CN107151671B - Method for improving resistance of spCEMA to phytopathogens and transgenic tobacco material - Google Patents
Method for improving resistance of spCEMA to phytopathogens and transgenic tobacco material Download PDFInfo
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Abstract
The invention discloses a method for improving the resistance of spCEMA to plant pathogenic bacteria, which comprises the steps of taking ergosterol on a plant pathogenic fungus cell membrane as a target point, fusing an ergosterol binding domain ErBD at the C terminal of the spCEMA by a molecular improvement means, obtaining a corresponding fused antibacterial peptide gene (such as a nucleotide sequence shown in SEQ ID NO.1 and an amino acid sequence shown in SEQ ID NO. 2), constructing a plant expression vector, and introducing the plant expression vector into wild tobacco for constitutive expression. Compared with wild spCEMA, the obtained spCEMA shows that the resistance of ErBD transgenic tobacco to plant pathogenic bacteria is obviously improved, and the molecular targeting technology is used for directionally improving the existing antibacterial peptide, so that the method has great application potential in plant disease-resistant genetic engineering.
Description
Technical Field
The invention relates to a method for improving the resistance of spCEMA to plant pathogenic bacteria, and also relates to a plant expression vector of spCEMA, ErBD. Belongs to the technical field of plant genetic engineering.
Background
In agricultural production, plant diseases, particularly fungal diseases, have been important factors limiting the improvement of crop yield. For a long time, the widely adopted prevention and treatment means are mainly chemical prevention and treatment, but pathogenic bacteria have high mutation speed, so that the pathogenic bacteria can generate drug resistance or drug resistance to various chemical pesticides, and meanwhile, the problems of environmental pollution and ecological balance caused by the pesticides cannot be ignored. Therefore, in order to ensure food safety and reduce the damage of chemical control methods to the environment, the development of efficient and environmentally safe means for controlling plant diseases is urgently needed.
In recent years, genetic engineering has made a preliminary progress in the study of plant disease resistance. Researches show that the antibacterial peptide genes from different sources can be expressed in plants to remarkably enhance the disease resistance of the plants to pathogenic bacteria (Gao et al, 2000; Jaynes et al, 1993; Jha et al, 2009). However, the transgenic plant material obtained by using the antibacterial peptide has poor disease resistance and can not meet the production requirement. In order to obtain efficient plant disease-resistant genes, except that antibacterial peptide genes are continuously screened and cloned in different species, the antibacterial peptide is directionally improved by utilizing a molecular evolution means, and the antibacterial effect of the antibacterial peptide is improved on the basis of the prior art, so that the method is a more effective way. It has been found that different antibacterial peptides have different bacteriostatic mechanisms, including binding to and disrupting fungal cell membranes, crossing cell membranes into cells to inhibit different targets, etc. Therefore, interaction with fungal cell membranes is an important step in the development of bacteriostatic activity of antimicrobial peptides. It is assumed that increasing the enrichment capacity of the antimicrobial peptide on the fungal cell membrane will increase the bacteriostatic effect of the antimicrobial peptide. In fungi, ergosterol is a unique lipid component on the cell membrane that regulates the fluidity and permeability of fungal cell membranes and is a key factor for cell survival (sangarder et al, 2003). CEMA is a cationic antibacterial peptide consisting of 8 amino acids at the N-terminal of ceropinA and the C-terminal of modified melittin, and we laboratory further added signal peptide at its N-terminal to obtain spCEMA (Niu nationality et al, 2002). Introduction of spCEMA into plants can improve plant disease resistance, but the improvement is still limited. In the invention, ergosterol on a fungal cell membrane is taken as a target, an ergosterol binding domain ErBD (Lucca et al, 1998) is added on the existing antibacterial peptide spCEMA by a molecular improvement means, so that the targeting property and the enrichment capacity to plant pathogenic fungi are improved, and the ideal antibacterial effect is achieved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for improving the resistance of spCEMA to phytopathogens, solves the problem that the bacteriostatic effect of the antibacterial peptide in the current plant disease-resistant genetic engineering is not ideal, and improves the targeting and enrichment capacity of the antibacterial peptide on the phytopathogen cell membrane by fusing an ergosterol binding domain ErBD at the C terminal of the spCEMA by a molecular improvement means, thereby further enhancing the resistance of transgenic plants to the phytopathogens.
