Phytophthora cinnamomum effector protein SCR93258 and application thereof
A technology of effector protein and Phytophthora, which is applied in the fields of application, angiosperm/flowering plants, and the use of vectors to introduce foreign genetic materials, etc. It can solve the problems of failure to achieve ideal control effects, difficulties in the prevention and control of Phytophthora camphora, and increase the burden on farmers, etc. question
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Embodiment 1
[0045] Example 1 Acquisition of the gene encoding the effector protein SCR93258 of Phytophthora cinnamomea
[0046] The strain of Phytophthora cinnamomum was selected as the test material, and the genome sequence of Phytophthora cinnamomum was analyzed according to the reported effector protein gene information, and the effector protein gene in the whole genome of Phytophthora cinnamomum was obtained. Then, primers p1 and p2 are designed according to the obtained gene fragments, and the obtained target gene fragments are amplified and screened.
[0047] 1. Using CTAB-SDS method to extract high-quality Phytophthora cinnamomum genomic DNA
[0048] Phytophthora cinnamomum strains were cultured on solid V8 medium plate (recipe: 170ml V8 vegetable juice plus 1.6g calcium carbonate, mixed, centrifuged at 2000rpm for 5min to take the supernatant, add purified water to 1L, add 15g agar powder, autoclave Bacteria pot for 20min standby), after culturing for 3 days in a 25°C biochemical...
Embodiment 2
[0062] Example 2 Analysis of the expression pattern of effector SCR93258 in the process of Phytophthora cinnamomea infecting apples
[0063] 1. Phytophthora cinnamomum strains infect apples
[0064] The mycelial blocks stored in the refrigerator at 4°C were placed on V8 solid medium for activation, and cultured at 25°C for 24-36 hours. A suitable size of sterile filter paper was placed at the bottom of the sterile petri dish (diameter 9 cm) and moistened with sterile water. Sterilize on sterile bench. Wipe the surface of the apple with alcohol cotton to sterilize it. Use the outer flame of an alcohol lamp to sterilize the edge of the cutter, cut the apple into small cubes of about 1cm2 on all four sides, and remove the pulp. Pick out the colonies and put them on sterilized filter paper and plug them into the incision on the surface of the apple. Only three sides are plugged, and the other side is used as a control of blank solid medium. Plug the incision with absorbent cot...
Embodiment 3
[0075] Example 3 Transient expression of effector SCR93258 in tobacco
[0076] 1. Construction of PVX recombinant expression vector
[0077] (1) The target gene fragment SCR93258 was amplified by SmaI digestion and PCR, and the inserted fragment was recovered with a size of about 291 bp.
[0078] Reaction system: ddH 2 O (33 μL), SmaI (2 μL), plasmid (10 μL), 10×cut Buffer (5 μL). 37℃, 30min.
[0079] (2) ligated with pGR107 vector digested by the same enzyme, and transformed into Escherichia coli DH5α.
[0080] (3) The transformed DH5α was screened for Kan resistance, and the colonies were obtained and the plasmids were extracted after shaking at 37°C overnight.
[0081] (4) The recombinant plasmid was identified by restriction endonuclease SmaI. The recombinant plasmids that were initially identified correctly by enzyme digestion (two target bands of about 10098 bp and 291 bp in size were obtained) were sent to GenScript Bio Co., Ltd. for sequencing. The recombinant pl...
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