Method for identifying disease resistant variety of cruciferous crop
A technology of cruciferous crops and cruciferous, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problems of long time spent and inadaptability to modern production and construction.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0053] Cloning of XC_0542 Gene and Construction of Expression Vector pBI0542
[0054] According to the gene sequence of XC_0542, design primers:
[0055] Forward primer: F, 5′-GGGTCTAGAATGGAGATCAAGAAAGCGCAGTCC-3′,
[0056] Reverse primer: R, 5'-GGGGGATCCTCAGCCGGAGCGCGGCGGGTC-3'.
[0057] Using the total DNA of Brassicaceae black rot fungus as a template, the full-length sequence of the gene was amplified by PCR, cloned into pBI121, and the expression vector pBI0542 was constructed and verified by restriction enzyme digestion. Please refer to figure 1 . The sequence was subcloned into pK18mob and verified by sequencing. The sequence was consistent with the sequence of XC_0542 gene (GeneID: 3381550).
[0058] figure 1 Electrophoretic pattern for PCR clone XC_0542 gene, M: 0321 standard DNA (fragment size from large to small: 3kb, 2kb, 1.5kb, 1.2kb, 1.0kb, 0.9kb, 0.8kb, 0.7kb, 0.6kb, 0.5 kb, 0.4kb, 0.3kb, 0.2kb, 0.1kb, ); Lane 1: PCR cloned fragment of XC_0542 gene.
Embodiment 2
[0059] Embodiment 2, introducing expression vector pBI0542 into Agrobacterium
[0060] The triparental pBI0542 was introduced into Agrobacterium tumefaciens EHA105, and the obtained triparental zygote was named EHA105 / pBI0542. For the PCR verification of the triparental zygote, please refer to figure 2 and image 3 .
[0061] figure 2 Restriction electrophoresis pattern of pBI0542 expression vector, M: 0321 standard DNA (fragment sizes from large to small: 3kb, 2kb, 1.5kb, 1.2kb, 1.0kb, 0.9kb, 0.8kb, 0.7kb, 0.6kb, 0.5kb, 0.4kb, 0.3kb, 0.2kb, 0.1kb, ); Lane 1: XbaI / BamHI fragment of gene clone pBI0542.
[0062] image 3 It is the PCR verification electrophoretic pattern of pBI0542 in EHA105, M: 0321 standard DNA (fragment sizes from large to small are: 3kb, 2kb, 1.5kb, 1.2kb, 1.0kb, 0.9kb, 0.8kb, 0.7kb, 0.6kb, 0.5kb, 0.4kb, 0.3kb, 0.2kb, 0.1kb, ); Lane 1: PCR verified fragment of pBI0542 in EHA105.
Embodiment 3
[0064] Agrobacterium Hypersensitivity Reaction (HR) Test
[0065] The plant materials used are cauliflower, A is a disease-resistant variety, and B is a disease-susceptible variety. Healthy cauliflower leaves were selected for testing.
[0066] (1) Inoculate Agrobacterium into LB medium supplemented with corresponding antibiotics and culture overnight at 28°C.
[0067] (2)OD 600 When the temperature is 0.4 to 0.5, collect 1 mL of the bacterial solution in a 1.5 mL EP tube, centrifuge at 5000 rpm for 5 min, pour off the supernatant, and use 2 mL of induction medium (10 mM MgCl 2 , 5mM MES pH5.6, 150μM AS) resuspended, induced at 28°C for 5h.
[0068] (3) Adjust OD with induction medium 600 to 0.4 to 0.5, the wild-type Xcc 8004 of the cruciferous black rot fungus was used as a positive control (such as Figure 4 Middle A1 and B1), EHA105 / pBI121 as negative control (such as Figure 4 in A3 and B3). Use a needle-free syringe to draw the bacterial solution and infuse it into...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com