Protein VaPBP2-L for enhancing drought resistance of plants and application of protein VaPBP2-L
A drought resistance, vapbp2-l technology, applied in the direction of application, plant peptides, plant products, etc., to achieve the effect of promoting the cultivation process, improving plant drought tolerance, and improving drought resistance
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Embodiment 1
[0022] Example 1 - Acquisition of protein VaPBP2-L and its coding gene and recombinant expression vector
[0023] (1) Using 9.0% mannitol stress treatment for 36h as material, extract total RNA, and obtain cDNA by reverse transcription, using the cDNA as a template, under the guidance of primer VaPBP2-L-F1 and primer VaPBP2-L-R1 , amplified by the conventional PCR method, after the reaction, the PCR amplified product was detected by 1% agarose gel electrophoresis, and a DNA fragment of about 1511bp was recovered and purified, such as figure 1 shown;
[0024] (2) Using LIC (non-ligation reaction cloning) reaction, the gene fragment is connected to the carrier PVX-LIC (the T-DNA fragment of the carrier PVX-LIC contains the lethal gene ccdB, and its two sides contain the recognition sequence of the LIC reaction, The vector is owned by the laboratory, literature: Zhao J, Liu Q, Hu P, et al (2016) An efficient Potato virus X-basedmicroRNA silencing in Nicotiana benthamiana. Sci Re...
Embodiment 2
[0056] Example 2 - Acquisition of recombinant Agrobacterium tumefaciens
[0057] The recombinant vector PVX-LIC-VaPBP2-L was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method to obtain Agrobacterium tumefaciens GV3101 containing the recombinant vector PVX-LIC-VaPBP2-L, and the recombinant Agrobacterium was named GV3101 / PV X- LIC-VaPBP2-L; (freeze-thaw method refers to Amanda M Davis, Anthony Hall, Andrew J Millar, ChiarinaDarrah and Seth J Davis, Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana, 2009, public available obtained from Yangtze University).
[0058] The empty vector PVX-LIC was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method to obtain Agrobacterium tumefaciens GV3101 containing the empty vector PVX-LIC, and the recombinant Agrobacterium was named GV3101 / PVX-LIC.
Embodiment 3
[0059] Example 3 - Acquisition and Identification of Transient Expression Transgenic Tobacco
[0060] (1) Obtaining genetically modified tobacco
[0061] Two kinds of recombinant Agrobacterium GV3101 / PVX-LIC-VaPBP2-L and GV3101 / PVX-LIC obtained in Example 2 were used to prepare an Agrobacterium suspension, and the volume ratio of the culture medium to the cell in the suspension was 1:1. The seeds of Nicotiana benthamiana were sown in the culture medium (peat: vermiculite: perlite mixed at a volume ratio of 1:3:0.5) and cultivated in an artificial greenhouse. When the tobacco grows to 4-5 leaves, start injecting the topmost fully expanded new leaves. Use a disposable syringe to absorb 1mL of the bacterial solution, remove the needle of the syringe, hold the lower part of the leaf with your fingers, and gently force the bacteria in the syringe to hydraulically send and penetrate into the leaf tissue. Inject 2 leaves per tobacco, GV3101 / PVX- Five plants were injected with LIC-V...
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