Peanut chitinase and application thereof
阅读说明：本技术 一种花生几丁质酶及其应用 (Peanut chitinase and application thereof ) 是由 赵小波 李春娟 王娟 闫彩霞 单世华 张�浩 谢宏峰 石程仁 于 2021-09-13 设计创作，主要内容包括：本发明涉及一种花生几丁质酶及其应用。所述花生几丁质酶CHI-ChBD-hinge的氨基酸序列如SEQ ID NO.1所示,其对应的核苷酸序列如SEQ ID NO.2所示。本发明所述的花生几丁质酶CHI-ChBD-hinge,其N端具有融合的ChBD结构域序列,可以增加几丁质的结合能力,同时融合后序列后面增加了一段具有5个氨基酸的铰链区,可以增加ChBD结构域的灵活性,增加几丁质酶的催化功能,可显著提高植物对病原真菌抗性的功能。本发明基因转入到烟草中过量表达,很强的体外抗真菌活性。同时,CHI-ChBD-hinge超表达的转基因烟草对胶孢炭疽菌的生长均具有很强的抑制作用。(The invention relates to peanut chitinase and application thereof. The amino acid sequence of the peanut chitinase CHI-ChBD-hinge is shown as SEQ ID NO.1, and the corresponding nucleotide sequence is shown as SEQ ID NO. 2. The N end of the peanut chitinase CHI-ChBD-hinge has a fused ChBD structure domain sequence, so that the binding capacity of chitin can be increased, and meanwhile, a section of hinge region with 5 amino acids is added behind the fused sequence, so that the flexibility of the ChBD structure domain can be increased, the catalytic function of the chitinase is increased, and the function of resisting pathogenic fungi of plants can be obviously improved. The gene of the invention is transferred into tobacco for over-expression, and has strong in vitro antifungal activity. Meanwhile, the transgenic tobacco over-expressed by CHI-ChBD-hinge has strong inhibition effect on the growth of colletotrichum gloeosporioides.)
1. An amino acid sequence of the peanut chitinase CHI-ChBD-hinge is shown as SEQ ID NO. 1.
2. The nucleotide sequence of claim 1 corresponding to peanut chitinase CHI-ChBD-hinge, which is represented by SEQ ID No. 2.
3. A vector carrying the gene of claim 2.
4. The peanut chitinase of claim 1, having a fused ChBD domain at the N-terminus.
5. The peanut chitinase of claim 4, wherein the N-terminal ChBD domain is fused to a hinge region sequence.
6. Use of the peanut chitinase CHI-ChBD-hinge of claim 1 for plant antimicrobial applications.
7. The use of a peanut chitinase according to claim 1 for increasing tobacco resistance to colletotrichum gloeosporioides.
8. The use of a peanut chitinase as claimed in claim 1 for increasing resistance of watermelon to blight and peanut resistance to Aspergillus flavus.
9. The method of cloning the peanut chitinase CHI-ChBD-change gene of claim 1, comprising:
(1) obtaining a peanut chitinase CHI;
(2) fusion of the N segment of the ChBD domain and the hinge region;
(3) constructing a recombinant vector;
(4) the mediated transfer and expression of plant.
Technical Field
The invention relates to peanut chitinase and application thereof, belonging to the technical field of biology.
Background
Plants are stressed by many biotic and abiotic factors during their growth and development, such as drought, cold, UV radiation, trauma, pathogen (fungal, bacterial, viral) infestation, etc., for which organisms have evolved a series of defense mechanisms to combat the adversity stresses, and activation and accumulation of pathogenesis-related Proteins (PR) are important components of the plant defense response (Singh a, et al, Heterologous expression of new antibacterial chitinase from protein expression.2007, 56(1): 100-) -109). Plants express a plurality of disease course related protein genes to deal with the harm of a plurality of adversity factors such as pathogenic bacteria, wherein chitinase (chitinase) coded by a PR3 gene family is concerned in plant disease-resistant defense reaction.
