阅读说明：本技术 黄瓜光合系统II析氧增强蛋白CsPSII-OEEP在抗瓜类疫病中的应用 (Application of cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in resistance to melon epidemic diseases ) 是由 金庆敏 吴廷全 杜虎 王瑞 钟玉娟 杨晓珊 于 2021-08-23 设计创作，主要内容包括：本发明属于植物分子生物学与植物遗传工程领域,公开了黄瓜光合系统II析氧增强蛋白CsPSII-OEEP在抗瓜类疫病中的应用。本发明首次发现黄瓜光合系统II析氧增强蛋白CsPSII-OEEP对瓜类疫病具有抗性：在过量表达黄瓜光合系统II析氧增强蛋白CsPSII-OEEP的植物子叶上接种瓜类疫霉菌,病斑面积明显减小,而在沉默黄瓜光合系统II析氧增强蛋白CsPSII-OEEP的植物子叶上接种瓜类疫霉菌,病斑面积明显增大；可见,黄瓜光合系统II析氧增强蛋白CsPSII-OEEP对植物抗瓜类疫病具有非常重要的作用,可用于抗瓜类疫病、制备抗瓜类疫病的产品、培育抗瓜类疫病转基因植物,具有广泛的应用前景。(The invention belongs to the field of plant molecular biology and plant genetic engineering, and discloses application of cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in resistance to melon epidemic diseases. The invention discovers that the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP has resistance to melon epidemic diseases for the first time: the phytophthora melonis inoculated on the plant cotyledon of the over-expression cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP, the scab area is obviously reduced, while the phytophthora melonis inoculated on the plant cotyledon of the silencing cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP, the scab area is obviously increased; therefore, the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP has very important function on plant resistance to the melon epidemic disease, can be used for resisting the melon epidemic disease, preparing products for resisting the melon epidemic disease and cultivating transgenic plants for resisting the melon epidemic disease, and has wide application prospect.)

1. The application of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

2. The application of a nucleic acid molecule for coding cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

3. Use of a vector comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

4. The application of an expression cassette containing a nucleic acid molecule for encoding the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

5. Use of a transgenic cell line comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

6. The application of a recombinant bacterium or a recombinant virus containing a nucleic acid molecule for encoding an oxygen evolution enhancement protein CsPSII-OEEP of a cucumber photosynthetic system II in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

7. The application of the reagent for regulating the expression quantity of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in a targeted manner or enhancing the activity of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

8. Use according to any one of claims 1 to 7, wherein:

the melon epidemic disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

9. Use according to any one of claims 2 to 6, wherein:

the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

10. A method for cultivating transgenic plants resistant to melon blight comprises the following steps: introducing a nucleic acid molecule for coding cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP into a target plant to obtain a transgenic plant for resisting the melon epidemic disease;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1;

preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis);

preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Technical Field

The invention belongs to the field of plant molecular biology and plant genetic engineering, and particularly relates to application of cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in resistance to melon epidemic diseases.

Background

The melon epidemic disease is a worldwide oomycete disease, and the pathogenic bacteria is Phytophthora melonis (Phytophthora melonis) which causes browning and death or death of leaves, stems and fruits of melons such as cucumbers, pumpkins, white gourds, balsam pears and the like. The disease occurs in all areas where melons are planted in China. The occurrence of heavy melons is severe in open field cultivation in summer and autumn and in protection in spring in the north, and severe in spring and high-temperature and high-humidity seasons in summer and autumn in the south. The melons can be damaged in the whole growth period and all parts, and the damaged growing points and the base parts of tender stems of the melons are contracted in a water stain shape, so that the melons die. The stem is damaged in the adult plant stage, the base part and the node are in the shape of water stain and black brown disease spots, the disease spots are gradually softened and contracted, the leaves are drooped, and the plant is wilted and dead; dark green large-scale spots with unclear edges appear on the near leaf edges of the leaves; the melon and fruit shows water stain-like, nearly circular concave spots at the affected part, and the melon and fruit quickly decay, and a white or grey-white sparse mildew layer grows on the surface. Once the disease occurs, the control effect of chemical agents is not ideal, resulting in economic loss. The most fundamental way for controlling the disease is to cultivate new melon species with epidemic disease resistance, and to cultivate new melon species with high epidemic disease resistance, melon disease resistance genes need to be excavated and applied. Previous researches show that the plant photosynthetic system II oxygen evolution enhancement protein has thioredoxin activity and plays an important role in abiotic stress, and reports of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in melon epidemic disease resistance are not found.