The invention also provides a plant expression vector of the fusion antibacterial peptide gene and a transgenic tobacco material.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for improving the resistance of spCEMA to plant pathogenic bacteria, which takes ergosterol on the cell membrane of plant pathogenic fungi as a target spot, fuses an ergosterol binding domain ErBD at the C terminal of spCEMA by a molecular improvement means, and obtains a corresponding fused antibacterial peptide gene; and constructing a plant expression vector by using the nucleotide sequence shown by SEQ ID number 1 and the amino acid sequence shown by SEQ ID number 2.
Further, the method comprises the following specific steps: because the molecular weight of the ergosterol binding domains ErBD and spCEMA is small, the two ends of the ergosterol binding domains ErBD and spCEMA are respectively added by adopting an artificial synthesis mode (Changzhou Jiyu Biotech limited)BamHI andEcoRI enzyme cutting sites, fusion antibacterial peptide connected by linker sequence in the middle, and connected to Escherichia coli cloning vector pUC57, named as pUC57-spCEMA:: ErBD. Subsequently, the process of the present invention,BamHI andEcoRI enzyme cuts pUC 57-spCEMA:ErBD, recovers spCEMA:ErBDfragment, and connects with plant expression vector to obtain correct spCEMA:ErBDplant expression vector.
A transgenic tobacco material fused with antibacterial peptide genes utilizes the constructed plant expression vector to be introduced into wild tobacco for constitutive expression after genetic transformation.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention utilizes a molecular improvement means to fuse an ergosterol binding domain ErBD at the C terminal of spCEMA and obtains a new fusion antibacterial peptide gene. Compared with wild antibacterial peptide, the improved antibacterial peptide can more effectively inhibit the germination and growth of plant pathogenic fungi.
2. The invention introduces the fused antibacterial peptide gene into wild tobacco to obtain corresponding transgenic tobacco. The results show that the obtained spCEMA shows a significantly improved resistance to phytopathogens for ErBD transgenic tobacco compared to wild-type spCEMA. The result shows that the molecular modified spCEMA shows that ErBD has good application prospect in plant disease-resistant gene engineering.
3. With the development of transgenic technology, more and more antibacterial peptides have been applied to plant disease-resistant genetic engineering. However, natural antimicrobial peptides only improve resistance of transgenic plants to pathogenic bacteria to a limited extent and do not meet production requirements. Therefore, the key of the plant disease-resistant gene engineering is to obtain high-efficiency and specific disease-resistant genes. According to the invention, the differences of the structure and the composition of plant pathogenic bacteria cells and plant cells are utilized, specific ergosterol on the plant pathogenic fungi cell membrane is taken as a target point, and an ergosterol binding domain ErBD is fused at the C terminal of spCEMA by a directional improvement means to obtain a corresponding fused antibacterial peptide, so that the targeting property and the enrichment capacity of the fused antibacterial peptide on plant pathogenic fungi are improved, the inhibition effect of the antibacterial peptide on the pathogenic fungi is enhanced under the same concentration, and the harm to non-target cells is reduced.
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FIG. 1 spCEMA restriction enzyme digestion verification of ErBD plant expression vector;
in the figure M2000Denotes marker2000 (Bioer Co.) M15Marker15 (Fermentas corporation); lanes 1-5 show different spCEMA:ErBDplant expression vector transformants with restriction enzymesBamHI andEcoresults of RI cleavage.
