CN116121252B - Nanocomposite for enhancing resistance of apple alternaria leaf spot and application thereof - Google Patents
Nanocomposite for enhancing resistance of apple alternaria leaf spot and application thereof Download PDFInfo
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Abstract
The invention relates to a nano-composite for enhancing resistance of apple alternaria leaf spot and application thereof. According to the invention, a novel 5' tsRNA named mdm-tsRMet is obtained by comparing the expression difference of tsRNA before and after apple inoculation apple alternaria leaf spot ALT 1. mdm-tsRMet up-regulates expression after ALT1 inoculation and cleaves the antipathogenic gene MdSTK1, thereby reducing resistance of apples to alternaria leaf spot. By designing the silencing sequence of mdm-tsRMet, the silencing sequence is further combined with the magnetic nanoparticle to be delivered into apples, and the expression of mdm-tsRMet is silenced and up-regulated, so that ALT1 resistance of apples is enhanced.
Description
Technical Field
The invention relates to the field of biological nano drug loading and delivery, in particular to a nano compound for enhancing resistance of apple alternaria leaf spot and application thereof.
Background
Transfer RNAs (tRNAs) are produced in the nucleus from precursor tRNAs (pre-tRNAs) by transcription of RNA polymerase III, and in addition to classical functions in protein synthesis, tRNA can be cleaved to produce tRNA-derived small RNAs, i.e., tsRNAs. tsRNAs can act as important regulatory factors in plants and are induced under adverse conditions such as biotic stress and abiotic stress of plants, however, there are few related studies on regulation of fungal disease resistance by tsRNAs.
The apple industry is seriously threatened by fungal diseases, and apple alternaria leaf spot is the most main fungal disease of apples, so that not only is the apple leaves endangered to cause blackish brown alternaria and fallen leaves, but also the yield is reduced, the fruits lose commodity value, the loss is huge, and the production safety of the fruits is seriously influenced. The traditional fungal disease prevention and treatment method mainly adopts chemical prevention and treatment, but the method can cause damage to the environment and also can influence the health of human bodies. There is an urgent need for a new method to study fungal diseases and their control.
The appearance of nanotechnology provides a new idea for sustainable treatment of harmful organisms. Nanomaterials as plant vehicles (nanocarriers) can protect loaded biomolecules from the threat of cellular metabolism and degradation, with unique advantages such as large specific surface area and biocompatibility for chemical modification, and have great potential in plant genetic engineering.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a nano-composite for enhancing the resistance of apple alternaria leaf spot and application thereof. According to the invention, a novel 5' tsRNA named mdm-tsRMet is obtained by comparing the expression difference of tsRNA before and after apple inoculation apple alternaria leaf spot Alternaria alternata sp.mali (ALT 1). It was found that mdm-tsRMet up-regulates expression after ALT1 inoculation and cuts the antipathogenic gene Malus domestica Serine/Threonine kinases 1 (MdSTK 1) to reduce apple resistance to alternaria leaf spot. By designing the silencing sequence of mdm-tsRMet, the magnetic nanoparticles (Magnetic nanoparticles) are further combined to deliver into apples, and ALT1 resistance of apples is enhanced.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a nanocomposite for enhancing resistance to apple alternaria leaf spot, characterized by: comprises a magnetic nanoparticle carrier and plasmid DNA;
the plasmid DNA is a recombinant plant expression vector which is a pFGC5941 vector containing artificial miRNA;
the nucleotide sequence of the artificial miRNA is shown as SEQ ID NO. 4.
Based on the scheme, the artificial miRNA comprises a reverse complementary sequence for silencing tsRNA, and the nucleotide sequence of the reverse complementary sequence is shown as SEQ ID NO. 3.
A tsRNA, characterized by: the nucleotide sequence is shown as SEQ ID NO. 1; the tsRNA is used for regulating and controlling apple alternaria leaf spot.
A precursor sequence of the tsRNA, characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.
An engineering bacterium is characterized in that: the engineering bacteria contain the recombinant plant expression vector.
The gene involved in regulation and control of apple alternaria leaf spot is characterized in that the gene is targeted by the tsRNA, and the nucleotide sequence of the gene is shown as SEQ ID NO. 5.
A method of preparing the nanocomposite described above, comprising the steps of:
step 1, modifying the surface of magnetic nano particles by using polyethyleneimine to obtain a nano particle carrier;
and 2, incubating the nanoparticle carrier and plasmid DNA to obtain a nanocomposite.
Based on the scheme, the magnetic nano particles are 10nm Fe 3 O 4 And (3) microspheres.
Based on the above scheme, step 2 specifically includes:
nanoparticle carriers were mixed with plasmid DNA in a mass ratio of 1:1 and incubated at 25 ℃ for 30min.
An application of the nanocomposite, which is characterized in that: the nano-composite and the surfactant are uniformly sprayed on apple leaves after being mixed, and the recombinant plant expression vector in the nano-composite is delivered into apple plants in the mode, so that the resistance of apple susceptibility varieties to alternaria leaf spot is enhanced.
Magnetic nanoparticle-based gene delivery system, 10nm Polyethyleneimine (PEI) -modified Fe is selected 3 O 4 Particles, binding to plasmid DNA containing an artifical miRNA. MNP-DNA complex and a surfactant Silwet L-77 are mixed at a ratio of 5000:1, sprayed on leaves of Jin Guanzu seedlings in a spraying mode, and after three days, the expression levels of mdm-tsRMet and MdSTK1 of apples are detected, and the apples are inoculated for detecting the disease resistance of ALT 1.
The nano-carrier for regulating and controlling the resistance of apple alternaria leaf spot and the application thereof have the beneficial effects that:
the invention provides a novel method for preventing and treating apple alternaria leaf spot, which uses Fe 3 O 4 The magnetic nanoparticle as carrier will contain an artificial miRNPlasmid DNA of A was delivered into apples for silencing mdm-tsRMet in vivo. It has high specific surface area and positive surface charge, can adsorb DNA with negative electricity, and is safe, environment friendly and pollution-free. Different from the traditional gene transfer mode, the magnetic nanoparticles are used for transferring genes into apple cells, so that the method is safe and harmless, has long duration, smoothly achieves the effect of preventing apple alternaria leaf spot, and has great potential for improving the development of apple industry.
Drawings
The invention has the following drawings:
FIG. 1 shows the mdm-tsRMet precursor MdtRNA-Met of the invention CAT Structure diagram: a is the tsRNA content of the golden crown endophyte and the unsplit strain in sRNA sequencing; b is MdtRNA-Met CAT A structural diagram;
FIG. 2 shows the PCR amplified MdtRNA-Met of the invention CAT And MdSTK1 agarose gel electrophoresis pattern: a is MdtRNA-Met CAT Amplifying the band; b is a target gene MdSTK1 amplification band;
FIG. 3 is a cleavage site map of mdm-tsRMet for its target gene MdSTK 1;
FIG. 4 shows that the amount of mdm-tsRMet increases after 12 days of inoculation with ALT, and that the control group is WT, H 2 0；
FIG. 5 is an overexpression of the precursor MdtRNA-Met CAT Effect on ALT1 resistance in the susceptible and resistant varieties corolla and Hanfu: a is the overexpression of MdtRNA-Met CAT After the last 4 days, the expression quantity of the golden crown and the Hanfu MdSTK1 is quantitatively detected by fluorescence, and the pFGC5941 no-load (EV) is reduced relative to the Wild Type (WT); b is the overexpression of MdtRNA-Met CAT Phenotype figures of golden crown and cold enrichment after 12 days of inoculation ALT on the last four days; c is Jin Guanhan rich in overexpressing MdtRNA-Met CAT Counting the area of the lesions after inoculating ALT for 12 days after 4 days;
FIG. 6 is a schematic design of the si-mdm-tsRMet vector: a is a structure diagram of premier 319a and the modified artificial miRNA; b is a schematic diagram of silencing mdm-tsRMet vector mode;
FIG. 7 is a transmission electron microscope view after magnetic nanoparticles bind to DNA: a is a magnetic nanoparticle; b is magnetic nano particles combined with DNA;
FIG. 8 is the effect of foliar application of MNP-DNA complex on apple resistance: a is Northern blot to detect the expression level of mdm-tsRMet in WT, EV, si-mdm-tsRMet and MNP-si-mdm-tsRMet, u6 was used as a control; b is RT-qPCR to detect the expression level of MdSTK1 in WT, EV, si-mdm-tsRMet and MNP-si-mdm-tsRMet after three days of foliar treatment;
FIG. 9 shows the absorption of MNP-si-mdm-tsRMet by apples: the MNP-DNA complex in the apple body can be observed by a transmission electron microscope;
fig. 10 is a graph of resistance to ALT1 of apple leaves after various treatments, specifically expressed on lesion area: a is a phenotype graph of administration of si-mdm-tsRMet and MNP-si-mdm-tsRMet, control group is WT, EV; b is the spot area statistics of WT, EV, si-mdm-tsRMet and MNP-si-mdm-tsRMet 7 days after inoculation.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings. The examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1tsRNA and discovery of target genes thereof
Screening and inoculating tsRNA with a disease-sensitive variety 'golden crown' as a test material, wherein the tsRNA is subjected to forward and backward expression difference of more than 2 times, and is compared with miR Base (http:// www.mirbase.org /) and apple genome (https:// www.rosaceae.org /), so as to obtain a novel tsRNA, the novel tsRNA is named mdm-tsRMet (SEQ ID NO: 1), and the precursor of the novel tsRNA is MdtRNA-Met through apple genome website (https:// www.rosaceae.org /) CAT The sequence is shown as SEQ ID NO. 2, and the target gene is further predicted by psRNAtarget (https:// www.zhaolab.org/psRNAtarget /), and the predicted target gene is serine/threonine protein kinase and is named MdSTK1 (SEQ ID NO. 5).
The mdm-tsRMet precursor tRNA structure is shown in FIG. 1.
EXAMPLE 2 cloning of tRNA and target Gene
The specific method comprises the following steps:
1. total RNA extraction from plants
(1) Rapidly grinding apple 'golden crown' tissue samples in liquid nitrogen;
(2) Adding 990 mu L of preheated CTAB solution and 10 mu L of beta-mercaptoethanol into plant tissues, and carrying out water bath at 65 ℃ for 10min after vortex for 30 s;
(3) 1000 μl CI (chloroform/isoamyl alcohol volume ratio=24:1) was added, and mixed upside down;
(4)4℃12000rpm 10min；
(5) Sucking 800 mu L of supernatant, adding an equal volume of CI, and mixing the mixture upside down;
(6)4℃12000rpm 10min；
(7) The supernatant was aspirated at approximately 650. Mu.L and 1000. Mu.L of isopropanol was added;
(8) Precipitating at-20deg.C for 30min;
(9)4℃12000rpm 10min；
(10) The supernatant was decanted and 1ml of 75% ethanol was added to wash the pellet;
(11)4℃12000rpm 10min；
(12) Pouring out the supernatant, and carrying out 2min at 12000rpm at 4 ℃;
(13) The supernatant was blotted dry and 40. Mu.L of RNase-free H was added 2 O is dissolved and precipitated;
(14) Detecting the integrity by 1% agarose gel electrophoresis, and measuring the absorbance at 260nm by an ultraviolet spectrophotometer to calculate the concentration of RNA;
(15) RNA samples were stored at-80 ℃.
2. Reverse transcription reaction system and step1st Strand cDNA Synthesis Kit,gDNA Purge,E042)
(1) The following reactants were added in order:
mix gently and centrifuge.
(2) Incubation at 42℃for 15-30 min (25℃for 5min followed by 42℃for 15-30 min if the RNA template does not contain poly A structure).
(3) Placed on ice.
Md-S provided on NCBI (https:// www.ncbi.nlm.nih.gov /)TK1 and MdtRNA-Met CAT Primers were designed in sequence.
