CN117843745A - Application of PpSWEET2a gene in regulation and control of gummosis resistance of peach - Google Patents
Application of PpSWEET2a gene in regulation and control of gummosis resistance of peach Download PDFInfo
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Abstract
The invention particularly relates to application of a PpSWEET2a gene in regulating and controlling gummosis resistance of peaches. The PpSWEET2a gene is a nucleotide sequence capable of encoding a protein of (a) or (b) below: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2; (b) The protein derived from (a) which has the same enzymatic activity and has the amino acid sequence in (a) substituted, deleted or added with one or more amino acid sequences. According to the invention, through agrobacterium-mediated transient overexpression, the gene is overexpressed in peach seedlings, and the resistance of leaves to gummosis of peach is obviously enhanced; the VI GS is used for silencing the target gene in peach seedlings, and the resistance is reduced, so that the important effect of the gene in peach gummosis resistance is proved.
Description
Technical Field
The invention relates to the field of methods for improving disease resistance of peaches, in particular to application of a PpSWEET2a gene in regulation and control of gummosis resistance of peaches.
Background
Peach gummosis has wide distribution range, serious disease conditions in Yangtze river basin and south China, seriously damages the cortex structure of the tree body, prevents nutrient substances from being transported, causes the weakening of tree vigor and even the death of branches, and influences the production life, the fruit yield and the quality of peach trees. At present, no resistant material with high resistance to gummosis of peach is found, and analysis of resistance genes of peach trees is a problem to be solved in peach production.
Disclosure of Invention
The invention screens out a gene PpSWEET2a responding to peach gummosis Lasiodiplodia theobromae strain through transcriptome data, and discovers that the expression level of the gene in different sensitive peach varieties has positive correlation with the resistance of peach gummosis. Further through agrobacterium-mediated transient overexpression, the gene is overexpressed in peach seedlings, and the resistance of leaves to gummosis of peach is obviously enhanced; the VI GS is used for silencing the target gene in peach seedlings, and the resistance is reduced, so that the important effect of the gene in peach gummosis resistance is proved.
One of the purposes of the present invention is the protection of the use of the ppwe 2a gene in the regulation of gummosis resistance in peaches, said ppwe 2a gene being a nucleotide sequence capable of encoding a protein of (a) or (b) below: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2; (b) The protein derived from (a) which has the same enzymatic activity and has the amino acid sequence in (a) substituted, deleted or added with one or more amino acid sequences.
Further, the nucleotide sequence of the PpSWEET2a gene is selected from any one of the following (a) or (b): (a) the nucleotide sequence is shown in SEQ ID NO:1, a DNA sequence shown in seq id no; (b) A nucleotide sequence which has 90% or more homology with the DNA sequence of (a) and which encodes the protein of claim 1.
The second purpose of the invention is to protect the application of the primer for detecting the PpSWEET2a gene in screening peach gummosis resistant plants.
The invention also aims to protect the application of the expression vector containing the PpSWEET2a gene and engineering bacteria in improving the peach gummosis resistance of peaches.
Further, the engineering bacteria include, but are not limited to, E.coli and Agrobacterium.
The fourth object of the invention is to protect a method for improving peach gummosis resistance, wherein the method is to overexpress the PpSWEET2a gene.
Further, the method comprises the steps of: an over-expression vector containing the PpSWEET2a gene is constructed, transformed into agrobacterium, and then injected into peach seedling leaves.
Further, the vector used in the construction of the overexpression vector is pSAK277 vector, and the primers are as follows: pp SWEET2a-OE-F: tccaaagaattcaaaaagcttATGTTGTCAACTGGGTTGTCTTCTG; ppSWEET2a-OE-R: gactctagaagtactctcgagTCACACATATGAAACTATCAATG GT.
The invention determines the application of the PpSWEET2a gene in regulating and controlling the disease resistance of peach trees in the process of responding to gummosis bacteria infection of the peach trees, and has important significance for improving the resistance of the peach trees to gummosis bacteria, obviously reducing the incidence of gummosis and reducing the economic loss of gummosis of the peach trees to industry by a transgenic means.