Chitin is a linear polymeric compound formed by connecting N-acetyl-D-glucosamine by beta-1, 4-glycosidic bonds, is a main component forming the skeleton of the fungal cell wall, and does not contain chitin in the cell wall of plant cells. Chitinase is an enzyme system that takes chitin as a substrate and hydrolyzes it into N-acetyl oligosaccharides and glucose. Chitinase is widely present in higher plants, and is distributed in various tissue parts of plants. Typical chitinases have an N-terminal signal peptide (signal peptide), a catalytic domain (catalytic domain) and a C-terminal domain (C-terminal extension) that is present only in vacuolar chitinases. Some chitinase N-terminal signal peptides contain one or more cysteine-rich chitin-binding domains (ChBD). The ChBD domain can enhance the binding capacity and activity to different chitins, and has a remarkable effect on the antifungal effect of the chitinase, while the peanut chitinase does not contain the ChBD domain. The method for improving the catalytic function of the antibacterial protein by a genetic engineering method is one of the key points of the current antibacterial protein research.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a cloning method of a peanut chitinase gene, wherein the N end of the peanut chitinase gene is fused with a ChBD structural domain, so that the binding capacity and activity to chitin are increased. Meanwhile, a hinge region sequence containing 5 amino acids is added behind the ChBD structure domain sequence, and the flexibility of the three-dimensional structure of the peanut chitinase is increased. Hinge region sequence as a Flexible linker (Glu)68-Lys72) On one hand, the swinging of the ChBD structure domain can be enhanced, and on the other hand, the distance between the ChBD structure domain and the catalytic structure domain can be reduced, so that the catalytic efficiency is improved. The invention provides a cloning method of a novel peanut chitinase gene, and defines the effects of the peanut chitinase CHI-ChBD-hinge on improving the resistance of tobacco to colletotrichum gloeosporioides and improving the resistance of watermelon to blight and peanut to aspergillus flavus. Based on the above, the invention provides a peanut chitinase, a sequence of a coding gene CHI-ChBD-hinge thereof and application thereof.
In one aspect, the invention provides a novel peanut chitinase CHI-ChBD-hinge, the amino acid sequence of which is shown as SEQ ID NO. 1.
SEQ ID NO.1：
MASNKSSSTHQPLTLLLFFLLTLSSAHAKGGIAIYDCGCTADLCCSRFGYCGNGTDYCGTGCQGGPCEA AAKWGQNNGDGNLTSTCDTGNYEIVLLAFLYTFGCGRTPDWNFAGHCGSWSPCDKLQPEIEHCQRNGVKVFLSLGGAVGPYSLCSPEDAKSVSDYLYNNFLTGQKGPLGSVYLDGIDFDIEGGSNLYWDDLARELDTRRKQDRYFYLSAAPQCFFTDYYLDTAIRTWLFDYLFVQFYNNPPCQYSNGDASLLLSSWNTWTSYVKINNTVFMGLPAAPDAAPSGGYISPQDLCTKVLPTIKHTPNYGGVMLWDRFRDVTNHYSDQIKDCVIVDDSVRVSQTVMATLSNTVSQCVSAAFNRIIPKLRPF
In another aspect, the invention also provides a nucleic acid sequence corresponding to the peanut chitinase CHI-ChBD-hinge, which is shown as SEQ ID NO. 2.
SEQ ID NO.2：
ATGGCTTCCAACAAGAGTAGTAGTACTCATCAACCATTAACATTGCTCCTCTTCTTCCTCCTCACCCTCTCCTCTGCACATGCCAAAGGTGGCATAGCCATCTACTTGCCGGAGAATTGATAACAGCTCAAGATTGTGGTTGCACC GCCGACCTATGTTGTAGTCGGTTTGGTTATTGTGGCAACGGCACAGATTACTGTGGAAGCGGCGGCGAAATGGGGCCAGAACAATGGCGACGGCAACTTAACCTCCACATGTGACACCGGAAACTACGAGATTGTGCTTCTCGCCTTTCTCTACACTTTCGGTTGTGGCAGAACTCCAGATTGGAACTTTGCTGGCCACTGTGGTTCATGGTCCCCTTGTGACAAACTACAACCAGAAATCGAACATTGCCAAAGAAACGGTGTGAAGGTGTTCCTCTCCCTTGGAGGAGCCGTTGGGCCCTACTCCTTATGCTCGCCGGAAGATGCCAAGAGCGTCTCCGACTACCTTTACAACAACTTCCTCACTGGCCAAAAGGGTCCATTAGGGAGTGTGTACCTTGATGGCATTGATTTCGACATTGAAGGTGGTTCAAACCTTTATTGGGATGACTTAGCTAGAGAACTCGATACACGCAGAAAGCAAGACAGGTACTTTTACTTGTCGGCAGCACCACAATGTTTCTTCACAGATTACTACCTTGATACCGCCATTAGAACTTGGCTTTTCGATTACCTCTTCGTCCAGTTCTACAACAACCCTCCATGCCAATATAGTAACGGCGACGCGTCTCTGCTCTTATCTTCTTGGAATACATGGACCTCCTATGTGAAGATCAACAACACGGTGTTTATGGGGCTACCCGCTGCACCTGATGCAGCTCCCAGCGGTGGCTATATTTCACCGCAAGATCTGTGTACTAAGGTTCTTCCAACCATCAAGCACACTCCTAACTATGGAGGCGTCATGCTATGGGATAGGTTCCGCGATGTTACTAACCACTACAGCGATCAAATCAAGGATTGTGTTATAGTTGATGATAGTGTTAGGGTGTCACAGACTGTGATGGCAACGTTATCAAACACTGTATCTCAATGTGTATCTGCAGCTTTCAACCGCATCATACCAAAACTAAGACCCTTT
On the other hand, the invention also provides a cloning method of the peanut chitinase CHI-ChBD-hinge gene. The method comprises the following steps:
(1) obtaining of peanut chitinase CHI: after the plant seedlings are subjected to low temperature, drought and salt stress, aspergillus flavus is inoculated before harvest to induce and express the expression of related genes, including chitinase. After total RNA was extracted, the coding region of chitinase CHI was amplified by RT-PCR. Connecting the product to a pGEM-T Easy vector, and obtaining a peanut chitinase sequence through orientation;
(2) fusion of segment N of the ChBD domain and hinge region: designing a sequence of peanut chitinase with an N-end fused with a ChBD structure domain and a hinge region, and then carrying out simulation analysis and three-dimensional modeling;
(3) constructing a recombinant vector: synthesizing a sequence in vitro, and connecting the sequence gene with a plant expression vector pCAMBIA2300s to construct a plant overexpression vector;
(4) the mediated transfer and expression of plants: the constructed plant overexpression vector is connected to the tobacco through agrobacterium tumefaciens to be transformed into the tobacco for expression.
Taking 2-5 peanut plants to cultivate in a field cultivation environment, carrying out stress treatment on the peanut plants by adopting a NaCl solution and a PEG6000 solution in the cultivation process, and inoculating aspergillus flavus to the peanut plants 24 days before harvest for three times, wherein the peanut plants are peanut seedlings in a trefoil stage growing for 2 weeks;
obtaining 2-10 g of peanut seed coat of peanut seeds of the peanut plant, grinding, extracting total RNA of the peanut seed coat, and synthesizing cDNA by adopting the RNA;
and amplifying a target gene J11-AhHevamine-A by using the synthesized first strand cDNA as a template. The primers used were Forword: 5'-CCAATATAGTAACGGCGACGCG-3' and Reverse: 5'-CATAAACACCGTGTTGTTGATC-3', respectively; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1min for 30 cycles; extending for 5min at 72 ℃; stabilizing at 4 deg.C for 15 min. The DNA polymerase used for the PCR reaction was Primerstar HS, available from Dalibao Bio. After the PCR was completed, 5. mu.L of the resulting mixture was subjected to agarose gel electrophoresis to examine the specificity and size of the amplified product. Connecting the purified product with a pGEM-T Easy vector, converting the purified product into competent escherichia coli, culturing the competent escherichia coli in an LB (lysogeny) culture medium for a preset time, randomly selecting a preset number of culture products for amplification culture after the culture for the preset time, and performing PCR (polymerase chain reaction) amplification detection and sequencing on the amplified culture products;
after sequencing to obtain a sequence, obtaining an amino acid sequence of the sequence, fusing a ChBD structure domain in the N section and simultaneously increasing a hinge region sequence, then performing three-dimensional modeling on the fused peanut chitinase sequence, combining the substrate pocket conformation of chitin, simulating the interaction of the substrate and enzyme molecules through calculation, and analyzing to obtain the fused ChBD sequence with the function of enhancing the chitin combination. The hinge region sequence is used as a flexible linker, so that the swinging property of the ChBD structure domain can be enhanced, the distance between the hinge region sequence and the catalytic structure domain of the peanut chitinase can be reduced, and the catalytic efficiency is improved;
synthesizing a nucleic acid sequence of the peanut chitinase CHI-ChBD-hinge, connecting the obtained gene segment with a plant expression vector pCAMBIA2300s, constructing a plant overexpression vector, and then transferring the constructed recombinant vector into tobacco for expression through agrobacterium tumefaciens mediation.
In another aspect, the invention also provides a vector carrying the peanut chitinase gene. The vector is constructed from the plant pCAMBIA2300s and the like.
On the other hand, the invention also provides application of the peanut chitinase CHI-ChBD-hinge in plant antibiosis. Further, the plant is selected from tobacco, watermelon or peanut; the bacteria are selected from colletotrichum gloeosporioides, fusarium wilt and aspergillus flavus. Preferably: the application of the peanut chitinase in improving the resistance of tobacco to colletotrichum gloeosporioides is provided. Preferably: the peanut chitinase has the function of improving the resistance of watermelons to blight and peanut aspergillus flavus resistance.