The primer sequences used for MdSTK1 are as follows:
an upstream primer: 5'-ATGAAAAACATGACTTCCGTTCTTT-3'
A downstream primer: 5'-TCAACGGGCC ACAACATTAG-3'
MdtRNA-Met CAT The primer sequences used were as follows:
an upstream primer: 5'-ATCAGAGTGGCGCAGCG-3'
A downstream primer: 5'-TGGTGTCTGTGCCTGGTTT-3'
PCR reaction System (CW 0682L):
the PCR reaction procedure was as follows:
pre-denaturation at 94℃for 2min;94 ℃ for 30s,61 ℃ for 30s and 72 ℃ for 1min, and 34 cycles are total; finally, the extension is carried out at 72 ℃ for 2min.
And (3) PCR product detection: 1% agarose gel, 0.1% TAE electrophoresis buffer, and 70-110v voltage electrophoresis for about 15min were prepared according to the size of the target fragment, and the PCR product fragment was detected under ultraviolet light for staining, and the result is shown in FIG. 2.
EXAMPLE 3 5' RACE analysis of tsRNA target genes
5' RACE experiments were performed on the target genes predicted from tsRNA.
The specific method comprises the following steps:
(1) Extraction of Jin Guanzong RNA
(2) The total RNA was reverse transcribed using specific primers to obtain cDNA.
(3) Ethanol was added to the cDNA for precipitation for 1 hour, centrifuged at 12000rpm at 4℃and DEPC was dissolved in water to purify the cDNA.
(4) The purified cDNA was subjected to terminal phosphorylase (TdT) treatment.
The system is as follows:
mixing, and treating at 37deg.C for 30min.
(5) After 150. Mu.L of RNase-free water was added, 200. Mu.L of CI was added thereto and mixed uniformly, and centrifuged at 12000rpm at 4℃for 10 minutes.
(6) Adding the supernatant into the equal volume CI, mixing, and centrifuging at 12000rpm at 4deg.C for 10min.
(7) The supernatant was taken and precipitated at-20℃for 1 hour by adding 5. Mu.L of 3M NaOAC (pH 5.2) and 2.5 volumes of absolute ethanol.
(8) Centrifuging at 12000rpm at 4deg.C for 20min;
(9) The supernatant was discarded, the precipitate was washed with 75% pre-chilled ethanol, dried and dissolved in 20. Mu.L sterile water, and stored at-70℃until use.
(10) The TdT treated C-tailed product was used as a template for PCR reactions using 3' anchor primers and specific primers for reverse transcription.
Adaptor：5’-TAATACGACTCACTATAGGGGGGGGGG-3’
F:5’-TAATACGACTCACTATAGGG-3’
PCR reaction system:
8) The PCR products are subjected to the next round of nested PCR reaction, and the primers are the conserved part in the anchor primers and the gene specific primers inside the reverse transcription primers.
MdSTK1-R1:5’-TAGAAGTCGTCCCCCTTGTTGTATTCTGG-3’
MdSTK1-R2:5’-GTCGTCCCCCTTGTTGTATTCTG-3’
The PCR product was recovered by agarose gel and the T-A cloned into Pmd19-T for sequencing.
The RACE sites are shown in figure 3.
Example 4 verification of tsRNA expression level before and after inoculation
And verifying the content change of tsRNA in the infectious disease variety Jin Guanzhong strain ALT 1.
The specific operation is as follows:
1. extracting Jin Guanzong RNA 48h after inoculating ALT1;
2. detection of tsRNA Northern method
Synthesizing 5' -end modified digoxin marked mdm-tsRMet-probe (5'-TCCGCTGCGCCACTCTGAT-3') and U6-probe, adding 20 mug RNA (CTAB extraction) into a 2 XLoading buffer at 95 ℃ for 5min, cooling at 4 ℃, loading on 15% polyacrylamide gel (containing 7M urea), and carrying out 220V electrophoresis in a 1 XTBS buffer for 1h; 220mA in a 1 XTBS buffer is electrified and transferred to a nylon membrane; 1200mJ ultraviolet cross-links for 3mins, prehybridization, hybridization, membrane washing and signal detection are performed by using a digoxin hybridization detection kit (Mylab corporation).
