CN113355302B - Application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein - Google Patents
Application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein Download PDFInfo
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Abstract
The invention belongs to the technical field of cultivation or production of agricultural disease-resistant crop lines, and particularly relates to application of CS10 protein or a coding gene of CS10 protein for negatively regulating plant immunity. The invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in cultivation or production of agricultural disease-resistant crop lines. The kinase gene CS10 can effectively play a role in negative regulation of resistance of phytophthora parasitica.
Description
Technical Field
The invention belongs to the technical field of cultivation or production of agricultural disease-resistant crop lines, and particularly relates to application of CS10 protein or a coding gene of CS10 protein for negatively regulating plant immunity.
Background
Oomycetes (oomycetes) are a class of eukaryotic microorganisms that are similar in morphological structure to fungi, while gold brown algae, which are phylogenetically and protozoa kingdom, are similar to different flagella. Oomycetes contain many animal and plant pathogens that can cause destructive hazards, such as Phytophthora (Phytophthora), downy mildew (downy milde), saprolegnia (Saprolegnia) and Pythium (Pythium), which can cause significant economic losses by infestation of plants, insects, fish, vertebrates, crustaceans, and a variety of microorganisms. Among them, phytophthora has identified more than 120 important plant pathogens, including the disease responsible for decay and subsequent loss of Irish potatoes in the 50 s of the 19 th century, causing the disease familial culprit potato late blight-phytophthora infestans (P.infestans); phytophthora sojae (P.sojae) which can cause rot of the root and stem of soybean and seriously affect soybean production; wherein, the phytophthora parasitica (P.paramedica), also called phytophthora nicotianae (P.icotinae), has the characteristic of wide host range and can cause root and stem diseases of various crops, including arabidopsis thaliana, tobacco and the like.
The main chemical pesticide used for preventing and controlling oomycetes in agricultural production is phenylamide bactericide metalaxyl, but various pathogenic oomycetes such as phytophthora infestans and phytophthora capsici have drug resistance to the main chemical pesticide. In addition, oomycetes are very powerful in their ability to overcome or delay the loss of disease resistance in crop varieties. For the prevention and treatment of oomycete diseases, the most economical and effective method is to cultivate disease-resistant varieties of crops at present, however, the resistance of the disease-resistant varieties is kept facing a great challenge due to the toxicity variation of pathogenic bacteria.
Disclosure of Invention
In order to solve the problems, the invention provides application of CS10 protein or a coding gene of CS10 protein for negatively regulating plant immunity. The CS10 protein for negative regulation of plant immunity or the coding gene of the CS10 protein in the technical scheme provided by the invention is a novel plant immunity negative regulation factor, and can be used for negative regulation of the resistance of plants to phytophthora parasitica, and effectively controlling the resistance of arabidopsis thaliana to phytophthora parasitica.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in cultivation or production of agricultural disease-resistant crop lines.
The invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in cultivating plants with resistance to phytophthora.
The invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in regulating plant and phytophthora affinity interaction.
The invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in regulating plant and phytophthora parasitica affinity interaction through cytoplasmic membrane localization.
The invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in regulating plant and phytophthora parasitica affinity interaction through two kinase domains, wherein in the application, when the plant is arabidopsis thaliana, amino acid sequences corresponding to the two kinase domains are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.
The invention provides application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein in regulating affinity interaction of Arabidopsis and phytophthora parasitica through the action of GDK domain related to downstream signal path.
Preferably, the CS10 protein includes CS10 protein in Arabidopsis thaliana, CS10 protein in Nicotiana benthamiana, and CS10 protein in Solanum tuberosum.
Preferably, the coding gene of the CS10 protein comprises a coding gene of CS10 protein in Arabidopsis thaliana, a coding gene of CS10 protein in Nicotiana benthamiana and a coding gene of CS10 protein in potato.
Preferably, the amino acid sequence of CS10 protein in the Arabidopsis thaliana is shown as SEQ ID NO. 3; the amino acid sequence of CS10 protein in Nicotiana benthamiana is shown as SEQ ID NO. 4 or SEQ ID NO. 5; the amino acid sequence of CS10 protein of potato species is shown as SEQ ID NO. 6.
Preferably, the nucleotide sequence of the coding gene of CS10 protein in the arabidopsis thaliana is shown as SEQ ID NO. 7; the nucleotide sequence of the coding gene of CS10 protein in Nicotiana benthamiana is shown as SEQ ID NO. 8 or SEQ ID NO. 9; the nucleotide sequence of the coding gene of CS10 protein in the potato is shown as SEQ ID NO. 10.
The invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in cultivation or production of agricultural disease-resistant crop lines. In the application of the invention, taking Arabidopsis as an example, the coding gene CS10 (called AtCS10 for short) in Arabidopsis plays a role in negative regulation of the resistance of phytophthora parasitica by participating in the affinity interaction of Arabidopsis and phytophthora parasitica. According to the embodiment, CS10 can effectively play a role in negative regulation of resistance of phytophthora parasitica; in Nicotiana benthamiana (CS 10 in Nicotiana benthamiana is abbreviated as NbCS10-1 and NbCS 10-1), over-expression of CS10 can promote infection and colonization of Nicotiana benthamiana by phytophthora parasitica; the ability to promote phytophthora parasitica infection is lost after the subcellular localization of CS10 is changed, and cytoplasmic membrane localization is necessary for CS10 to negatively regulate the resistance of Arabidopsis to phytophthora parasitica; CS10 functions in promoting infection of Arabidopsis by phytophthora parasitica and related to key enzyme activity sites, and the coding gene CS10 is also called kinase gene; the function of CS10 to promote phytophthora parasitica infection is related to the GDK domain of its downstream signal pathway.