Disclosure of Invention

The first aspect of the invention aims to provide the application of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP.

The object of the second aspect of the present invention is to provide the use of a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-OEEP.

The object of the third aspect of the present invention is to provide the use of a vector comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep.

The fourth aspect of the present invention aims to provide the use of an expression cassette comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep.

The object of the fifth aspect of the present invention is to provide the use of a transgenic cell line comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep.

The sixth aspect of the present invention aims to provide the use of a recombinant bacterium comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep.

The seventh aspect of the present invention aims at providing the use of a recombinant virus comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep.

The eighth aspect of the present invention is directed to the use of an agent for the targeted modulation of the expression level of the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep or the enhancement of the activity of the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep.

The ninth aspect of the invention aims at providing a method for cultivating the transgenic plant resisting the melon blight.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in the first aspect of the invention, the application of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in any one of (1) to (3) is provided;

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

In a second aspect of the present invention, there is provided a use of a nucleic acid molecule encoding cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

In a third aspect of the present invention, there is provided a use of a vector comprising a nucleic acid molecule encoding cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

In a fourth aspect of the present invention, there is provided a use of an expression cassette comprising a nucleic acid molecule encoding a cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

In a fifth aspect of the present invention, there is provided the use of a transgenic cell line comprising a nucleic acid molecule encoding the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the transgenic cell line does not comprise propagation material.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

In a sixth aspect of the invention, the application of the recombinant bacterium containing the nucleic acid molecule for coding the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in any one of (1) to (3) is provided;

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

In a seventh aspect of the present invention, there is provided a use of a recombinant virus comprising a nucleic acid molecule encoding cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) cultivating transgenic plants resisting melon epidemic diseases;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

The eighth aspect of the present invention provides the use of an agent for the targeted regulation of the expression level of the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep or the enhancement of the activity of the cucumber photosynthetic system II oxygen evolution enhancing protein CsPSII-oep in any one of (1) to (3);

(1) resisting melon epidemic disease;

(2) preparing a product for resisting the melon epidemic disease;

(3) culturing transgenic plant resisting melon epidemic disease.

The amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

In a ninth aspect of the present invention, there is provided a method for breeding transgenic plants against melon blight, comprising the steps of: introducing a nucleic acid molecule for coding cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP into a target plant to obtain a transgenic plant for resisting the melon epidemic disease;

the amino acid sequence of the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP is shown in SEQ ID NO. 1.

Preferably, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the melon disease is a disease caused by Phytophthora melonis (Phytophthora melonis).

Preferably, the plant is at least one of cucumber, pumpkin, wax gourd, balsam pear, towel gourd and Japanese pumpkin; further at least one of cucumber and pumpkin.

The invention has the beneficial effects that:

the invention discovers that the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP has resistance to melon epidemic diseases for the first time: the phytophthora melonis inoculated on the plant cotyledon of the over-expression cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP, the scab area is obviously reduced, while the phytophthora melonis inoculated on the plant cotyledon of the silencing cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP, the scab area is obviously increased; therefore, the cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP has very important function on plant resistance to the melon epidemic disease, can be used for resisting the melon epidemic disease, preparing products for resisting the melon epidemic disease and cultivating transgenic plants for resisting the melon epidemic disease, and has wide application prospect.

Drawings

FIG. 1 is a map of pGWB5 plasmid.

FIG. 2 is a map of pK7GWIW plasmid.

FIG. 3 is a diagram showing the preparation process of the CsPSII-OEEP monoclonal antibody.

FIG. 4 is a graph showing the results of detecting the expression level of CsPSII-OEEP protein in cucumber leaves treated differently.

Fig. 5 is a functional result of the resistance of the different treated cucumbers to melon blight: wherein A is an intuitive picture of resistance functions of cucumbers treated differently to melon blight; b is a disease spot area statistical result chart of cucumber leaves treated differently; different letters represent significant differences (p < 0.05).

FIG. 6 is a graph showing the results of measurement of the expression level of CsPSII-OEEP protein in differently treated leaves of pumpkin seeds.

FIG. 7 is a functional diagram of resistance of different treated pumpkins to melon blight: wherein A is an intuitive picture of resistance functions of the pumpkins treated differently to the melon epidemic disease; b is a statistical result chart of lesion areas of the pumpkin leaves treated differently; different letters represent significant differences (p < 0.05).

