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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

Inactive Publication Date: 2013-04-10
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, many genes related to cold resistance have been cloned, and the results show that changing one or several functional genes alone does not significantly improve the cold resistance of plants

Method used

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  • Dendranthema dichrum cold-resistant gene DdICE1 as well as plant expression vector and constructing method thereof
  • Dendranthema dichrum cold-resistant gene DdICE1 as well as plant expression vector and constructing method thereof
  • Dendranthema dichrum cold-resistant gene DdICE1 as well as plant expression vector and constructing method thereof

Examples

Experimental program
Comparison scheme
Effect test

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 ...

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Abstract

The invention relates to a dendranthema dichrum cold-resistant gene DdICE1 as well as a plant expression vector and constructing method thereof, belonging to the field of molecular biology. The sequence of the gene DdICE1 is shown in SEQ ID NO.1, and the expression vector is obtained by inserting the gene DdICE1 between the Sal I and Not I endonuclease sites of a gateway vector pENTRlA and enabling recombined pENTRlA-DdICEl plasmid Nsi I single endonuclease digestion to be linearized and then subjected to LR recombination with a pEarleyGate103 vector plasmid. The dendranthema dichrum cold-resistant gene DdICE1 and plant expression vector provided by the invention can be directly used for carrying out agrobacterium-mediated genetic transformation, creating new cold-resistant germplasm, improving the cold resistance of plants, and improving breeds of the plants.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a cold-resistant gene DdICE1 of the genus Chrysanthemum chrysanthemum, a plant expression vector and a construction method thereof. Background technique [0002] Low temperature is the main limiting factor affecting the geographical distribution, yield and quality of plants. Therefore, the research on plant cold resistance has always been one of the hot spots in the field of botany research. Chrysanthemum is native to my country and is one of the top ten traditional famous flowers in my country and one of the four major cut flowers in the world. It has high ornamental and economic value and occupies an important position in flower production. Low temperature is the main limiting factor that restricts the annual supply of chrysanthemums. In order to meet market demand, production needs to be heated in winter. However, expensive heating costs and huge energy consumption have become...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/66
Inventor 陈素梅陈煜陈发棣刘兆磊房伟民蒋甲福管志勇滕年军
Owner NANJING AGRICULTURAL UNIVERSITY
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