Special carrier for improving utilization ratio of plant nitrogen nutrition and use thereof
A plant nitrogen, plant expression vector technology, applied in the field of special vector construction, can solve the problems of variable amino acid sequence and non-conservation, and achieve the effect of promoting growth and improving utilization efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0069] Embodiment 1, construction of intermediate vector pUC118-PrbcS-T
[0070] Purify (according to the instructions of the kit) pUC118-PrbcS-T-rbcS-3C with a plasmid extraction kit (Broad Tech), and pUC118-PrbcS-T-rbcS-3C (formed by Sugita et al. constructed and provided, rbcS-3C in Sugita et al., 1987, MGG, 209:247-256) was cut out, and the cut vector 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 ligase kit of TaKaRa to ligate (operate according to the kit instructions) the vector DNA fragment without rbcS-3C to generate an intermediate vector pUC118-PrbcS -T (Figure 1), conversion of high efficiency (10 8 ) Escherichia coli competent cells (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on a plate added with ampicillin (Amp, 100 μg / ml), cultivate overnight at 37°C, and screen Amp-resistant recombinants The plasmid was extracted from the Amp-resi...
Embodiment 2
[0071] Example 2, using point mutation technology to introduce NcoI site in the intermediate vector pUC118-PrbcS-T
[0072] Using the purified plasmid pUC118-Prbcs-T as a template, a pair of complementary primers (NcoI5 and NcoI3, Figure 1) for point mutations were designed according to the chloroplast localization sequence, and TaKaRa was commissioned to synthesize them. Add 25ng of purified plasmid pUC118-PrbcS-T to the point mutation reaction mixture as a template, and at the same time add 125ng of point mutation primers NcoI5 and NcoI3, 1μldNTP (2.5mM), 5μl of 10×KOD reaction buffer and 1μl of KOD polymerase (Toyobo Japan), add double distilled water to make the final reaction volume 50 μl. Heated at 95°C for 30 seconds on a PCR instrument, followed by 15 cycles of reaction at 95°C for 30 seconds, 55°C for 1 minute, 68°C for 10 minutes, and finally extended the reaction at 68°C for 10 minutes to synthesize The child chain of the mutation site. After the reaction is compl...
Embodiment 3
[0073] Example 3, construction of special vector pPZP221-PrbcS-Dof1
[0074] The construction strategy of the special vector pPZP221-PrbcS-Dof1 is shown in Figure 2. The first choice is to search for the full-length gene sequence of Arabidopsis Dof1 from GenBank, and design a pair of specific primers to amplify the Arabidopsis first-strand cDNA as a template. The full-length cDNA of Dof1 was obtained, the full-length gene fragment of Dof1 was recovered and purified, and connected to pMD18-T (Treasure Bioengineering Co., Ltd. (Dalian), TaKaRa) vector to obtain pMD-Dof1. Digest pMD-Dof1 and pUC118-PrbcS-*T with NcoI and XbaI, recover and purify the Dof1 gene fragment and the large vector fragment pUC118-PrbcS, and then connect and obtain the recombinant plasmid pUC118-PrbcS-Dof1. Digest pUC118-PrbcS-Dof1 and pPZP221 with HindIII and XbaI, recover and purify the small fragment PrbcS-Dof1 and the large fragment pPZP221, and then connect to obtain the recombinant vector pPZP221-Prb...
PUM
![No PUM](https://static-eureka.patsnap.com/ssr/23.2.0/_nuxt/noPUMSmall.5c5f49c7.png)
Abstract
Description
Claims
Application Information
![application no application](https://static-eureka.patsnap.com/ssr/23.2.0/_nuxt/application.06fe782c.png)
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap