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Application of cspsi-n protein cspsi-n in cucumber photosynthetic system i reaction center in resistance to melon blight

A technology of photosynthetic system and reaction center, applied in the field of plant molecular biology and plant genetic engineering, can solve the problem that the N subunit protein of cucumber photosynthetic system I reaction center has not yet been discovered, and achieve the effect of wide application prospects

Active Publication Date: 2022-07-12
INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that the photosynthetic system I reaction center subunit protein plays an important role in plant photosynthesis, but there is no report on the cucumber photosynthetic system I reaction center N subunit protein CsPSI-N in the resistance to melon blight

Method used

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  • Application of cspsi-n protein cspsi-n in cucumber photosynthetic system i reaction center in resistance to melon blight
  • Application of cspsi-n protein cspsi-n in cucumber photosynthetic system i reaction center in resistance to melon blight
  • Application of cspsi-n protein cspsi-n in cucumber photosynthetic system i reaction center in resistance to melon blight

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Construction of cucumber CsPSI-N gene overexpression vector

[0093] The cucumber CsPSI-N gene belongs to the N subunit protein gene of the photosynthetic system I reaction center. The full-length CDS of the cucumber CsPSI-N gene (SEQ ID NO. 2) was constructed into pGWB5 (the plasmid map of pGWB5 is as follows: figure 1 The specific position is between attR1 and attR2 after the 35S promoter (the ccdB gene was replaced by gene recombination exchange) to obtain an overexpression vector (pGWB5-CsPSI-N); the CDS non-conserved region of the cucumber CsPSI-N gene was The fragment of 200bp length (CDS108-307bp, SEQ ID NO.4) and its complementary fragment were constructed on pK7GWIW (pK7GWIW plasmid map such as figure 2 shown), the specific position is between the two attR1 and attR2 after the 35S promoter (the ccdB gene was replaced by gene recombination exchange), and the gene silencing vector (pK7GWIW-CsPSI-N) was obtained.

[0094] The amino acid sequence of cu...

Embodiment 2

[0102] Example 2 Construction of a transient overexpression / gene silencing cucumber cotyledon model

[0103] (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 hours.

[0104] (2) Pick a single clone and add it to 4 mL of LB medium containing the corresponding antibiotics and rifampicin, shake the bacteria at 28° C. and 180 rpm for 24 h.

[0105] (3) Add in fresh LB medium containing corresponding antibiotics and rifampicin at a ratio of 1:100, shake the bacteria at 28°C and 180 rpm until the OD600 value is about 3.0.

[0106] (4) The cells were collected by centrifugation at 3000 rpm for 5 minutes, and the cells were collected with a suspension (10 mM MES, 10 mM MgCl) 2 ) to resuspend the cells, adjust the OD600 value to about 0.4, and add 200 mM acetosyringone.

[0107] (5) Stand at room temperature for 3h.

[0108] (6) Use a syr...

Embodiment 3

[0121] Example 3 Disease resistance experiment of cucumber cotyledons

[0122] For the cucumber cotyledon model with transient overexpression of CsPSI-N, the cucumber cotyledon model with CsPSI-N gene silencing, and the wild-type cucumber cotyledon successfully constructed in Example 2, the injected cucumber seedlings were cultured in the dark for 12 hours and then incubated in the light at 22°C for 3 days. Days later, the cotyledons of the cucumbers were isolated and inoculated with Phytophthora cucurbita, and then the cotyledons of the cucumbers were cultured in a Petri dish at 28°C for 24h in the dark.

[0123] The resistance function of the target gene to the disease was judged according to the size of the isolated cotyledon of cucumber with different treatments. The results are shown in the appendix. Figure 5 shown.

[0124] The results showed that transient overexpression of CsPSI-N gene (pGWB5-CsPSI-N) in cucumber cotyledons could significantly enhance the resistance ...

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Abstract

The invention belongs to the field of plant molecular biology and plant genetic engineering, and discloses the application of the N subunit protein CsPSI-N of the I reaction center of the cucumber photosynthetic system in the resistance to melon blight. The present invention discovers for the first time that the cucumber photosynthetic system I reaction center N subunit protein CsPSI-N has resistance to cucurbit blight: inoculate Phytophthora cucurbita on the cotyledons of plants overexpressing the cucumber photosynthetic system I reaction center N subunit protein CsPSI-N , the lesion area was significantly reduced, while the cotyledons of plants in which the N subunit protein CsPSI-N of the cucumber photosystem I reaction center was silenced were inoculated with Phytophthora cucurbita, and the lesion area increased significantly; it can be seen that the cucumber photosystem I reaction center N The subunit protein CsPSI-N plays a very important role in plant resistance to melon blight, and can be used for melon blight resistance, preparation of melon blight-resistant products, and cultivation of melon blight-resistant transgenic plants, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of plant molecular biology and plant genetic engineering, and in particular relates to the application of CsPSI-N, an N subunit protein of cucumber photosynthetic system I reaction center, in resistance to melon blight. Background technique [0002] Melon blight is a worldwide oomycete disease. The pathogen is Phytophthora melonis, which causes the leaves, stems and fruits of cucumbers, pumpkins, wax gourds, bitter gourds and other melons to turn brown and kill or kill vaccines. The disease occurs in all areas where melons are grown in my country. In the north, the occurrence of open field cultivation in summer and autumn and the protection of sweet potatoes in spring are heavier. The whole growth period and various parts of melons can be damaged. The damaged growth point and the base of the tender stem of the seedlings are water-soaked, constricted, wilted and die. The stems are damaged at the adult stage, the base...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/34
CPCC07K14/415C12N15/8282
Inventor 王瑞吴廷全杜虎林毓娥徐晓美金庆敏杨晓珊
Owner INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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