Northen results showed that after inoculation with apple alternaria leaf spot pathogen (ALT 1), mdm-tsRMet levels were up-regulated in the gold crowns, as shown in FIG. 4.
EXAMPLE 5 overexpression of MdtRNA-Met CAT Effects on ALTL resistance in apples
MdtRNA-Met CAT The sequence was constructed on pFGC5941 vector (between NcoI and BamHI cleavage sites), the recombinant plasmid was transformed into GV3101 Agrobacterium strain, transiently transformed into golden crown and Hanfu tissue culture seedlings.
The specific operation is as follows:
1. agrobacterium transformation (Shanghai Weidi organism, CAT#: AC 1001)
(1) Taking a tube of competent cells, and slightly suspending the cells after complete dissolution on ice;
(2) Adding 5 mu L of plant expression vector plasmid, gently mixing, and standing on ice for 5min;
(3) Cooling in liquid nitrogen for 5min;
(4) Heat shock at 37 ℃ for 5min;
(5) Placing on ice for 5min.
(6) Adding 500 μLYEP culture medium, and shaking culturing at 28deg.C and 180rpm for 2-3 hr;
(7) Centrifuge at 10000rpm for 1min at room temperature, remove about 400. Mu.L of supernatant and suspend cells with the remaining medium.
(8) Bacteria were plated on solid YEP medium with antibiotics (50 mg/L Kana,20mg/L Rif).
(9) The plates were incubated upside down at 28℃for 24-48 h.
2. Agrobacterium-mediated transient expression
The apple golden crown and the Hanfu tissue culture seedlings are cultivated in an illumination incubator with the temperature of 25+/-1 ℃ and the humidity of 60 percent, the length of day and night is kept to be 16-8 hours, and the illumination intensity is 200 mu mol m & lt-2 & gt s & lt-1 & gt. And (5) performing agrobacterium injection when 5-6 true leaves of the plants are unfolded. Agrobacterium injection methods are described in Bai et al (Bai et al 2011).
(1) Pre-culturing: the agrobacterial plaque or glycerol bacteria are added with YEP 2ml (50 mg/L Km,20mg/L Rif), cultured overnight at 28 ℃ and 180 rpm;
(2) The culture method comprises the following steps: 4ml of YEP culture medium (corresponding antibiotics and 10 mu M acetosyringone are added), 1/50 volume of bacterial liquid (80 mu L) is added, and the culture is carried out at 28 ℃ at 180rpm for 12-16 hours;
(3) The culture medium was removed by centrifugation at 10000rpm at 1min, and the cells were suspended (vortex shaking was possible) in 1-2ml of the suspension. 10. Mu.L of the bacterial liquid was added to 990. Mu.L of the suspension, and the OD600 was measured by a spectrophotometer to adjust the bacterial suspension to OD 600=1.0. Standing at room temperature for 2-5h;
suspension: (10 mM MES-KOH (pH 5.2), 10mM MgCl2, 100. Mu.M acetosyringone).
(4) Vortex vibration or gun sucking suspended bacteria before the bacteria liquid is used, and sucking the bacteria liquid by a 1ml syringe without a needle;
(5) Avoiding veins, using an injector needle to open small holes on blades, using an injector filled with bacterial liquid to press the small holes, using fingers of the other hand to press the small holes in the opposite direction of the blades, slowly applying force, injecting bacterial liquid into the blades, and injecting 2 holes into each blade;
(6) After 4 days of culture, the expression levels of MdSTK1 and mdm-tsRMet were measured;
the specific operation is as follows:
1. extraction of Jin Guanzong RNA by ctab
2. Reverse transcription reaction System and procedure
The reverse transcription reaction system is as follows:
(1) DNA removal from Total RNA
RNA 2μg
gDNA purge 1μL
DEPC water make-up to 10. Mu.L
Adding the sample on ice, mixing, centrifuging briefly, standing at 42 deg.C for 5min, and immediately placing on ice.
