2017，http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html)。由于Bt杀虫蛋白质控制虫害的专一性和高度选择性,对植物和包括人在内的动物没有毒害,而且对环境友好，因此得到了广泛应用，曾经是广为应用的一种生物杀虫剂。目前，转基因技术为防治虫害给农业生产造成严重损失这一世界性难题提供了更为理想的解决方案。人们可通过种植Bt转基因抗虫作物防治虫害。2016年全球转基因作物种植面积1.851亿公顷，其中Bt抗虫作物的种植比例约占53％，包括复合或单一的抗虫性状转基因作物，即2016年Bt抗虫转基因作物的种植面积约9810万公顷(Clive J.,ISAAA Briefs,2016)。
Cry2Aa Bt杀虫蛋白对鳞翅目害虫具有抗性，是抗虫转基因植物研究领域研究较为深入的一种Bt杀虫蛋白。例如，2008年，崔洪志等人改造合成了CmCry2Aa和Cm1Cry2Aa杀虫基因并在模式植物烟草中进行了功能验证(ZL200810065477.2)。但本领域中仍然需要杀虫活性提高、效果改良的新Bt抗虫基因，以提高抗虫植物的抗虫效率、延缓害虫对抗虫植物的抗性演化速度。
发明内容
根据发明人对Bt杀虫蛋白结构和功能的独特理解，本发明进行了一系列突变设计，并利用基因定点突变技术，对cmCry2Aa杀虫基因(ZL200810065477.2)进行了突变以及功能筛选鉴定，获得了W78F、L144A和R213E三个杀虫活性提高的单点突变，这三个突变与N端28个氨基酸的缺失突变的组合运用，可提高Cry2Aa杀虫蛋白的杀虫效果。
因此，在第一方面，本发明提供了一种突变Cry2Aa杀虫蛋白质，所述突变Cry2Aa杀虫蛋白质的氨基酸序列在SEQ ID NO.1所示的氨基酸序列基础上，具有如下一种、两种或三种突变：
A)W78F；B)L144A和C)R213E。
结合本发明第一方面，在第一方面的第一可能实现方式中，
D)所述氨基酸序列N端28个氨基酸缺失。
在第二方面，本发明提供了本发明第一方面的突变Cry2Aa杀虫蛋白质的编码基因。
在第三方面，本发明提供了一种重组表达载体，其含有本发明第二方面的编码基因并且所述编码基因的核苷酸序列与所述表达载体的表达控制序列可操作地连接。
在第四方面，本发明提供了一种改善植物抗虫效果的方法，包括：将本发明第二方面的编码基因或者本发明第三方面的重组表达载体导入植物或植物组织并使所述编码基因表达；优选地，所述植物是烟草。
在第五方面，本发明提供了一种制备转基因植物的方法，包括：在有效产生植物的条件下培养含有本发明第二方面的编码基因或者本发明第三方面的重组表达载体的植物或植物组织。
在第六方面，本发明提供了本发明第二方面的编码基因或本发明第三方面的重组表达载体用于改善植物抗虫效果以及用于植物育种的用途。
在第七方面，本发明提供了一种制备突变基因的方法，所述突变基因编码本发明第一方面的突变Cry2Aa杀虫蛋白质，
所述方法包括用如下三组引物中的1组、2组或3组依次突变SEQ ID NO.2所示的序列的DNA样品：
(1)5′-GATCTTGTCAGAACTTTTTGGTATTATCTTTCC-3′
5′-GGAAAGATAATACCAAAAAGTTCTGACAAGATC-3′
(2)
5’-CCTACTCAAAACCCAGTTCCTGCTAGCATTACTTCTTCAGTGAATAC-3’
5’-GTATTCACTGAAGAAGTAATGCTAGCAGGAACTGGGTTTTGAGTAGG-3’
(3)5′-ATAGAGACTATCTTGAGAATTACACAAGAGA-3′
5′-TCTCTTGTGTAATTCTCAAGATAGTCTCTAT-3′。
在本发明中，1mCry2Aa、W78FCry2Aa、R213ECry2Aa和L144ACry2Aa四个突变及其组合突变可显著提高对棉铃虫的杀虫活性。
附图说明
图1A-C示出了W78F、L144A和R213E突变cmCry2Aa杀虫基因。(A)W78F突变cmCry2Aa杀虫基因，示出W78F突变的核苷酸变化；(B)L144A突变cmCry2Aa杀虫基因，示出L144A突变的核苷酸变化；(C)R213E突变cmCry2Aa杀虫基因，示出R213E突变的核苷酸变化。
图2示出突变cmCry2Aa-pET30a原核表达载体的PCR及酶切鉴定情况：(A)PCR鉴定：1～11：突变cmCry2Aa-pET30a克隆样品、+：cmCry2Aa-pET30a；(B)酶切鉴定：1、5、8：突变cmCry2Aa-pET30a/NcoI+SacI，M：λDNA marker。
图3示出W78F、L144A和R213E等突变cmCry2Aa杀虫基因原核表达可溶性蛋白SDS-PAGE电泳分析。(A)1：A460L；2：W78F；3：L144A；4：cmCry2Aa；5：T615TFNP；6：ATNL353AVF；7：Loop3；8：G324V；9：pET30a(空载体对照)；10：中分子量蛋白Marker；(B)1：1mCry2Aa；2：2mCry2Aa；3：R213A； 4：R213E；5：R213K；6：T439R；7：cmCry2Aa；8：pET30a；9：BSA(2μg)；10：中分子量蛋白Marker。
