Plant expression vector for SGF14a gene of Tanba black soybean and application of plant expression vector
A plant expression vector, pk-35s-sgf14a technology, applied in the fields of application, plant products, genetic engineering, etc., can solve the problem of lack of experimental data such as 14-3-3 protein
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Embodiment 1
[0039] Embodiment 1: Obtaining of exogenous SGF14a gene
[0040] (1) Detection of the expression level of SGF14a gene under aluminum stress
[0041] Select Danba Black Soybean (RB) seedlings with consistent growth in hydroponics for 2 weeks, and first use 0.5mM CaCl at pH 4.3 2 After overnight pretreatment, and then placed in 50 μM AlCl 3 solution (containing 0.5mM CaCl 2 , pH 4.3) after treatment for 0 (CK), 2, 4, 8, 12 and 24 h respectively, 0.3 g of 1-2 cm root tip samples were collected, quickly frozen in liquid nitrogen, and root tips were extracted using Trolz kit For total RNA, the possible residual DNA in the RNA was digested with DNase, and extracted and purified by phenol:chloroform:isoamyl alcohol (25:24:1). Take 5 μg from each RNA sample, use M-MLV Reverse Transcriptase (Promega Company) to synthesize cDNA by reverse transcription, use the cDNA synthesized by reverse transcription as a template for RT-PCR amplification, and use 28s rRNA as an internal refere...
Embodiment 2
[0044] Example 2: Plant expression vector pK-35S- SGF14a build
[0045] (1) Construction of T / A cloning vector
[0046] Strategies for constructing plant expression vectors such as image 3 As shown, the PCR product recovered from the gel (with HindIII and XhoI restriction sites at both ends) was ligated with the pMD-18T vector under the action of T4-DNA ligase to obtain the recombinant vector pMD18T- SGF14a . Then the recombinant vector pMD18T- SGF14a Is it connected correctly. because SGF14a Both ends of the gene contain HindIII and XhoI restriction sites. Use these two enzymes to digest the plasmid of the recombinant vector. If the connection is correct, a fragment 0.8Kb in size similar to the target gene can be cut out ( Figure 4 A). At the same time, through bacterial liquid and plasmid PCR detection, it was found that a fragment 0.8Kb similar in size to the target gene could be amplified ( Figure 4 B), while the negative control cannot amplify fragments of ...
Embodiment 3
[0051] Example 3: Transformation of plant expression vector pK-35S-SGF14a into Agrobacterium and screening of transgenic tobacco
[0052] Take a small amount of detected correct plant expression vector pK-35S- SGF14a Add the plasmid into the Agrobacterium competent cells and mix gently; add the mixture to the pre-cooled electroporation cup, tap the cup body gently to make the mixture reach the bottom of the cup; place the electroporation cup on the chute of the electrotransformer After the electric shock, immediately take out the electroporation cup and quickly add 0.5mL SOC medium, mix well, and transfer to a 1.5ml centrifuge tube; incubate on a shaker at 200rpm at 28°C for 3-5h; at room temperature, centrifuge at 7500rpm for 1min, discard it Clean up to 100 μl and suspend the cells; spread the bacteria on LB solid medium with antibiotics, and incubate at 28°C for 2 days; select positive clones, and perform bacterial liquid PCR detection. It can be seen from the bacterial l...
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