Drawings
FIG. 1 is a schematic representation of an Arabidopsis T-DNA insertion mutant of AtCS10 against phytophthora parasitica infection; wherein (a) is a schematic representation of the T-DNA insertion site of mutant M13-4; (b) For the detection of the expression level of AtCS10 in the Arabidopsis T-DNA insertion mutant M13-4, atUBC9 was used as an Arabidopsis internal reference gene in a quantitative experiment; (c) The experimental result of inoculating zoospores of phytophthora parasitica to the isolated leaves of the Arabidopsis T-DNA insertion mutant shows that; (d) Quantitative detection results of parasitic phytophthora biomass in infected leaves;
FIG. 2 is a schematic representation of infection of phytophthora parasitica by homozygous stable transgenic plants CS10OE53 and CS10OE69 obtained by resistance screening identification to present a disease phenotype; (a) is a statistical graph of the relative expression level of AtCS 10; (b) is a relative expression statistical graph of phytophthora parasitica; (c) Results after inoculation of Col-0, CS10OE53 and CS10OE69 respectively;
FIG. 3 is a schematic representation of overexpression of AtCS10 to promote infection of P.parasiticus on Nicotiana benthamiana; (a) The inoculation result after transient expression of tobacco (Flag-GFP is expressed on the left side of leaf blades and CS10-GFP is expressed on the right side); (b) For the plaque diameter statistics of the experimental group and the control group two days after inoculation, "x" indicates P <0.05; (c) Quantitative determination of the amount of mycelium colonization in infected leaves (pptg_ 09948/NbActin), "x" indicates P <0.01;
FIG. 4 is a schematic representation of an AtCS10 whose altered localization has lost its ability to promote the infestation of Nicotiana benthamiana by phytophthora parasitica; (a) To inoculate zoospores of phytophthora parasitica on leaves transiently overexpressing CS10M6-30 and CS10CGFP, trypan blue staining of leaves after 3 days of infestation; (b, e) is the statistical analysis result of the diameter of the lesions of the infected leaves, "x" indicates P <0.05; (c, f) is quantitative detection result of mycelium colonization amount in infected leaves (PPTG_ 09948/NbActin); (d) To inoculate zoospores of Phytophthora parasitica on leaves transiently overexpressing CS10M7-12 and CS10CGFP, trypan blue staining of leaves after 3 days of infection;
FIG. 5 is a schematic diagram showing the function of AtCS10 in promoting infection of Arabidopsis by phytophthora parasitica and the key enzyme activity sites thereof; (a) Schematic representation of expression levels of CS10M2 and CS10M3 in complementation transformants, atUBC9 was used as an arabidopsis internal reference gene in quantitative experiments ("x" indicates P <0.01, "x" indicates P < 0.001); (b) showing the result of the in vitro leaf inoculation of arabidopsis thaliana; (c) Quantitative detection results of the colonization amount of hyphae in infected leaves;
FIG. 6 is a schematic diagram of the GDK domain necessary for the AtCS10 protein to promote phytophthora parasitica infection function; (a) For inoculation results after transient expression of tobacco (leaf left hand side expression of CS10M4, right hand side expression of CS10 CFlag), "x" indicates P <0.01; (b) counting the diameter of the lesion in the experimental group and the control group; (c) Quantitative determination of the amount of mycelium colonization in infected leaves (pptg_ 09948/NbActin), "x" indicates P <0.05;
FIG. 7 is a diagram of silencing NbCS10 enhancement by R3a/Avr3a KI Schematic representation of recognition of induced cell necrosis; (a) Is INF1, BAX, NIP and gene pair R3a/Avr3a KI Necrosis of Rb/Avrblb1 and R1/Avr1 on the leaves of TRV-GFP and TRV-NbCS10 gene-silenced plants; (b) is the statistics of the injection spot necrosis in (a);
FIG. 8 is a schematic representation of an alignment of AtCS10, nbCS10-1, nbCS10-2 and potato CS10 protein sequences of the invention.
Detailed Description
The invention provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in cultivation or production of agricultural disease-resistant crop lines. In the present invention, the CS10 protein preferably includes CS10 protein in Arabidopsis thaliana, CS10 protein in Nicotiana benthamiana, and CS10 protein in Solanum tuberosum; the coding genes of CS10 protein preferably comprise coding genes of CS10 protein in Arabidopsis thaliana, coding genes of CS10 protein in Nicotiana benthamiana and coding genes of CS10 protein in potato, and the coding genes of CS10 protein in Nicotiana benthamiana and coding genes of CS10 protein in potato are homologous genes of the coding genes of CS10 protein in Arabidopsis thaliana in corresponding plants.
In the present invention, the amino acid sequence of CS10 protein in Arabidopsis thaliana is preferably as shown in SEQ ID NO. 3.
In the invention, the coding gene of CS10 protein in the Arabidopsis is preferably shown as SEQ ID NO. 7.
In the invention, the amino acid sequence of CS10 protein in the Nicotiana benthamiana is preferably shown as SEQ ID NO. 4,
or preferably SEQ ID NO 5.
In the invention, the coding gene of CS10 protein in the Nicotiana benthamiana is preferably shown as SEQ ID NO. 8,
or preferably as shown in SEQ ID NO. 9.
In the invention, the amino acid sequence of CS10 protein in potato is preferably shown as SEQ ID NO. 6.
In the invention, the coding gene of CS10 protein in the potato is shown as SEQ ID NO. 10.
The invention also provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in cultivating plants with resistance to phytophthora. In a specific embodiment of the invention, the T-DNA of Arabidopsis thaliana of AtCS10, after insertion into the inactivating mutant, exhibits resistance to infection by Phytophthora parasitica.
The invention also provides application of CS10 protein for negative regulation of plant immunity or a coding gene of CS10 protein in regulating plant and phytophthora affinity interaction. In the specific embodiment of the invention, the colonization amount of the phytophthora parasitica in the infected leaves of M13-4 is obviously smaller than that of the wild Col-0, and the inactivated mutants M9-6 and M13-4 of the AtCS10 are resistant to the infection of the phytophthora parasitica, which indicates that the Arabidopsis kinase gene AtCS10 is really involved in the affinity interaction of the Arabidopsis-phytophthora parasitica.
The invention also provides application of the CS10 protein for negatively regulating plant immunity or the coding gene of the CS10 protein in regulating plant and phytophthora parasitica affinity interaction through cytoplasmic membrane positioning. In a specific embodiment of the invention, the subcellular localization of AtCS10 is altered to lose the ability to promote phytophthora parasitica infection.
The invention also provides application of the CS10 protein for negatively regulating plant immunity or the coding gene of the CS10 protein in regulating plant and parasitic phytophthora through two kinase domains, wherein in the application, when the plant is arabidopsis, the amino acid sequences corresponding to the two kinase domains are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. In the specific embodiment of the invention, known active sites (K155 and G252) in the two kinase domains are mutated, and base mutations are respectively and independently introduced into the amino acid sites related to the activity of the 2 proteases to mutate the amino acids of the corresponding sites into asparagine and arginine (K115N and G252R) and obtain 2 mutant forms of AtCS10 protein, wherein the mutation site of the mutant CS10M2 is lysine at 155 and the mutation site of the CS10M3 is glycine at 252. The complementary transgenic plants of mutant proteins CS10M2 and CS10M3 are obtained by an Arabidopsis thaliana flower dipping method with the Arabidopsis thaliana T-DNA insertion mutant M13-4 of AtCS10 as a background. The water stain of CM-line leaf is obviously weaker than that of the leaf of wild Col-0 of the control plant, and the colonization amount of hypha is smaller.
The invention also provides application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein in regulating affinity interaction of Arabidopsis and phytophthora parasitica by virtue of GDK domain related to downstream signal path. Specific examples of the present invention the vector CS10M4 was constructed by introducing base mutations into the amino acid sites (G241, D242, K243) in the motifs individually to mutate the amino acids at the corresponding sites to serine and alanine (G241S, D242A, K243A). By using an agrobacterium-mediated transient expression system, transient over-expression post-inoculation experiments are carried out on the Nicotiana benthamiana leaves, and the experiment shows that the function of AtCS10 for promoting phytophthora parasitica infection is related to the GDK domain of a downstream signal path.
For further explanation of the present invention, the CS10 protein or the gene encoding the CS10 protein for negative regulation of plant immunity provided by the present invention will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
The resistance of the AtCS10 negative regulation plant to phytophthora parasitica is determined by an in vitro leaf inoculation test, the resistance is specifically expressed as an Arabidopsis T-DNA insertion inactivation mutant of the AtCS10, M13-4 (SALK-107785C) is purchased from an Arabidopsis biological resource center (Arabidopsis Biological Resource Center, ABRC)), the insertion site is shown in (a) in figure 1, and (b) is the detection of the expression level of the AtCS10 in the Arabidopsis T-DNA insertion mutant M13-4, and AtUBC9 is used as an Arabidopsis internal reference gene in a quantitative experiment. The insertion inactivated mutant showed resistance to infection by Phytophthora parasitica, and the test results are shown in (c) and (d) in FIG. 1. As can be seen from (d) in FIG. 1, the leaf of insertion mutant M13-4 had a significantly lower degree of susceptibility than wild-type Col-0, and a significant difference from wild-type Col-0.