FIG. 2 spCEMA: ErBD plant expression vector map;
wherein GRP-GusPlus-His6 represents GUS Plus reporter gene, GRP signal peptide is fused at N terminal of the gene, and His6 sequence label is fused at C terminal of the gene; NPTII represents a neomycin phosphotransferase gene, and has kanamycin resistance; CaMV35S: a plant constitutive promoter derived from cauliflower mosaic virus; LB: the T-DNA left border; RB: right border of T-DNA.
FIG. 3 GUS staining of transgenic tobacco;
the Transgenic represents the GUS staining result of the Transgenic tobacco; non-transgenic represents GUS staining results of Non-transgenic tobacco.
FIG. 4 screening of expression level of transgenic tobacco;
the abscissa indicates the transformants of the different transgenic tobacco plants, where spCEMA:: ErBD-4 and spCEMA:: ErBD-42 represents the spCEMA:: ErBD transgenic line, spCEMA-44 and spCEMA-46 represent the spCEMA transgenic line, WT indicates the wild-type tobacco plant; the ordinate represents the relative expression levels of the foreign gene in different plants.
FIG. 5 comparison of in vitro disease resistance of transgenic tobacco leaves;
a: comparing the resistance of the transgenic tobacco leaves to verticillium dahliae; b: and (3) comparing the resistance of the transgenic tobacco leaves to the alternaria alternate. WT denotes wild type tobacco leaf; spCEMA refers to spCEMA transgenic tobacco leaf; ErBD transgenic tobacco leaf; water represents Water-treated wild-type leaves.
FIG. 6 comparison of the resistance of total protein extract of transgenic tobacco leaves to Protovora brassicae;
WT represents the total protein extract of wild type tobacco leaf; spCEMA represents extract of total protein from spCEMA transgenic tobacco leaf; ErBD represents the extract of total protein from leaf of tobacco transgenic tobacco; KPB denotes phosphate buffer.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.
The invention provides a method for improving the resistance of spCEMA to phytopathogens, which fuses an ergosterol binding domain ErBD to the C-terminal of spCEMA by using a molecular improvement means, wherein the ergosterol binding domain ErBD has a nucleotide sequence shown as SEQ ID number 1 and an amino acid sequence shown as SEQ ID number 2.
[ example 1 ] acquisition of spCEMA:ErBDGene
Because the molecular weight of the ergosterol binding domains ErBD and spCEMA is small, the two ends of the ergosterol binding domains ErBD and spCEMA are respectively added by adopting an artificial synthesis mode (Changzhou Jiyu Biotech limited)BamHI andEcoRI enzyme cutting site,The middle of the fusion antibacterial peptide is connected by a linker sequence, and 1-165bp shown as a sequence 1 is a spCEMA sequence; 166-189bp is an linker sequence; 190-; 241-243bp is a stop codon sequence, and 1-55aa is spCEMA coding as shown in a sequence 2; 56-63aa is linker code; 64-80 are ErBD codes. And ligated to the E.coli cloning vector pUC57, designated pUC 57-spCEMA:ErBD.
Example 2 construction of spCEMA ErBD plant expression vectors
In order to verify that the molecular improvement of spCEMA is passed, ErBD can further improve the disease resistance of plants, a plant expression vector fused with antibacterial peptide is constructed, and the method comprises the following steps:
the plant expression vector is a basic vector for binary expression obtained by the autonomous modification of pCambia2300 and pBI121 by the biotechnology center of southwest university. The HPT II gene of the vectorXhoReplacing the single enzyme digested product I with NPT II gene (obtained by amplifying designed primer from pBI121 vector, and designing primer with two end bandsXhoThe plant expression vector contains 1 set of 2 × plant expression elements of the CaMV35S promoter for controlling NPTII genes, 1 set of plant expression elements of the CaMV35S promoter for controlling a reporter gene GRP-GusPlus-His6 and a set of plant expression elements of the CaMV35S promoter for controlling a target gene, and can realize double-marker screening of Kan and GUS activities.A foreign gene spCEMA:ErCEMA: (ErBD) can be inserted into a Multiple cloning site (Multiple cloning site, MCS), and the over-expression of the spCEMA:: ErBD can be realized (figure 2).