First Strand Synthesis of cDNA
The above system was added with 10. Mu.L of 2 Xmix, 15min at 50℃and 5min at 75℃and stored at-80℃after cooling.
3. Fluorescent quantitative reaction system
Md-STK1 specific primers (F: 5'-TGCTTCCGAGTTGTAGCGTT-3', R: 5'-TTGCACTCGTCAGTGGAGAC-3') were designed and fluorescent quantitation was performed at Applied Biosystems 7500 using SYBR Green fluorescent quantitation premix (Vazyme, Q341V21.1) and the PCR reaction procedure was as follows: 95 ℃ and 3mins;95℃for 10sec and 60℃for 30sec for 40 cycles. Results were statistically analyzed using the 2- ΔΔct method (Livak and Schmittgen, 2001).
(6) Inoculating bacteria ALT1 after 4 days;
(7) Phenotypes were observed after 2 days of dark culture.
Overexpression of MdtRNA-Met CAT After that, the expression level of MdSTK1 in golden crown and frigid-rich was decreased, 0.1 times WT and EV at Jin Guanzhong, 0.3 times in frigid-rich, and resistance to ALT1 was decreased, as shown in fig. 5.
At Jin Guanzhong, the size of the lesions treated with WT and EV was about 30%, and MdtRNA-Met was overexpressed CAT About 60% of the lesion area, and about 14% of the lesion area treated with WT and EV in Hanfu, and overexpress MdtRNA-Met CAT The lesion area of (a) was about 43%, as shown in fig. 5.
EXAMPLE 6 silencing tsRMet sequence design
The silencing mdm-tsRMet sequence is designed, an Arabidopsis thaliana mature MIR319a is taken as a framework, an artificial miRNA is constructed, and the sequence is synthesized and constructed on a pFGC5941 vector (between NcoI and BamHI cleavage sites) and is transferred into escherichia coli DH5 alpha.
The transformation method is as follows:
(1) DH5 alpha competent cells are taken out from the temperature of minus 80 ℃, quickly inserted into ice, after 5 minutes, the fungus blocks are melted, plasmids are added and the EP tube bottom is stirred by hands and gently mixed (avoiding sucking by a gun), and the mixture is kept stand in the ice for 25 minutes;
(2) Heat shock in 42 deg.c water bath for 45 sec, fast returning to ice and standing for 2min;
(3) To the centrifuge tube, 700. Mu.l of sterile medium (2 YT or LB) without antibiotics was added, and after mixing, resuscitated at 37℃for 60 minutes at 200 rpm.
(4) The cells were collected by centrifugation at 5000rpm for 1 minute, and about 100. Mu.l of the supernatant was gently swirled to resuspension the pellet and spread on 2YT or LB medium containing the corresponding antibiotic.
(5) The plates were placed in an incubator at 37℃overnight.
(6) The precursor of Arabidopsis preMIR319a was engineered into the front-back structure and the silencing mdm-tsRMet vector pattern is shown in FIG. 6.
EXAMPLE 7 construction of MNP-DNA
The plasmid DNA containing the artificial miRNA was purified and bound to Magnetic Nanoparticles (MNPs).