To further confirm the disease resistant phenotype of the mutants, the biomass of Phytophthora parasitica in the infected leaves of Col-0 and M13-4 was quantified. As shown in FIG. 1 (e), the quantitative results showed that the amounts of phytophthora parasitica colonization in mutant M13-4 were significantly smaller than that in wild-type Col-0 as shown in FIGS. 1 (b) to (e). The disease grade of each group of leaves is consistent with the result of detecting the colonization amount of phytophthora parasitica, and the result shows that the inactivated mutant M13-4 of the AtCS10 is resistant to the infection of the phytophthora parasitica, which proves that the Arabidopsis kinase gene AtCS10 is really involved in the affinity interaction of the Arabidopsis and the phytophthora parasitica.
The method comprises the steps of carrying pART27, designing a full-length gene primer according to a CDS sequence of Arabidopsis CS10 published in a Tair website by a conventional method, amplifying cDNA of wild type Arabidopsis Col-0 by using the primer, purifying and recovering the PCR product after electrophoresis, carrying out enzyme digestion on the vector pART27 connected with GFP by using endoenzymes EcoR1 and BamHI, inserting a CS10 fragment into the vector for connection, infecting the wild type Arabidopsis Col-0 by agrobacterium, and obtaining homozygous stable transgenic plants CS10OE53 and CS10OE69 which overexpress AtCS10 through resistance screening identification to present a disease-sensitive phenotype to infection of phytophthora parasitica by adopting a method of inoculating zoospores of the phytophthora parasitica by using isolated Arabidopsis leaves of about 4 weeks of seedlings, and analyzing the resistance of the CS10OE53 and CS10OE69 to the phytophthora parasitica by using the control group of wild type Arabidopsis Col-0. The results show that after 3 days of single-sided scratch inoculation, the CS10OE53 and CS10OE69 leaves are found to have extremely serious water-borne lesions compared with Col-0 and almost spread to most of the leaves of Phytophthora parasitica to infect wild-type Arabidopsis thaliana Col-0, CS10OE53 and CS10OE69, the results are shown in FIG. 2, and FIG. 2 is a schematic diagram showing the infection of Phytophthora parasitica by homozygous stable transgenic plants CS10OE53 and CS10OE69 obtained by resistance screening identification to overexpress AtCS 10; wherein (a) is a statistical graph of the relative expression amount of AtCS 10; wherein (b) is a relative expression statistical graph of phytophthora parasitica; wherein (c) is the result of inoculation of Col-0, CS10OE53 and CS10OE69, respectively, and it is known from FIG. 2 (a) that the expression level of AtCS10 in the overexpressed transformants CS10OE53 and CS10OE69 was confirmed by real-time fluorescence quantitative PCR, and that the AtCS10 gene was up-regulated to a higher level in both the overexpressed lines CS10OE53 and CS10OE 69; (b) Analysis of biomass of phytophthora parasitica infection of arabidopsis thaliana shows that the colonization amount of phytophthora parasitica in the over-expression lines CS10OE53 and CS10OE69 is obviously higher than that of the wild-type Col-0; (c) It is seen that the lesions on the leaves of CS10OE53 and CS10OE69 were larger in diameter and the water spots were more pronounced.
The method comprises the steps of utilizing an agrobacterium-mediated transient expression system to transiently overexpress AtCS10 on leaf blades of Nicotiana benthamiana, inoculating zoospores of phytophthora parasitica on two sides of the back surface of an in-vitro injection leaf blade after 2 days of injection, and enabling the overexpression of the AtCS10 to promote the infection and colonization of the Nicotiana benthamiana by the phytophthora parasitica, wherein a result is shown in a diagram in FIG. 3, and a diagram in FIG. 3 shows that the overexpression of the AtCS10 promotes the infection of the Nicotiana benthamiana by the phytophthora parasitica; wherein (a) is the inoculation result after transient expression of tobacco (Flag-GFP is expressed on the left side of leaf blade, CS10-GFP is expressed on the right side); wherein (b) is the statistical result of the diameter of the lesion in the experimental group and the control group after two days of inoculation; as shown in FIG. 3 (a), the quantitative determination result of the amount of colonization of mycelium in infected leaves (PPTG_ 09948/NbActin) shows that the diameter of the lesion is larger and the water lesion is more remarkable than that of the control leaves (leaves over-expressing Flag-GFP). (b) The results show that the leaf spot diameter of the transient over-expressed CS10-GFP on the right side of the tobacco is larger than that of the left control group (transient over-expressed Flag-GFP); (c) The colonization amount of phytophthora parasitica on the side of the over-expressed CS10-GFP was found to be significantly higher than that of the side of the over-expressed Flag-GFP, which is in good agreement with the lesion statistics.
Example 2
The AtCS10 protein is positioned in a cytoplasmic membrane, the myristoylation site and 2 palmitoylation sites of the AtCS10 gene are related to the subcellular localization of the AtCS10, the subcellular localization of the AtCS10 is changed by mutating the sites, and a nuclear localization sequence (Nuclearlocalization signal, NLS) is added at the N end to localize the protein in a cell nucleus so as to explore whether the change of the subcellular localization affects the gene AtCS10 to regulate the plant immune function.
The results of the inoculation test performed after over-expression on Nicotiana benthamiana leaves using the Agrobacterium-mediated transient expression system are shown in FIG. 4. FIG. 4 (a) shows trypan blue staining of leaves after 3 days of infection by inoculating zoospores of Phytophthora parasitica onto leaves transiently overexpressing CS10M6-30 and CS10 CGFP; statistical analysis of the diameter of lesions of (e, h) infected leaves in fig. 4, "x" indicates P <0.05; FIG. 4 (f, i) quantitative determination of mycelium colonization in infected leaves (PPTG_ 09948/NbActin); FIG. 4 (d) shows trypan blue staining of leaves after 3 days of infection by inoculating zoospores of Phytophthora parasitica onto leaves transiently overexpressing CS10M7-12 and CS10 CGFP. The results showed that the lesion diameter was smaller and the amount of colonization by hyphae was reduced as compared with the control leaf (leaf expressing AtCS 10-GFP). The above results indicate that the ability to promote phytophthora parasitica infection is lost after the subcellular localization of AtCS10 is changed, and cytoplasmic membrane localization is necessary for AtCS10 to negatively regulate phytophthora nicotianae resistance.
Example 3
By performing sequence alignment and secondary structure prediction, it was found that the protein sequence of AtCS10 contains two typical kinase domains. The known active sites (K155, G252) in the two kinase domains were mutated, and base mutations were introduced separately at the 2 protease activity-related amino acid sites, respectively, to mutate the amino acids at the corresponding sites to asparagine and arginine (K115N, G252R) and to obtain 2 mutant forms of AtCS10 protein, wherein the mutant CS10M2 mutation site was lysine at position 155 and the CS10M3 mutation site was glycine at position 252. Then, by using the Arabidopsis T-DNA insertion mutant M13-4 of AtCS10 as a background, the complementation transgenic plants of the mutant proteins CS10M2 and CS10M3 were obtained by an Arabidopsis dip-in method. Then, experiments of inoculating zoospores of phytophthora parasitica on the in-vitro leaves are carried out, and the results are shown in figure 5. Wherein (a) in FIG. 5 is that the expression levels of CS10M2 and CS10M3 were significantly up-regulated in the complementation transformants. AtUBC9 as an arabidopsis internal reference gene in quantitative experiments ("x" means P <0.01, "x" means P < 0.001); the results of in vitro leaf inoculation of Arabidopsis thaliana in FIG. 5 (b) show that CS10M2CM and CS10M3CM showed disease resistance to Phytophthora parasitica infection compared to wild type Arabidopsis thaliana. At least 10 leaves from different plants were tested in each experiment with 3 independent biological replicates; FIG. 5 (c) shows quantitative determination of the amount of colonization of hyphae in infected leaves. As can be seen from FIG. 5, the water stain of CM-line leaf is significantly weaker than that of the leaf of the wild type Col-0 of the control plant, and the result of the smaller colonization amount of hypha indicates that the function of AtCS10 for promoting the infection of the Arabidopsis by the phytophthora parasitica is related to the key enzyme activity site.
