Dendranthema dichrum cold-resistant gene DdICE1 as well as plant expression vector and constructing method thereof
A plant expression vector and heterochromatic technology, applied in the field of molecular biology, can solve the problem that the improvement of plant cold resistance is not significant, and achieve the effect of improving cold resistance
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0041] Example 1 Cloning of DdICE1:
[0042] Choose Dendranthema dichrum as the material, select healthy cuttings, and the seedlings (20 days) that survived the cuttings were treated with continuous stress at low temperature (4°C) for 7 days, and 0.05g of young leaves were taken, and extracted with reference to Trizol RNA Kit (TaKaRa) manual method, extract total RNA from leaves, take 1 μg total RNA reverse transcription into cDNA according to M-MLV Reverse Transcription Kit (TaKaRa), digest cDNA product with RNase, refer to Arabidopsis ICE1 sequence information ( NCBI accession number: AT3G26744), analyzed and designed primers to amplify DdICE1 by Oligo6.0 software;
[0043] Upstream primer DdICE1-F: 5′-ATGCTACCGGAAAACGACA-3′ (SEQ ID NO.2)
[0044] Downstream primer DdICE1-R: 5′-AATGGCACCATGATAACCT-3′ (SEQ ID NO.3)
[0045] Using the extracted leaf cDNA as a template, carry out PCR reaction,
[0046]50 μL reaction system: 5.0 μL of 10×RCR Buffer, 1.0 μL each of DdICE1-F an...
Embodiment 2
[0047] Example 2 Construction of plant expression vector pEarleyGate103-DdICE1
[0048] Design primers for PCR reaction, introduce restriction sites Sal I and Not I at the upstream and downstream of the target gene DdICE1 respectively, connect the PCR product to the pMD19-T Simple vector, transform TOP10 competent cells, and extract positive plasmids, Sal I and Not I double-digested DdICE1 fragment was ligated with Sal I and Not I double-digested pENTR1A, and transformed. The extracted positive plasmid was linearized by NsiI single-enzyme digestion, and then carried out LR recombination reaction with pEarleyGate103 vector plasmid (Invitrogen, USA), and transformed. The positive plasmid was extracted, detected by electrophoresis and verified by sequencing as SEQ ID NO.1.
[0049] Upstream primer DdICE1-gateway-F: 5′-GCGTCGACATGCTACCGGAAAACGACA-3′ (SEQ ID NO.4), downstream primer DdICE1-gateway-R: 5′-TTGCGGCCGCGAAATGGCACCATGATAACC
[0050] T-3' (SEQ ID NO. 5).
[0051] (1) Usi...
Embodiment 3
[0054] Example 3 Genetic transformation of Arabidopsis thaliana with plant expression vector pEarleyGate103-DdICE1 and identification of its cold resistance
[0055] (1) Competent preparation and freeze-thaw transformation of Agrobacterium strain EHA105
[0056] Pick a single colony of EHA105 from the YEB (50 μg / mL rifampicin) plate, inoculate it in 50 mL of YEB liquid medium containing 50 μg / mL rifampicin, cultivate at 200 rpm, 28°C until the OD value is 0.5, and then Ice-bath the bacterial solution for 30 min, collect the bacterial cells by centrifugation, and suspend in 2 mL of pre-cooled 100mM CaCl 2 (20% glycerol) solution, aliquot 200 μL / tube and set aside.
[0057] Take 10 μL pEarleyGate103-DdICE1 vector plasmid, add 200 μL competent cells, ice-bath for 30 min, freeze in liquid nitrogen for 5 min, 37°C for 5 min, add 800 μL YEB liquid medium, pre-cultivate at 28°C 200 rpm for 4 h, apply bacterial solution Plate on YEB (50 μg / mL rifampicin + 50 μg / mL kanamycin) solid ...
PUM
Abstract
Description
Claims
Application Information
- 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, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com