CN114854752B - Double-stranded RNA molecule of targeted silencing phytophthora capsici cellulose synthase 3 and application thereof - Google Patents
Double-stranded RNA molecule of targeted silencing phytophthora capsici cellulose synthase 3 and application thereof Download PDFInfo
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Abstract
The invention discloses a double-stranded RNA molecule capable of efficiently inhibiting phytophthora capsici cellulose synthase 3 gene expression, belonging to the technical field of agricultural biology. The double-stranded RNA molecule consists of the nucleic acid sequences shown in SEQ ID No.1 and SEQ ID No. 2. The invention also discloses application of the double-stranded RNA molecule in preventing and controlling plant diseases caused by phytophthora capsici. The double-stranded RNA molecules of the invention can inhibit phytophthora capsici infection by directly treating phytophthora capsici or spraying on plants. Meanwhile, the double-stranded RNA related by the invention has strong specificity on the essential genes for growth and development of phytophthora capsici, has no drug resistance problem, and can provide an effective way for preventing and treating related diseases caused by phytophthora capsici when used for preparing biopesticide.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a double-stranded RNA molecule capable of inhibiting phytophthora capsici cellulose synthase 3 gene expression and application of the molecule in preventing and controlling plant diseases caused by phytophthora capsici.
Background
Phytophthora capsici (Phytophthora capsici) is an important plant pathogen oomycete which is distributed around the world and can infect more than 70 plants of the Solanaceae, leguminosae and most cucurbitaceae, cause cataplexy, wilting and rotting of roots, stems and fruits, and can be ill from seedling stage to fruit stage, thus causing serious economic loss. The phytophthora capsici leonia caused by phytophthora capsici is a destructive disease, can be transmitted through rain, soil, air flow and other ways, causes symptoms such as leaf withering, fruit rot, even complete plant withering and the like, has great influence on crop yield and quality, and can cause yield reduction of more than 50% when serious.
The control of phytophthora capsici mainly depends on chemical control, but due to the fact that the number of types of oobactericides is small, the single use of chemical pesticides for a long time causes the phytophthora capsici to generate drug resistance to a plurality of chemical pesticides, and the chemical pesticides are continuously applied in increased doses to cause serious environmental pollution. In addition, plant species having resistance to phytophthora capsici are scarce, and development of novel means for controlling phytophthora capsici-induced plant diseases is highly demanded.
RNA interference (RNA INTERFERENCE, RNAI) is a phenomenon of gene silencing induced by double-stranded RNA that can inhibit gene expression by blocking transcription or translation of a particular gene. RNAi is ubiquitous in organisms, and RNA sequences of double-stranded or hairpin structures formed by transcription of DNA can be processed into small RNAs of between 20 and 30bp that specifically recognize the complementary pairing target gene DNA or mRNA sequence by mediating the gene silencing complex, resulting in cleavage of mRNA, translational inhibition or DNA methylation, ultimately inhibiting normal expression of the gene (Fire, A. Et al 1998.Nature.391 (6669): 806). RNAi technology has achieved a great deal of effort in recent years in plant disease control, and direct spraying of double-stranded RNA molecules can interfere with the critical genes of pathogens, resulting in a decrease in pathogen fitness and a decrease in pathogenicity, called SIGS (spread-Induced GENE SILENCING).
SIGS is a hot spot in recent years for controlling crop disease by directly interfering with the expression of important genes of pathogenic bacteria through double-stranded RNA. Spraying double-stranded RNA of the essential gene CYP51 and the RNAi important element DCL and the AGO gene for synthesizing targeted fusarium sterol on the barley leaves can remarkably reduce the pathogenicity of fusarium graminearum on the barley leaves (Koch, A. Et al 2016.PLoS pathogens.12 (10): e1005901; werner, B.T. Et al 2020.Frontiers in plant science.11:476). The use of double stranded RNA targeting different cotton leaf curl virus (CLCuV) genes on cotton can significantly reduce the expression of these genes, successfully controlling the incidence of lice-transmitted CLCuV (Verma, P. Et al 2018.Journal of entomology and zoology studies.6 (4): 1055-1060). The above studies demonstrate that SIGS-based cross-border gene silencing is effective for plant disease control.
Cellulose synthase 3 protein (CesA 3) is the target of CAA-like bactericides, and the amino acid mutation of PcCesA can lead to high level resistance of phytophthora capsici to the medicament. Through searching, no relevant literature for preventing and controlling plant diseases caused by phytophthora capsici based on an RNA silencing technology is found at present.