Example 4
The arabidopsis gene AtCS10 was subjected to transient over-expression experiments on tobacco.
By performing sequence alignment and secondary structure prediction, the AtCS10 protein sequence was found to contain a GDK domain associated with downstream signaling pathways. The construction of the vector CS10M4 was carried out by introducing the amino acid sites (G241, D242, K243) in the motifs individually into base mutations to mutate the amino acids at the corresponding sites to serine and alanine (G241S, D242A, K243A). The transient over-expression post-inoculation experiment is carried out on the leaf of Nicotiana benthamiana by using an agrobacterium-mediated transient expression system, the result is shown in (a) in fig. 6, and (a) in fig. 6 is the inoculation result after transient expression of tobacco (CS 10M4 is expressed on the left side of the leaf and CS10CFlag is expressed on the right side), and the area of the water stain on one side of the leaf in which CS10M4 is expressed in fig. 6 is obviously reduced compared with that on the other side of the leaf in which CS10CFlag is over-expressed. Statistical results of lesion diameters as shown in fig. 6 (b), which shows that leaf lesion diameters of the transient over-expressed CS10M4 on the left side of tobacco are smaller than those of the control group on the right side (transient over-expressed CS10 CFlag) and there are significant differences. The quantitative determination result of the parasitic phytophthora biomass in the infected leaf is shown in (c) in fig. 6, and the (c) in fig. 6 shows that the colonization amount of the parasitic phytophthora on the side of the over-expression CS10M4 is lower than that of the side of the over-expression CS10CFlag, and the result is in close agreement with the disease spot statistical result, which shows that the function of the AtCS10 for promoting the parasitic phytophthora infection is related to the GDK domain of a downstream signal channel.
Example 5
By utilizing the Benshi tobacco genome sequence provided by the Solanaceae plant website Sol Genomics Network, 2 sequences with higher homology with Arabidopsis thaliana CS10 protein are found in the Benshi tobacco genome through amino acid sequence comparison, the homology with AtCS10 is between 70% and 80%, the protein sequences of the two homologous genes are different from each other by only one amino acid, the genes encoding the two protein sequences are respectively named as NbCS10-1, nbCS10-2, the amino acid sequences of CS10 proteins in Benshi tobacco are respectively SEQ ID NO 4 and SEQ ID NO 5, and the nucleotide sequences of NbCS10-1 and NbCS10-2 are respectively SEQ ID NO 8 and SEQ ID NO 9. The 2 homologous genes are simultaneously silenced by using the VIGS technology, and then the zoospore of the phytophthora parasitica and the zoospore of the phytophthora infestans are carried out, and the result shows that the NbCS10-1 and NbCS10-2 silenced plants are more resistant to the infection of the phytophthora parasitica and the phytophthora infestans compared with control plants, and the result further confirms that the kinase gene CS10 negatively regulates the resistance of the plants to the phytophthora parasitica.
Example 6
2 tobacco homologous genes NbCS10-1 and NbCS10-2 are simultaneously silenced by injecting agrobacterium carrying silencing NbCS10-1 and NbCS10-2 gene vectors on Nicotiana benthamiana seedlings in a 5-6 leaf stage by using a virus-mediated gene silencing technique (VIGS). Necrosis-inducing factor INF, BAX, NIP was transiently expressed on flattened leaves in the same leaf position as the albino injection of TRV-PDS, and on the gene pairs R3a/Avr3aKI, rb/Avrblb1 and R1/Avr1, obtained on control plants TRV-GFP and NbCS10-1 and NbCS10-2 silenced Nicotiana benthamiana. The injection spot necrosis was observed after about 5 days of injection, and the results are shown in FIG. 7. FIG. 7, which shows that the degree and frequency of the injection spot necrosis of INF, BAX and NIP on the leaf of the silencing plant TRV-NbCS10 were not significantly different from those of the corresponding injection spot on the leaf of the control plant TRV-GFP, and the above results indicate that silencing NbCS10-1 and NbCS10-2 did not affect the PTI defense response of the plant. The extent and frequency of allergic cell necrosis induced by recognition of potato disease-resistant protein R3a and phytophthora infestans effector protein Avr3aKI on leaves of the gene-silenced plant TRV-NbCS10 are particularly serious compared with those of control plant leaves, rb/Avrblb1 and R1/Avr1 are seriously necrotized on the silenced plant and the control plant leaves, and no obvious difference exists between the two, so that the effect of enhancing the allergic cell necrosis induced by recognition of potato disease-resistant protein R3a and phytophthora infestans effector protein Avr3aKI by silencing the homologous gene NbCS10 of AtCS10 in Nicotiana benthamiana by using a VIGS technology is shown that the ETI disease-resistant pathway of plants is influenced by NbCS10-1 and NbCS 10-2.
Example 7
FIG. 8 is a schematic diagram showing the alignment of AtCS10 and NbCS10-1, nbCS10-2 and potato XP_006359071.1 (StCS 10) proteins of the present invention.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Sequence listing
<110> university of agriculture and forestry science and technology in northwest