Has the advantages that:
the N end of the peanut chitinase CHI-ChBD-hinge provided by the invention is provided with a fused ChBD structure domain and a hinge region thereof. The fused structural domain increases the assembly rate of the chitinase on the basis of not damaging the structure of the chitinase, thereby showing better chitin binding activity; the fusion hinge region sequence is used as a flexible linker, so that on one hand, the swinging property of the ChBD structure domain can be enhanced, and the binding capacity of the ChBD for binding chitin is further increased; on the other hand, the distance between the catalytic structure domain and the catalyst can be reduced, and the catalytic efficiency is improved. The combination ability and the catalytic activity are considered comprehensively, and the synergy of the ChBD structure domain and the hinge region is fused, so that the catalytic activity of the peanut chitinase is improved, and the resistance of the plant to pathogenic fungi can be enhanced.
Drawings
FIG. 1 is a simulated 3D structure of the peanut chitinase CHI-ChBD-change of the present invention;
FIG. 2 is a graph showing the results of analysis of the transcription level expression of the chitinase CHI-ChBD-hinge in the positive transgenic tobacco of the present invention, wherein the Marker is DL2000 DNA Marker (Dalibao organism); WT is a PCR product with non-transgenic tobacco total RNA reverse transcription cDNA as a template;
FIG. 3 shows the bacteriostatic effect of the transgenic tobacco in vitro antifungal activity of the peanut chitinase CHI-ChBD-hinge of the present invention.
Detailed Description
Example 1 full-Length peanut chitinase cDNA cloning and sequence analysis
Taking 2-5 peanut plants to cultivate in a field cultivation environment, and carrying out stress treatment on the peanut plants in the cultivation process. Specifically, NaCl solution and PEG6000 solution are adopted to carry out stress treatment on the peanut plants. The three-leaf period peanut seedlings are placed in a low-temperature illumination incubator at 4 ℃ for low-temperature treatment to realize the selection of low-temperature resistant genes from the three-leaf period peanut seedlings, then the three-leaf period peanut seedlings are subjected to salt-tolerant treatment by adopting a NaCl solution, namely the roots of the three-leaf period peanut seedlings are soaked in a 250mM NaCl solution for 6 hours after soil is removed; and finally, carrying out drought resisting treatment on the peanut seedlings in the trefoil stage by adopting a PEG6000 solution, namely, removing soil from the roots of the peanut seedlings in the trefoil stage, soaking the peanut seedlings in a 20 percent PEG6000 solution for 6 hours, and inoculating aspergillus flavus to the peanut plants 24 days before harvesting for three times. Selecting 2-5 peanut plant seedlings cultivated in advance, and cultivating in a fertile field in a natural environment, wherein preferably, the peanut plant seedlings in the trefoil stage growing for 2 weeks are adopted for field cultivation. 24 days before peanut plants are harvested, furrows with the depth of 3-4cm are arranged among the peanut ridges, and the arranged furrows can not touch the roots of the peanut plants. After the furrow is set, the aspergillus flavus is scattered in the furrow, and a soil layer with the thickness of 3-4cm is covered on the upper part of the aspergillus flavus; then adopting a water spraying kettle according to the proportion of 100-2Water is uniformly sprayed on the soil layer covered on the upper part of the aspergillus flavus so as to create a damp and warm environment suitable for the growth of the aspergillus flavus, and during the execution of the process, the water is required to be sprayed on the soil layerThe temperature of the soil layer covered on the upper part of the aspergillus flavus is kept within the range of 15-30 ℃, preferably, the temperature of the soil layer covered on the upper part of the aspergillus flavus is kept at 25 ℃, the relative humidity of the soil layer covered on the part of the aspergillus flavus is more than 50%, the soil layer under the temperature and the humidity is the temperature and humidity environment which is most suitable for the aspergillus flavus to propagate, and the rapid propagation of the aspergillus flavus can be ensured.
Further, after the inoculation of the aspergillus flavus for the first time in the above steps is completed, according to 8 days as a period, the inoculation of the aspergillus flavus for the second time is performed according to the above steps again, that is, after 8 days are respectively separated, the inoculation of the aspergillus flavus for the second time and the third time is performed according to the above steps.
Obtaining 2-10 g of peanut seed coat of peanut seeds of the peanut plants, grinding, extracting RNA of the peanut seed coat, and synthesizing cDNA by adopting the RNA.