Detailed Description

The present invention will be described in further detail with reference to specific examples.

These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available. The experiment uses cucumber material as the source of the unit, the teachers of forest concer in 1995 month 2, introduced from the vegetable center (mang in Thailand), and the self-bred seed reservation is disclosed in the patent literature: CN 112175988B. The pumpkin material is Guangmei No.1, and is purchased from Guangdong agricultural vegetable species company. Phytophthora melonis isolated from cucumber strains in the white cloud area of Guangzhou in 2012 in the laboratory and is disclosed in the literature: wang Rui, Linyue, Liangzhao, jin Qingmin, separation and identification of pathogenic bacteria of cucumber epidemic disease in Guangdong region of Wuting Teng Guangdong region [ J ]. report of agriculture in China 2016,32(01):76-80.

Example 1 construction of cucumber CsPSII-OEEP Gene overexpression vector

The cucumber CsPSII-OEEP gene belongs to a cucumber photosynthetic system II member protein gene. Constructing CDS full length (SEQ ID NO.2) of cucumber CsPSII-OEEP gene on pGWB5(pGWB5 plasmid map is shown in figure 1), wherein the specific position is between attR1 and attR2 (ccdB gene is replaced by gene recombination exchange) after 35S promoter, and obtaining an overexpression vector (pGWB 5-CsPSII-OEEP); a239 bp fragment (CDS 1-239bp, SEQ ID NO.4) of a CDS non-conserved segment of a cucumber CsPSII-OEEP gene and a complementary fragment thereof are constructed on pK7GWIW (a pK7GWIW plasmid map is shown in figure 2), and a gene silencing vector (pK7GWIW-CSPSII-OEEP) is obtained by specifically positioning between two attR1 and attR2 behind a 35S promoter (the ccdB gene is replaced by gene recombination exchange).

The amino acid sequence of the cucumber CsPSII-OEEP protein is (332 aa):

MAASVQAAAATLMQPSKLASRTTTSHLRSSQSLSKAFGLESSGPRLTCSLHSDLKDVSRKFADAAKIAGFALATSALVVSGAGAEGVPKRLTFDEIQSKTYLEVKGTGTANQCPTIDGGVDSFAFKAGKYQAKKFCLEPTSFTVKAEGVSKNAPPEFQNTKLMTRLTYTLDEIEGPFEVGADGSIKFEEKDGIDYAAVTVQLPGGERVPFLFTIKQLVASGKPESFSGDFLVPSYRGSSFLDPKGRGGSTGYDNAVALPAGGRGDEEELAKENIKNASSSTGKITLSVTKSKPETGEVIGVFESIQPSDTDLGAKAPKDVKIQGVWYAQLDS(SEQ ID NO.1)。

the CDS (999bp) sequence of the cucumber CsPSII-OEEP gene is as follows:

ATGGCGGCTTCAGTTCAGGCGGCCGCGGCTACCCTTATGCAACCCTCCAAGTTGGCTTCAAGAACTACCACCTCTCACCTCAGATCTTCTCAATCCCTTTCTAAAGCCTTTGGCCTTGAATCTTCCGGACCTAGACTCACCTGCTCTCTTCACTCCGATCTCAAAGACGTTTCTCGCAAGTTTGCCGATGCTGCTAAGATTGCCGGCTTCGCTCTTGCCACTTCCGCTCTTGTCGTTTCGGGAGCTGGTGCTGAAGGAGTACCAAAGAGACTCACCTTTGATGAAATTCAGAGCAAGACATACCTTGAAGTTAAGGGAACTGGAACAGCTAACCAGTGCCCAACCATTGATGGAGGAGTTGATTCATTTGCCTTCAAGGCAGGCAAATATCAAGCCAAGAAATTCTGTCTTGAGCCAACTTCTTTCACTGTGAAGGCTGAAGGAGTGAGCAAGAACGCCCCACCAGAATTCCAAAACACCAAGCTCATGACTCGTCTAACTTACACACTAGACGAGATTGAAGGACCCTTTGAAGTTGGTGCTGATGGTTCGATCAAGTTTGAGGAGAAAGATGGAATTGACTATGCTGCTGTCACTGTCCAGTTGCCGGGAGGTGAGCGTGTCCCGTTCCTCTTCACCATCAAACAATTGGTGGCTTCTGGAAAACCAGAGAGCTTTAGTGGAGACTTTTTAGTCCCATCTTACCGTGGCTCGTCTTTCTTGGACCCAAAGGGAAGAGGAGGTTCAACTGGATATGATAATGCTGTTGCATTGCCTGCTGGAGGGAGAGGTGATGAAGAAGAACTTGCTAAAGAAAACATAAAGAATGCTTCATCTTCAACAGGGAAAATCACTTTGAGTGTGACCAAGAGCAAGCCTGAGACTGGTGAAGTTATTGGTGTGTTTGAGAGTATTCAGCCTTCTGATACCGATTTGGGAGCAAAAGCTCCCAAGGATGTGAAGATCCAGGGTGTTTGGTATGCTCAGCTTGACTCTTAG(SEQ ID NO.2)。