<120> negative control of plant immunity CS10 protein or application of CS10 protein coding gene
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 269
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Leu Gly Glu Gly Gly Phe Gly Arg Val Tyr Lys Gly Arg Leu Asp Ser
1 5 10 15
Thr Gly Gln Val Val Ala Val Lys Gln Leu Asp Arg Asn Gly Leu Gln
20 25 30
Gly Asn Arg Glu Phe Leu Val Glu Val Leu Met Leu Ser Leu Leu His
35 40 45
His Pro Asn Leu Val Asn Leu Ile Gly Tyr Cys Ala Asp Gly Asp Gln
50 55 60
Arg Leu Leu Val Tyr Glu Phe Met Pro Leu Gly Ser Leu Glu Asp His
65 70 75 80
Leu His Asp Leu Pro Pro Asp Lys Glu Ala Leu Asp Trp Asn Met Arg
85 90 95
Met Lys Ile Ala Ala Gly Ala Ala Lys Gly Leu Glu Phe Leu His Asp
100 105 110
Lys Ala Asn Pro Pro Val Ile Tyr Arg Asp Phe Lys Ser Ser Asn Ile
115 120 125
Leu Leu Asp Glu Gly Phe His Pro Lys Leu Ser Asp Phe Gly Leu Ala
130 135 140
Lys Leu Gly Pro Thr Gly Asp Lys Ser His Val Ser Thr Arg Val Met
145 150 155 160
Gly Thr Tyr Gly Tyr Cys Ala Pro Glu Tyr Ala Met Thr Gly Gln Leu
165 170 175
Thr Val Lys Ser Asp Val Tyr Ser Phe Gly Val Val Phe Leu Glu Leu
180 185 190
Ile Thr Gly Arg Lys Ala Ile Asp Ser Glu Met Pro His Gly Glu Gln
195 200 205
Asn Leu Val Ala Trp Ala Arg Pro Leu Phe Asn Asp Arg Arg Lys Phe
210 215 220
Ile Lys Leu Ala Asp Pro Arg Leu Lys Gly Arg Phe Pro Thr Arg Ala
225 230 235 240
Leu Tyr Gln Ala Leu Ala Val Ala Ser Met Cys Ile Gln Glu Gln Ala
245 250 255
Ala Thr Arg Pro Leu Ile Ala Asp Val Val Thr Ala Leu
260 265
<210> 2
<211> 197
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Phe Leu Gly Glu Gly Gly Phe Gly Arg Val Tyr Lys Gly Arg Leu Asp
1 5 10 15
Ser Thr Gly Gln Val Val Ala Val Lys Gln Leu Asp Arg Asn Gly Leu
20 25 30
Gln Gly Asn Arg Glu Phe Leu Val Glu Val Leu Met Leu Ser Leu Leu
35 40 45
His His Pro Asn Leu Val Asn Leu Ile Gly Tyr Cys Ala Asp Gly Asp
50 55 60
Gln Arg Leu Leu Val Tyr Glu Phe Met Pro Leu Gly Ser Leu Glu Asp
65 70 75 80
His Leu His Asp Leu Pro Pro Asp Lys Glu Ala Leu Asp Trp Asn Met
85 90 95
Arg Met Lys Ile Ala Ala Gly Ala Ala Lys Gly Leu Glu Phe Leu His
100 105 110
Asp Lys Ala Asn Pro Pro Val Ile Tyr Arg Asp Phe Lys Ser Ser Asn
115 120 125
Ile Leu Leu Asp Glu Gly Phe His Pro Lys Leu Ser Asp Phe Gly Leu
130 135 140
Ala Lys Leu Gly Pro Thr Gly Asp Lys Ser His Val Ser Thr Arg Val
145 150 155 160
Met Gly Thr Tyr Gly Tyr Cys Ala Pro Glu Tyr Ala Met Thr Gly Gln
165 170 175
Leu Thr Val Lys Ser Asp Val Tyr Ser Phe Gly Val Val Phe Leu Glu
180 185 190
Leu Ile Thr Gly Arg
195
<210> 3
<211> 456
<212> PRT
<213> Artificial sequence (Artificial Sequence)
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Met Gly Cys Phe Ser Cys Phe Asp Ser Ser Asp Asp Glu Lys Leu Asn
1 5 10 15
Pro Val Asp Glu Ser Asn His Gly Gln Lys Lys Gln Ser Gln Pro Thr
20 25 30
Val Ser Asn Asn Ile Ser Gly Leu Pro Ser Gly Gly Glu Lys Leu Ser
35 40 45
Ser Lys Thr Asn Gly Gly Ser Lys Arg Glu Leu Leu Leu Pro Arg Asp
50 55 60
Gly Leu Gly Gln Ile Ala Ala His Thr Phe Ala Phe Arg Glu Leu Ala
65 70 75 80
Ala Ala Thr Met Asn Phe His Pro Asp Thr Phe Leu Gly Glu Gly Gly
85 90 95
Phe Gly Arg Val Tyr Lys Gly Arg Leu Asp Ser Thr Gly Gln Val Val
100 105 110
Ala Val Lys Gln Leu Asp Arg Asn Gly Leu Gln Gly Asn Arg Glu Phe
115 120 125
Leu Val Glu Val Leu Met Leu Ser Leu Leu His His Pro Asn Leu Val
130 135 140
Asn Leu Ile Gly Tyr Cys Ala Asp Gly Asp Gln Arg Leu Leu Val Tyr
145 150 155 160
Glu Phe Met Pro Leu Gly Ser Leu Glu Asp His Leu His Asp Leu Pro
165 170 175
Pro Asp Lys Glu Ala Leu Asp Trp Asn Met Arg Met Lys Ile Ala Ala
180 185 190
Gly Ala Ala Lys Gly Leu Glu Phe Leu His Asp Lys Ala Asn Pro Pro
195 200 205
Val Ile Tyr Arg Asp Phe Lys Ser Ser Asn Ile Leu Leu Asp Glu Gly
210 215 220
Phe His Pro Lys Leu Ser Asp Phe Gly Leu Ala Lys Leu Gly Pro Thr
225 230 235 240
Gly Asp Lys Ser His Val Ser Thr Arg Val Met Gly Thr Tyr Gly Tyr
245 250 255
Cys Ala Pro Glu Tyr Ala Met Thr Gly Gln Leu Thr Val Lys Ser Asp
260 265 270
Val Tyr Ser Phe Gly Val Val Phe Leu Glu Leu Ile Thr Gly Arg Lys
275 280 285
Ala Ile Asp Ser Glu Met Pro His Gly Glu Gln Asn Leu Val Ala Trp
290 295 300
Ala Arg Pro Leu Phe Asn Asp Arg Arg Lys Phe Ile Lys Leu Ala Asp
305 310 315 320
Pro Arg Leu Lys Gly Arg Phe Pro Thr Arg Ala Leu Tyr Gln Ala Leu
325 330 335
Ala Val Ala Ser Met Cys Ile Gln Glu Gln Ala Ala Thr Arg Pro Leu
340 345 350
Ile Ala Asp Val Val Thr Ala Leu Ser Tyr Leu Ala Asn Gln Ala Tyr
355 360 365
Asp Pro Ser Lys Asp Asp Ser Arg Arg Asn Arg Asp Glu Arg Gly Ala
370 375 380
Arg Leu Ile Thr Arg Asn Asp Asp Gly Gly Gly Ser Gly Ser Lys Phe
385 390 395 400
Asp Leu Glu Gly Ser Glu Lys Glu Asp Ser Pro Arg Glu Thr Ala Arg
405 410 415
Ile Leu Asn Arg Asp Ile Asn Arg Glu Arg Ala Val Ala Glu Ala Lys
420 425 430
Met Trp Gly Glu Ser Leu Arg Glu Lys Arg Arg Gln Ser Glu Gln Gly
435 440 445
Thr Ser Glu Ser Asn Ser Thr Gly
450 455
<210> 4
<211> 448
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Met Gly Cys Phe Ser Cys Phe Asp Ser Lys Glu Glu Glu Lys Leu Asn
1 5 10 15
Pro Gln Arg Asp Asp Arg Lys Glu Val His Leu Thr Ala Pro Ser Asn
20 25 30
Ile Ser Arg Leu Ser Ser Gly Ala Asp Arg Leu Lys Thr Arg Ser Ile
35 40 45
Asn Gly Ser Lys Arg Glu Phe Leu Gly Leu Lys Asp Ala Pro Asp Val
50 55 60
Gln Ile Ala Ala His Thr Phe Thr Phe Arg Glu Leu Ala Ala Ala Thr
65 70 75 80
Ser Asn Phe Arg Pro Glu Ser Phe Ile Gly Glu Gly Gly Phe Gly Arg
85 90 95
Val Tyr Lys Gly Arg Leu Pro Ser Gly Gln Val Val Ala Val Lys Gln
100 105 110
Leu Asp Arg Asn Gly Leu Gln Gly Asn Arg Glu Phe Leu Val Glu Val
115 120 125