图4示出cmCry2Aa部分突变杀虫蛋白对棉铃虫生测结果。
图5示出部分植物表达载体构建过程中PCR及酶切鉴定情况：(A)PCR鉴定：1-10:p2300载体自连克隆子；11-16:L144A克隆子；17-22:D108L克隆子；23-28:QFQ163LFR克隆子；29-34:MH183LR克隆子、35-40:ATNL353AVF克隆子；41:L144A-pET30a正对照、42:p2300载体质粒负对照、43:空白负对照；(B)酶切鉴定：1，2:MH183LR；3,4 ATNL353AVF；5,6 SQF163LFR；7,8 D108L(3、5)；9,10 L144A。
图6示出35S-cmCry2Aa-Pnos-NPTII-p2300质粒图谱。
图7示出Cry2Aa Elisa蛋白标准曲线。
图8示出转基因烟草杀虫叶片蛋白表达量情况。
图9示出R213E突变cmCry2Aa与cmCry2Aa烟草棉铃虫生测对比情况(接虫4天)。
图10示出了植物表达载体35S-Cry2Aa-Pnos-NPTII-2300构建。
图11转基因拟南芥抗性生测过程中棉铃虫危害典型情况：WL、WLR和WLRN分别代表W78F、L144A、R213E和NS336LR不同聚合情况。
具体实施方式
在本发明中，Cry2Aa杀虫蛋白质具有SEQ ID NO.1所示的氨基酸序列，根据Cry2Aa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cmCry2Aa杀虫基因的3个基因片段，人工合成后分别克隆到载体pGEM和pUC57中，并进一步拼接完整cmCry2Aa Bt杀虫基因。按照Cry2Aa杀虫蛋白的功能结构域分别合成并克隆基因片段，是考虑方便后续对该基因的结构和功能进一步研究。cmCry2Aa杀虫基因的核苷酸序列与天然的Cry2Aa(genebank AY496458，http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi？db＝nuccore&id＝45685585)基因对应部分的核苷酸序列对比，其同源性为75.97％。根据Cry2Aa杀虫蛋白的分子结构，N端存在长度为29个氨基酸的Coli结构片段，位于结构域1α-螺旋1的前端，原毒素活化时需要被昆虫消化道胰蛋白酶水解去除。设计如下引物，以cmCry2Aa基因为模板，直接突变缺失了该片段，所得基因命名为1mCry2Aa。以下文本中BIR2＝cmCry2Aa；BIR2-1＝1mCry2Aa(N端缺失突变，N端缺失28个a.a.)；BIR2-2＝2mCr2Aa(N端缺失突变，N端缺失43个a.a.)。BIR2(cmCry2Aa)序列如SEQ ID NO.2所示，突变W78F、L144A和R213E对应的基因突变分别是：233-234GG变成TT；430-431CT变成GC；637-638AG变成GA。所述氨基酸序列N端28个氨基酸缺失，对应的基因突变是SEQ ID NO.2的5’端计4-87位核苷酸缺失；所述氨基酸序列N端43个氨基酸缺失，对应SEQ ID NO.2的5’端计4-132位核苷酸缺失。
实施例一 突变基因克隆
1.1突变方法 采用设计突变引物，利用PCR进行基因定点突变。根据设计，三个单点突变设计的突变引物分别见SEQ ID NO.3～8如下：
(1)W78F 5′-GATCTTGTCAGAACTTTTTGGTATTATCTTTCC-3′
5′-GGAAAGATAATACCAAAAAGTTCTGACAAGATC-3′
(2)L144A
5’-CCTACTCAAAACCCAGTTCCTGCTAGCATTACTTCTTCAGTGAATAC-3’
5’-GTATTCACTGAAGAAGTAATGCTAGCAGGAACTGGGTTTTGAGTAGG-3’
(3)R213E 5′-ATAGAGACTATCTTGAGAATTACACAAGAGA-3′
5′-TCTCTTGTGTAATTCTCAAGATAGTCTCTAT-3′
质粒CmCry2Aa-pUC57，携带cmCry2Aa Bt杀虫基因，由创世纪种业有限公司生物技术中心人工设计合成，已获得专利授权：人工改造合成的杀虫基因及其编码的蛋白质与应用(ZL 200810065477.2)、(PCT/CN2009/070560)。
以质粒CmCry2Aa-pUC57为模板，利用突变引物进行基因定点突变的PCR反应体系如下：
以携带BIR2杀虫基因的CmCry2Aa-pUC57质粒为模板，利用所设计的突变引物，通过PCR单点突变实验分别对cmCry2Aa基因进行定点突变。PCR反应体系如下：
Figure PCTCN2018078572-appb-000001
反应程序：95℃预变性4min，94℃变性30sec，54℃退火30sec，68℃延伸15min，16个循环。
