Plant expression vector containing chloroplastic glutamine synthetase genes, construction and use thereof
A technology of plant expression vectors and chloroplasts, applied in the field of plant expression vectors, can solve the problems of low nitrogen utilization, difficulties in meeting the needs of crop growth and development, environmental hazards, etc., and achieve the effect of enhancing nitrogen utilization
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Embodiment 1
[0048] Embodiment 1: GS2 gene cDNA amplification and TA cloning
[0049] First, search for the full-length gene cDNA sequence of Arabidopsis chloroplast type GS2 from GenBank, and design a pair of primers with the following sequence:
[0050] GS25: 5'-caccgcATGcCTCAGATCTTAGCAGCTTC-3'
[0051] GS23: 5'-gaattcTTAAACATTCAAAGAAAGCTTTT-3'
[0052] Primer GS25 at the 5' end, add the characteristic sequence of caccgc at the end, and change the G after ATG to c to form a SphI restriction site; primer GS23 at the 3' end, add an EcoRI site at the end;
[0053] Use TRIzoL Reagent (Invitrogen) to extract total RNA from Arabidopsis thaliana seedlings, take 0.1g of young leaves of Arabidopsis thaliana, add 1ml of TRIzoL RNA extraction solution, grind in a mortar, let stand at room temperature for 5min, and then transfer to a centrifuge tube , add 0.2ml of chloroform, shake and mix, and centrifuge at 12000rpm / min for 15min at 4°C. Transfer the supernatant, add 0.5ml of isopropanol, mix we...
Embodiment 2
[0055] Example 2: Construction of Gateway entry cloning vector pENTR*-PRbcS-*T-GS2
[0056] The construction strategy of the entry cloning vector pENTR*-PRbcS-*T-GS2 is as follows: Figure 4 As indicated, the purified plasmid vectors pENTR*-PrbcS-*T-GFP and pMD18-GS2 were cut with SphI (Fermentas) and EcoRI (Fermentas), and the cut vectors and inserts were separated by agarose gel electrophoresis. The vector fragment pENTR*-PrbcS-*T (4.0kb) produced after pENTR*-PrbcS-*T-GFP was cleaved and the DNA fragment (1.2kb) of the GS2 gene produced by cleavage of pMD18-GS2 were recovered in the gel, and then The DNA fragments of pENTR*-PrbcS-*T and GS2 genes were ligated with the ligase kit of TaKaRa to generate the entry vector pENTR*-PRbcS-*T-GS2. Conversion of high efficiency (10 8 ) Escherichia coli competent (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on the LB plate added with kanamycin (Km, 50μg / ml), cultivate overnight at 37°C, and screen f...
Embodiment 3
[0057] Embodiment 3: Construction of GS2 gene plant expression vector pK2-35S-Prbcs-*T-GS2
[0058] The construction strategy of GS2 gene plant expression vector is as follows: Image 6 As shown, pENTR*-PrbcS-*T-GS2 was subcloned into the plant expression vector pK2GW7 (the destination vector of Gateway, purchased from Belgium VIB / Gent Company) through the LR reaction of Gateway technology. The specific method is: use the plasmid extraction kit to purify Gateway’s target vector pK2GW7, add 150ng each of pENTR*-PrbcS-*T-GS2 and pK2GW7 to the LR reaction system of Gateway, 1μl LR ClonaseII Enzyme Mix (Invitrogen), mix After reacting overnight at 25°C, PrbcS-*T-GS2 was integrated into pK2GW7 by the action of integrase to obtain the plant expression vector plasmid pK2-35S-Prbcs-*T-GS2 of GS2. Conversion of high efficiency (10 8 ) Escherichia coli competent (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on an LB plate added with spectinomycin (Spe...
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