Disclosure of Invention
The invention aims to provide a double-stranded RNA molecule capable of inhibiting phytophthora capsici cellulose synthase 3 gene expression.
A second object of the present invention is to provide the use of the double-stranded RNA molecule described above for inhibiting the expression of Phytophthora capsici cellulose synthase 3 gene.
A third object of the present invention is to provide the use of the double-stranded RNA molecule described above for controlling plant diseases caused by phytophthora capsici.
It is a fourth object of the present invention to provide a pesticide comprising the double stranded RNA molecule described above.
The fifth object of the present invention is to provide the use of a pesticide containing the double-stranded RNA molecule described above for controlling phytophthora capsici-induced plant diseases.
The sixth object of the present invention is to provide a method for controlling plant diseases caused by phytophthora capsici using the double-stranded RNA molecule.
The technical scheme for realizing the invention is as follows:
a double-stranded RNA molecule capable of inhibiting phytophthora capsici cellulose synthase 3 gene expression has a sequence shown in SEQ ID No.1 on one strand and a sequence shown in SEQ ID No.2 on the other strand.
The double-stranded RNA molecule is applied to inhibiting the expression of phytophthora capsici cellulose synthase 3 genes.
The double-stranded RNA molecule is applied to preventing and controlling plant diseases caused by phytophthora capsici.
Pesticides containing the double-stranded RNA molecules.
The pesticide containing the double-stranded RNA molecule is applied to the prevention and treatment of plant diseases caused by phytophthora capsici.
The method for preventing and controlling plant diseases caused by phytophthora capsici by utilizing the double-stranded RNA molecules comprises the steps of directly spraying a solution or a preparation containing the double-stranded RNA molecule carrier on the surface of plant tissues or performing soil treatment (such as root irrigation).
The double-stranded RNA molecules of the present invention may be produced by means of prokaryotic expression.
The invention relates to a double-stranded RNA molecule using method: the aqueous solution of double-stranded RNA molecules with proper concentration (such as about 100 nM) is sprayed on the surfaces of plant leaves or root irrigation treatment is carried out on plant rhizosphere soil, so that infection and expansion of phytophthora capsici in plant leaves or root stems can be effectively inhibited.
The plant is preferably tobacco or capsicum.
The invention has the advantages or beneficial effects that: (1) The double-stranded RNA molecule provided by the invention has obvious inhibition effect on the expression of phytophthora capsici cellulose synthase 3, can effectively inhibit the infection of phytophthora capsici to plants, and provides a new effective path for preventing and controlling plant diseases caused by phytophthora capsici. (2) The double-stranded RNA molecule has strong specificity to the cellulose synthase 3 gene of phytophthora, can inhibit a plurality of plant pathogenic phytophthora including phytophthora capsici in theory, and has no drug resistance problem. (3) The target gene related by the invention is also an action target of CAA medicaments, and can be used for treating the drug resistance of phytophthora groups which generate drug resistance due to the mutation of amino acid, or delaying the drug resistance of medicaments with high drug resistance risks when being compounded with bactericides. (4) The double-stranded RNA molecule related by the invention is safe to human or animals, can effectively reduce the use amount of chemical pesticides, has no environmental pollution problem, and is beneficial to environmental protection.
Drawings
FIG. 1 is a gel electrophoresis diagram of a double stranded RNA molecule according to the present invention. Drawing and annotating: the gel bands are, left to right, double stranded RNA molecule dsCes, blank vector control L4440, negative control double stranded RNA molecule dsGFP, and Maker, respectively.
FIG. 2 is a photograph showing infection of phytophthora capsici after the double-stranded RNA molecules of the present invention are applied to the isolated leaves. The protection effect of the double-stranded RNA on tobacco leaves is achieved; and B, the double-stranded RNA has a protective effect on pepper leaves. In fig. 2A: spraying dsRNA crude extract to the isolated tobacco leaves for 1d, and then inoculating phytophthora capsici zoospore (10 4/mL) for 3d to obtain diseased tobacco leaves (clear water control, blank carrier control L4440, negative control double-stranded RNA molecule dsGFP and double-stranded RNA molecule dsCes5 from left to right); in fig. 2B: the dsRNA crude extract was sprayed to the isolated pepper leaves for 1d, and the pepper leaves developed after 3d inoculation with phytophthora capsici zoospores (10 4/mL) (clear water control, blank vector control L4440 and double-stranded RNA molecule dsCes, respectively, from left to right).