Specifically, after the cultivated peanut plant grows to be mature, selecting 2-10 g of peanut seed coat of peanut seeds of the peanut plant, grinding the peanut seed coat by using a bowl, and separating and extracting RNA of the ground peanut seed coat by using a MiniBEST Universal RNA Extraction Kit method of TAKARA; after the RNA of the extracted peanut seed coat is removed from DNA pollution, cDNA synthesis is carried out by a SMART-RACE kit method, and a cDNA synthesis product is stored in a low-temperature refrigerator at the temperature of minus 20 ℃ for standby, wherein a vessel used in the processes of RNA extraction and cDNA synthesis is soaked in 0.1 percent DEPC for 12 hours and sterilized at high temperature.
Specifically, the MiniBEST Universal RNA Extraction Kit and the SMART-RACE Kit are adopted to extract the RNA of the peanut seed coat and synthesize the cDNA respectively, so that the finally obtained cDNA synthetic product has better gene integrity and gene length, and the improvement of the anti-mould property of the finally cloned peanut chitinase gene is facilitated.
And amplifying a target gene J11-AhHevamine-A by using the synthesized first strand cDNA as a template. The primers used were Forword: 5'-CCAATATAGTAACGGCGACGCG-3' and Reverse: 5'-CATAAACA CCGTGTTGTTGATC-3', respectively; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1min for 30 cycles; extending for 5min at 72 ℃; stabilizing at 4 deg.C for 15 min. The DNA polymerase used for the PCR reaction was Primerstar HS, available from Dalibao Bio. After the PCR was completed, 5. mu.L of the resulting mixture was subjected to agarose gel electrophoresis to examine the specificity and size of the amplified product. And connecting the purified product with a pGEM-T Easy vector, converting the purified product into competent escherichia coli, culturing the competent escherichia coli in an LB (lysogeny) culture medium for a preset time, randomly selecting a preset number of culture products for amplification culture after the culture for the preset time, and performing PCR (polymerase chain reaction) amplification detection and sequencing on the amplified culture products.
Furthermore, the open reading frame of the peanut chitinase gene obtained by cloning through the method for cloning the peanut chitinase gene is 1020bp, and the encoding of the peanut chitinase gene has 339 amino acid sequences.
Example 2 Synthesis of peanut chitinase CHI-ChBD-hinge and construction of plant overexpression vector
The N-terminal of the peanut chitinase obtained in the example 1 is fused with ChBD structure domain and hinge region sequences, and then simulation analysis is carried out. The fused structural domain increases the assembly rate of the chitinase on the basis of not damaging the structure of the chitinase, thereby showing better chitin binding activity; the fusion hinge region sequence is used as a flexible linker, so that on one hand, the swinging property of the ChBD structure domain can be enhanced, and the binding capacity of the ChBD for binding chitin is further increased; on the other hand, the distance between the catalytic domains can be reduced, and the catalytic efficiency is improved (figure 1).
The peanut chitinase sequence CHI-ChBD-Hinge fusing the ChBD domain and the Hinge region was synthesized in vitro, while CHI-ChBD fusing the ChBD domain without the Hinge region and CHI-Hinge fusing the Hinge region sequence without the ChBD domain were synthesized, and then the synthesized sequence and the wild type peanut chitinase (CHI-wt) obtained in example 1 were ligated to the plant expression vector pCAMBIA2300s using T4 DNA Ligase (TaKaRa) (plasmid pCAMBIA2300s was double-digested with restriction enzymes Bam HI (TaKaRa) and Eco RI (TaKaRa)). The ligation product was then transferred into E.coli DH 5. alpha. by heat shock transformation, and positive clones were selected on a solid medium containing 50mg/L kanamycin (Km). Selecting single colony shake bacteria, carrying out PCR by using a specific primer for amplifying CHI-ChBD-hige by using a bacterial liquid as a template, selecting clones successfully connected with the CHI-wt, the CHI-ChBD, the CHI-hige and the CHI-ChBD-hige and pCAMBIA2300s, and adding glycerol and storing at-80 ℃ for later use if the detected strains are positive.
The amino acid sequence of the novel peanut chitinase gene CHI-ChBD-hinge obtained in the embodiment 2 is shown as SEQ ID NO.1, and the corresponding nucleotide sequence is shown as SEQ ID NO. 2.
