CN117757802A - Vitis spinosa VdERF054 gene and encoding protein and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a VdERF054 gene of grape, and a coding protein and application thereof. The VdERF054 gene of the spiny grape is located on chromosome 12 of the spiny grape and distributed in 5138849-5140246 area. The amino acid sequence of the protein coded by the gene is shown as SEQ ID No. 2. And the application of the VdERF054 gene of the grape in improving the resistance of plants to pathogenic bacteria after silencing. The VdERF054 gene provided by the invention can improve the resistance of Kyoho grape to grape anthracnose after silencing, and provides a theoretical basis for grape anthracnose resistance breeding.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a VdERF054 gene of grape, and a coding protein and application thereof.
Background
Grape (grape vinifera l.) is one of the oldest tree species in the world, cultivated in the western part of asia, and distributed in the northern hemisphere in a concentrated manner about 95% of the grapes in the world, which is one of the most widely planted fruits in the world. Anthracnose (Anthracnose) is caused by infection of fungus of the genus Cephalosporium of the subdivision Deuteromycotina, and is a worldwide important disease on grapes, and seriously affects the yield and quality of the grapes. Anthracnose of grape mainly occurs on the fruit cob, and can also attack the parts of leaf, young shoot, tendril, fruit stem and the like, but the symptoms are not obvious as those on the fruit and the cob.
At present, in grape cultivation production, a bactericide is mainly used for controlling anthracnose germs, but the use of the bactericide is harmful to the environment and health, and the drug resistance of pathogenic germs can be enhanced. Due to the adverse effects of using bactericides to control the spread of anthracnose, there is an increasing interest in breeding anthracnose resistant fresh grapes by hybridization. Early studies showed that the defense response mechanism of grape in response to anthracnose infection has only few reported functions of disease-resistant genes, and especially fewer reports on the interaction network of grape plants and anthracnose, the separation of key response genes and the functional analysis.
Genes are vectors of genetic information and can determine the traits and functions of organisms. However, sometimes the expression of certain genes can lead to negative effects, such as over-expression may trigger disease or abnormal growth. To avoid this adverse effect, organisms evolved a mechanism of gene silencing, the vigs gene silencing principle. The invention provides basis for enriching grape anthracnose stress resistance theory and grape anthracnose resistance breeding by analyzing the functional characteristics and action mechanism of the transcription factors involved in anthracnose stress response.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the thorn grape VdERF054 gene and the coded protein thereof, and the gene can improve the resistance of grape fruits to anthracnose after silencing.
The invention is realized in the following way:
the invention firstly provides a VdERF054 gene of the spiny grape, which is positioned on a 12 # chromosome of the spiny grape and distributed in a 5138849-5140246 area.
Specifically, the nucleotide sequence of the VdERF054 gene of the thorn grape is shown as SEQ ID No. 1.
The invention also provides a protein coded by the gene VdERF054 of the Vitis Viniferae, the amino acid sequence of which is shown as SEQ ID NO.2, contains an AP2 structural domain and has the highest homology with European Vitis Viniferae.
Finally, the invention provides application of the VdERF054 gene of the grape in improving the resistance of plants to pathogenic bacteria after silencing. The expression level of the VdERF054 gene is down-regulated under the stress of pathogenic bacteria, and the grape after the VdERF054 is silenced enhances the resistance to the pathogenic bacteria.
Further, the pathogenic bacteria are pathogenic anthrax bacteria.
Further, the plant comprises grape.
Further, vdERF054 was used to silence the Vitis Viniferae VdERF054 gene by constructing silencing vector TRV.
Still further, the silencing vector is pTRV2 and pTRV2 carrying the gene of interest VdERF054.
The invention has the following advantages:
the invention provides a thorn grape VdERF054 gene, the nucleic acid sequence of which is shown as SEQ ID No.1, the full length of which is 1398bp, the encoded 466 amino acids of which the amino acid sequence is shown as SEQ ID No.2, contains an AP2 structural domain and is highly homologous with European grape, and the encoded protein is positioned on the cell nucleus.
According to the invention, after the grape fruits are infected by the anthracnose, the expression condition of the VdERF054 in the peel is analyzed, and experiments prove that the VdERF054 expression level is regulated downwards in the later period along with the infection, so that the VdERF054 can respond to the anthracnose infection of the grape.
According to the invention, through cloning VdERF054 sequences, a silencing vector TRV:VdERF 054 is constructed, and after grape anthracnose is inoculated to transformed Kyoho fruits, compared with TRV:00 (control), the onset symptoms are lighter. It was verified that VdERF054 silencing enhances resistance of the megalobum grape to grape anthracnose. Therefore, after the VdERF054 gene provided by the invention is silenced, the resistance of the Kyoho grape to grape anthracnose can be improved, and a theoretical basis is provided for grape anthracnose resistance breeding.
Drawings
The invention will be further described with reference to examples of embodiments with reference to the accompanying drawings.