the full-length sequence of the cucumber CsPSII-OEEP gene (2166bp) is as follows:

GAATTAAAAAGTATATCCAAGTAGAAAATGAGTTTGTATGCCACGTGGAAGAAAATTGAGGCAAAGAATGATGAGGGAATCATCCAATGAGATTTTTCTTATCCAATTCATTATCTTACTCTAACCAAAACCACATAATTCATTTCCTAAATCCTCTCTACTCTCCGTTATAGTTTCAGTATCATTCCCCGCACAAAATTTCACTTCTCCTCTCTTACACTCAATCCGGATACAACAATGGCGGCTTCAGTTCAGGCGGCCGCGGCTACCCTTATGCAACCCTCCAAGTTGGCTTCAAGAACTACCACCTCTCACCTCAGATCTTCTCAATCCCTTTCTAAAGCCTTTGGCCTTGAATCTTCCGGACCTAGACTCACCTGCTCTCTTCACTCCGATCTCAAAGACGTTTCTCGCAAGTTTGCCGATGCTGCTAAGATTGCCGGCTTCGCTCTTGCCACTTCCGCTCTTGTCGTTTCGGTATGTTCTTATCGTACTTTCCTTCCACGATTACTTTCTTCTTATGATTCTCCACGATGCTTTATATTTGCGTTTGGATGTGAATGTCTGTATTGTTTTAGTTGCTGATATTGAATTGAGTTCTTGGAGAAAATAATTATAGTTCAGACTTAGAATGGGTAATTGATAATCTCCGAATTTGTTTAACTTAGTGGCAATTTCTTGCCAAATAAAGGATCAAACTACTTAGATCCAAGTGGAATAATCCATTTGATTCTCTGTGACTTAATAAAGTACCTGTTGTTCTTAATTTAACTTGTAATTCATTTTTACATTAATGATCCCATAACATTTTCATTATACTATTCAATCAAGGATTATTTGAAAATCAGTCCACCACATAGTCCACTGATGATATATGGTTAGTGTGTTACGGTAAGTCTTAAATGACTTAAATATTAGATACAGACTTTAAAGTTTTAAAGCGTTAAAGAAAATCTCACTTCAAGGACTTAATTAGTCTGAGTACACTTGATATATGAGATCTAAGAATGTCTAGACTGATTAACACTTGATATATACAAAGTGTAATCCTACAACTTGGTGAGCATCTTAATTGTTCGATACTTTTCATGTATTTCCAGGGAGCTGGTGCTGAAGGAGTACCAAAGAGACTCACCTTTGATGAAATTCAGAGCAAGACATACCTTGAAGTTAAGGGAACTGGAACAGCTAACCAGTGCCCAACCATTGATGGAGGAGTTGATTCATTTGCCTTCAAGGCAGGCAAATATCAAGCCAAGAAATTCTGTCTTGAGCCAACTTCTTTCACTGTGAAGGCTGAAGGAGTGAGCAAGAACGCCCCACCAGAATTCCAAAACACCAAGCTCATGACTCGTCTAACTTACACACTAGACGAGATTGAAGGACCCTTTGAAGTTGGTGCTGATGGTTCGATCAAGTTTGAGGAGAAAGATGGAATTGACTATGCTGCTGTCACTGTCCAGTTGCCGGGAGGTGAGCGTGTCCCGTTCCTCTTCACCATCAAACAATTGGTGGCTTCTGGAAAACCAGAGAGCTTTAGTGGAGACTTTTTAGTCCCATCTTACCGTGGCTCGTCTTTCTTGGACCCAAAGGGAAGAGGAGGTTCAACTGGATATGATAATGCTGTTGCATTGCCTGCTGGAGGGAGAGGTGATGAAGAAGAACTTGCTAAAGAAAACATAAAGAATGCTTCATCTTCAACAGGGAAAATCACTTTGAGTGTGACCAAGAGCAAGCCTGAGACTGGTGAAGTTATTGGTGTGTTTGAGAGTATTCAGCCTTCTGATACCGATTTGGGAGCAAAAGCTCCCAAGGATGTGAAGATCCAGGGTGTTTGGTATGCTCAGCTTGACTCTTAGGCCCCCCTTTCTTCTTTTATGATTGTGTAATATTGTTCCTTGTAAGTTAAAAGAGAGAGTTGCTAAACTTTCTTATGGAGAGAGACGCTAGCAGGGGTTCTATTTTTATCCAAGTTTGCATATAAAATCTTGTGCCTTTTCACTCTTTCTCAAACATACATTTGCTTAACTTCATAACTTTAGAGGAAGAGATGGAATCATCAATCTTTGAGATGATTTAGAGGAATGAATAGAATATATAAAACTTTTTAGATGAAATCTAAAGCAATTTATGAGAATTATGTTCAAAAGTAGACAATATCATACA(SEQ ID NO.3)。