The specific operation is as follows:
1. extraction of plasmids (Vazyme, fastPure Plasmid Mini Kit, DC 201)
(1) Taking 1-5ml of bacterial liquid cultured overnight (12-16 h), adding into a 2ml centrifuge tube, and centrifuging at 10000rpm for 1min. Discarding the culture medium and sucking out residual liquid;
(2) Adding 250 μl Buffer P1 (RNase A is added) into the centrifuge tube, and mixing by vortex oscillation;
(3) Adding 250 μl Buffer P2 into the step 2, and mixing gently upside down for 8-10 times;
(4) Adding 350 μl Buffer P3 into the step 3, immediately gently reversing upside down for 8-10 times, and centrifuging at 12000rpm for 10min;
(5) FastPure DNA Mini Columns adsorption columns were placed in Collection Tube2ml Collection tubes. The supernatant from step 4 was carefully transferred to an adsorption column by a pipette and centrifuged at 12000rpm for 30sec. Pouring out the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;
(6) 600 μl Buffer PW2 (diluted with absolute ethanol) was added to the column. Centrifuge at 12000rpm for 30sec. Discarding the waste liquid, and putting the adsorption column back into the collecting pipe;
(7) Repeating the step 6;
(8) Placing the adsorption column back into a collecting pipe, centrifuging at 12000rpm for 1min, and drying the adsorption column;
(9) The column was placed in a fresh sterilized 1.5ml centrifuge tube. Add 30-100. Mu.l of the Elution Buffer to the center of the membrane of the column. Standing at room temperature for 2min, and centrifuging at 12000rpm for 1min to elute DNA;
(10) Discarding the adsorption column, storing DNA product at-20deg.C,
2. magnetic nanoparticle modified Polyethylenimine (PEI)
And (3) ultrasonically dispersing the 10nm magnetic nano particles washed by deionized water in oxalic acid solution (5 g/L) for 60min to surface-modify negative charge, washing and re-suspending, and then dripping 2.0ml of 20% PEI solution to obtain the surface-modified PEI magnetic nano particles.
3. Binding of MNP to DNA
Mixing the purified DNA and MNP in a mass ratio of 1:1, vortex shaking for 1 minute, and standing at room temperature of 25 ℃ for 30 minutes for combination.
An electron micrograph of MNP without DNA binding and MNP after DNA binding is shown in fig. 7.
Example 8MNP-DNA spraying apple tissue culture seedling
The MNP-DNA complex of example 7 was mixed with a surfactant Silwet L-77 at a ratio of 5000:1 at a concentration of 100 ng/. Mu.l, and the mixture was uniformly sprayed on Jin Guanzu seedlings, each of which was applied at about 200. Mu.l, and three days later, the MNP-DNA complex was observed in apples by a transmission electron microscope, while the nodhen detected the mdm-tsRMet content, and the expression level of MdSTK1 was quantitatively detected by fluorescence in real time.
The specific operation is as follows:
1. extraction of Jin Guanzong RNA by ctab
2. Reverse transcription reaction System and procedure
The reverse transcription reaction system is as follows:
(1) DNA removal from Total RNA
RNA 2μg
gDNA purge 1μL
DEPC water make-up to 10. Mu.L
Adding the sample on ice, mixing, centrifuging briefly, standing at 42 deg.C for 5min, and immediately placing on ice.
(2) First Strand Synthesis of cDNA
The above system was added with 10. Mu.L of 2 Xmix, 15min at 50℃and 5min at 75℃and stored at-80℃after cooling.
3. Fluorescent quantitative reaction system
The Md-STK1 specific primer (F: 5'-TGCTTCCGAGTTGTAGCGTT-3', R: 5'-TTGCACTCGTCAGTGGAGAC-3') was designed,
fluorescence quantification was performed with SYBR Green fluorescent quantitation premix (Vazyme, Q341V21.1) at Applied Biosystems 7500, and the PCR reaction procedure was as follows: 95 ℃ and 3mins;95℃for 10sec and 60℃for 30sec for 40 cycles. Results were statistically analyzed using the 2- ΔΔct method (Livak and Schmittgen, 2001).
4. Northen detects the expression of mdm-tsRMet
After spraying of the si-mdm-tsRMet and MNP-si-mdm-tsRMet, the mdm-tsRMet content was detected after 3 days, and the mdm-tsRMet content was significantly reduced relative to WT and EV, as shown in FIG. 8, the expression level of mdSTK1 was quantitatively detected by real-time fluorescence, and the expression level of mdSTK1 was increased after silencing mdm-tsRMet, as shown in FIG. 8.