PCR后加入1μl Dpn Ⅰ限制性内切酶，置于37℃水浴中消化甲基化的模板质粒DNA，时间1-2h。取1μl Dpn Ⅰ处理过的PCR产物，转化感受态细胞，并对克隆子依次进行PCR鉴定和测序分析，分别获得了系列突变的BIR2杀虫基因，突变部分的测序结果分别如下，表明已经按照设计方案，分别获得了正确的突变BIR2杀虫基因，突变内容如标题所示：数字代表突变的氨基酸位置。数字签名是突变前的氨基酸，数字后面是突变后的氨基酸。图1A-C示出了W78F、L144A和R213E突变BIR2杀虫基因。
实施例二 突变基因原核表达及功能鉴定
1. 单点突变基因原核表达
首先利用携带各突变cmCry2Aa杀虫基因的质粒，进一步构建如下其原核表达载体：BIR2-pET30a(对照1)、BIR2-1-pET30a、L144A-pET30a、R213E-pET30a和W78F-pET30a等。构建实施步骤如下：
载体酶切：将pET30a表达质粒用限制性内切酶(同引物的酶切位点NdeI+NotI)进行酶切，酶切产物经琼脂糖电泳，用磁珠法回收载体DNA片段；
突变杀虫基因PCR扩增与酶切：以克隆的突变杀虫基因质粒为模板，设计在5’端增加NcoI，在3’端增加SacI的BIR2杀虫基因扩展引物，扩增后的PCR产物NcoI+SacI酶切后电泳回收。引物序列如下SEQ ID NO.9和10
BRI2NcoI5`：5’-tcacccatggttgctcctgtggttggaac-3’
BRI2SacI3`：5’-gagctcttagtacaagggt-3’
连接与转化：将回收后的片段与载体以合适的比例混合，16℃连接3个小时，转化E.coli.Rosetta感受态细胞并涂板，提取质粒DNA PCR及酶切鉴定，获得 携带目的基因，可进行原核表达的基因工程菌株。
每个质粒均分别通过PCR鉴定和双酶切鉴定，如图2所示，确保每个菌株正确无误。
获得原核表达菌株后，根据如下方法步骤进行突变杀虫基因的原核表达：
取单菌落接种到5mL含100μg/ml Kan的LB液体培养基中37℃震荡培养过夜；按1∶100比例将培养过夜的菌液接种到含100μg/ml Kan的LB液体培养基中，37℃条件下培养到OD 600≌0.6；加入IPTG至终浓度为0.6mM，28℃条件下震荡培养4hr；将诱导好的菌液在4℃，12000r/m离心5min收集细胞；倒掉培养基上清，吸干液体，加入1/10体积的1×PBS(PH7.4)重悬沉淀以洗涤菌体，4℃，12000r/m离心5min后倒掉上清并重复一次；吸干液体，加入适当体积的灭菌1×PBS(PH7.4)重悬，然后冰浴超声破碎(功率400W，破碎10sec，间隔10sec，20次)，4℃、12000rpm离心破碎好的溶液10min；将上清倒入三角瓶中保存在4℃，取适当的体积进行SDS-PAGE分析。
原核表达样品电泳结果可参见图3A、B所示，在66KD左右大小处均有一条目的带。根据电泳结果，通过与标准BSA蛋白参比，每条带上样的杀虫蛋白量估算约1μg。
2. 突变杀虫基因原核表达鉴定
本实施例对部分单点突变BIR2杀虫蛋白进行了棉铃虫生测，包括：W78FCry2Aa、L144ACry2Aa、A460LCry2Aa、T615TFNPCry2Aa、ATNL353AVFCry2Aa、Loop3Cry2Aa、G324VCry2Aa、MH183LRCry2Aa、P616QCry2Aa、D578RCry2Aa、NS336LACry2Aa、QFQ163LFRCry2Aa十二个单点突变BIR2杀虫蛋白以及Cry1Ab/Cry1Ac、Cry2Aa、1mCry2Aa和pET30a四个对照样品。其中pET30a是空载体表达的阴性对照。将原核表达获得的突变杀虫蛋白以不同的剂量参入饲料中进行抗虫性测试，具体实施方式如下：
待新鲜配制好的饲料温度降到50℃左右时加入欲验证功能的蛋白(每一蛋白样品以6ml、2ml、660μl、220μl、73μl、24μl 6个梯度稀释各加入20g饲料)迅速混合均匀，然后倒入托盘并尽量弄平整，冷却后用手术刀划成1cm宽的条状，然后再将条状切成1cm的立方小块，用手术刀尖，将切好的饲料块挑入24孔养虫盒内接虫，用BT-pET30a，Cry2Aa-pET30a做阳性对照，以pET30a做阴性对照，接虫后根据试验情况在第4～7天分别检查幼虫取食状况和幼虫死亡状况。记录幼虫死亡虫数和活虫数，目测幼虫取食状况。
幼虫死亡率按公式(1)计算：
Figure PCTCN2018078572-appb-000002
式中：X：幼虫死亡率，单位为％；n：死虫数，单位为头；A：接虫数，单位为头。
校正死亡率按公式(2)计算：
Figure PCTCN2018078572-appb-000003
式中：Xt：幼虫校正死亡率，单位为％；X1：处理死亡率，单位为％；X0：对照死亡率，单位为％。
结果见表4-5或图4。
表4.cmCry2Aa突变杀虫蛋白对棉铃虫生测结果
Figure PCTCN2018078572-appb-000004
Figure PCTCN2018078572-appb-000005
