CN113046366A

CN113046366A - StCAD7 gene and application of protein encoded by gene as negative regulator in improving potato late blight resistance

Info

Publication number: CN113046366A
Application number: CN202110371112.8A
Authority: CN
Inventors: 单卫星; 王小霞
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-04-07
Filing date: 2021-04-07
Publication date: 2021-06-29
Anticipated expiration: 2041-04-07
Also published as: CN113046366B

Abstract

The invention belongs to the technical field of agricultural disease-resistant crop strain cultivation or production, and particularly relates to an application of StCAD7 gene and coded protein thereof as a negative regulation factor in improving potato late blight resistance. The gene StCAD7 promotes the infection of late blight bacteria to plants, and three conserved structural domains of the protein coded by the gene StCAD7 are necessary for exerting the immune function and can play a role in negatively regulating and controlling the resistance of the late blight bacteria.

Description

StCAD7 gene and application of protein encoded by gene as negative regulator in improving potato late blight resistance

Technical Field

The invention belongs to the technical field of agricultural disease-resistant crop strain cultivation or production, and particularly relates to an application of StCAD7 gene and coded protein thereof as a negative regulation factor in improving potato late blight resistance.

Background

Potato is the fourth largest food crop in the world, second only to rice, wheat and corn in planting area and yield. Late blight caused by phytophthora infestans is a main disease in the production process of potatoes, and is considered to be the most serious crop disease in all countries in the world. According to statistics, late blight in arid regions can cause the yield reduction of potatoes by 10-20 percent; in humid areas, the yield of the potatoes is reduced by about 80 percent, even the potatoes are dead, and the late blight causes serious reduction of the yield and the quality of the potatoes. Like the prevention and control of other crop diseases, the cultivation and the utilization of disease-resistant varieties for resisting the crop diseases are the most economic and effective way for preventing and controlling the potato late blight at present. However, due to the toxic variation of late blight bacteria, the problems of loss of disease resistance of varieties and the like are very prominent, and the sustainable production of potatoes is seriously threatened. Therefore, the method has important significance for exploring a novel crop disease-resistant way and identifying novel disease-resistant gene resources.

During the interaction between plants and pathogenic bacteria, some genes are often up-regulated and expressed in response to the induction of the pathogenic bacteria, so as to promote the infection of the plants by the pathogenic bacteria, and the up-regulated and expressed genes are called as plant immune negative regulatory factors. If the immune negative regulatory factor is mutated or deleted, the infection of pathogenic bacteria to plants is inhibited, namely the plants obtain disease resistance, and the disease resistance has broad spectrum. Although the immune negative regulatory factor of the plant has been identified at present, the research on the resistance gene of phytophthora infestans is still less, and the application of the immune negative regulatory factor in breeding for disease resistance is more flexible.

Disclosure of Invention

In order to solve the problems, the invention provides an application of StCAD7 gene and its coded protein as a negative regulator in improving potato late blight resistance. According to the technical scheme provided by the invention, the StCAD7 gene is a novel negative regulation factor, can effectively play a role in negatively regulating the resistance of the potato late blight, and provides a basis for cultivating the potato variety resistant to the late blight.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a gene StCAD7 for negatively regulating plant immunity, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

The invention also provides a protein coded by the gene StCAD7 for negatively regulating plant immunity, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention also provides the application of the gene or the protein in breeding of plant varieties resisting late blight.

The invention also provides the application of the gene or the protein in improving the late blight resistance of plants.

The invention also provides an application of the protein in improving the late blight resistance of plants by inhibiting or blocking the protein in the positioning of cell nucleuses.

The invention also provides an application of the plant late blight resistance improvement compound in plants by inhibiting the functions of the Zn1 domain, the Zn2 domain and/or the NADPH domain of potato StCAD7 or deleting the functions of the Zn1 domain, the Zn2 domain and/or the NADPH domain of potato StCAD 7.

Preferably, the plant comprises potato.

The invention provides a gene StCAD7 for negatively regulating plant immunity, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1. The gene StCAD7 promotes the infection of late blight bacteria to plants, and three conserved structural domains of the protein coded by the gene StCAD7 are necessary for the immune function and can play a role in negatively regulating the resistance of the late blight bacteria. As can be seen from the examples, the StCAD7 gene in potato leaves was up-regulated in response to induction by Phytophthora infestans after inoculation; the StCAD7 gene is transiently overexpressed on the potato through an agrobacterium-mediated transient expression system, so that the StCAD7 gene is proved to promote the infection of late blight bacteria to plants; the three conserved domains of the potato StCAD7 protein are essential for its immune function; the construction of fusion protein carriers of different subcellular localization of StCAD7 proves that the negative regulation immunity of potato StCAD7 gene depends on the nuclear localization, and the application of StCAD7 gene as a negative regulation factor in improving the potato late blight resistance is proved, and the method can be used for breeding the potato late blight resistant variety.