the 239bp length fragment of the CDS non-conserved segment of the cucumber CsPSII-OEEP gene is shown as follows:

ATGGCGGCTTCAGTTCAGGCGGCCGCGGCTACCCTTATGCAACCCTCCAAGTTGGCTTCAAGAACTACCACCTCTCACCTCAGATCTTCTCAATCCCTTTCTAAAGCCTTTGGCCTTGAATCTTCCGGACCTAGACTCACCTGCTCTCTTCACTCCGATCTCAAAGACGTTTCTCGCAAGTTTGCCGATGCTGCTAAGATTGCCGGCTTCGCTCTTGCCACTTCCGCTCTTGTCGTTTC(SEQ ID NO.4)。

example 2 construction of transient overexpression/Gene silencing cucumber cotyledon model

(1) The overexpression vector and the silencing vector in example 1 were transformed into Agrobacterium GV3101, respectively, and cultured upside down on the corresponding resistant medium for 48 h.

(2) Single clones were picked and added to 4mL LB medium containing the corresponding antibiotic and rifampicin, shaken at 180rpm for 24h at 28 ℃.

(3) Adding into LB culture medium containing corresponding antibiotic and rifampicin at a ratio of 1:100, shaking at 28 deg.C and 180rpm to OD600 of about 3.0.

(4) The cells were collected by centrifugation at 3000rpm for 5 minutes and suspendedSupernatant (10mM MES,10mM MgCl)₂) Resuspend the cells, adjust OD600 to about 0.4, and add 200mM acetosyringone.

(5) Standing at room temperature for 3 h.

(6) A needle hole is respectively pricked at two sides of the main vein of the cucumber seed leaf by using a syringe needle.

(7) And sucking the equivalent amount of the bacteria solution after standing by using a 1mL syringe, aligning the syringe to a pinhole on the back, blocking the front by hand, and injecting the bacteria solution.

(8) The injected cucumber seedlings are cultured in the dark for 12 hours and then in the light at 22 ℃ for 3 days.

And detecting the expression quantity of the CsPSII-OEEP protein in the cucumber isolated cotyledon with transient over-expression, silence and wild control of the CsPSII-OEEP protein by Western Blot.

The method comprises the following specific steps:

(1) the collected cucumber cotyledons were liquid nitrogen-frozen, ground, added with a protein extract (50mM Tris-HCl pH 7.5,150mM NaCl, 0.1% Triton X-100, 0.2% ethylphenylpolyethylene glycol (NP-40),1mM phenylmethylsulfonyl fluoride (PMSF),1 XPlast protease inhibitor cocktail (Sigma, P8340-5ML)), mixed well and placed on ice for 30 minutes.

(2) Centrifuging at 14000rpm at 4 ℃ for 10min, collecting supernatant, adding 80uL of the supernatant into 20uL of protein loading buffer solution, uniformly mixing, and carrying out boiling water bath for 10 min.

(3) 20uL samples were run on SDS-PAGE gels.

(4) The protein samples after electrophoresis were transferred to PVDF membrane, and 5% PBST milk was used to incubate the membrane.

(5) Primary antibody (CsPSII-OEEP monoclonal antibody, preparation shown in FIG. 3) was added to the blocking solution and incubated for 2 hours, and the membrane was washed with PBST for 5min 4 times.