*线性回归拟合度
表5.cmCry2Aa突变杀虫蛋白对棉铃虫校正死亡率与对照对比情况
Figure PCTCN2018078572-appb-000006
上述对15个不同杀虫蛋白的生测研究结果表明：以突变的原始样本Cry2Aa为参照，杀虫活性可能提高的单点突变设计有五个，分别为：L144A、D578R(与本发明申请保护的R213E为近义突变)，G324V、T615TFNP和ATNL353AVF；杀虫活性基本没变化的有五个，分别为：P616Q、W78F、QFQ163LFR、A460L和NS336LA；MH183LR杀虫活性降低；loop3突变导致杀虫活性完全丧失。
W78F突变杀虫活性没有变化，却符合预先设计，即在不影响杀虫活性的情况下，该突变可提高杀虫蛋白对自然环境中紫外线照射的耐性，需进一步测试在转基因植物中的抗虫效果。
实施例三 以烟草为模式植物的突变基因功能鉴定
1.突变杀虫基因植物表达载体构建
在实施例一获得各突变杀虫基因的基础上，构建了如下15个突变杀虫基因植物表达载体：35S-cmCry2Aa-Pnos-NPTII-p2300、35S-SG324Cry2Aa-Pnos-NPTII-p2300、35S-R213ACry2Aa-Pnos-NPTII-p2300、 35S-R213ECry2Aa-Pnos-NPTII-p2300、35S-R213KCry2Aa-Pnos-NPTII-p2300、35S-SQF163Cry2Aa-Pnos-NPTII-p2300、35S-Loop3Cry2Aa-Pnos-NPTII-p2300、35S-L148Cry2Aa-Pnos-NPTII-p2300、35S-SW78Cry2Aa-Pnos-NPTII-p2300、35S-SATN353Cry2Aa-Pnos-NPTII-p2300、35S-D578RCry2Aa-Pnos-NPTII-p2300、35S-SMH183Cry2Aa-Pnos-NPTII-p2300、35S-SD108Cry2Aa-Pnos-NPTII-p2300、35S-SNS336Cry2Aa-Pnos-NPTII-p2300、35S-p616Cry2Aa-Pnos-NPTII-p2300。构建步骤如下：
I：载体酶切：将35S-cmCry2Aa-Pnos-NPTII-p2300质粒用限制性内切酶(同引物的酶切位点PstI和KpnI)进行酶切，酶切产物经琼脂糖电泳，用磁珠法回收载体片段35S-Pnos-NPTII-p2300载体片段；
II：突变基因的PCR扩增与酶切：利用高保真Taq DNA聚合酶，合成如下引物进行各突变基因的扩增，SEQ ID NO.11和12
cmCry2Aa-pstI：5’-ATATCTGCAGGAATAACGTTCTTAATTCTGG-3’
cmCry2Aa-KnpI：5’-AAGGGGTACCTGAGTTGAGAGTCAC-3’
将扩增后的PCR产物回收，再进行PstI和KpnI的酶切；
III：连接与转化：将回收后的片段与载体以合适的比例混合，16℃连接3个小时，转化涂板克隆含有目的基因的植物表达载体。
利用PCR或酶切鉴定所构建的载体，如图5所示。图6是35S-cmCry2Aa-Pnos-NPTII-p2300质粒图谱，其他突变杀虫基因植物表达载体是构建方式均与之类同。鉴定正确的植物表达载体均通过测序确保正确无误。
2.模式植物烟草遗传转化
用农杆菌LBA4404介导的遗传转化方法，将所构建的突变cmCry2Aa杀虫基因植物表达载体转入模式植物烟草Xanthi，获得转基因烟草植株。具体方法如下：
农杆菌感受态制备：挑取LBA4404单菌落接种于5ml YEB(含链霉素100μg/ml)中，28℃，250rpm培养过夜。吸取2ml菌落加入50ml YEB培养基中，继续培养至OD值约为0.6。将菌液转至无菌离心管中，冰浴30分钟，5000rpm离心5分钟，用2ml 20mM的CaCl2重悬菌体，按每管200μl分装于无菌小离心管中。
杀虫基因植物表达载体转化农杆菌：在200μl LBA4404感受态细胞中加入2ug植物表达载体质粒DNA，置冰浴5分钟，然后转至液氮中冷冻8分钟，迅速于37℃水浴中温育5分钟后，加入800μl YEB培养基，28℃250rpm预表达培养4～5小时，然后涂铺含有卡那霉素的YEB固体平板，28℃培养24～48小时。
农杆菌介导转化烟草：28℃过夜培养带有植物表达载体的农杆菌50ml，5000rpm离心5分钟，收集菌体沉淀，用液体MS培养液洗涤一次，再用MS重悬至OD600＝0.2～0.4；取烟草无菌苗叶片，用刀切成边长约0.5厘米小方块，在农杆菌悬液中浸泡5～10分钟，然后用灭菌滤纸吸干；将叶块摆放在不加抗生素的共培养培养基中(上面垫二层滤纸)于25％避光共培养3天；将叶片继代到选择培养基中在光照培养箱中(光照12小时，黑暗12小时)培养至抗性芽的出现；将抗性芽移至生根培养基因，每隔二周继代培养一次，种植到小塑料钵中。
获得T0代转基因烟草植株后，首先进行Elisa检测，为统一进行抗虫性鉴定，选择高表达转基因烟草移栽温室，收获种子。转基因烟草转化汇总情况见表6。