Drawings

FIG. 1 shows the expression of StCAD7 gene in potato leaf of example 1 after inoculation with Phytophthora infestans;

FIG. 2 is a map of the pKANNibal vector provided in example 2;

FIG. 3 is a map of the pART27 vector provided in example 2;

FIG. 4 shows that overexpression of potato StCAD7 promotes infection by Phytophthora infestans as provided in example 2;

FIG. 5 is a schematic diagram of M1, M2 and M3 in example 3;

FIG. 6 shows that the overexpression of M1, Myc, M2, Myc and M3, Myc, provided in example 3 has no effect on the colonization of Phytophthora infestans in potato;

FIG. 7 is a confocal laser diagram of GFP fusion expression vector NLS 7:GFP, NES 7:GFPand Myr 7:GFPprovided in example 4.

Detailed Description

The invention provides a gene StCAD7 for negatively regulating plant immunity, the nucleotide sequence of the gene is shown in SEQ ID NO. 1:

ATGGAAAAAACACATGAAAATGTTGAGGCATTTGGATGGGCAGCTAGAGATACTTCTGGGGTACTTTCTCCTTTCAACTTCTCAAGAAGGGTGACTGGTGAAAAAGATGTGCAATTCAAAGTTATGTATTGTGGAATTTGTCATTCTGATCTTCATCAACTCAAGAATGAATGGAGCAATAGTATATATCCAATGGTACCAGGGCATGAAGTTGTTGGTGTAGTAACTGAAGTTGGTAGCAAAGTCGAGAAATTCAAAATTGGTGACAAAGTTGGTGTTGGATGTTTAGTAGGATCATGTAGAAAATGTGAAAATTGTGACAACGATCTTGAAAATTACTGTCGCGATCAGATCATGACATACAATGGTGTTTACACCGATGGAATCCCCACGTATGGTGGTTACTCCGATATAATGGTAACCAACGAGCACTACGTGGTCCATTGGCCCGAAAATTTACCAATGGAAGCAGCTTCCCTGTTATGTGCAGGGATCACAACTTATAGTCCATTGAGATATTTTGGACTTGACAAGCCTGGAATGCACATTGGTGTTGTTGGTCTAGGTGGTTTGGGACATATGGCTGTGAAATTTGCTAAGGCGTTCGGAACCAAAGTTACTGTGATAAGTACATCTGTTAGTAAGAAAGACGAAGCAATTGATCGTTTAGGGGCAGACTCGTTTTTGGTCAGTCGTGATCCTGACCAAATGCAGGGTGCAGCGGGGTCACTAGATGGCATCATCGATACTGTATCCGCGATTCATCCTCTTCTTCCATTGATTAATTTGTTGAAAACTCATGGGAAGCTTGTGATGGTTGGTGCCCCTGAAAAACCACTAGAGTTACCTGTATTTCCCCTGCTTTTAGGAAGGAAGCTAGTGGCGGGGAGCGCGATAGGAGGGATGAAGGAGACACAAGAGATGGTAGATTTCGCGGCAAAGCATAACATAACACCAGATGTTGAAGTTGTGCCAATGGACTATGTGAATAAAGCGTTGGAACGCCTTTTGAAATCGGATGTGAAGTATCGTTTTGTGCTTGACATTGGAAACACGTTGAACAAGAAT。

the StCAD7 gene can negatively regulate plant immunity, the overexpression of the StCAD7 gene can enhance the susceptibility of phytophthora infestans to plants, and the negatively regulated StCAD7 gene can provide a basis for cultivating potato varieties resistant to late blight.

The invention also provides a protein coded by the gene StCAD7 for negatively regulating plant immunity, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 2:

MEKTHENVKAFGWAARDTFGVLSPFNFSRRVTGEKDVQFKVVYCGICHTDLHQLKNEWSNSIYPMVPGHEVVGVVTEIGTKVEKFKIGDKVGVGGLVESCRKCENCDNDLENYCRDQIMTYNGVYTDGTTTYGGYSDIMVTNEHYVVHWPENLPMEAAPLLCAGITIYSPLKYFGLDKPGMHIGVVGLGGLGHMAVKFAKVFGTKVTVISTSLSKKDEAIEHLGADSFLVSRDPDQMQGAAGSLDGIIDTVSVIHPLLPLINLLKTRGKLAMLGAPEKPLELPVFPLLLGRKLVAGSFIGGMKETQEMVDFAAKHSITPDVEVVPMDYVNTAFERLLKSNVKYRFVLDIGNTLNKN。

the invention provides the application of the gene or the protein in breeding of a plant variety resisting late blight. In the present invention, the plant preferably comprises potato.

The invention provides application of the gene or the protein in improving the late blight resistance of plants. In the present invention, the plant preferably comprises potato.