(6) Secondary antibodies (Invitrogen, Goat anti-Mouse IgG, IgM, IgA (H + L) Secondary Antibody, HRP, A-10668) were then added, incubated for 30min, and the membrane was washed with PBST for 5min and 4 times.

(7) The film was subjected to scanning color development, and the results are shown in FIG. 4.

The result shows that the CsPSII-OEEP protein expression level in cucumber cotyledon with transient overexpression of CsPSII-OEEP (pGWB5-CsPSII-OEEP) is obviously higher than that of a wild type Control (Control), while the CsPSII-OEEP protein expression level in cucumber cotyledon with silencing of CsPSII-OEEP (pK7GWIW W-CsPSII-OEEP) is obviously lower than that of the wild type Control (Control). The cucumber cotyledon model with CsPSII-OEEP transient overexpression and the cucumber cotyledon model with CsPSII-OEEP gene silencing are successfully constructed.

EXAMPLE 3 cucumber cotyledon disease resistance test

For the cucumber cotyledon model of CsPSII-OEEP transient overexpression, the cucumber cotyledon model of CsPSII-OEEP gene silencing and the wild cucumber cotyledon successfully constructed in the example 2, after the injected cucumber seedlings are cultured in the dark for 12 hours, the cucumber cotyledons are isolated after being cultured in the light at 22 ℃ for 3 days, and after phytophthora melonis inoculated, the cucumber cotyledons are cultured in the dark for 24 hours in a culture dish at 28 ℃.

The resistance function of the target gene to epidemic diseases is judged according to the sizes of the disease spots of the excised cotyledons of the cucumbers treated differently, and the result is shown in the attached figure 5.

The results show that the transient over-expression of the CsPSII-OEEP gene (pGWB5-CsPSII-OEEP) in the leaves of cucumber seeds can obviously enhance the resistance of the cucumber cotyledons to the epidemic disease compared with the Control (Control), and the transient silencing of the CsPSII-OEEP gene (pK7GWIW-CsPSII-OEEP) in the leaves of cucumber seeds can obviously weaken the resistance of the cucumber cotyledons to the epidemic disease compared with the Control (Control). The 50 leaf lesion areas for each treatment were statistically averaged (using photoshop pixel count instead of area) and subsequently, significance analysis and comparison were performed using over-expressed, silenced, and control leaf areas. The results showed that the lesion area of the over-expressed CsPSII-OEEP gene leaf was significantly reduced compared to the control, while the lesion area of the silenced CsPSII-OEEP gene leaf was significantly increased (FIG. 5).

Example 4 disease resistance test of cotyledon of pumpkin

By the same operation steps as the method in example 2, the cotyledons of the pumpkin with transient overexpression of CspsPSII-OEEP are also constructed, the transient overexpression of the CspPSII-OEEP protein is detected by Western Blot, and the expression level of the CspPSII-OEEP protein is detected in the excised cotyledons of the wild-type control pumpkin, and the result is shown in the attached figure 6: the expression level of CsPSII-OEEP protein in the south cucumber cotyledon (pGWB5-CsPSII-OEEP) with transient over-expression of CsPSII-OEEP gene is obviously higher than that of wild Control (Control).

Culturing the injected pumpkin seedling in the dark for 12h, culturing at 22 ℃ for 3 days by illumination, then separating the pumpkin cotyledon in vitro, inoculating the phytophthora melonis, then placing the pumpkin cotyledon in a culture dish at 28 ℃ and culturing in the dark for 24 h.

The resistance function of the target gene to epidemic diseases is judged according to the sizes of the lesion spots of the excised cotyledons of the pumpkins treated differently, and the result is shown in the attached figure 7.

The results show that: compared with a control, the lesion area of the transient overexpression CsPSII-OEEP gene leaf is obviously reduced, and the disease resistance is obviously enhanced.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> vegetable research institute of academy of agricultural sciences of Guangdong province

<120> application of cucumber photosynthetic system II oxygen evolution enhancement protein CsPSII-OEEP in resistance to melon epidemic diseases

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 332

<212> PRT

<213> Cucumis sativus Linn.