表6.转基因烟草转化情况汇总表
Figure PCTCN2018078572-appb-000007
Figure PCTCN2018078572-appb-000008
3.转基因烟草杀虫蛋白表达情况的Elisa检测
利用印度Amar公司的Elisa检测试剂盒进行杀虫蛋白表达的Elisa检测。具体实验操作步骤如下：
称取0.01g待测烟草叶片装eppendorf管。需准备相同背景的阴性对照。将装有材料的离心管用液氮冷冻，研磨仪进行研磨。
每管加入500ul蛋白提取液(PBS缓冲液)，混匀。4℃放置30min。12000rpm，4℃离心10min，吸取50ul上清置96孔板(含有抗体的酶标板，购自印度Amar公司)中。然后加50ul conjucate(酶标二抗结合目标蛋白)置96孔板中。室温在摇床上摇40min。
用洗板机洗板4次。每孔加入100ul substrate(底物)显色，室温在摇床上摇20min至显色反应完成。
显色完成后，每孔加入100ul试剂盒自带stop solution终止。
终止完成后，用酶标仪在450nm吸光度下读取OD值。根据样品OD值比较不同材料的蛋白表达量。
需要获得准确杀虫蛋白表达量数据时，可利用标准蛋白制作OD值和蛋白浓度标准曲线，根据检测的OD值计算杀虫蛋白表达量。制作标准曲线是利用不同浓度的Cry2Aa蛋白标样稀释液作为样品，浓度从156.252ng/mL做2倍连续稀释至0.153ng/mL，各稀释度平行加入酶标板中。检测抗体为最佳稀释浓度的抗体，按上述Elisa实验步骤进行操作。以重组Cry2A蛋白质的标准品浓度为纵坐标,OD值为横坐标做出双抗体夹心Elisa标准曲线(图7)。多项式方程为y＝6.407.6x5-28.79x4+47.91x3-29.5x2+33.25x-7.037。决定系数R2＝1。
在对转基因烟草进行抗虫性检测之前，跟踪检测其杀虫蛋白表达情况，选择杀虫蛋白稳定表达，表达水平相当的转基因植株进行棉铃虫抗性生物测定分析， 可提高抗性分析的准确性。表7是一组转基因烟草叶片杀虫蛋白表达量检测数据。
表7.部分转基因烟草Elisa蛋白表达情况
Figure PCTCN2018078572-appb-000009
Figure PCTCN2018078572-appb-000010
Figure PCTCN2018078572-appb-000011
注：表中材料编号中字母所对应的基因型分别是:“D”-R213A；“F”-D108L；“G”-BIR2-1；“H”-BIR2-2；“I”-BIR1+BIR2-1。
检测结果表明：大部分转基因烟草可有效表达出所编码的cmCry2Aa(突变)杀虫蛋白；同一个基因的不同转基因植株间杀虫蛋白表达水平存在差异，杀虫蛋白表达水平在整体上呈线性分布(图8)，高表达植株叶片杀虫蛋白表达水平可超过1000ng/g。转基因植株杀虫蛋白表达的差异对通过生测比较不同突变基因的杀虫效果带来了一定困难。项目后续生测将首先通过Elisa检测，选择蛋白表达水平相当的转基因植株，可提高抗虫性生测数据的可靠性。
1.1.1.1 4.单点突变转基因烟草抗虫鉴定实例一
利用Elisa检测，杀虫蛋白表达阳性的转基因烟草陆续进行了抗虫性生测鉴 定试验。第一批抗虫鉴定材料包括TPGGA456～460SSSSV(Loop3)等8个单点突变的cmCry2Aa转基因烟草，以未突变的cmCry2Aa(BIR2)和2mCry2Aa(BIR2-2，N端缺失43个a.a.)为对照。具体材料和鉴定结果见表8。
表8.第一次转基因烟草抗性生测情况
Figure PCTCN2018078572-appb-000012
表8生测实验中每个测试基因的转基因烟草数目3～22株，每株两个重复，每个实验接虫数8头。校正死亡率是接虫三天后数据。因试验体系问题，对照测试棉铃虫死亡率偏高，5天死亡率大部分接近100％，未作分析。试验数据显示标准误较高，数据变异幅度较大，说明试验系统误差较大。原因可能是棉铃虫活性不均一、试验条件控制等问题造成，也可能与受体烟草品种Xanthi本身有一定棉铃虫抗性有关，因为后续试验发现对照烟草Xanthi的死亡率一直偏高。
本次试验结果表明：R213E突变是唯一一个比对照BIR2抗虫有所提高的材料，其次表现较好的突变是NS336LA，与对照情况基本相当；而其余均不同程度差于BIR2的情况。其中TPGGA456～460SSSSV(Loop3)单点突变抗性最低，与前面杀虫蛋白原核表达鉴定的结果一致。但前期原核表达鉴定中，抗性提高的ATNL353AVF和G324V在本次试验中表现抗性一般。
1.1.1.2 5.单点突变转基因烟草抗虫鉴定实例二
鉴定材料包括W78F、R213E、D578R和MH183LR四个单点突变的cmCry2Aa转基因烟草。分别于接虫2天和4天进行了幼虫存活情况调查，鉴定结果见表9。
表9.第二次转基因烟草抗性生测情况
Figure PCTCN2018078572-appb-000013