The invention provides an application of inhibiting or blocking the protein in improving the late blight resistance of plants by positioning cell nucleuses. In the specific embodiment of the invention, a fusion protein carrier of different subcellular localization of StCAD7 is constructed, which shows that the negative regulation immunity of potato StCAD7 gene depends on the nuclear localization, and proves the application of the StCAD7 gene as a negative regulation factor in improving the potato late blight resistance, and the method can be used for breeding the anti-late blight variety. In the present invention, the plant preferably comprises potato.

The invention provides an application of the plant late blight resistance improvement through inhibiting the functions of a Zn1 domain, a Zn2 domain and/or an NADPH domain of potato StCAD7 or deleting the functions of a Zn1 domain, a Zn2 domain and/or an NADPH domain of potato StCAD 7. In a particular embodiment of the invention, the three conserved domains of the potato StCAD7 protein are essential for its immune function. In the present invention, the plant preferably comprises potato.

For further illustration of the present invention, the StCAD7 gene and its encoded protein provided by the present invention are described in detail below as a down-regulator for improving potato late blight resistance with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

The expression condition of the gene StCAD7 in the potato leaves after inoculation of phytophthora infestans,

the method comprises the following steps:

the method comprises the following steps: obtaining plant materials, respectively inoculating phytophthora infestans on the potato leaves of the Atlantic variety, and collecting the potato leaves after 0h, 3h, 6h, 24h, 48h and 72h of inoculation;

step two: extracting RNA, extracting RNA by using an RNA extraction kit (OMGA), identifying the integrity of the RNA by using agarose gel electrophoresis, and then determining the purity and concentration of the RNA on a spectrophotometer;

step three: obtaining cDNA of the potato by using a reverse transcription kit (TaKaRa);

step four: the real-time fluorescence quantitative PCR is carried out on a real-time fluorescence quantitative PCR instrument of an instrument Bio-Rad iQ7, the experimental result is shown in Table 1 (0 in Table 1 refers to the expression condition of StCAD7 in potato leaves after 0h inoculation of phytophthora infestans, W0 refers to the expression condition of StCAD7 in the potato leaves after 0h inoculation of water, 3/W3, 6/W6, 24/W24, 48/W48, 72/W72 and the like, wherein StEF1 alpha is an internal reference gene of the potato, the expression of the internal reference gene in each tissue of the potato is relatively constant, the expression level of StEF1 alpha gene is used as a reference substance when the expression level change of the StCAD7 gene is detected, qPCR-StCAD7 refers to the expression condition of the StCAD7 gene in a sample, and qPCR-StEF1 alpha refers to the expression condition of the StEF1 alpha gene in the sample, and FIG. 1 is the expression condition of the phytophthora infestans 7 in the potato leaves after inoculation of the phytophthora infestans.

TABLE 1 expression of StCAD7 gene and StEF1 alpha gene in potato leaves after inoculation with Phytophthora infestans

As can be seen from Table 1 and FIG. 1, the relative expression level of the potato StCAD7 gene began to increase at 3h after phytophthora infestans inoculation, reached the maximum at 48h, and then began to decrease. The potato StCAD7 gene upregulated expression in response to induction by late blight.

Example 2

1. The cloning of the potato negative regulatory factor StCAD7 gene comprises the following steps:

the method comprises the following steps: obtaining plant material, leaves of an atlantic variety of potatoes (available through open channels);

step two: extracting RNA, extracting RNA by using an RNA extraction kit (OMGA, Lot #: R6827-01), identifying the integrity of the RNA by agarose gel electrophoresis, and then determining the purity and the concentration of the RNA on a spectrophotometer;

step three: gene cloning, using reverse transcription kit (TaKaRa, Lot #: AHE3187A) to obtain potato Atlantic cDNA, according to Sol genetics network: designing an upstream primer and a downstream primer for the full-length coding sequence of PGSC0003DMC400009489, and amplifying by taking cDNA as a template; the PCR product is subjected to enzyme digestion, connection and bacterial liquid PCR verification to construct a vector pKANNIBAL-StCAD7 (the map of the pKANNIBAL plasmid vector is shown in figure 2), sequencing is performed and compared with a published sequence, and the vector pART27-pKANNIBAL-StCAD7 (the map of the pART27 plasmid vector is shown in figure 3) is constructed after the correct plasmid is subjected to enzyme digestion, connection and bacterial liquid PCR verification.