<400> 1

Met Ala Ala Ser Val Gln Ala Ala Ala Ala Thr Leu Met Gln Pro Ser

1 5 10 15

Lys Leu Ala Ser Arg Thr Thr Thr Ser His Leu Arg Ser Ser Gln Ser

20 25 30

Leu Ser Lys Ala Phe Gly Leu Glu Ser Ser Gly Pro Arg Leu Thr Cys

35 40 45

Ser Leu His Ser Asp Leu Lys Asp Val Ser Arg Lys Phe Ala Asp Ala

50 55 60

Ala Lys Ile Ala Gly Phe Ala Leu Ala Thr Ser Ala Leu Val Val Ser

65 70 75 80

Gly Ala Gly Ala Glu Gly Val Pro Lys Arg Leu Thr Phe Asp Glu Ile

85 90 95

Gln Ser Lys Thr Tyr Leu Glu Val Lys Gly Thr Gly Thr Ala Asn Gln

100 105 110

Cys Pro Thr Ile Asp Gly Gly Val Asp Ser Phe Ala Phe Lys Ala Gly

115 120 125

Lys Tyr Gln Ala Lys Lys Phe Cys Leu Glu Pro Thr Ser Phe Thr Val

130 135 140

Lys Ala Glu Gly Val Ser Lys Asn Ala Pro Pro Glu Phe Gln Asn Thr

145 150 155 160

Lys Leu Met Thr Arg Leu Thr Tyr Thr Leu Asp Glu Ile Glu Gly Pro

165 170 175

Phe Glu Val Gly Ala Asp Gly Ser Ile Lys Phe Glu Glu Lys Asp Gly

180 185 190

Ile Asp Tyr Ala Ala Val Thr Val Gln Leu Pro Gly Gly Glu Arg Val

195 200 205

Pro Phe Leu Phe Thr Ile Lys Gln Leu Val Ala Ser Gly Lys Pro Glu

210 215 220

Ser Phe Ser Gly Asp Phe Leu Val Pro Ser Tyr Arg Gly Ser Ser Phe

225 230 235 240

Leu Asp Pro Lys Gly Arg Gly Gly Ser Thr Gly Tyr Asp Asn Ala Val

245 250 255

Ala Leu Pro Ala Gly Gly Arg Gly Asp Glu Glu Glu Leu Ala Lys Glu

260 265 270

Asn Ile Lys Asn Ala Ser Ser Ser Thr Gly Lys Ile Thr Leu Ser Val

275 280 285

Thr Lys Ser Lys Pro Glu Thr Gly Glu Val Ile Gly Val Phe Glu Ser

290 295 300

Ile Gln Pro Ser Asp Thr Asp Leu Gly Ala Lys Ala Pro Lys Asp Val

305 310 315 320

Lys Ile Gln Gly Val Trp Tyr Ala Gln Leu Asp Ser

325 330

<210> 2

<211> 999

<212> DNA

<213> Cucumis sativus Linn.

<400> 2

atggcggctt cagttcaggc ggccgcggct acccttatgc aaccctccaa gttggcttca 60

agaactacca cctctcacct cagatcttct caatcccttt ctaaagcctt tggccttgaa 120

tcttccggac ctagactcac ctgctctctt cactccgatc tcaaagacgt ttctcgcaag 180

tttgccgatg ctgctaagat tgccggcttc gctcttgcca cttccgctct tgtcgtttcg 240

ggagctggtg ctgaaggagt accaaagaga ctcacctttg atgaaattca gagcaagaca 300

taccttgaag ttaagggaac tggaacagct aaccagtgcc caaccattga tggaggagtt 360

gattcatttg ccttcaaggc aggcaaatat caagccaaga aattctgtct tgagccaact 420

tctttcactg tgaaggctga aggagtgagc aagaacgccc caccagaatt ccaaaacacc 480

aagctcatga ctcgtctaac ttacacacta gacgagattg aaggaccctt tgaagttggt 540

gctgatggtt cgatcaagtt tgaggagaaa gatggaattg actatgctgc tgtcactgtc 600

cagttgccgg gaggtgagcg tgtcccgttc ctcttcacca tcaaacaatt ggtggcttct 660

ggaaaaccag agagctttag tggagacttt ttagtcccat cttaccgtgg ctcgtctttc 720

ttggacccaa agggaagagg aggttcaact ggatatgata atgctgttgc attgcctgct 780

ggagggagag gtgatgaaga agaacttgct aaagaaaaca taaagaatgc ttcatcttca 840

acagggaaaa tcactttgag tgtgaccaag agcaagcctg agactggtga agttattggt 900

gtgtttgaga gtattcagcc ttctgatacc gatttgggag caaaagctcc caaggatgtg 960

aagatccagg gtgtttggta tgctcagctt gactcttag 999

<210> 3

<211> 2166

<212> DNA

<213> Cucumis sativus Linn.