FIG. 1 is a cluster analysis of homologous protein sequences of Vitis vinifera and part of the species VdERF054, wherein red font represents Vitis vinifera (Vit is davidii) VdERF054.
FIG. 2 is a schematic diagram of the construction of VdERF054 gene onto pCambia2300-GFP and pTRV2-GATEWAY vector.
FIG. 3 is a graph of analysis of the VdERF054 protein domain of Vitis vinifera.
FIG. 4 is a diagram showing the VdERF054 chromosome localization of Vitis vinifera.
FIG. 5 is a graph showing the analysis of the induced expression level of VdERF054 in response to grape anthracnose.
FIG. 6 is a chart of VdERF054 subcellular localization analysis (25 μm scale) and yeast transcriptional activation activity analysis.
FIG. 7 is a graph showing analysis of the RT-qPCR detection results of Kyoho grape after interference and resistance to anthrax; a, RT-qPCR is used for detecting the interference efficiency of the grape; b is the phenotype after virus-induced gene silencing VdERF054 Kyoho fruit is inoculated with anthrax bacteria for 5d (left is control, right is silencing VdERF 054), wherein white part is inoculated bacterial mass, and red coiled part is lesion.
Detailed Description
The present invention will now be described in more detail by way of examples with reference to the accompanying drawings, which are not intended to limit the invention thereto, but are illustrative only.
The test methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The species and plasmids used in the examples:
the overexpression vector pCambia2300-GFP used in the test interferes with the vector pTRV2-GATEWAY and the yeast auto-excitation vector PGBKT7; the infection agrobacterium used in the tobacco epidermic cell localization analysis and virus induction gene silencing experiment is GV3101 (p19+pSoup) strain; coli Top10 competent cells were used and purchased from the company TianGen.
The main reagents used in the examples include reverse transcription kit, RT-qPCR supplmix (Transgen Co.), plasmid extraction kit, plant RNA extraction kit, gel recovery kit, LA high-fidelity enzyme, sucrose, MS powder, tris, agar powder, CTAB,75% ethanol, absolute ethanol.
Medium during the induction experiments in the examples: 1/2MS liquid medium.
EXAMPLE 1EFR054 CDS sequence cloning and vector construction
1. Cloning of the full Length and specific fragments of ERF054 in the Vitis Viniferae cDNA library Using the following primers, respectively
SEQ ID No.3：
p2300-VdERF054-BamHI-F:
tcggtacccggggatccATGGATGCAGCTAAGGACGGTG
SEQ ID No.4：
p2300-VdERF054-SalI-R:
gctcaccatggtgtcgacCTTTTGGAGGGATCAAGATCAAGAT
SEQ ID No.5：
Attb1-VdERF054:
ggggacaagtttgtacaaaaaagcaggcttc GAACCAGCCTCAACAAGCTC
SEQ ID No.6：
Attb2-VdERF054:
ggggaccactttgtacaagaaagctgggtcAAACCGACTCTGGTGGTGAC
Note that: the lower case sequence is the adapter primer (vector sequence) and the upper case is the gene sequence.
2. Construction of vectors
The homologous recombination reaction (see FIG. 2) was performed with reference to the seamless cloning kit of Bomeide company and Yingfei Jiegui company, the gel recovered product obtained above was mixed with linearization vectors (pCambia 2300-GFP and pTRV2 vector), and the reaction solution was added at the same time, the reaction system was 10. Mu.L/1. Mu.L, reacted at 37℃C/250℃for 60min, and ice-water bath for 15min, followed by a subsequent reaction, and the sequence was detected correctly by PCR verification and sequencing for the subsequent experiments.
Example 2VdERF054 bioinformatics analysis
The amino acid sequences of VdERF054 were placed on the BLAST website for sequence alignment and homologous sequences from different species were downloaded for use in constructing phylogenetic trees (fig. 1). Amino acid sequence similarity of VdERF054 was performed using MEGA6 for multiple comparisons. The VdERF054 gene was located on chromosome 12 of vitis amurensis (fig. 4), and distributed in 5138849-5140246 region. The construction of the phylogenetic tree is carried out by using a maximum likelihood method (NJ). The Clustw program was used to align with European grape, arabidopsis, oryza sativa, tobacco, tomato, and soybean using the VdERF054 amino acid sequence. The result is shown in FIG. 3, vdERF054 full length 1498bp, encoding 465 amino acids.
EXAMPLE 3 analysis of expression Pattern of VdERF054 Gene
Inoculating anthrax (colletotrichum gloeosporioides) to the grape by adopting a needling method, and collecting grape peel after the inoculation of the anthrax as a test material. RT-qPCR was performed and the expression pattern of VdERF054 gene was analyzed. As shown in FIG. 5, the expression levels of VdERF054 in the peel of 0d, 1d, 2d, 4d and 6d after the grape anthracnose inoculation of the grape became the lowest value at the 6d as the infection proceeded. It was shown that VdERF054 could induce expression in response to grape anthracnose.