The primer sequence is as follows: StCAD 7-F: CCGCTCGAGATGGAAAAAACACATG AAAATGTAAAGG, SEQ ID NO: 3;

StCAD7-R：GCTCTAGAATTCTTGTTCAACGTGTTTCCAATG，SEQ ID NO:4。

2. the StCAD7 gene over-expression plant is obtained, the embodiment utilizes the Agrobacterium tumefaciens mediated transient expression technology to increase the expression of the target gene, and the specific implementation is as follows:

the method comprises the following steps: converting the prepared overexpression vector pART27-pKANNibal-StCAD7 into agrobacterium-infected competent cells, and selecting successfully-converted clones to a liquid LB culture medium for culture for 24-36 h;

step two: collecting thallus at low rotation speed, re-suspending thallus with MES solution containing acetosyringone, and taking part of bacteria liquid to diluteOD detection with spectrophotometer after release₆₀₀The concentration of (c);

step three: adjusting the bacterial liquid to a proper concentration;

step four: the potato leaves are injected, the inoculation experiment is carried out on the potato leaves 2-3 days later, and the experimental result is shown in figure 4. FIG. 4 shows that overexpression of StCAD7 in potato promotes the infestation of late blight bacteria on plants.

As shown in FIG. 4, the gene StCAD7 of the negative regulatory factor of the invention is cloned as a gene for controlling plant phytophthora root rot of solanaceae crops, and the gene StCAD7 is transiently overexpressed on potatoes through an agrobacterium-mediated transient expression system, so that the gene StCAD7 promotes the infection of late blight bacteria on plants.

Example 3

Since all CAD proteins have Zn1, Zn2 and NADPH domains, and the potato StCAD7 protein also has these 3 conserved domains, mutant vectors M1:: Myc, M2:: Myc and M3:: Myc were constructed based on this. Wherein M1 is a mutation of all amino acids in Zn1 to alanine; m2 is the mutation of cysteine at positions 99, 102, 105 and 113 in Zn2 to alanine, respectively; m3 is obtained by mutating all amino acids in NADPH to alanine, the schematic diagram is shown in FIG. 5, and a fusion expression vector containing Myc gene, M1:: Myc, M2:: Myc and M3:: Myc, was constructed.

M1 Myc, M2 Myc, M3 Myc and GFP are expressed on the potatoes in a transient mode respectively, then an in vitro leaf inoculation test is carried out, the result is shown in figure 6, and as can be seen from figure 6, after the Zn1 structural domain of the potato StCAD7 gene is mutated, the overexpression StCAD7 protein does not enhance the susceptibility of phytophthora infestans to the potatoes any more; similarly, overexpression of StCAD7 protein no longer enhances susceptibility of phytophthora infestans to potato after mutation of Zn2 domain and NADPH domain of potato StCAD7 protein, which demonstrates that the three conserved domains of potato StCAD7 protein are essential for its immune function.

Example 4

The construction method of the fusion protein carrier of different subcellular localization of StCAD7 gene is as follows: a nuclear input Signal (NLS), a nuclear output Signal (NES) and a Myristoylation Signal (Myr) are added to the N-terminal of the StCAD7 gene, and a GFP fusion expression vector NLS7:: GFP, NES7:: GFP and Myr7:: GFP are constructed. The results of laser confocal measurements are shown in FIG. 7, and it can be seen from FIG. 7 that NLS 7:GFPis localized in the nucleus, NES 7:GFPand Myr 7:GFPis localized in the cell membrane. The transient expression technology of the Nicotiana benthamiana and the in vitro leaf inoculation are used for testing, and the results show that the over-expression of NLS7 on the Nicotiana benthamiana enhances the susceptibility of the Nicotiana benthamiana to phytophthora infestans, the over-expression of NES7 and the over-expression of Myr7 show that GFP enhances the resistance of the Nicotiana benthamiana to the phytophthora infestans, so that the negative regulation and control immunity of the StCAD7 gene depends on the nuclear localization. The construction of fusion protein carriers of different subcellular localization of StCAD7 proves that the negative regulation immunity of potato StCAD7 gene depends on the nuclear localization, and the application of StCAD7 gene as a negative regulation factor in improving the potato late blight resistance is proved, and the method can be used for breeding the potato late blight resistant variety.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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<120> StCAD7 gene and application of protein coded by gene as negative regulator in improving potato late blight resistance

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Claims

1. The gene StCAD7 for negatively regulating plant immunity is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The protein encoded by gene StCAD7 for negatively regulating plant immunity is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. Use of the gene of claim 1 or the protein of claim 2 for breeding plant varieties resistant to late blight.

4. Use of the gene according to claim 1 or the protein according to claim 2 for increasing the resistance of a plant to late blight.

5. Use of the protein according to claim 2 for increasing the resistance of plants to late blight by inhibiting or blocking the nuclear localization.

6. The application of the plant late blight resistance improvement through inhibiting the functions of a Zn1 domain, a Zn2 domain and/or an NADPH domain of potato StCAD7 or deleting the functions of a Zn1 domain, a Zn2 domain and/or an NADPH domain of potato StCAD 7.

7. Use according to any one of claims 3 to 6, wherein the plant comprises potato.