Clone and expression of maize FK506 binding protein gene and application of maize FK506 binding protein gene
A technology that combines protein and corn, applied in the fields of molecular biology and biology, can solve problems such as improvement, inability to solve the negative effects of soil salinity and cold seeds, and inability to achieve crop yield, and achieve the effect of improving drought resistance.
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Embodiment 1
[0042] Example 1: Obtaining the gene ZmFKBP16-2 encoding the maize FK506 binding protein
[0043] 1. Treatment of corn seedlings: Take corn Chang 7-2 seedlings grown for 10 days, treat them with 400mM mannitol for 18 hours, and collect the leaves;
[0044] 2. Extraction of total RNA: RNAeasyplantminikit (promoga product) was used to extract total RNA;
[0045] 3. Use SMARTcDNALibraryConstructionKit (purchased from Clontech Company) to construct a maize drought stress cDNA library and a control cDNA library;
[0046] 4. Use the reversed-phase Northern differential screening method to obtain the differentially expressed cDNA fragment pTriplEx2; the specific steps refer to "Principles and Techniques of Plant Genetic Engineering" (Wang Guanlin, Fang Hongdu, Science Press, 1998);
[0047] 5. Transform the pTriplEx2 vector carrying cDNA fragments with differential expression into Escherichia coli BM25.8; refer to "Principles and Techniques of Plant Genetic Engineering" for specific...
Embodiment 2
[0050] Embodiment 2: Construction of recombinant expression vector
[0051] Take 2 μl of the maize FK506 binding protein coding gene ZmFKBP16-2 obtained in Example 1 above and connect it to the pMD18-T vector, and the operation steps are carried out according to the instructions of the product pMD18-TVectorsystem of Promega Company. Then transform Escherichia coli DH5α strain, transfer to the LB plate containing ampicillin (100μg / ml) coated with 5-bromo-4-chloro-3-indole-β-D-galactoside and X-gal overnight. White colonies were picked, cultured overnight in LB liquid medium, and identified by colony PCR; at the same time, the plasmid DNA was extracted by alkaline method for sequence determination, and the recombinant pMD18-T vector was obtained.
[0052] Digest the recombinant pMD18-T vector with restriction endonuclease BamHI and restriction endonuclease SalI, and then connect with the vector PBI121 digested with restriction endonuclease BamHI and restriction endonuclease Sal...
Embodiment 3
[0053] Embodiment 3: Obtaining of transgenic Arabidopsis
[0054] The recombinant expression vector PBI121-ZmFKBP16-2 was transformed into the competent cells of Agrobacterium GV3101, and the single clone of Agrobacterium transferred into the recombinant expression vector PBI121-ZmFKBP16-2 was picked and inoculated in LB liquid culture containing 50 mg / L kanamycin culture medium at 28°C for two days with shaking. The fermentation broth was centrifuged at 3000rpm / min for 5 minutes, and the obtained Agrobacterium precipitate was suspended with an infection solution containing 5wt% sucrose and 0.03wt% SilwetL-77 (see also for the specific process: Cheng Zhendong, Wei Zhiming, Xu Zhihong, Genknong Bacillus transformation of Brassica napus and regeneration of transgenic plants, Acta Bot, 1994, 36(9): 657-663).
[0055] Columbia ecotype wild-type Arabidopsis thaliana (Col-0) was transformed by dipping method (purchased from American Arabidopsis Biological Resource Center, ABRC,
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