Drawings
FIG. 1 is a phylogenetic tree and amino acid sequence alignment analysis of PpSWEET2 a;
FIG. 2 is a PpSWEET2a promoter cis-acting element analysis;
FIG. 3 is an electrophoresis photograph of the full length of the PpSWEET2a gene;
FIG. 4 is subcellular localization of PpSWEET2 a;
FIG. 5 shows the relative expression levels of peach plants overexpressing the PpSWEET2a gene, and the disease symptoms and lesion areas after inoculation with gummosis bacteria;
FIG. 6 shows the silencing efficiency of peach plants silencing the PpSWEET2a gene, and the disease symptoms and lesion areas after inoculation with gummosis bacteria;
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
Key experimental materials:
vector pK7GWR2: the professor task group was presented by the university of agriculture in China Wang Pengwei, and related published articles are as follows: gong et al, red light-induced citrus fruit colouration is attributable to increased carotenoid metabolism regulated by FcrNAC, journal of Experimental Botany,2021,72:6274-6290;
pCaRNA1&2, pCaRNA3 vector: the university of hainan Cui Gongguang teaches that the subject group gives away, and related published articles are as follows: cui et al An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene, plant Biotechnology Journal,2017,15:344-356 and Zhang et al Two interacting ethylene response factors negatively regulate peach resistance to Lasiodiplodia theobromae, plant Physiology,2023,192:3134-3151;
PNRSV-PpPDS vector: prepared and stored by the laboratory, related published articles see: zhang et al, two interacting ethylene response factors negatively regulate peach resistance to Lasiodiplodia theobromae, plant Physiology,2023,192:3134-3151 and Pan Jiajia, et al, optimization and validation of the plum necrosis ringspot virus-induced peach PpPDS gene silencing system, gardening journal, 2023, 50:1587-1600;
the pBlunt-Zero vector was purchased from TransGen company;
the construction method of the pBlunt-Zero-PpSWEET2a plasmid is as follows: the phloem of the 'spring snow' branch, which is inoculated with JMB-122 in vitro, is used as a material, RNA is extracted after liquid nitrogen grinding, and the RNA is reversely transcribed into cDNA. The sequences were found on GDR and NCBI according to the numbers, the full-length primers of the PpSWEET2a gene were designed by using Primer5, the CDS sequence of PpSWEET2a was cloned by Vazyme high fidelity enzyme using the cDNA as a template, and the PCR product was recovered by using SanPrep column PCR product purification kit to obtain the full-length CDS of PpSWEET2a, which was stored at-20 ℃. The product of interest was ligated into the Blunt-Zero cloning vector using the TransGen cloning kit.
Example 1
(1) Cloning and analysis of the PpSWEET2a Gene
Cloning and obtaining PpSWEET2a CDS full-length sequence from peach cDNA, and analyzing cDNA sequence by bioinformatics shows that the full length of the gene is 708bp, which comprises 235 amino acids, isoelectric point is 9.00, and predicted molecular weight is 26.0kDa. The amino acid multiple sequence alignment by MEGA7 is utilized to construct a phylogenetic tree, and the phylogenetic tree is found to be closest to PbSWEET2a in Arabidopsis thaliana, and the specific reference can be seen in FIG. 1A. In the NCBI database, the PpSWEET2a gene is located on chromosome 4, contains 6 exons, and contains multiple Substrate Recognition Sites (SRS), see fig. 1B.
The PpSWEET2a promoter fragment (2000 bp) was cloned from peach DNA and the promoter cis-regulatory element was analysed using the plant CARE database. The PpSWEET2a promoter contains 9 MYB recognition elements, 2 MYC recognition elements, and various hormone response elements, see fig. 2, which can be bound by MYB, MYC proteins, and other proteins.
Primers were designed at both ends of the ORF according to the full length sequence using Primer5, the Primer sequences were: ppSWEET2a-Full-length-F,5'-CTTCTGTTTGGTGTTGCGTAG-3'; ppSWEET2a-Full-length-R:5'-ATTGAGTATTAGGCTGGT-3' the tissue of phloem of current year branch inoculated with gummosis bacteria is taken as a sample, total RNA is respectively extracted by using an EASY spin Plus plant RNA rapid extraction kit (Aidlab, beijing), the concentration and quality of RNA are detected by using a Nanodrop one (Thermo, USA), and the reverse transcription kit is utilizedRT Reagent Kit with gDNA Eraser(TaKaRa,Dalian,China) instructions, reverse transcription to obtain cDNA, finally using the obtained cDNA as template, using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, nanjing) to amplify the ORF sequence of the protein coding gene in 'Dahongpao' and 'Chun Xue'. The amplified product was subjected to DNA gel recovery kit (Sangon Biotech, shanghai) to recover the desired fragment, and after the positive clone was obtained by ligating the vector with Zero Background pTOPO-Blunt Cloning Kit (Aidlab, beijing), the sequence was obtained by sequencing the DNA fragment in TSINGKE company (TSINGKE, wuhan), and the nucleotide sequence of the gene was found to be identical in different resistant varieties of peach, namely 'spring snow' and 'Dahongpao'.