5. Apple leaf for transmission electron microscope observation
The samples were fixed in 2.5% glutaraldehyde solution at 4 ℃ overnight and then treated as follows: pouring out the fixing solution, and rinsing the sample three times with 0.1M phosphate buffer solution with pH of 7.0 for 15min each time; fixing the sample with 1% osmium acid solution for 1-2h; carefully remove the osmium acid waste solution, rinse the sample three times with 0.1M phosphate buffer, pH7.0, 15min each time; dehydrating the sample with ethanol solutions of gradient concentration (including 30%,50%,70%,80%,90% and 95% concentration), each concentration being treated for 15min, and then 100% ethanol for 20min; finally, the mixture is transited to pure acetone for 20min. Treating the sample with a mixed solution of a sparr embedding agent and acetone (V/v=1/1) for 1h; treating the sample with a mixed solution of a sparr embedding agent and acetone (V/v=3/1) for 3h; treating the sample with pure embedding medium overnight; embedding the sample subjected to the permeation treatment, and heating at 70 ℃ overnight to obtain the embedded sample. The sample is sliced in an LEICA EM UC7 type ultrathin slicer to obtain 70-90nm slices, and the slices are respectively dyed for 5-10min by lead citrate solution and uranyl acetate 50% ethanol saturated solution, so that the slices can be observed in a Hitachi H-7650 type transmission electron microscope.
The MNP-DNA complexes inside apples were observed through TEM, and complexes in apples were observed, demonstrating their successful absorption, as shown in fig. 9.
Example 9 inoculation ALT1 verifies the effect of silencing mdm-tsRMet on resistance to alternaria leaf spot
The specific method comprises the following steps:
the golden crown apple tissue culture seedlings treated for three days in example 8 were inoculated with ALT1, the veins were avoided, after the leaves were perforated with a syringe needle, ALT1 spore suspension was sprayed on the back of the leaves, the phenotype was observed for about 200. Mu.l each plant for 7 days, the plaque area was counted for 14 days, the plaque areas for WT and EV treatments were 40-50% or so on day 7, while the siI-mdm-tsRMet and MNP-siI-mdm-tsRMet were only 10% or so on day 14, the WT and EV incidence areas were 100%, and siI-mdm-tsRMet was 50% or so on day 7, while the MNP-siI-mdm-tsRMet still did not appear as shown in FIG. 10.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
What is not described in detail in this specification is prior art known to those skilled in the art.
Claims (7)
1. A nanocomposite for enhancing resistance to apple alternaria leaf spot, characterized by: comprises a magnetic nanoparticle carrier and plasmid DNA;
the plasmid DNA is a recombinant plant expression vector which is a pFGC5941 vector containing artificial miRNA;
the nucleotide sequence of the artificial miRNA is shown as SEQ ID NO. 4.
2. A nanocomposite for enhancing resistance to apple alternaria leaf spot according to claim 1, wherein: the artificial miRNA comprises a reverse complementary sequence for silencing tsRNA, and the nucleotide sequence of the reverse complementary sequence is shown as SEQ ID NO. 3.
3. An engineering bacterium is characterized in that: the engineering bacteria contain the recombinant plant expression vector of claim 1.
4. A method of preparing the nanocomposite of claim 1, comprising the steps of:
step 1, modifying the surface of magnetic nano particles by using polyethyleneimine to obtain a nano particle carrier;
and 2, incubating the nanoparticle carrier and plasmid DNA to obtain a nanocomposite.
5. A method of preparing a nanocomposite as claimed in claim 4 wherein said magnetic nanoparticles are 10nm Fe 3 O 4 And (3) microspheres.
6. A method of preparing a nanocomposite as claimed in claim 4 wherein: the step 2 is specifically as follows:
nanoparticle carriers were mixed with plasmid DNA in a mass ratio of 1:1 and incubated at 25 ℃ for 30min.
7. Use of a nanocomposite according to claim 1, characterized in that: the nano-composite and the surfactant are uniformly sprayed on apple leaves after being mixed, and the recombinant plant expression vector in the nano-composite is delivered into apple plants in the mode, so that the resistance of apple susceptibility varieties to alternaria leaf spot is enhanced.
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