FIG. 3 is a bar graph of the area of an affected lesion formed by the inoculation of P.capsici after the application of the double stranded RNA molecules of the present invention to an isolated leaf. The protection effect of the double-stranded RNA on tobacco leaves is achieved; and B, the double-stranded RNA has a protective effect on pepper leaves. In fig. 3A: the diameter of the diseased tobacco lesions (clear water control, blank vector control L4440, negative control double-stranded RNA molecule dsGFP and double-stranded RNA molecule dsCes respectively from left to right) after spraying the dsRNA crude extract for 1d and inoculating phytophthora capsici cake for 3 d. In fig. 3B: the diameter of the diseased hot pepper lesions (clear water control, blank carrier control L4440, and double-stranded RNA molecules dsCes from left to right) after 1d of the dsRNA crude extract was sprayed and inoculated with phytophthora capsici leonian cake for 3 d.
FIG. 4 is a photograph showing the efficacy of the double stranded RNA molecules of the present invention against Phytophthora capsici after application to living plants. Drawing and annotating: the pepper plants were irrigated with 1d of a crude extract of root dsRNA and then with 3mL of a zoospore suspension of Phytophthora capsici (10 4/mL), and 7d of a suspension of the zoospore of Phytophthora capsici was examined for the disease states of the pepper (clear water control, blank vector control L4440, negative control double-stranded RNA molecule dsGFP and double-stranded RNA molecule dsCes, respectively, from left to right).
FIG. 5 is a bar graph of disease index caused by the inoculation of P.capsici after the application of the double stranded RNA molecules of the present invention to living plants.
Drawing and annotating: the pepper plants were irrigated with 1d of the crude extract of root dsRNA and then with 3mL of zoospore suspension of Phytophthora capsici (10 4/mL), and 7d of the disease index of the pepper plants (clear water control, blank vector control L4440, negative control double-stranded RNA molecule dsGFP and double-stranded RNA molecule dsCes, respectively, from left to right).
Detailed Description
The reagents used in the examples described below are all commercially available, and the techniques used in the examples are conventional, as known to those skilled in the art, unless otherwise indicated.
EXAMPLE 1 double stranded RNA molecule Source inhibiting Phytophthora capsici cellulose synthase 3 Gene expression according to the present invention
(1) The sequence of the double stranded RNA molecule of the present invention is derived from the cDNA of PcCesA. PcCesA 3A cDNA sequence is derived from the gene numbered JX905357 in the database of Phytophthora capsici (GenBank).
(2) A total of 555 base sequences of No. 3005-3559 of JX905357 in the cDNA sequence database (GenBank) of PcCesA was selected as the sequence of the double-stranded RNA molecule according to the present invention. The sequence of the sense strand is shown as sequence 1 in the sequence table, and the antisense strand is shown as sequence 2 in the sequence table.
(3) The sequences are respectively aligned with genome sequences (NCBI) of common hosts of phytophthora capsici such as tobacco, tomatoes, peppers and the like, and the possibility of nonspecific silencing of plant genes is detected. The comparison result shows that the maximum continuous matching of the sequence related to the invention and the plant genome sequence is less than 10 bases, and the siRNA prediction potentially generated by the sequence shows that the siRNAs generated by the sequence can not reach the effective length of gene silencing, thereby eliminating the possibility of silencing host plant genes.
EXAMPLE 2 preparation of double-stranded RNA molecules of the invention
(1) The double-stranded RNA expression plasmid L4440 contains two bidirectional T7 promoters, and can be transcribed into double-stranded RNA in RNase III-deficient escherichia coli. The target fragment was amplified by PCR, and the amplification primers are shown in Table 1. cDNA of phytophthora capsici standard strain LT1534 (doi: 10.1094/MPMI-02-12-0028-R) is used as template, the amplified fragment is inserted between two T7 promoters (between Sac II and Xba I enzyme recognition sites) of L4440 plasmid in enzyme cutting connection mode, and the plasmid capable of expressing double-stranded RNA molecule is obtained after PCR and sequencing verification.
TABLE 1 primer sequences for construction of double stranded RNA molecules
(2) The plasmid obtained in step (1) and capable of expressing double-stranded RNA molecules is transformed into RNase III-deficient E.coli strain HT115 to induce expression of dsRNA.