The specific nucleotide sequence is shown below: ATGTTGTCAACTGGGTTGTCTTCTGTTTATTTAGGTTTCAGTACTGCAGCCGGCATTGCTGGGAATATCTTTGCCTTCGTGTTGTTTGTGTCACCACTGCCAACATTCAAGAGAATCATTAGAAACAAGTCAACAGAACAATTCTCTGGATTGCCTTACATATATGCCTTTTTGAATTGCTTGATATGTCTTTGGTATGGCATGCCTGTAGTGAAGACTGGTATTATATTGGTGGCTACAGTCAATTCATTTGGGGCTGTTTTCCAGTTAGTCTACTTGAGCATTTTCATTACGTACGCTGAAAGAGCAACTAAGCTTAGGATGTTGGGATTATTAGGGGCAGTTGCTGTTGTATTTGCGTTCGTTGTCTTTGTGAGCTTAGGAGTTTTAGAGTATGATGACAGGCAAACGTTCGTTGGATATTTGAGTGTCGCTTCACTTATTTCAATGTTTGCTTCGCCGCTGTTTATCATAAAACTGGTTATCAAAACAAGGAGTGTCGAATTCATGCCATTTAATCTCTCTTTTGCAACTTTCTTGATGAGTCTCTCTTTCTCTGCCTATGGAATATTCAAGGAAGACCCCTTCCTTTATATCCCAAATGGTATTGGAACAATTTTAGGCCTTGTCCAGTTGGCTCTCTACTCCTACTATAGTAAGATATCTGGAGAAGACTCAAGAGAACCATTGATAGTTTCATATGTGTGA (SEQ ID NO: 1);
the amino acid sequence is: MLSTGLSSVYLGFSTAAGIAGNIFAFVLFVSPLPTFKRIIRNKSTEQFSGLPYIYAFLNCLICLWYGMPVVKTGIILVATVNSFGAVFQLVYLSIFITYAERATKLRMLGLLGAVAVVFAFVVFVSLGVLEYDDRQTFVGYLSVASLISMFASPLFIIKLVIKTRSVEFMPFNLSFATFLMSLSFSAYGIFKEDPFLYIPNGIGTILGLVQLALYSYYSKISGEDSREPLIVSYV (SEQ ID NO: 2).
(2) PpSWEET2a protein subcellular localization
Constructing a subcellular localization vector by using a gateway method according to the gene sequence of PpSWEET2a, and firstly performing BP recombination reaction, wherein the primer sequence is PpSWEET2a-attB 1-F5'-aaaaagcaggc tATGTTGTCAACTGGGTTGTCTTCTG-3'; ppSWEET2a-attB 1-R5'-agaaagc tgggtTCACACATATGAAACTATCAATGGT-3'.
After the first round of PCR reaction, the PCR product is used as a template, and a universal primer attB1-F/R and an entry vector PDONR221 are added for carrying out a second round of PCR reaction, wherein the sequence of the primer is attB 1-F5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3'; attB 1-R5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'.
Then constructing the gene sequence constructed on the PDONR221 vector on a subcellular localization vector pK7GWR2 through LR recombination reaction, and finally transferring 1-5 mu L of LR reaction liquid into escherichia coli competent Trans5α through thermal excitation to carry out positive identification on pK7GWR2-PpSWEET2a, wherein the identification primer sequence is RFP-F:5-GCGCCTACAAGACCGACATCAAG-3'; RFP-R:5'-GAACCCTAATTCCCTTATCTGGGAAC-3'.
Positive clones were obtained and sequenced by company, the correctly sequenced monoclonal rocked bacteria were used to extract the recombinant vector plasmid using Omega plasmid miniprep kit, and then the plasmid was freeze-thawed and transferred into Agrobacterium GV3101 competent (indigenous organism, china) for positive detection.
