Short peptide capable of improving expression of protein in plant cell and application thereof
A plant cell and plant expression vector technology, applied in the field of short peptides that can increase protein expression in plant cells, and can solve problems such as low target protein expression
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Embodiment 1
[0029] Embodiment 1 Cloning Sali3-2 gene and the nucleotide sequence fragment of coding SP short peptide
[0030] Using the cDNA of immature seeds of soybean (Glycine max.L) Bainong No. 6 (provided by Jilin Province Baicheng Agricultural Science Research Institute) as a template, PCR was performed to obtain the inserted target gene fragment Sali3-2. The fragment contains a nucleotide fragment encoding the SP short peptide.
[0031] PCR primers are as follows:
[0032] SF: 5′-cacaggat aatttcgatgctcag-3′ with Nco I restriction site
[0033] SR: 5′-tgcgc aacaacaacgttagtctgatagga-3′, with restriction site for XbaI
Embodiment 2
[0034] Example 2 Construction of SP-GFP fusion protein plant expression vector pCAMBIA1302-SP-GFP First, construct plant expression vector pCAMBIA1302-SALI3-2-GFP:
[0035] Set primer SF (5′-cacaggat aatttcgatgctcag-3′, with Nco I restriction site) and SR (5′-tgcgc aacaacaacgttagtctgatagga-3′, with XbaI restriction site), using the cDNA of immature seeds of soybean (Glycine max. Amplify the reaction to obtain the inserted target gene fragment Sali3-2. The obtained Sali3-2 target gene fragment was double-digested with Nco I and Xba I and obtained the digestion product; the pCAMBIA1302 plasmid (plasmid map as shown in figure 1 shown) to perform double enzyme digestion, and obtain the vector digestion product; connect the insert fragment Sali3-2 and the vector digestion fragment, transform TOP10 Escherichia coli, and identify and obtain the Sali3-2-GFP fusion expression vector pCAMBIA1302-Sali3-2-GFP ( Plasmid map such as figure 2 shown), the Sali3-2 gene fragment is locate...
Embodiment 3
[0039] The transformation of embodiment 3 tobacco suspension cells
[0040] The pCAMBIA1302-SP-GFP vector was transformed into Agrobacterium LB4404, and the Agrobacterium transformed with pCAMBIA1302-SP-GFP was screened and identified. Pick the positive single colony of Agrobacterium tumefaciens and inoculate it in LB liquid medium (containing 50mg / L Kan and 50mg / LRif), and culture it with shaking at 28°C for 36-48h; Medium (containing 50mg / L Kan and 50mg / L Rif), cultured with shaking at 28°C until OD 600 Take 1mL of Agrobacterium bacteria liquid at about 0.5, centrifuge at 4000rpm for 2min, discard the supernatant; add an equal volume of tobacco suspension cell culture medium to resuspend the pellet, take 200μL of Agrobacterium into 5mL of tobacco suspension cells cultured for 4 days, and then add 5μL 100μM acetosyringone (AS), mix well, culture statically at 26°C, collect the co-culture in 1.5mL EP tube after two days, centrifuge at low speed, then wash the cells 3 times wi...
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