(3) Single colonies of double stranded RNA molecule expression strain HT115, which were verified by PCR and sequenced successfully, were selected, shake-cultured overnight in LB liquid medium containing ampicillin, and stored in a-80℃freezer.
(4) Double-stranded RNA molecule expression strains were streaked on LB plates with resistance markers, and single colonies were dipped into 5mL of liquid LB medium and shake-cultured overnight at 37℃at 200 rpm.
(5) And (3) taking 500 mu L of shaking culture bacteria liquid into 10mL of LB liquid culture medium, shaking culture at 37 ℃ and 230rpm for about 2.5 hours until the bacterial liquid OD 600 is between 0.5 and 0.8.
(6) Adding 2mM IPTG to the bacterial liquid, shaking and culturing for 8h at 230rpm at 37 ℃.
(7) 2ML of the cultured bacterial liquid is taken into an enzyme-free centrifuge tube, and the total RNA is extracted by a Triozol method.
(8) And (3) carrying out agarose gel electrophoresis on the extracted RNA, and determining whether the size of the double-stranded RNA band meets the expectations according to the electrophoresis result. As shown in FIG. 1, the double-stranded RNA molecule electrophoresis strip prepared by the invention is clear and has the size of the intended purpose. Sequencing shows that the sense strand sequence of the obtained double-stranded RNA molecule is shown as sequence 1 in a sequence table, and the antisense strand is shown as sequence 2 in the sequence table.
EXAMPLE 3 evaluation of disease resistance of plants to Phytophthora capsici by application of double stranded RNA molecules according to the invention
The patent is characterized in that double-stranded RNA molecules are directly sprayed on the surface of a host plant (in-vitro leaves of the benthamia tinctoria and the capsicum) for 1d, then zoospores of phytophthora capsici are inoculated, and after 3d, the diameter of leaf lesions is observed and measured. b. Firstly, double-stranded RNA molecules are used for root irrigation of plants, then zoospore inoculation is carried out on pepper seedlings in a simulated field environment, and the disease index of the pepper seedlings is measured. The influence of double-stranded RNA on phytophthora capsici growth and pathogenicity is clear according to the two evaluation modes.
Zoospores of Phytophthora capsici BYA (a pepper plant from the development of pepper epidemic disease in Guangdong white cloud, which is separated from the pathogenic bacteria drug resistance laboratory by the agricultural university of China, has been identified by morphology and molecular biology, and confirmed as Phytophthora capsici) strain were prepared as described in example 3, and the concentration of zoospores was adjusted to 10 5 per mL for standby after counting by a hemocytometer.
Crude extraction of double-stranded RNA: the dsRNA of interest was induced to prokaryotic express in E.coli according to the 100mL system under the induction conditions described in example 1. And (3) centrifuging the induced bacterial liquid at 4 ℃ and 4000rpm for 30min to collect bacterial sediment. The suspension was resuspended in 50ml of TE buffer, and the OD 600 of the resuspended suspension was adjusted to about 1.5. The bacterial liquid was crushed by a high-pressure cytoclasis machine at a pressure of 0.5kPa for 2.5 minutes. The bacterial liquid after crushing should be clear and transparent, and has strong fluidity.
A. Directly spraying double-stranded RNA molecules on the surface of a host plant (in-vitro leaves of the benthamia and the capsicum), inoculating phytophthora capsici zoospore after 1d, and observing and measuring the diameter of leaf lesions after 3 d;
Collecting leaf pieces of the leaf of the Bensheng tobacco with similar growth state for 6-8 weeks, spraying 5mL double-stranded RNA crude extract to the front side and the back side of the leaf of the in-vitro Bensheng tobacco and the leaf of the capsicum by using a 20mL spray pot after high-temperature high-pressure sterilization. And (5) placing the leaves in a moisturizing culture dish after the leaves are naturally dried. 10 leaves were treated per dsRNA crude extract. After the dsRNA is sprayed for 1d, 10 mu L of phytophthora capsici zoospore suspension (10 4/mL) is inoculated to the back of the leaf blade, the leaf blade is placed in a moisturizing culture dish, and after 3d, the diameter of the lesion is measured by adopting a crisscross method. The activity of double stranded RNA on phytophthora capsici was averaged according to the average diameter of lesions on the isolated leaves.