Leu Met Leu Ser Leu Leu His His Pro Asn Leu Val Asn Leu Ile Gly
130 135 140
Tyr Cys Ala Asp Gly Asp Gln Arg Leu Leu Val Tyr Glu Phe Met Pro
145 150 155 160
Leu Gly Ser Leu Glu Asp His Leu His Asp Leu Pro Pro Asp Lys Glu
165 170 175
Pro Val Asp Trp Asn Thr Arg Met Lys Ile Ala Ala Gly Ala Ala Lys
180 185 190
Gly Leu Glu Tyr Leu His Asp Lys Ala Asn Pro Pro Val Ile Tyr Arg
195 200 205
Asp Phe Lys Ser Ser Asn Ile Leu Leu Glu Glu Asn Phe Phe Pro Lys
210 215 220
Leu Ser Asp Phe Gly Leu Ala Lys Leu Gly Pro Thr Gly Asp Lys Ser
225 230 235 240
His Val Ser Thr Arg Val Met Gly Thr Tyr Gly Tyr Cys Ala Pro Glu
245 250 255
Tyr Ala Met Thr Gly Gln Leu Thr Val Lys Ser Asp Val Tyr Ser Phe
260 265 270
Gly Val Val Phe Leu Glu Leu Ile Thr Gly Arg Lys Ala Ile Asp Ser
275 280 285
Thr Met Pro Gln Gly Glu Gln Asn Leu Val Ala Trp Ala Arg Pro Leu
290 295 300
Phe Asn Asp Arg Arg Lys Phe Ala Lys Leu Ala Asp Pro Arg Leu Gln
305 310 315 320
Gly Gln Phe Pro Met Arg Gly Leu Tyr Gln Ala Leu Ala Val Ala Ser
325 330 335
Met Cys Ile Gln Glu Gln Ala Ala Ala Arg Pro Leu Ile Gly Asp Val
340 345 350
Val Thr Ala Leu Ser Tyr Leu Ala Asn Gln Val Asp Asp Lys Arg Asn
355 360 365
Lys Asp Asp Arg Gly Gly Arg Ile Cys Arg Asn Glu Asp Gly Ala Gly
370 375 380
Gly Gly Ser Gly Arg Lys Trp Pro Asp Leu Asp Gly Gly Ser Glu Lys
385 390 395 400
Glu Asp Ser Pro Arg Glu Thr Ala Arg Met Leu Asn Arg Asp Leu Asp
405 410 415
Arg Glu Arg Ala Val Ala Glu Ala Lys Met Trp Gly Glu Asn Trp Arg
420 425 430
Glu Lys Arg Arg Gln Asn Ala Gln Gly Ser Phe Asp Gly Thr Asn Gly
435 440 445
<210> 5
<211> 448
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Met Gly Cys Phe Ser Cys Phe Asp Ser Lys Glu Glu Glu Lys Leu Asn
1 5 10 15
Pro Gln Arg Asp Asp Arg Lys Glu Val His Leu Thr Ala Pro Ser Asn
20 25 30
Ile Ser Arg Leu Ser Ser Gly Ala Asp Arg Leu Lys Thr Arg Ser Ile
35 40 45
Asn Gly Ser Lys Arg Glu Phe Leu Gly Leu Lys Asp Ala Pro Asp Val
50 55 60
Gln Ile Ala Ala His Thr Phe Thr Phe Arg Glu Leu Ala Ala Ala Thr
65 70 75 80
Ser Asn Phe Arg Pro Glu Ser Phe Ile Gly Glu Gly Gly Phe Gly Arg
85 90 95
Val Tyr Lys Gly Arg Leu Pro Ser Gly Gln Val Val Ala Val Lys Gln
100 105 110
Leu Asp Arg Asn Gly Leu Gln Gly Asn Arg Glu Phe Leu Val Glu Val
115 120 125
Leu Met Leu Ser Leu Leu His His Pro Asn Leu Val Asn Leu Ile Gly
130 135 140
Tyr Cys Ala Asp Gly Asp Gln Arg Leu Leu Val Tyr Glu Phe Met Pro
145 150 155 160
Leu Gly Ser Leu Glu Asp His Leu His Asp Leu Pro Pro Asp Lys Glu
165 170 175
Pro Leu Asp Trp Asn Thr Arg Met Lys Ile Ala Ala Gly Ala Ala Lys
180 185 190
Gly Leu Glu Tyr Leu His Asp Lys Ala Asn Pro Pro Val Ile Tyr Arg
195 200 205
Asp Phe Lys Ser Ser Asn Ile Leu Leu Glu Glu Asn Phe Phe Pro Lys
210 215 220
Leu Ser Asp Phe Gly Leu Ala Lys Leu Gly Pro Thr Gly Asp Lys Ser
225 230 235 240
His Val Ser Thr Arg Val Met Gly Thr Tyr Gly Tyr Cys Ala Pro Glu
245 250 255
Tyr Ala Met Thr Gly Gln Leu Thr Val Lys Ser Asp Val Tyr Ser Phe
260 265 270
Gly Val Val Phe Leu Glu Leu Ile Thr Gly Arg Lys Ala Ile Asp Ser
275 280 285
Thr Met Pro Gln Gly Glu Gln Asn Leu Val Ala Trp Ala Arg Pro Leu
290 295 300
Phe Asn Asp Arg Arg Lys Phe Ala Lys Leu Ala Asp Pro Arg Leu Gln
305 310 315 320
Gly Gln Phe Pro Met Arg Gly Leu Tyr Gln Ala Leu Ala Val Ala Ser
325 330 335
Met Cys Ile Gln Glu Gln Ala Ala Ala Arg Pro Leu Ile Gly Asp Val
340 345 350
Val Thr Ala Leu Ser Tyr Leu Ala Asn Gln Val Asp Asp Lys Arg Asn
355 360 365
Lys Asp Asp Arg Gly Gly Arg Ile Cys Arg Asn Glu Asp Gly Ala Gly
370 375 380
Gly Gly Ser Gly Arg Lys Trp Pro Asp Leu Asp Gly Gly Ser Glu Lys
385 390 395 400
Glu Asp Ser Pro Arg Glu Thr Ala Arg Met Leu Asn Arg Asp Leu Asp
405 410 415
Arg Glu Arg Ala Val Ala Glu Ala Lys Met Trp Gly Glu Asn Trp Arg
420 425 430
Glu Lys Arg Arg Gln Asn Ala Gln Gly Ser Phe Asp Gly Thr Asn Gly
435 440 445
<210> 6
<211> 468
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Met Gly Cys Phe Ser Cys Phe Asp Ser Lys Glu Asp Glu Lys Leu Asn
1 5 10 15
Pro Gln Lys Asp Arg Asp Asp Ser Asn Arg Lys Gln Pro His Leu Thr
20 25 30
Ala Pro Ser Asn Ile Ser Arg Leu Ser Ser Gly Ala Asp Arg Leu Lys
35 40 45
Thr Arg Ser Thr Asn Cys Ser Lys Arg Glu Phe Leu Gly Ile Lys Asp
50 55 60
Ala Pro Asp Val Gln Ile Ala Ala His Thr Phe Thr Phe Arg Glu Leu
65 70 75 80
Ala Ala Ala Thr Asn Asn Phe Lys Pro Glu Ser Phe Ile Gly Glu Gly
85 90 95
Gly Phe Gly Arg Val Tyr Lys Gly Gln Leu Pro Ser Gly Gln Val Val
100 105 110
Ala Val Lys Gln Leu Asp Arg Asn Gly Leu Gln Gly Asn Arg Glu Phe
115 120 125
Leu Val Glu Val Leu Met Leu Ser Leu Leu His His Pro Asn Leu Val
130 135 140
Ser Leu Ile Gly Tyr Cys Ala Asp Gly Asp Gln Arg Leu Leu Val Tyr
145 150 155 160
Glu Phe Met Pro Leu Gly Ser Leu Glu Asp His Leu His Asp Leu Pro
165 170 175
Pro Asp Lys Glu Pro Leu Asp Trp Asn Thr Arg Met Thr Ile Ala Ser
180 185 190
Gly Ala Ala Lys Gly Leu Glu His Leu His Asp Lys Ala Asn Pro Pro
195 200 205
Val Ile Tyr Arg Asp Phe Lys Ser Ser Asn Ile Leu Leu Lys Asp Asn
210 215 220
Phe Phe Pro Lys Leu Ser Asp Phe Gly Leu Ala Lys Leu Gly Pro Thr
225 230 235 240
Gly Asp Lys Ser His Val Ser Thr Arg Val Met Gly Thr Tyr Gly Tyr
245 250 255
Cys Ala Pro Glu Tyr Ala Met Thr Gly Gln Leu Thr Val Lys Ser Asp
260 265 270
Val Tyr Ser Phe Gly Val Val Phe Leu Glu Leu Ile Thr Gly Arg Lys
275 280 285
Ala Ile Asp Ser Thr Lys Pro Gln Gly Glu Gln Asn Leu Val Ala Trp
290 295 300
Ala Arg Pro Leu Phe Asn Asp Arg Arg Lys Phe Ala Lys Leu Ala Asp
305 310 315 320
Pro Ser Leu Gln Gly Gln Phe Pro Met Arg Gly Leu Tyr Gln Ala Leu
325 330 335
Ala Val Ala Ser Met Cys Ile Gln Glu Gln Ala Ala Gly Arg Pro Leu
