Plant expression vector of citric acid synthesis gene and application thereof
A plant expression vector and gene technology, applied in the field of plant genetic engineering, can solve the problems of no tissue specificity, etc., and achieve the effect of improving resistance, improving tolerance, and good root growth
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[0057] Example 1. Construction of intermediate vector pUC118-PrbcS-T
[0058] Purify pUC118-PrbcS-T-rbcS-3C (constructed and provided by Sugita et al., Sugita et al., 1987, MGG, 209: 247-256) , the rbcS-3C in pUC118-PrbcS-T-rbcS-3C was cut out with the restriction enzyme SphI (Fermentas), and the cut vectors pUC118-PrbcS-T and rbcS-3C were separated by agarose gel electrophoresis Fragment, recover the 4.6kb vector pUC118-PrbcS-T, and then use the TaKaRa ligase kit to ligate (according to the kit instructions) the vector DNA fragment without rbcS-3C to generate an intermediate vector pUC118-PrbcS -T( figure 1 ), using the ligation reaction mixture to transform high efficiency (10 8 ) of Escherichia coli competent cells (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on a plate with ampicillin (Amp, 100 μg / ml), culture at 37°C overnight, and screen for Amp-resistant recombination The plasmid was extracted from the Amp-resistant recombinant sub...
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[0059] Example 2. Introduction of NcoI site into intermediate vector pUC118-T-PrbcS using point mutation technology
[0060] Using the purified plasmid pUC118-Prbcs-T as a template, a pair of complementary primers for point mutation (NcoI5 and NcoI3, figure 1 ), entrusted to TaKaRa synthesis. 25ng of purified plasmid pUC118-PrbcS-T was added to the point mutation reaction mixture as a template, 125ng of point mutation primers NcoI5 and NcoI3, 1 μl dNTP (2.5 mM), 5 μl of 10×KOD reaction buffer and 1 μl of KOD polymerase were added at the same time (Toyobo, Japan), and double-distilled water was added to make the final volume of the reaction 50 μl. Heating at 95°C for 30 seconds on a PCR machine, followed by 15 cycles of reaction at 95°C, 30 seconds, 55°C, 1 minute, 68°C, 10 minutes, and finally prolonging the reaction at 68°C for 10 minutes. daughter strand at the mutated site. After the reaction was completed, the reaction mixture was placed on ice for 2 minutes, and 1 μl ...
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[0061] Example 3. Construction of plant expression vector pPZP211-PrbcS-cs
[0062] The construction strategy of plant expression vector pPZP211-PrbcS-cs is as follows: figure 2 As shown, the full-length gene sequence of tobacco cs is firstly searched from GenBank, and a pair of specific primers are designed to amplify the first-strand cDNA of tobacco to obtain the full-length cDNA of cs, recover and purify the full-length gene fragment of cs, It was ligated into pMD18-T vector to obtain pMD-cs. The pMD-cs and pUC118-PrbcS-*T were digested with NcoI and XbaI, the cs gene fragment and the large vector fragment pUC118-PrbcS were recovered and purified, and then the recombinant plasmid pUC118-PrbcS-cs was obtained by ligation. The pUC118-PrbcS-cs and pPZP211 were digested with HindIII and XbaI, the small fragment PrbcS-cs and the large fragment pPZP211 were recovered and purified, and then ligated to obtain the recombinant vector pPZP211-PrbcS-cs, which contains the promoter Pr...
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