Subcellular localization experiments were performed in tobacco, by first amplifying GV3101 positive clones in LB with antibiotics at 220rpm,28℃to cell concentration OD 600 After centrifugation at 8000rpm for 5 minutes to collect cells, MMA (10 mM MgCl) was used 2 150. Mu.L acetosyringone,10mM 2-morpholinoethanesulfonic acid, pH 5.6) resuspended cells, OD 600 Adjusting to about 1.0, mixing pK7GWR2-PpSWEET2a (RFP mark is contained in pK7GWR2 vector) plasmid and Y-TIP-GFP (tonoplast membrane positioning mark) plasmid in equal volume, standing at room temperature in dark place for 3h, and injecting the Nicotiana benthamiana plant.
By adopting a leaf injection method, a 1mL injector without a needle head is used for carefully pushing bacterial liquid to the 3 uppermost fully-spread functional leaves from the back of the leaves until the whole leaves are water-stained, the leaves are placed in a 28 ℃ artificial climate chamber, the culture condition is that the illumination is 16h and the darkness is 8h, after 2-3 days of injection, part of the injection leaves are taken to observe the fluorescence effect under a laser confocal microscope (Leica), and the result is shown in figure 4, so that the PpSWEET2a protein is positioned in an endoplasmic reticulum.
Example 2
(1) Construction of the overexpression vector
According to the gene sequence of the PpSWEET2a, constructing a pSAK277-PpSWEE T2a overexpression vector by using a homologous recombination method, wherein the enzyme cutting sites are HindIII and XhoI, and the amplification primer is PpSWEET 2a-OE-F:5'-tccaaagaattcaaaaagcttATGTTGTCAACTGGGTTGTCTTCTG-3'; ppSWEET2a-OE-R:5'-gactctagaagtactctcgagTCACACATATGAAACTATCAATGGT-3'; the positive identification primers are: pSAK 277-F5'-GAAGACCAAAGGGCTATTGAGAC-3'; pSAK 277-R5'-GATGTCGCTATAAACCTATTCAGCA-3', the recombinant plasmid was transferred into the competence of Agrobacterium GV3101 for use.
(2) Agrobacterium-mediated transient overexpression of peach seedlings
Peach seedling transient over-expression method is referred to Zhang Dongmei (2021; doctor's article of China university of agriculture), ppSWEET2a over-expression strain is taken for expansion culture, pSAK277 empty control bacteria are simultaneously cultured, 220rpm is carried out, and the culture is carried out at 28 ℃ in 50mL LB (containing spectinomycin and rifampin) liquid culture medium for overnight. Bacterial liquid treatment As described above, OD 600 Adjusting to about 1.0, standing at room temperature in dark for 3h, injecting bacterial liquid from the back of the leaf into the fully-unfolded leaf of the peach seedling with the size of about 4 weeks by using a 1mL disposable medical injector without a needle head until the whole leaf is water-stained, and placing the leaf into a growth chamber for dark culture for 2 days after injection. The empty control group and the PpSWEET2a over-expression group are cultured for 2 days in a dark place, a small amount of injected leaves are ground into powder through liquid nitrogen, RNA is extracted by the method in the embodiment 1, the RNA is reversely transcribed into cDNA, qRT-PCR analysis is carried out on the change of the expression quantity of the PpSWEET2a gene, and the result shows that the expression quantity of the PpSWEET2a in the over-expression leaves is obviously increased as shown in the figure 5B, so that the method can effectively over-express the PpSWEET2a gene in peach leaves.
(3) Identification of resistance to Thermomyces lanuginosus strain L.theobromae of peach seedlings transiently overexpressing PpSWEET2a
The method of reference Zhang Dongmei (2021) uses the injected isolated peach leaves as a material, and uses the peach gummosis bacterial strain L.theobromae to inoculate the leaves, and disease symptoms are observed after 1 day and 2 days of inoculation, and the result is shown in fig. 5A, the leaf spot area of the transient over-expression of PpSWEET2a in peach seedlings is obviously smaller than that of a control EV (no-load control), which indicates that the over-expression of the PpSWEET2a obviously enhances the disease resistance of the peach to the gummosis bacterial strain.
Example 3
(1) VIGS-mediated silencing of peach seedling PpSWEET2a gene
The specific fragment (250 bp) of the PpSWEET2a gene is amplified by designing a primer by taking the pBlunt-Zero-PpSWEET2a plasmid as a template, and the fragment sequence is as follows: GAGAAATCGAAAAAGGTGAGGATGCTGGGATTTCTGTTGGCAGATTTTGGTCTATTTGCAATCATAGTTTTTGGGAGCTTGCAAATGACTGACCTTGTTATGCGCCGGCTGATTGTTGGACTGCTTAGTTGTGTTTCTCTCATATCAATGTTTGCTTCTCCCATGTTTATAATTAACTTGGTGATCCGGACAAAGAGTGTCGAATTTATGCCATTTTATCTCTCCCTTTCTACCTTCCTAATGAGCACGT specific primers were designed and the desired fragment of VIGS-PpSWEET2a was amplified by the method described in example 1, the amplified primer sequences being VIGS-PpSWEET2a-F:5'-aagacctgcttgagatctagaATGTTGTCAACTGGGTTG-3'; VIGS-PpSWEET2a-R:5'-gtgtgcttatctcactctagaTATGAAACTATCAATGGT-3'.