Culture medium for tobacco genetic transformation
Co-culture medium (ph 5.4): MS inorganic + B5 organic +30g/L sucrose +2.0 mg/L6-BA (6-benzylaminopurine) +0.1mg/L NAA (indoleacetic acid) +100 μ M AS (acetosyringone) +2g/L Gelrite;
screening medium (ph 5.8): MS inorganic + B5 organic +30g/L sucrose +2.0 mg/L6-BA (6-benzylaminopurine) +0.1mg/L NAA (indoleacetic acid) +400mg/L Cef (cefotaxime sodium) +100 mg/L Km (kanamycin) +2 g/LGelrite;
rooting medium (ph 5.8): MS inorganic + B5 organic +30g/L sucrose +200mg/L Cef (cefotaxime sodium) +2g/L Gelrite.
Example 3 genetic transformation of tobacco
⑴ preparation of Agrobacterium-infected liquid for transformation
Single colonies of Agrobacterium tumefaciens containing the fused antimicrobial peptide gene were picked up and inoculated into 20mL of YEB (5 g/L sucrose, 1g/L yeast extract, 10g/L tryptone, 0.5g/L MgSO)4·7H2O, pH7.0; 50mg/L Km (kanamycin), 125mg/L Sm (streptomycin)), cultured overnight at 28 ℃ and 200rpm, and then the bacterial suspension was inoculated into 20mL of antibiotic-free liquid YEB at a ratio of 5%, cultured at 28 ℃ and 200rpm until the OD600 was about 0.8 to 1.2. Collecting bacterial liquid, centrifuging, removing supernatant, re-suspending the bacteria by using MSB liquid with the same volume, collecting all the bacterial liquid into a sterile triangular flask, and placing the sterile triangular flask on a shaking table for shake culture for 1h to prepare the agrobacterium tumefaciens staining solution.
⑵ genetic transformation of tobacco
With reference to the method of Horsch et al (Science, A simple and general method for transferringgenes intro plants, 1985, 227: 1229-1231), the young leaves of the sterilized seedlings were used as explants for genetic transformation using Agrobacterium tumefaciens mediated method.
The specific operation is as follows: sterilizing tobacco seeds with 1% sodium hypochlorite solution for 10min, washing with sterile tap water for 5-6 times, culturing for solid germination at 25 deg.C under 16h illumination/8 h dark photoperiod, and taking young leaf as explant for agrobacterium-mediated genetic transformation when sterile seedling grows to 5 leaves. After the explants are impregnated with the agrobacterium impregnation liquid for 30min, the bacterial liquid is removed, the excess bacterial liquid on the surfaces of the explants is absorbed by sterile absorbent paper, and then the explants are inoculated into a co-culture medium paved with a layer of sterile filter paper and are subjected to dark co-culture at 25 ℃ for 2 d. After the co-culture is completed, the explants are inoculated into a screening culture medium for differentiation culture, and the differential culture is carried out at 25 ℃ for 16h light/8 h dark photoperiod culture, and the subculture is carried out once every 2 weeks. After the Km resistant sprouts are generated, the sprouts are cut off and inoculated into a rooting culture medium to obtain Km resistant regeneration plants. And (5) when the roots grow to 3-5cm, performing GUS (glucuronidase) staining confirmation on the regenerated plants, and transplanting the positive plants into a greenhouse to grow into seedlings.
GUS staining fluid formula
500mg/L X-Gluc (X-Gluc dissolved in DMSO at a concentration of 12.5 mg/mL), Na2EDTA（0.01 mol/L），K3Fe(CN)6（0.1mol/L），K4Fe (CN)6(0.1 mol/L), Triton X-1001% (v/v), PBS buffer (0.14 mol/L pH 7).