The four plasmids of the E.coli were extracted and purified using a SanPrep column plasmid extraction kit (Shanghai Biotech), which was pCAMBIA2300s-CHI-wt, pCAMBIA2300s-CHI-ChBD, pCAMBIA2300s-CHI-hinge and pCAMBIA2300s-CHI-ChBD-hinge, respectively. Then, the four plant expression vectors constructed above were transferred into Agrobacterium tumefaciens LBA4404 competent cells by a liquid nitrogen freeze-thaw method. The operation steps are as follows: 2 mu g of the four plasmids are respectively added into a centrifuge tube containing 200 mu L of competent cells, the mixture is gently mixed and then is subjected to ice bath for 5min, then the mixture is transferred into liquid nitrogen to be frozen for 1min, then the mixture is rapidly placed into a water bath at 37 ℃ for 5min, then is subjected to ice bath for 2min immediately, and is added with 800 mu L of LB liquid medium to be cultured for 4h under the condition of shaking at 28 ℃. The activated agrobacterium is smeared on LB solid culture medium containing 50mg/L Km and is statically cultured at 28 ℃. Selecting single colony shake bacteria, carrying out PCR by using specific primers for amplifying CHI-ChBD-hige, respectively detecting whether four plasmids pCAMBIA2300s-CHI-wt, pCAMBIA2300s-CHI-ChBD, pCAMBIA2300 s-CHI-hige and pCAMBIA2300 s-CHI-ChBD-hige are transferred into agrobacterium, and adding glycerol into positive clones, and storing at-80 ℃ for later use.
Example 3 Agrobacterium-mediated genetic transformation of plants and transgenic plant selection
The transgenic recipient in this experiment was tobacco, tobacco seeds were soaked in 75% ethanol for 30s, washed with sterile water and then washed with 0.1% HgCl2Soaking for 8min, washing with sterile water for several times, sowing on 1/2MS culture medium, dark culturing at 28 deg.C for 6d, germinating, transferring to light incubator (25 deg.C, 16h/d light), and subculturing with 1/2MS culture medium once a month.
Taken out of refrigerator at-80 deg.C for storage and respectively containAgrobacterium LBA4404 strain of pCAMBIA2300s-CHI-wt, pCAMBIA2300s-CHI-ChBD, pCAMBIA2300s-CHI-hinge and pCAMBIA2300s-CHI-ChBD-hinge plasmid was inoculated in 5mL LB liquid medium containing 50mg/L Km and 20mg/L rifampicin, and cultured at 28 ℃ until the medium became turbid. Sucking 1mL of turbid bacterial liquid to an LB solid culture medium containing 50mg/L Km, and culturing for 48h at 28 ℃; then, appropriate amount of the agrobacteria on LB solid medium was scraped and inoculated into MGL liquid medium supplemented with 20mg/L acetosyringone, and shake-cultured at 28 ℃ for 2-3h to activate the agrobacteria. Cutting leaves of aseptic seedling of tobacco into 1cm2And completely soaking the left and right leaf discs in the MGL liquid culture medium containing the activated agrobacterium for 15min, sucking bacterial liquid on the surfaces of the leaves by using sterile filter paper, placing the leaf discs on a co-culture medium for room-temperature culture, wherein the co-culture medium for tobacco transformation is MS +0.02 mg/L6-BA +2.1mg/L NAA +30g/L sucrose +6g/L agar, and co-culturing for 2 days at 22 ℃ in the dark. Transferring the co-cultured leaf discs to an MS screening culture medium added with antibiotics to be divided into seedlings, and screening transgenic plants. The tobacco screening culture medium is MS +0.5mg/L6-BA +0.1mg/L NAA +30g/L sucrose +6g/L agar +50mg/L Km +200mg/L cephamycin (cefixime sodium salt, Cef); during screening culture, the culture bottle is transferred to an illumination culture box for culture (25 ℃, 16h/d illumination and 8h/d darkness), after the tobacco grows out of buds, the MS culture medium containing 50mg/L Km and 200mg/L Cef is used for subculture, the regeneration plant needs to be further screened because the callus differentiation rate of the tobacco is higher, the tobacco regeneration seedling is transferred to the MS culture medium containing 50mg/L Km to root the tobacco regeneration seedling, and finally the regeneration seedling with better rooting is selected for further detection.
Extracting genome DNA of transgenic tobacco plant leaves by a CTAB method, taking 1 mu L of the extracted genome DNA, detecting the integrity and the concentration of the extracted genome DNA by agarose gel electrophoresis, carrying out PCR by using the genome DNA of the transgenic plant as a template and a specific primer for amplifying CHI-ChBD-hinge, and taking 8 mu L of a product after the PCR is finished, and using the product in the agarose gel electrophoresis to detect the positive transgenic plant of the gene.
