Proteins, genes, recombinant vectors, expression cassettes, hosts and uses
A technology of recombinant vectors and expression cassettes, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., to achieve the effect of improving plant photosynthetic efficiency and enzyme activity
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Embodiment 1
[0068] Conservative primers were designed based on the wheat mRNA sequence (Genebank NO. DQ456872.1), as shown in SEQ ID NO:5 and SEQ ID NO:6 in Table 1, and Xiaoyan 54 wheat material was prepared using the whole gold RNA-cDNA one-step kit The cDNA was used as a template for PCR amplification of its 6-phosphoglucose protein gene using FastPfu enzyme.
[0069] The PCR amplification reaction system is 40 μl, including: cDNA 100ng, FastPfu high-fidelity enzyme 1μl, FastPfuPCR buffer (10 ) 4 μl, pHUE-TaPGI1-For final concentration 40pM, pHUE-TaPGI1-rev final concentration 40pM, dNTPs (2.5mM each) 4μl, and the rest ddH 2 O. The PCR reaction program was: 95°C for 30s, 56°C for 30s, 72°C for 60s, 35 cycles; 72°C for 10 minutes to end the reaction.
[0070] After the PCR amplification, the amplification results were sequenced. The sequencing results showed that the amplified glucose-6-phosphate isomerase protein gene had five amino acid correspondences compared with the glucose-6-p...
Embodiment 2
[0079] This example is used to illustrate the in vitro expression and purification of wheat TaPGI1 protein and TaPGI1mT521E protein:
[0080] The pHUE-TaPGI1 recombinant expression vector and the pHUE-TaPGI1mT521E recombinant expression vector were transformed into BL21 (DE3) Escherichia coli by heat shock transformation, and two recombinant bacteria were obtained. The two recombinant bacteria were inoculated into 100 ml of LB liquid medium containing ampicillin resistance (100 μg / ml), and cultured overnight at 37°C and 220 rpm with shaking. On the next day, transfer 20 ml of recombinant bacteria to 1 liter of fresh LB liquid medium containing ampicillin resistance (100 µg / ml), and continue to shake at 37°C and 220 rpm until OD 600nm =1.0 (detected by spectrophotometer), add IPTG with a final concentration of 0.2mmol / L to the culture medium, and induce culture at 18°C for 16 hours, and collect the bacteria by centrifugation at 4000 rpm after the induction culture .
[0081...
Embodiment 3
[0086] This example is used to detect the enzyme activity of the TaPGI1 protein and TaPGI1mT521E protein purified in Example 2:
[0087] (1) Establishment of BCA standard curve
[0088] In order to compare the enzymatic activity of two proteins, it is first necessary to perform a quantitative experiment on the target protein so that the two proteins have the same concentration. Accurate quantification of TaPGI1 protein and TaPGI1mT521E protein was performed using the recognized BCA protein quantification kit (Pierce). image 3 In order to use the protein standard curve established by the BCA protein quantification method to the standard protein (BSA), the TaPGI1 protein and TaPGI1mT521E protein were accurately quantified based on this standard curve.
[0089] (2) Detection and comparison of in vitro enzyme activity of TaPGI1 protein and TaPGI1mT521E protein
[0090] Standard experimental methods were used to detect the enzyme activity of the purified TaPGI1 protein and TaPGI...
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