本次试验发现W78F突变与未突变的cmCry2Aa对照抗虫性基本相当，再次验证了该突变不但不降低杀虫蛋白活性。而且在自然多光照条件下可提高杀虫蛋白杀虫活性。
在上次试验中R213E抗虫性突出。本次试验对R213E进行了重复鉴定，结果表明，从对棉铃虫幼虫校正死亡率数据看，R213E与未突变的cmCry2Aa对照抗虫性也基本相当，但从抗性效果上，R213E抗性表现更优，棉铃虫危害叶片的程 度较轻。见图9。
1.1.1.3 6.单点突变转基因烟草抗虫鉴定实例三
鉴定材料连同对照转基因烟草材料合计22份，合计共477个转基因株系，包括了所有突变基因材料进行集中鉴定(见表10)。杀虫鉴定的试验方法进行了优化，每个转基因株系设置四个重复，每个重复接初孵棉铃虫幼虫9头。实验结果见表11所示。
表10：第四次转基因烟草抗虫鉴定材料清单
基因名称 event数量 基因名称 event数量
P616Q 29 Cry1A121C3* 16
Cry1A1C3* 22 D578R 33
L144A 38 R213A 25
ANTL353AVF 24 W78F 21
Loop3 19 G324V 10
QFQ163LFR 29 NS336LA 17
BIR2-2 18 D108L 23
R213K 13 R213E 30
MH183LR 18 BIR2-1 20
BIR1+BIR2-1 24 BIR1+BIR2 13
BIR2 23 BIR1 12
表11.第三次转基因烟草抗性生测情况
Figure PCTCN2018078572-appb-000014
Figure PCTCN2018078572-appb-000015
本次试验包括所获得的所有待测试材料。对照死亡率仍然偏高，但由于试验重复多，试验条件控制较严格，经统计学分析，部分突变的试验结果与对照表现了显著性差异。本次鉴定数据表明，L144A、R213E和W78F具有较好的抗虫性，平均校正死亡率超过90％；尤其L144A和R213E两个突变，与突变对照BIR2(突变前的cmCry2Aa)差异显著。表明这两个突变可以显著性提高杀虫蛋白的杀虫活性。此外ANTL353AVF、NS336LA和D578R等突变也表现了杀虫活性的提高，但差异不显著。R213A、R213K、Loop3和G324V则表现抗虫性下降。
1.1.1.4 7.抗虫鉴定实施例小结
在以原核表达的杀虫蛋白进行抗虫性生测分析时，初步完成了对L144A等突变的功能鉴定，同时也验证了BIR2-1缺失突变杀虫活性有较大幅度的提高。进一步以烟草为模式植物，进行了单点突变杀虫基因在转基因植物中杀虫活性的比较鉴定。在棉铃虫初孵幼虫取食对照非转基因烟草Xanthi死亡率较高的情况下，连续开展了多次转基因烟草抗虫性生测鉴定。通过Elisa杀虫蛋白表达定量检测，克服了转基因烟草表达水平差异的影响，完成了项目突变杀虫基因抗虫效果鉴定工作。具体结果汇总于表12中。
表12.单点突变杀虫基因抗虫性鉴定情况汇总一览表
Figure PCTCN2018078572-appb-000016
注：表中数据均为校正死亡率与对照相比的倍数；数据中“()”标注的是该突变在本次试验抗虫性表现的排名。
根据鉴定结果，BIR2-1缺失突变杀虫活性显著提高；L144A突变和R213E突变可显著提高cmCry2Aa杀虫活性；同时，研究表明W78F突变也具有提高杀虫蛋白活性的效果。
实施例四.聚合突变基因克隆
1.聚合突变杀虫基因植物表达载体构建
根据本发明前述实施例研究数据，克隆构建了七个聚合突变杀虫基因植物表达载体，连同对照，包括如下九个载体：(1)cmCry2Aa(BIR2，对照)；(2)1mCry2Aa(BIR2-1，对照)；(3)1mCry2Aa+W78F(聚合突变1)；(4)1mCry2Aa+L144A(聚合突变2)；(5)1mCry2Aa+R213E(聚合突变3)；(6)1mCry2Aa+NS336LA(聚合突变4)；(7)1mCry2Aa+W78F+L144A(聚合突变5)；(8)1mCry2Aa+W78F+L144A+R213E(聚合突变6)；(9)1mCry2Aa+W78F+L144A+R213E+NS336LA(聚合突变7)。上述载体按照分子克隆标准方法进行构建。构建过程分别如下：
(1)对照cmCry2Aa植物表达载体的构建：将cry2Aa用Sal1+SacI酶切构建到拟南芥转化载体35S-Pons-NPTII-2300上，构建获得cmCry2Aa植物表达载体35S-Cry2Aa-Pnos-NPTII-2300，质粒图谱如图10所示。利用其它杀虫基因的载体构建方式与之类同。
(2)对照1mCry2Aa植物表达载体的构建：以cry2Aa为模板加酶切位点扩增得到1mCry2Aa，引物如下SEQ ID NO.13和14：1m2AAFW：GTCGACATGTCTTTGGACACTATCCAAAAG；2AARv：GCGAGCTCTTAGTACAAGGGTGGAAGGTTAGT；将扩增的1mcry2Aa克隆到T载体上，并进行测序验证，验证正确后将1mCry2Aa的T载体，用Sal1+SacI酶切后将目的片段连到2300的载体上，构建得到35S-BIR2-1-Pnos-NPTII-2300。