340 345 350
Ile Gly Asp Val Val Thr Ala Leu Ser Tyr Leu Ala Asn Gln Ser Tyr
355 360 365
Asp Pro Gly Thr Val Pro Gly Gln Ile His Arg Phe Gly Ala Asp Ser
370 375 380
Val Asp Arg Arg Asn Lys Asp Asp Arg Val Gly Arg Ile Phe Arg Asn
385 390 395 400
Glu Asp Gly Ala Gly Gly Gly Ser Gly Gln Lys Trp Asp Val Asp Gly
405 410 415
Gly Ser Glu Lys Glu Asp Ser Pro Arg Glu Thr Ala Arg Met Leu Asn
420 425 430
Arg Asp Leu Asp Arg Glu Arg Ala Val Ala Glu Ala Lys Met Trp Gly
435 440 445
Glu Asn Trp Arg Asp Lys Arg Arg Gln Asn Gly Gln Gly Ser Phe Asp
450 455 460
Gly Gly Asn Glu
465
<210> 7
<211> 1371
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgggttgtt tctcgtgttt tgattcgagt gatgacgaga agctgaatcc agttgatgaa 60
tctaatcatg gtcagaagaa acaatcacaa ccgacagtat ccaataacat atctggactc 120
ccttcaggtg gggagaagct tagctcaaag accaatggag gatcaaaaag ggagctactg 180
cttccaaggg atggacttgg acaaattgct gctcatacat ttgctttccg cgagcttgct 240
gctgcaacta tgaactttca tcctgacact ttcttaggcg aaggtggatt tggacgtgtc 300
tacaaaggaa ggcttgacag caccggtcag gttgttgctg ttaaacaact agacaggaat 360
ggtctacaag gtaacagaga atttctggta gaggttctta tgctcagtct tcttcatcat 420
cccaacttag tcaaccttat tggttattgt gctgatggag atcaacgcct cttggtctac 480
gagtttatgc cgttaggatc attggaagat cacctccacg atcttccacc ggataaggag 540
gccttagatt ggaacatgag aatgaaaata gctgctggtg cggcgaaagg attggaattt 600
ctacatgata aggcaaaccc tccggttatt tatagagatt ttaagtcatc aaatatttta 660
ctggatgagg gtttccaccc taagctttct gattttggac ttgctaaact cggaccaacg 720
ggagacaaat ctcatgtctc cactagagtt atgggaactt atggttattg tgctcccgag 780
tacgcaatga cgggacaatt gacagtaaaa tcagatgtct acagttttgg tgtggttttt 840
ctcgagctca ttactggtcg caaagctata gacagcgaga tgcctcatgg agagcagaac 900
ctggtggctt gggctcgccc attgttcaac gacaggcgaa agttcataaa actggctgat 960
ccaaggttaa aggggcggtt tccaacgcgt gcactctacc aagctttagc tgtggcatca 1020
atgtgcatcc aagaacaggc ggctacacgt cctctcatag cagatgttgt cactgcactc 1080
tcctatcttg caaaccaagc ttatgatcca agtaaagatg atagtagaag aaaccgggat 1140
gaaagaggtg caaggttaat aacaaggaac gacgatggag gtggctcggg aagtaaattc 1200
gatttagaag gttcagagaa agaagattca ccgagagaga cagctcggat attgaaccga 1260
gatatcaata gggagcgtgc ggttgcagag gctaagatgt ggggagagag tttgagggag 1320
aaacgaagac agagcgagca gggtacttca gagagcaaca gtaccgggta g 1371
<210> 8
<211> 1347
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atgggttgct tctcctgttt tgattcaaag gaagaggaga agctgaatcc tcaaagagat 60
gatcgcaagg aagtccatct gacggctcct tcaaatatat ccaggttatc ttcaggagca 120
gacagactga aaacaagaag tatcaatggt tcgaagagag agtttttggg gctaaaggat 180
gcacctgacg ttcagattgc tgcacacaca ttcactttcc gtgagcttgc agctgctaca 240
agtaacttca ggccagaatc ttttataggt gaaggagggt ttggacgtgt gtataaaggg 300
agactaccca gcggtcaggt tgttgcagta aagcaattgg ataggaatgg gcttcagggg 360
aatagagaat ttttagtgga ggtgcttatg cttagtcttc ttcatcatcc taacttggtg 420
aacttaattg gttattgtgc tgacggggac cagaggcttc ttgtctacga gttcatgcct 480
ttgggatcac tagaggatca tcttcatgat ctccctcctg ataaagaacc agtagattgg 540
aacacgagaa tgaagatagc agctggtgca gcaaaaggtt tggagtacct tcatgataag 600
gcaaaccctc ctgttattta tagggacttc aagtcatcca acatattgct cgaggaaaat 660
tttttcccga agctttcaga ttttgggctt gctaaacttg gtcctactgg agacaagtca 720
catgtgtcca caagggtcat gggaacttat ggttactgtg cccctgagta tgccatgact 780
ggacaattga ctgtaaaatc tgatgtctat agttttgggg ttgtcttctt ggagcttatc 840
actgggcgta aggctattga cagcaccatg cctcagggag agcaaaacct tgtcgcatgg 900
gctaggccac tgtttaatga tcgtaggaag tttgcaaaat tagcagatcc aaggctgcaa 960
ggacaatttc caatgagagg tctataccag gctttagctg tggcgtccat gtgtatccaa 1020
gaacaggctg ctgctcgtcc tctaatcggg gatgtagtta ctgccctttc ttatctagca 1080
aatcaggtag atgataagag aaataaagat gatagaggtg ggagaatatg caggaatgaa 1140
gatggggcag gaggaggatc aggacggaaa tggccggact tggatggagg atctgagaag 1200
gaggattctc caagggaaac agcaaggatg ttaaacaggg atttggaccg agaaagagca 1260
gttgcggagg ctaaaatgtg gggcgaaaat tggagagaaa agagaagaca aaatgcgcaa 1320
ggcagttttg atggcactaa tggatag 1347
<210> 9
<211> 1347
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atgggttgct tctcctgttt tgattcaaag gaagaggaga agctgaatcc tcagagagat 60
gatcgaaagg aagtccatct gacggctcct tcaaatatat ccaggttatc ttcaggtgca 120
gacagactga aaacaagaag tatcaatggt tcgaagagag agtttttggg gctaaaggat 180
gcacctgacg ttcagattgc tgcacacaca ttcactttcc gtgagcttgc agctgctaca 240
agtaacttca ggccagaatc atttataggt gaaggagggt ttggacgtgt ttataaaggg 300
cgactaccta gcggtcaggt tgttgcagta aagcaattgg atagaaatgg gcttcagggg 360
aatagagaat ttttagtgga ggtgcttatg cttagtcttc ttcatcatcc taacttggtg 420
aacttaattg gttattgtgc tgacggggac cagaggcttc ttgtctacga gttcatgcct 480
ttgggatcac tagaggatca ccttcatgat ctccctcctg acaaagagcc actagattgg 540
aacacgagaa tgaagatagc agctggtgca gcaaaaggtt tggagtacct tcatgataag 600
gcaaaccctc ctgttattta tagggacttc aagtcatcca acatattgct cgaggaaaat 660
tttttcccga agctttcaga ttttgggctt gcaaaacttg gtcctactgg agacaagtca 720
catgtgtcca caagggtcat gggaacttat ggttactgtg cccctgagta tgctatgact 780
ggacaattga ctgtaaaatc tgatgtctat agttttgggg ttgtcttctt ggagctcatc 840
actgggcgta aggctattga cagcaccatg cctcagggag agcaaaacct tgtcgcatgg 900
gctaggccac tgtttaatga tcgtaggaag tttgcaaaat tagcagatcc aaggctgcaa 960
ggacaatttc caatgagagg tctataccag gctttagctg tggcgtccat gtgtatccaa 1020
gaacaggctg ctgctcgtcc tctaatcggg gatgtagtta ctgccctttc ttatctagca 1080
aatcaggtag atgataagag aaataaagat gatagaggtg ggagaatatg caggaatgaa 1140
gatggggcag gaggaggatc aggacggaaa tggccggact tggatggagg atctgagaag 1200
gaggattctc caagggaaac agcaaggatg ttaaacaggg acttggaccg agaaagagca 1260