The VIGS vector was linearized by FastDiget Xba I fast cutting enzyme from Thermo company, the homologous recombinant vector pCaRNA3-PpSWEET2a was obtained by ClonExpress IIOne Step Cloning Kit (Vazyme Nanjing), transferred into Agrobacterium GV3101 and checked for use. The peach seedlings were subjected to VIGS transient transformation by the method of reference Cui and Wang (2017). That is, the pCaRNA3-PpSWEET2a and pCaRNA1 will be carried&2 were cultured overnight with MMA (10 mM MgCl) 2 150. Mu.L of Acetostinone, 10mM MES, pH 5.6) to OD 600 About 1.0, uniformly mixing according to the volume ratio of 1:1, standing for 3 hours at room temperature in a dark place, injecting bacterial liquid into fully-unfolded peach seedling leaves of 6-8 leaves of seedling age from the back of the leaves by using a 1mL disposable medical injector with a needle removed, keeping away from light for 2 days after injection is completed, and then transferring into a 22 ℃ growth chamber for normal culture. With pCaRNA3 and pCaRNA1&2 as negative control, PNRSV-PpPDS and pCaRNA1&2, mixing the injected peach seedlings to be positive control. After 4 weeks, a small amount of uninjected functional leaves with fully developed silent peach seedlings were cut, ground into powder by liquid nitrogen, RNA was extracted using the method described in example 1, reverse transcribed into cDNA, and qRT-PCR analysis was performed, and as a result, as shown in fig. 6B, the method was effective in silencing the expression level of the peach PpSWEET2a gene.
(2) Identification of resistance of silencing plants to the Comamia parapsilosis strain L.theobromae
In vitro leaves of the silent peach plants and the control are used as materials, the leaves are inoculated with the peach gummosis bacterial strain L.theobromae, and disease symptoms are observed after 1 and 2 days of inoculation, so that as shown in the results of figures 6A and 6C, the area of the disease spots of the silent PpSWEET2a peach plants is obviously larger than that of the control EV (no-load control), and the resistance to the gummosis bacterial strain is obviously reduced.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
- Use of the PpSWEET2a gene for modulating gummosis resistance in peach, characterized in that the PpSWEET2a gene is a nucleotide sequence capable of encoding a protein of (a) or (b) below:(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2;(b) The protein derived from (a) which has the same enzymatic activity and has the amino acid sequence in (a) substituted, deleted or added with one or more amino acid sequences.
- 2. The use according to claim 1, wherein the nucleotide sequence of the PpSWEET2a gene is selected from any one of the following (a) or (b):(a) The nucleotide sequence is shown in SEQ ID NO:1, a DNA sequence shown in seq id no;(b) A nucleotide sequence which has 90% or more homology with the DNA sequence of (a) and which encodes the protein of claim 1.
- 3. Use of a primer for detecting the PpSWEET2a gene of claim 1 for screening plants against gummosis peach.
- 4. The use of an expression vector containing the ppwe 2a gene of claim 1, engineering bacteria in improving peach gummosis resistance.
- 5. The use according to claim 4, wherein the engineering bacteria include but are not limited to escherichia coli, agrobacterium.
- 6. A method for increasing resistance of peach to gummosis, wherein the method is overexpression of the ppwe 2a gene of claim 1.
- 7. The method according to claim 6, comprising the steps of:an overexpression vector containing the ppwe 2a gene as claimed in claim 1 was constructed and transformed into agrobacterium, after which it was injected into peach leaves.
- 8. The method according to claim 6, wherein the vector used in the construction of the overexpression vector is pSAK277 vector, and the primers are:PpSWEET2a-OE-F：tccaaagaattcaaaaagcttATGTTGTCAACTGGGTTGTCTTCTG；PpSWEET2a-OE-R：gactctagaagtactctcgagTCACACATATGAAACTATCAATGGT。
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