(3)聚合突变1～7植物表达载体的构建：W78F(原TCG-突变成TTT)突变基因载体前期已经获得，直接用1m2AAFW和2AARv引物直接扩增W78F载体质粒，扩增后片段克隆到T载体上得到W78F的T载体，用Sal1+SacI酶切此载体后将目的片段连到2300的载体上，构建得到35S-W78F/BIR2-1-Pnos-NPTII-2300。进一步结合与前面实施例相同的PCR突变方法，分别以类同方式构建获得植物表达载体35S-L144A/BIR2-1-Pnos-NPTII-2300、35S-R213E/BIR2-1-Pnos-NPTII-2300、35S-NS366LA/BIR2-1-Pnos-NPTII-2300、35S-W78F/L144A-Pnos-NPTII-2300、35S-W78F/L144A/R213E/BIR2-1-Pnos-NPTII-2300和35S-W78F/L144A/R213E/NS336LR/BIR2-1-Pnos-NPTII-2300。
实施例五 聚合突变基因拟南芥转化
将所构建的植物表达载体分别转化农杆菌菌株GV3101，具体方法步骤如下：
1.1.1.5(1)农杆菌感受态细胞制备
将农杆菌GV3101在含50μg/ml利福平和50μg/ml庆大霉素的YEB固体培养基上划单斑接种，28℃培养1至2天。挑取单菌落接种于5ml含50μg/ml利福平和50μg/ml庆大霉素的YEB液体培养基中，28℃下摇动培养过夜(约12-16小时)至OD600值为0.4，形成种子菌液。取5ml培养活化后的菌液(1∶20的比例)接种于100ml含50μg/ml利福平和50μg/ml庆大霉素的LB液体培养基中，28℃摇动培养2-2.5小时至OD600＝0.8。冰浴菌液10分钟，每隔3分钟摇匀一次，使所述细菌均匀进入休眠状态。于4℃下4000g离心10分钟，弃上清液；加入1ml冰预冷的10％(体积比)甘油重悬浮菌体，4℃下4000g离心10分钟，收集沉淀；用冰预冷的10％(体积比)甘油重复洗3-4次；然后加入适量冰预冷的10％(体积比)甘油重新悬浮细菌沉淀，即制得GV3101感受态细胞，以40μl/管将其分装，于-70℃保存备用。
1.1.1.6(2)转化农杆菌细胞
在冰上融化所述的GV3101感受态细胞，向40μl的所述感受态细胞中加入1μl待转化杀虫基因植物表达载体质粒DNA，混匀后冰浴约10分钟。将冰浴后的感受态细胞和35S-BgVP2-2300质粒的混合物用微量移液器转移到冰预冷的0.1cm规格的电击杯(购自Bio-Rad)中，轻敲使悬浮液到达电击杯底部(注意不要有气泡)。将所述电击杯放到电击室的滑道上，推动滑道将电击杯放至电击室基座电极处。将MicroPulser(购自Bio-Rad)的程序设置为“Agr”，电击一次。立即取出电击杯，加入28℃预热的200μl LB培养基。快速而轻柔的用微量移液器将感受态细胞打匀。将悬浮液转入1.5ml的离心管，在28℃下225rpm摇动培养1小时。取100-200μl的菌液涂布于相应的抗性筛选培养基平板上(LB固体培养基，含50μg/ml利福平、50μg/ml庆大霉素、50μg/ml卡那霉素)，28℃培养。筛选阳性转化克隆，并将其菌液于-70℃保存备用。
1.1.1.7(3)受体材料拟南芥培养
选择吸水性好，土质松软的蛭石配合营养土(1∶1)作为拟南芥种植土壤。使用直径9cm的花盆，每盆播种20-30颗拟南芥种子(哥伦比亚型，来自美国俄亥俄州立大学的拟南芥生物资源中心)。播种以后在花盆上罩上薄膜，给植株的生长提供一个湿润的环境。恒温22℃，光照强度3500-4000lx，光照周期为12小时黑暗/12小时光照培养，每7天浇灌一次1/2MS液体培养基。培养30天后，每盆保留4-5棵植株，光照周期调整为8小时黑暗/16小时光照培养，待大部分植株都抽苔之后，在花序基部剪掉整个主苔，去其顶端优势，约1周后在腋芽部位长出4-6个新生侧苔，待侧苔花序形成花蕾并部分开花或形成1-2个角果时，便可用于转化。
1.1.1.8(4)拟南芥花浸转化
将转化表达载体的GV3101农杆菌菌液接种至含有含50μg/ml利福平、50μg/ml庆大霉素、50μg/ml卡那霉素的LB液体培养基中培养过夜，第二天早上按1∶50接种至含有含50μg/ml利福平、50μg/ml庆大霉素、50μg/ml卡那霉素的新的LB培养基(1L)中，培养约8个小时，至农杆菌液OD600在1.0到1.2之间。室温5000rpm离心5分钟，弃上清，将农杆菌沉淀悬浮于浸染培养基(1/2MS液体培养基，并含有5％蔗糖；用KOH调至pH5.7；0.02％Silwet L-77)中，使OD600在0.8左右。将实施例5制备的用于转化的拟南芥的上部缓缓、螺旋式浸入所述含农杆菌的浸染培养基内，轻轻顺时针晃动，约2分钟，用透明塑料罩盖严以保持湿度，放入温室过夜。24小时后移去塑料透明罩，用水浇透。之后2-3周，保证植株水分充足。当植株停止开花，第一个果荚成熟变黄时，用纸袋套住，当纸袋内的所有果荚变黄后，停止浇水，1-2周干燥后收取种子，进行转化子筛选，同时取未经转化处理的拟南芥果荚作为对照。