Example 4 GUS staining of transgenic tobacco
Young leaves of regenerated tobacco plants were sampled with a punch and subjected to GUS histochemical staining in a 37 ℃ incubator. After 3 hours the stained sample was destained with 75% alcohol and photographed under a stereoscope. The results showed that the gene of interest had been successfully transferred into wild type tobacco (FIG. 3).
Example 5 expression level detection of transgenic tobacco
In order to detect the expression condition of exogenous genes in transgenic tobacco, RNA is extracted from each transformant plant in a greenhouse and is reversely transcribed into cDNA, RT-PCR is utilized to detect the expression quantity, and corresponding data analysis is carried out based on Bio-Rad CFX Manager and Excel. The primers used were (P1: 5'-tgcttccttttcttggttc-3'; P2: 5'-caacttcaaagctggcaat-3'). RT-PCR reaction system: 5.0. mu.L univeral SYBR Green Supermix (Bio-RAD), 4.0. mu.L cDNA, 0.5. mu. L P1, 0.5. mu. L P2, in a total volume of 10. mu.L. RT-PCR reaction parameters: 95 deg.C (3 min); 40 cycles: 94 deg.C (10 sec), 56 deg.C (30 sec), 72 deg.C (30 sec). The results are shown in FIG. 4, and 2 spCEMA strains of ErBD and spCEMA transgenic tobacco with higher expression level are obtained by screening through RT-PCR expression level detection analysis.
In order to compare the disease resistance difference of transgenic tobacco of spCEMA, ErBD and the wild-type antibacterial peptide spCEMA at the same level, the plants of spCEMA, ErBD-4 and spCEMA-44 with the expression level as consistent as possible are selected for disease resistance identification analysis.
Example 6 comparison of in vitro disease resistance of transgenic tobacco leaves
First, the resistance of transgenic tobacco leaves to verticillium dahliae was examined. Slight wound was created on tobacco excised leaf with a 1ml syringe without needle, followed by pipetting 10 mul concentration with pipette gun7spore/mL spore suspension of Verticillium dahliae of the falling leaf type was injected into the wound and the petioles were wrapped with wet absorbent paper. The treated leaves were placed in a plastic basket and covered with a transparent film to maintain humidity, and cultured in a thermostat at 28 ℃ for 15 days to observe the onset of disease. The result is shown in fig. 5A, the wild type tobacco leaves inoculated with pathogenic bacteria have serious disease, the leaves at the inoculated position have obvious browning phenomenon and have a large amount of fungal hyphae, while the wild type tobacco leaves inoculated with water have no diseases, thus eliminating the possibility of leaf lesion caused by mechanical damage; wild spCEMA transgenic tobacco has slight pathological symptoms, leaves at the inoculated part begin to brown, and a small amount of verticillium dahliae hyphae can be observed; compared with wild spCEMA, the ErBD transgenic tobacco leaves have no obvious lesion and hyphae found at the inoculated part. The fusion antibacterial peptide is shown to have stronger resistance to verticillium dahliae.
Meanwhile, resistance identification of alternaria alternate is also carried out. Inoculating the third leaf of healthy tobacco in greenhouse by leaf spraying method to obtain the third leaf with the inoculation concentration of 107The spore/mL of the alternaria alternate spore suspension is wrapped with wet absorbent paper at the petiole. The treated leaves were placed in a plastic basket and covered with a transparent film to maintain humidity, and cultured in a thermostat at 28 ℃ for 15 days to observe the onset of disease. The results are shown in fig. 5B, the inoculated wild type tobacco leaves are seriously ill, and have obvious withering symptoms; wild-type spCEMA transgenic tobacco is slightly diseased, and the edges of the leaves begin to discolor and wither; in contrast to the wild-type spCEMA, no obvious lesions were found in ErBD transgenic tobacco leaves. The fusion antibacterial peptide is shown to have stronger resistance to alternaria alternate.