As shown in fig. 2, the lesions formed by phytophthora capsici infection on leaves sprayed with double-stranded RNA molecules targeting the phytophthora capsici cellulose synthase 3 gene were significantly reduced relative to the control. The statistical result is shown in figure 3, and the infection capability of phytophthora capsici after the double-stranded RNA treatment of the leaves is obviously reduced, which indicates that the double-stranded RNA molecules have obvious inhibition effect on the growth and pathogenicity of the phytophthora capsici.
B. Firstly, double-stranded RNA molecules are used for root irrigation of plants, then zoospore inoculation is carried out on pepper seedlings in a simulated field environment, and the disease index of the pepper seedlings is measured.
Root irrigation treatment is carried out on the tobacco plants with similar growth states and in 6-8 weeks, and treatment is carried out on 3mL of double-stranded RNA crude extract for each plant. After 1d, 3mL of phytophthora capsici zoospore suspension (10 4/mL) was irrigated. The disease index was investigated 7d after inoculation and the disease index grading criteria were referenced in table 2. The average disease index was calculated for about 10 biological duplicate plants per treatment.
TABLE 2 Phytophthora capsici disease incidence grading Standard
As a result, as shown in FIG. 4, the plants sprayed with the double-stranded RNA molecules of the present invention were significantly healthier than the control plants sprayed with sterile water or E.coli strain-deposited disruption solution, and no significant constriction, necrosis, or lodging of the stem base occurred. The statistical result is shown in fig. 5, and the average disease index of the plant sprayed with the double-stranded RNA molecule of the invention is obviously lower than that of the control plant, which indicates that the spraying of the double-stranded RNA molecule of the invention obviously inhibits the infection of phytophthora capsici to the stem base of the plant.
EXAMPLE 4 determination of efficacy of plants applying double stranded RNA molecules according to the invention against Phytophthora capsici
In the greenhouse, 80-hole trays are used for planting pepper seedlings. After the pepper seedlings grow for 1.5 months, 3mL double-stranded RNA molecule crude extract obtained by induction is directly sprayed to the root and stem parts of plants. After 1d, inoculation is carried out by adopting a mode of root irrigation of phytophthora capsici zoospore suspension, 3mL of spore suspension with the concentration of 10 4/mL is sucked by a gun and directly applied to soil near the root base of the pepper plant. The disease condition of the capsicum was investigated 10 days after inoculation, and disease progression (0, 1,2,3,4,5 grade) was counted according to table 3, and disease index and dsRNA crude extract control efficiency were calculated.
Control efficiency = (control disease index-treatment disease index)/control disease index x 100%
As shown in Table 3, the double-stranded RNA molecules of the invention show 56.23% of control effect on phytophthora capsici after spraying, and the results show that the double-stranded RNA molecules of the invention can be used as green and safe novel pesticides for controlling plant diseases caused by phytophthora capsici.
TABLE 3 statistical chart of greenhouse effect test of double-stranded RNA molecules of the invention on pepper epidemic disease
Sequence listing
<110> Chinese university of agriculture
<120> Double-stranded RNA molecule for targeted silencing of phytophthora capsici leonian synthase 3 and application thereof
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Claims (3)
1. The application of a double-stranded RNA molecule of a targeted silencing phytophthora capsici cellulose synthase 3 gene in preventing and controlling plant diseases caused by phytophthora capsici is disclosed, wherein the double-stranded RNA molecule has a sequence shown in SEQ ID No. 1 on one strand and a sequence shown in SEQ ID No. 2 on the other strand.
2. The application of a double-stranded RNA molecule of a targeted silencing phytophthora capsici cellulose synthase 3 gene in preparing a pesticide for controlling plant diseases caused by phytophthora capsici is disclosed, wherein the double-stranded RNA molecule has a sequence shown in SEQ ID No. 1 on one strand and a sequence shown in SEQ ID No. 2 on the other strand.
3. The method for preventing and controlling plant diseases caused by phytophthora capsici by utilizing double-stranded RNA molecules of targeted silencing phytophthora capsici cellulose synthase 3 genes is characterized in that a solution or a preparation containing the double-stranded RNA molecules is directly sprayed on the surface of plant tissues or subjected to soil treatment; wherein, the double-stranded RNA molecule has a sequence shown in SEQ ID No. 1 on one strand and a sequence shown in SEQ ID No. 2 on the other strand.
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