1.1.1.9(5)拟南芥转基因阳性转化子的筛选
种子消毒：先用70％乙醇浸泡10分钟，并不时地使种子悬浮；然后用无菌水洗四次，并不时地使种子悬浮。然后，将处理后的种子均匀涂布在含50μg/ml卡那霉素的1/2MS固体筛选培养基表面上(一块150mm直径的平皿最多播种1500粒种子)，4℃春化2天，然后在恒温22℃、光照强度3500-4000lx、光照周期为12小时黑暗/12小时光照条件下培养7-10天。转基因种子在所述筛选培养基上萌发2周以后，将能够萌发并正常生长的植株转入土壤继续培养。
实施例六 转聚合突变杀虫基因拟南芥抗虫性鉴定
浸染转化后的拟南芥种子在卡那平板上萌发，可筛选到绿色的卡那霉素抗性转基因拟南芥植株。然后将转基因拟南芥植株移栽到营养钵中，待小苗8～10叶 片后，用吸水脱脂棉球裹根，移至培养瓶中，每瓶接8头初孵棉铃虫幼虫进行抗虫性生物测定。
各类转抗虫基因的拟南芥和对照相比均具有显著的对棉铃虫初孵幼虫的抗性，从鉴定情况可以看出，对照普遍取食较多，甚至可将整个叶片吃光；而取食转基因拟南芥只形成一些小洞后，棉铃虫会死亡。转基因拟南芥抗性生测过程中棉铃虫危害典型情况如图11所示。
从表17的试验数据可以发现，数据的标准差非常高，这个结果是预料之中的。因为不同转基因拟南芥转化事件中外源杀虫基因的整合与表达情况都是差异化的，因此，数据的离散程度会很高。但利用较大群体的平均校正死亡率仍然可以对不同杀虫基因的效果进行有效地评价。我们可以看到L144A/W78F/R213E/BIR2-1聚合突变cmCry2Aa杀虫基因的平均校正死亡率最高，为75.0％，比对照提高了37.2％。在结果中，也可以观察到WLR中的两个实验重复中，转基因拟南芥的杀虫效果与对照BIR2相比，棉铃虫取食形成的小洞更小，说明突变后的杀虫蛋白抗虫能力得到了有效提升。
表17.转基因拟南芥抗性生测平均校正死亡率情况
Figure PCTCN2018078572-appb-000017
本发明以提升cmCry2Aa Bt杀虫蛋白对棉铃虫杀虫活性为研究目标，通过原核表达鉴定和转模式植物生测鉴定，发现1mCry2Aa、W78FCry2Aa、R213ECry2Aa和L144ACry2Aa四个突变及其组合突变可显著提高对棉铃虫的杀虫活性。本发明可用于抗虫转基因植物研究。

Claims (10)

  1. 一种突变Cry2Aa杀虫蛋白质，所述突变Cry2Aa杀虫蛋白质的氨基酸序列是在SEQ ID NO.1所示的氨基酸序列的基础上，具有如下一种、两种或三种突变：
    A)W78F；
    B)L144A；
    C)R213E。
  2. 根据权利要求1所述的突变Cry2Aa杀虫蛋白质，其特征在于，
    D)所述氨基酸序列N端28个氨基酸缺失。
  3. 权利要求1或2所述的突变Cry2Aa杀虫蛋白质的编码基因。
  4. 根据权利要求3所述的编码基因，所述编码基因的序列是在SEQ ID NO.2的基础上，具有如下一种、两种或三种突变：233-234GG变成TT；430-431CT变成GC；637-638AG变成GA。
  5. 根据权利要求4所述的编码基因，所述编码基因5’端计4-87位核苷酸缺失。
  6. 一种重组表达载体，其含有权利要求3-5任一项所述的编码基因并且所述编码基因的核苷酸序列与所述表达载体的表达控制序列可操作地连接。
  7. 一种改善植物抗虫效果的方法，包括：将权利要求3-5任一项所述的编码基因或者权利要求6所述的重组表达载体导入植物或植物组织并使所述编码基因表达；优选地，所述植物是烟草或拟南芥。
  8. 一种制备转基因植物的方法，包括：在有效产生植物的条件下培养含有权利要求3-5任一项所述的编码基因或者权利要求6所述的重组表达载体的植物或植物组织。
  9. 权利要求3-5任一项所述的编码基因、权利要求6所述的重组表达载体用于改善植物抗虫效果以及用于植物育种的用途。
  10. 一种制备突变基因的方法，所述突变基因编码根据权利要求1所述的突变Cry2Aa杀虫蛋白质，所述方法包括用如下三组引物中的1组、2组或3组依次突变SEQ ID NO.2所示的序列的DNA样品：
    (1)5'-GATCTTGTCAGAACTTTTTGGTATTATCTTTCC-3'
    5'-GGAAAGATAATACCAAAAAGTTCTGACAAGATC-3'
    (2)
    5’-CCTACTCAAAACCCAGTTCCTGCTAGCATTACTTCTTCAGTGAATAC-3’
    5’-GTATTCACTGAAGAAGTAATGCTAGCAGGAACTGGGTTTTGAGTAGG-3’
    (3)5'-ATAGAGACTATCTTGAGAATTACACAAGAGA-3'
    5'-TCTCTTGTGTAATTCTCAAGATAGTCTCTAT-3'。
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