Example 7 comparison of resistance of Total protein extracts of transgenic tobacco plants to pathogenic bacteria
In order to more intuitively compare the resistance differences between ErBD and wild-type spCEMA transgenic tobacco, in vitro bacteriostatic experiments were performed using 0.03M PBS buffer (pH 5.8) to extract total plant protein. The Bradford method measures the content of total protein in each sample, unifies the concentration of the total protein of each plant to 1mg/ml by using a protein extract solution, and then compares the bacteriostatic effects on the bacterial spores of the alternaria alternata.
A layer of filter paper with proper size is laid in a sterilized 90mm culture dish, sterilized water is added for wetting, then two toothpicks are placed on the filter paper, and a sterilized glass slide is placed on the filter paper. 6 mul of tobacco protein extracting solution (1 mg/ml) and 1 mul of rape black spot spore resuspension (10)7Pieces/ml) and 3 mul PDB were dropped in the middle of the slide glass, and after co-cultivation for 10h at 26 ℃, electron microscopy was performed. As a result, it was found (fig. 6): the rape black spot spores can normally germinate and grow in a PBS buffer solution and a wild type tobacco total protein extracting solution; in the wild spCEMA transgenic tobacco total protein extract, most spores can normally germinate, but the growth of hyphae is inhibited; the fusion antibacterial peptide transgenic tobacco total protein extract can obviously inhibit the germination of the bacterial spores. The results show that the addition of the ergosterol binding domain can further improve the bacteriostatic effect of the spCEMA on the bacterial spores of the alternaria alternata.
SEQUENCE LISTING
<110> university of southwest
<120> a method for improving resistance of spCEMA to phytopathogens and transgenic tobacco material
<160>2
<170>PatentIn version 3.5
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<212>DNA
<213> Artificial sequence
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<223> nucleotide sequence shown as SEQ ID NO.1
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atggagaaga agtctcttgc tggcttgtgc ttccttttct tggttctttt tgttgctcaa 60
gaaattgtgg tgactgaagc taagtggaag ttgttcaaga agatcggtat cggtgctgtt 120
ttgaaggttt tgactactgg attgccagct ttgaagttga ccaagggtgg ttctggtggt 180
ggttctggtt ttaagttgag agctaagatt aaggttagat tgagagctaa gattaagttg 240
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MEKKSLAGLC FLFLVLFVAQ EIVVTEAKWK LFKKIGIGAV LKVLTTGLPA LKLTKGGSGG 60
GSGFKLRAKI KVRLRAKIKL 80
Claims (2)
1. A method for improving the resistance of spCEMA to plant pathogenic bacteria is characterized in that ergosterol on the cell membrane of plant pathogenic fungi is taken as a target point, an ergosterol binding domain ErBD is fused at the C terminal of spCEMA by a molecular improvement means, and a corresponding fused antibacterial peptide gene is obtained; constructing a plant expression vector by using a nucleotide sequence shown as SEQ ID number 1 and an amino acid sequence shown as SEQ ID number 2;
the method comprises the following specific steps: because the molecular weight of the ergosterol binding domains ErBD and spCEMA is small, the two ends are obtained by adopting an artificial synthesis mode and are respectively addedBamHI andEcoRI enzyme cutting site, fusion antibacterial peptide connected by linker sequence in the middle, and connected to Escherichia coli cloning vector pUC57, named pUC 57-spCEMA:ErBD, and then,BamHI andEcoRI digested pUC 57-spCEMA:ErBD, recovered spCEMA:ErBDfragment, and ligated with plant expression vector to obtain correct transformant.
2. A method for culturing tobacco resistant to fungal pathogens, characterized in that the plant expression vector containing spCEMA:ErBDconstructed in claim 1 is introduced into wild tobacco for constitutive expression; the pathogenic bacteria are verticillium dahliae, alternaria alternata or alternaria alternata.
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| 《Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1》;De Lucca等;《CANADIAN JOURNAL OF MICROBIOLOGY》;19980630;第44卷(第6期);参见对比文件2摘要、第515页左栏第1-3段、第516页左栏第2段、第519页左栏第5-6段 * |
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