gttgcggagg ctaaaatgtg gggtgaaaat tggagagaaa agagaagaca aaatgcgcaa 1320
ggcagttttg atggcactaa tggatag 1347
<210> 10
<211> 1407
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atgggttgct tctcctgttt tgattctaaa gaggatgaga agttgaatcc ccaaaaagat 60
agagatgatt ctaaccgcaa gcaaccccat ctcaccgctc cctcaaatat atccaggtta 120
tcttcaggag cagacagact taaaacaaga agtaccaatt gttcaaagag agaatttttg 180
gggataaagg atgcacctga tgttcagatt gctgcacata catttacttt ccgtgagctt 240
gcagctgcta ctaataactt caagccagaa tcttttatag gtgaaggagg gtttggtcgt 300
gtgtataaag ggcaattacc tagcggtcag gttgttgcag ttaagcaatt ggataggaat 360
gggcttcagg ggaatagaga atttttagtg gaggttctta tgcttagtct tcttcatcat 420
cctaacttgg tgagtttaat tggttactgt gctgatgggg accagaggct tcttgtctat 480
gagttcatgc ctttgggatc actagaggat caccttcatg atctccctcc tgataaagag 540
ccactagatt ggaacacgag aatgacgatt gcatctggtg cagcaaaagg tttggagcac 600
cttcatgata aggcgaaccc tcctgttatt tatagggact tcaagtcatc caacatattg 660
cttaaagata actttttccc gaagctttct gattttgggc tagcaaaact tggtcctact 720
ggagacaagt cacatgtgtc cacaagggtc atgggaactt atggttactg tgcccctgag 780
tatgccatga ctggacaatt gactgtcaaa tctgatgtct atagttttgg ggttgtgttc 840
ttggagctta tcactggacg taaggctatt gacagcacca aacctcaggg agaacaaaac 900
cttgtcgcat gggctaggcc actgtttaat gatcgtcgga aatttgcaaa attagctgat 960
ccaagtcttc aaggacaatt tccaatgaga ggtctgtacc aggctttagc tgtggcctcc 1020
atgtgtatcc aagaacaggc tgctggtcgt ccgctaattg gggatgtggt cactgccctt 1080
tcttatcttg caaatcagtc atatgatcct ggtacagttc ctgggcaaat tcatagattt 1140
ggggcagaca gtgttgatag gagaaataaa gatgatagag ttggaagaat atttaggaat 1200
gaagatgggg caggaggagg atcaggacag aaatgggacg tggatggagg atctgagaag 1260
gaagattctc caagggaaac agcaaggatg ttaaacaggg atttggatag agaaagagca 1320
gttgcagagg ctaaaatgtg gggcgagaat tggagagaca agagaagaca aaatggtcaa 1380
ggcagttttg atgggggtaa cgaatag 1407
Claims (6)
1. Application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein in cultivating arabidopsis with resistance to phytophthora;
the CS10 protein is CS10 protein in Arabidopsis thaliana;
the coding gene of the CS10 protein is the coding gene of the CS10 protein in the Arabidopsis thaliana;
the amino acid sequence of CS10 protein in the Arabidopsis thaliana is shown as SEQ ID NO. 3;
the phytophthora is parasitic phytophthora.
2. Application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein in culturing Nicotiana benthamiana with resistance to phytophthora;
the CS10 protein is CS10 protein in Nicotiana benthamiana;
the coding gene of the CS10 protein is the coding gene of the CS10 protein in Nicotiana benthamiana;
the application is that CS10 protein in Nicotiana benthamiana with amino acid sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5 is simultaneously silenced;
the phytophthora is phytophthora parasitica and phytophthora infestans.
3. Application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein in improving resistance of arabidopsis thaliana to phytophthora infection;
the CS10 protein is CS10 protein in Arabidopsis thaliana;
the coding gene of the CS10 protein is the coding gene of the CS10 protein in the Arabidopsis thaliana;
the amino acid sequence of CS10 protein in the Arabidopsis thaliana is shown as SEQ ID NO. 3;
the phytophthora is parasitic phytophthora.
4. Application of CS10 protein for negative regulation of plant immunity or coding gene of CS10 protein in improving resistance of Nicotiana benthamiana to phytophthora infestations;
the CS10 protein is CS10 protein in Nicotiana benthamiana;
the coding gene of the CS10 protein is the coding gene of the CS10 protein in Nicotiana benthamiana;
the application is that CS10 protein in Nicotiana benthamiana with amino acid sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5 is simultaneously silenced;
the phytophthora is phytophthora parasitica and phytophthora infestans.
5. The use according to claim 1 or 3, wherein the nucleotide sequence of the gene encoding CS10 protein in arabidopsis thaliana is shown in SEQ ID No. 7.
6. The use according to claim 2 or 4, wherein the nucleotide sequence of the gene encoding CS10 protein in nicotiana benthamiana is shown in SEQ ID No. 8 and SEQ ID No. 9.
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| CN101107362A (en) * | 2004-10-21 | 2008-01-16 | 文甘扎公司 | Methods and materials for conferring resistance to pests and pathogens of plants |
| CN104812901A (en) * | 2012-09-13 | 2015-07-29 | 美国印第安纳大学研究和技术公司 | Compositions and systems for conferring disease resistance in plants and methods of use thereof |
| WO2020219879A1 (en) * | 2019-04-25 | 2020-10-29 | Indiana University Research And Technology Corporation | Use of pbs1 genes from plant species to engineer disease resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101107362A (en) * | 2004-10-21 | 2008-01-16 | 文甘扎公司 | Methods and materials for conferring resistance to pests and pathogens of plants |
| CN104812901A (en) * | 2012-09-13 | 2015-07-29 | 美国印第安纳大学研究和技术公司 | Compositions and systems for conferring disease resistance in plants and methods of use thereof |
| WO2020219879A1 (en) * | 2019-04-25 | 2020-10-29 | Indiana University Research And Technology Corporation | Use of pbs1 genes from plant species to engineer disease resistance |
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| Title |
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| 基于模式识别受体和人工设计诱饵蛋白扩展植物识别特异性;孙丽璠 等;《中国科学:生命科学》;20170913;第47卷(第9期);第893-902页 * |
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