<400> 3

gaattaaaaa gtatatccaa gtagaaaatg agtttgtatg ccacgtggaa gaaaattgag 60

gcaaagaatg atgagggaat catccaatga gatttttctt atccaattca ttatcttact 120

ctaaccaaaa ccacataatt catttcctaa atcctctcta ctctccgtta tagtttcagt 180

atcattcccc gcacaaaatt tcacttctcc tctcttacac tcaatccgga tacaacaatg 240

gcggcttcag ttcaggcggc cgcggctacc cttatgcaac cctccaagtt ggcttcaaga 300

actaccacct ctcacctcag atcttctcaa tccctttcta aagcctttgg ccttgaatct 360

tccggaccta gactcacctg ctctcttcac tccgatctca aagacgtttc tcgcaagttt 420

gccgatgctg ctaagattgc cggcttcgct cttgccactt ccgctcttgt cgtttcggta 480

tgttcttatc gtactttcct tccacgatta ctttcttctt atgattctcc acgatgcttt 540

atatttgcgt ttggatgtga atgtctgtat tgttttagtt gctgatattg aattgagttc 600

ttggagaaaa taattatagt tcagacttag aatgggtaat tgataatctc cgaatttgtt 660

taacttagtg gcaatttctt gccaaataaa ggatcaaact acttagatcc aagtggaata 720

atccatttga ttctctgtga cttaataaag tacctgttgt tcttaattta acttgtaatt 780

catttttaca ttaatgatcc cataacattt tcattatact attcaatcaa ggattatttg 840

aaaatcagtc caccacatag tccactgatg atatatggtt agtgtgttac ggtaagtctt 900

aaatgactta aatattagat acagacttta aagttttaaa gcgttaaaga aaatctcact 960

tcaaggactt aattagtctg agtacacttg atatatgaga tctaagaatg tctagactga 1020

ttaacacttg atatatacaa agtgtaatcc tacaacttgg tgagcatctt aattgttcga 1080

tacttttcat gtatttccag ggagctggtg ctgaaggagt accaaagaga ctcacctttg 1140

atgaaattca gagcaagaca taccttgaag ttaagggaac tggaacagct aaccagtgcc 1200

caaccattga tggaggagtt gattcatttg ccttcaaggc aggcaaatat caagccaaga 1260

aattctgtct tgagccaact tctttcactg tgaaggctga aggagtgagc aagaacgccc 1320

caccagaatt ccaaaacacc aagctcatga ctcgtctaac ttacacacta gacgagattg 1380

aaggaccctt tgaagttggt gctgatggtt cgatcaagtt tgaggagaaa gatggaattg 1440

actatgctgc tgtcactgtc cagttgccgg gaggtgagcg tgtcccgttc ctcttcacca 1500

tcaaacaatt ggtggcttct ggaaaaccag agagctttag tggagacttt ttagtcccat 1560

cttaccgtgg ctcgtctttc ttggacccaa agggaagagg aggttcaact ggatatgata 1620

atgctgttgc attgcctgct ggagggagag gtgatgaaga agaacttgct aaagaaaaca 1680

taaagaatgc ttcatcttca acagggaaaa tcactttgag tgtgaccaag agcaagcctg 1740

agactggtga agttattggt gtgtttgaga gtattcagcc ttctgatacc gatttgggag 1800

caaaagctcc caaggatgtg aagatccagg gtgtttggta tgctcagctt gactcttagg 1860

cccccctttc ttcttttatg attgtgtaat attgttcctt gtaagttaaa agagagagtt 1920

gctaaacttt cttatggaga gagacgctag caggggttct atttttatcc aagtttgcat 1980

ataaaatctt gtgccttttc actctttctc aaacatacat ttgcttaact tcataacttt 2040

agaggaagag atggaatcat caatctttga gatgatttag aggaatgaat agaatatata 2100

aaacttttta gatgaaatct aaagcaattt atgagaatta tgttcaaaag tagacaatat 2160

cataca 2166

<210> 4

<211> 239

<212> DNA

<213> Cucumis sativus Linn.

<400> 4

atggcggctt cagttcaggc ggccgcggct acccttatgc aaccctccaa gttggcttca 60

agaactacca cctctcacct cagatcttct caatcccttt ctaaagcctt tggccttgaa 120

tcttccggac ctagactcac ctgctctctt cactccgatc tcaaagacgt ttctcgcaag 180

tttgccgatg ctgctaagat tgccggcttc gctcttgcca cttccgctct tgtcgtttc 239