Example 4 analysis of CHI-ChBD-hinge expression in transgenic tobacco and analysis of antifungal Activity of transgenic plants
Taking positive CHI-wt, CHI-ChBD, CHI-hige and CHI-ChBD-hige transgenic single strains and tender leaves of non-transgenic tobacco (wild type) to extract total RNA, carrying out reverse transcription to generate a first strand of cDNA, and PCR was performed using the primers specific for amplification of CHI-ChBD-change (including CHI-wt, CHI-ChiBD and CHI-change) using this as a template, the expression of CHI-ChBD (including CHI-wt, CHI-ChiBD and CHI-hige) transcript level, total RNA extraction and RT-PCR in each transgenic individual was analyzed based on the PCR results in the same manner as in example 1, and after completion of PCR, mu.L of the resulting sample was subjected to agarose gel electrophoresis, and the results of the detection of a part of the individual plants are shown in FIG. 2, and it was detected that CHI-ChBD-hinges (including CHI-wt, CHI-ChiBD and CHI-hinges) were expressed in large amounts at the transcriptional level in the transgenic individual plants.
Inoculating colletotrichum gloeosporioides stored in a laboratory on a PDA (potato dextrose agar) solid medium (200g/L, 15g/L and 20 g/L), carrying out dark culture at 28 ℃, adding protein when bacterial colonies grow to the diameter of about 2-3cm, and analyzing the in vitro antifungal activity of a transgenic plant. In order to prevent other infectious microbes from polluting extracted protein, the whole plant protein extraction process is aseptic operation, firstly, 1g of single transgenic tobacco plant and wild type leaves are put into a mortar, 1mL of protein extracting solution (1M NaCl, 0.1M sodium acetate, 1% PVP, pH6) is added, and the mixture is fully ground; transferring into 1.5mL centrifuge tube, mixing, standing overnight at 4 deg.C, centrifuging at 4 deg.C for 30min (12,000g/min), collecting supernatant, and determining total protein concentration with ultraviolet spectrophotometer. Adjusting the total protein concentration of the transgenic and wild-type plants to 0.2. mu.g/. mu.L, then dropping 20. mu.L onto sterile filter paper of each fungus culture medium, adding the total protein of the transgenic tobacco plant on the fungus plate, simultaneously adding a blank control (solution for extracting protein) in parallel, observing the growth of each treatment fungus after culturing for several days at 28 ℃, and accordingly evaluating the in vitro antifungal activity of CHI-wt, CHI-ChBD, CHI-hige and CHI-ChBD-hige transgenic tobaccos, and the results are shown in FIG. 3, wherein the CHI-wt, CHI-ChBD, CHI-hige and CHI-ChBD-hige transgenic tobaccos have the in vitro antifungal activity; CHI-ChBD-hige transgenic tobacco proteins have an enhanced inhibitory effect on the growth of colletotrichum gloeosporioides compared to CHI-wt, CHI-ChBD and CHI-hige.
The invention extracts the peanut chitinase gene with mould resistance and stress resistance, and is used for further improvement to obtain the novel peanut chitinase CHI-ChBD-hinge, and the N end of the novel peanut chitinase CHI-ChBD-hinge is provided with a fused ChBD structural domain and hinge region sequence. The fused structural domain increases the assembly rate of the chitinase on the basis of not damaging the structure of the chitinase, thereby showing better chitin binding activity; the fused hinge region sequence is used as a flexible linker, so that on one hand, the swinging property of the ChBD structure domain can be enhanced, and the binding capacity of the ChBD for binding chitin is further increased; on the other hand, the distance between the catalytic structure domain and the catalytic structure domain can be reduced, the catalytic efficiency is improved, and the anti-mould property of crops (tobacco, peanut, watermelon and the like) is improved, so that the stress resistance of the crops is improved, and the anti-mould infection capacity is enhanced.