Example 4 tobacco subcellular localization and transcriptional self-activation analysis
The recombinant plasmid with correct sequence is transferred into agrobacterium GV3101 by using an electric excitation method, and the correct bacterial liquid is amplified and cultured in 10mL LB liquid medium containing the same antibiotics. Centrifugation at 5000rpm for 5min, the supernatant was discarded, the cells were resuspended in a heavy suspension (MES 2.130g/L+MgCl 22.03 g/L+sucrose 20 g/L) and washed 3 times. The resuspended bacteria solution was diluted to OD600 = 0.4, then AS (200 mmol.L-1) was added and the mixture was allowed to stand at room temperature for 3h to activate Agrobacterium. The suspension was injected into the back of healthy leaf of Nicotiana benthamiana with a 1mL syringe without needle, cultured in a light incubator for 72 hours, and the distribution of GFP fusion protein in the leaf of Nicotiana was observed under a laser confocal microscope (Lycra TCSSP 8), and the pictures were saved. The results are shown in FIG. 6, which demonstrate that VdERF054-GFP protein is localized on the nucleus.
To verify whether VdERF054 has transcriptional activation activity, the present invention uses a yeast system for transcriptional activation activity. The CDS of full length and five fragments of different lengths of VdERF054 was constructed on pGBKT7 vector, transformed into yeast strain Y2H Gold and grown on tryptophan-deleted medium, and the full length of VdERF054 and whether those fragments have the ability to activate the expression of reporter genes ADE2 and HIS3 were examined, with empty pGBKT7 as negative control (binding region for GAL4 expression alone). The results showed that fragments 2 and 4 had no self-activating activity, fragments 1,3,5 were inhibited from self-activating activity AT 10mM 3-AT, while VdERF054 was inhibited from self-activating activity AT 35mM 3-AT over its full length.
Example 5 resistance analysis of Virus-induced Gene silencing VdERF054 on grape and anthracnose
The gene silencing used in the invention is that a tobacco mosaic virus (TRV) mediated VIGS is adopted, namely, a specific segment of a gene is designed, and VdERF054 cross 3-UTR specific part segment is finally connected to pTRV2 vector by using a Gateway vector construction method. And (3) through agrobacterium transformation, selecting monoclonal for verification and shaking. The final re-suspended agrobacterium concentration was adjusted to around od600=1, and then empty pTRV2 and pTRV2 carrying the target gene VdERF054 were combined with pTRV1 at 1:1, mixing evenly at 28 ℃,80-100rpm, mixing evenly for 3-4h. The color-changed Kyoho grape fruits were then soaked in a separate mixture of pTRV1 and pTRV2-ERF054 for 10 minutes under vacuum. Empty pTRV2 and pTRV1 vectors co-infiltrated as a negative control experiment. The grape fruit surface was wiped with clean absorbent paper to remove any residual bacterial liquid, incubated at 26℃and 60% relative humidity in the dark for 24h, and then incubated at 26℃for 3d at 100. Mu. Mol m-2s-1, 16/8h (light/dark) for subsequent testing. Inoculating a dish long spore-shaped dish spore block with the diameter of 5mm to a needle-punched part of a fruit by adopting a needle-punching inoculation method, placing the fruit in a constant-temperature incubator at 25 ℃ for culturing and keeping humidity, observing the disease symptoms of the grape fruit and the size of lesions on the fruit after 5 days, and evaluating the incidence degree of anthracnose on the grape.
To gain insight into the tolerance of VdEFRO54 to pathogenic bacteria in grapes, gene silencing of VdEFRO54 by virus induction was performed in the post-transfer megalobum grape fruits. RT-qPCR results showed that the expression level of VdFERO54 was significantly reduced in the silenced plants (TRV:: EFRO 54) (FIG. 7A); the results after 5d inoculation of anthracnose on the silenced and control grape fruits showed that the silenced fruits with TRV:: EFRO54 had less visible lesions (B in FIG. 7) than the control (TRV:: 00), indicating that silencing TRV:: EFRO54 enhances resistance of grape fruits to anthracnose.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Claims (8)
1. A VdERF054 gene of grape, characterized in that: is located on chromosome 12 of Vitis Viniferae and distributed in 5138849-5140246 region.
2. The VdERF054 gene of grape according to claim 1, wherein: the nucleotide sequence of the thorn grape VdERF054 gene is shown as SEQ ID No. 1.
3. The protein encoded by the VdERF054 gene of grape according to claim 1 or 2, wherein: the amino acid sequence is shown as SEQ ID NO. 2.
4. Use of the VdERF054 gene of vitis amurensis as claimed in claim 1 or 2 for increasing resistance of plants to pathogenic bacteria after silencing.
5. The use according to claim 4, characterized in that: the pathogenic bacteria are pathogenic anthrax bacteria.
6. The use according to claim 4, characterized in that: the plant comprises grape.
7. The use according to claim 4, characterized in that: vdERF054 was used to silence the Vitis vinifera VdERF054 gene by constructing silencing vector TRV.
8. The use according to claim 7, characterized in that: the silencing vector is pTRV2 and carries a target gene VdERF054.
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