Sequence listing
<110> institute for peanut research in Shandong province
<120> peanut chitinase and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 376
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ala Ser Asn Lys Ser Ser Ser Thr His Gln Pro Leu Thr Leu Leu
1 5 10 15
Leu Phe Phe Leu Leu Thr Leu Ser Ser Ala His Ala Lys Gly Gly Ile
20 25 30
Ala Ile Tyr Asp Cys Gly Cys Thr Ala Asp Leu Cys Cys Ser Arg Phe
35 40 45
Gly Tyr Cys Gly Asn Gly Thr Asp Tyr Cys Gly Thr Gly Cys Gln Gly
50 55 60
Gly Pro Cys Glu Ala Ala Ala Lys Trp Gly Gln Asn Asn Gly Asp Gly
65 70 75 80
Asn Leu Thr Ser Thr Cys Asp Thr Gly Asn Tyr Glu Ile Val Leu Leu
85 90 95
Ala Phe Leu Tyr Thr Phe Gly Cys Gly Arg Thr Pro Asp Trp Asn Phe
100 105 110
Ala Gly His Cys Gly Ser Trp Ser Pro Cys Asp Lys Leu Gln Pro Glu
115 120 125
Ile Glu His Cys Gln Arg Asn Gly Val Lys Val Phe Leu Ser Leu Gly
130 135 140
Gly Ala Val Gly Pro Tyr Ser Leu Cys Ser Pro Glu Asp Ala Lys Ser
145 150 155 160
Val Ser Asp Tyr Leu Tyr Asn Asn Phe Leu Thr Gly Gln Lys Gly Pro
165 170 175
Leu Gly Ser Val Tyr Leu Asp Gly Ile Asp Phe Asp Ile Glu Gly Gly
180 185 190
Ser Asn Leu Tyr Trp Asp Asp Leu Ala Arg Glu Leu Asp Thr Arg Arg
195 200 205
Lys Gln Asp Arg Tyr Phe Tyr Leu Ser Ala Ala Pro Gln Cys Phe Phe
210 215 220
Thr Asp Tyr Tyr Leu Asp Thr Ala Ile Arg Thr Trp Leu Phe Asp Tyr
225 230 235 240
Leu Phe Val Gln Phe Tyr Asn Asn Pro Pro Cys Gln Tyr Ser Asn Gly
245 250 255
Asp Ala Ser Leu Leu Leu Ser Ser Trp Asn Thr Trp Thr Ser Tyr Val
260 265 270
Lys Ile Asn Asn Thr Val Phe Met Gly Leu Pro Ala Ala Pro Asp Ala
275 280 285
Ala Pro Ser Gly Gly Tyr Ile Ser Pro Gln Asp Leu Cys Thr Lys Val
290 295 300
Leu Pro Thr Ile Lys His Thr Pro Asn Tyr Gly Gly Val Met Leu Trp
305 310 315 320
Asp Arg Phe Arg Asp Val Thr Asn His Tyr Ser Asp Gln Ile Lys Asp
325 330 335
Cys Val Ile Val Asp Asp Ser Val Arg Val Ser Gln Thr Val Met Ala
340 345 350
Thr Leu Ser Asn Thr Val Ser Gln Cys Val Ser Ala Ala Phe Asn Arg
355 360 365
Ile Ile Pro Lys Leu Arg Pro Phe
370 375
<210> 2
<211> 1128
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggcttcca acaagagtag tagtactcat caaccattaa cattgctcct cttcttcctc 60
ctcaccctct cctctgcaca tgccaaaggt ggcatagcca tctacttgcc ggagaattga 120
taacagctca agattgtggt tgcaccgccg acctatgttg tagtcggttt ggttattgtg 180
gcaacggcac agattactgt ggaagcggcg gcgaaatggg gccagaacaa tggcgacggc 240
aacttaacct ccacatgtga caccggaaac tacgagattg tgcttctcgc ctttctctac 300
actttcggtt gtggcagaac tccagattgg aactttgctg gccactgtgg ttcatggtcc 360
ccttgtgaca aactacaacc agaaatcgaa cattgccaaa gaaacggtgt gaaggtgttc 420
ctctcccttg gaggagccgt tgggccctac tccttatgct cgccggaaga tgccaagagc 480
gtctccgact acctttacaa caacttcctc actggccaaa agggtccatt agggagtgtg 540
taccttgatg gcattgattt cgacattgaa ggtggttcaa acctttattg ggatgactta 600
gctagagaac tcgatacacg cagaaagcaa gacaggtact tttacttgtc ggcagcacca 660
caatgtttct tcacagatta ctaccttgat accgccatta gaacttggct tttcgattac 720
ctcttcgtcc agttctacaa caaccctcca tgccaatata gtaacggcga cgcgtctctg 780
ctcttatctt cttggaatac atggacctcc tatgtgaaga tcaacaacac ggtgtttatg 840
gggctacccg ctgcacctga tgcagctccc agcggtggct atatttcacc gcaagatctg 900
tgtactaagg ttcttccaac catcaagcac actcctaact atggaggcgt catgctatgg 960
gataggttcc gcgatgttac taaccactac agcgatcaaa tcaaggattg tgttatagtt 1020
gatgatagtg ttagggtgtc acagactgtg atggcaacgt tatcaaacac tgtatctcaa 1080
tgtgtatctg cagctttcaa ccgcatcata ccaaaactaa gacccttt 1128
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccaatatagt aacggcgacg cg 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cataaacacc gtgttgttga tc 22