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Expression vector of arabidopsis gene REM16 and application to regulation and control of flowering stages of plants thereof

A technology of REM16 and overexpression vector, which is applied to the expression vector of Arabidopsis gene REM16 to regulate the flowering period of plants, and can solve the problems of unclear key regulatory factors and molecular regulation mechanism of target genes

Active Publication Date: 2017-06-13
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These studies have shown that ADF protein is directly involved in the regulation of plant growth and flowering, but the target genes it regulates, the key regulatory factors involved, and the molecular regulatory mechanisms are not clear.

Method used

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  • Expression vector of arabidopsis gene REM16 and application to regulation and control of flowering stages of plants thereof
  • Expression vector of arabidopsis gene REM16 and application to regulation and control of flowering stages of plants thereof
  • Expression vector of arabidopsis gene REM16 and application to regulation and control of flowering stages of plants thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, REM16 gene cloning

[0023] 1. Planting of Arabidopsis

[0024] Surface disinfection of Arabidopsis seeds: Put 50 Arabidopsis seeds to be sterilized into a 1.5 ml centrifuge tube, add 1 ml of 75% ethanol (containing 0.03% TritonX-100 by volume) to sterilize by shaking for 1 min, and then Sterilize with 70% ethanol for 1 min (twice), and finally use a suction tip to suck the seeds onto sterile filter paper and dry them, then use a sterile toothpick to spot them into 1 / 2 MS medium, and put them in the refrigerator for 4 After vernalization at ℃ for 3 days, they were taken out and placed in a light incubator for cultivation.

[0025] The culture conditions were as follows: 16 h light / 8 h dark alternating light, temperature 22 °C, light intensity 100 μmol m -2 ·s -1 . Then, after 7-10 days of light growth, the Arabidopsis seedlings were moved to the soil, covered with plastic wrap for 3-7 days and removed, and then cultivated in the cultivation room under...

Embodiment 2、35

[0066] Embodiment 2, 35S::REM16 CDS expression vector construction

[0067] 1. Obtaining the target gene

[0068] Primer design and PCR reaction system and conditions:

[0069] (1) Using the correctly sequenced recombinant plasmid as a template and KpnI-REM16-For and XbaI-REM16 Rev as primers, the 861 bp full-length REM16 gene coding (CDS) sequence was amplified by PCR. The primer sequences used were as follows:

[0070] KpnI-REM16-For: 5'-CGGGGTACCCCG ATG GCT GAC GAT AGC GAAT-3' (SEQ ID No: 6);

[0071] XbaI-REM16-Rev: 5'-GCTCTAGAGC TCA AGC ATC TCT ACG CAA GAC ATG-3' (SEQ IDNo: 7);

[0072] The PCR reaction system and conditions are the same as in Example 1.

[0073] 2. Construction of expression vector

[0074] The product recovered from the gel in the previous step and the pCambia1300-221-HA vector with XbaI and KpnI restriction sites were subjected to double digestion with KpnI and XbaI respectively, and then carried out under the catalysis of T4 ligase at a ratio of...

Embodiment 3

[0081] Embodiment 3, REM16- RNAi vector construction

[0082] 1. Obtaining target fragments containing fixed adapters (attB1, attB2)

[0083] Will sequence correctly contain the target fragment REM16 The 18-T recombinant plasmid of the gene was diluted 1000 times, and the diluted plasmid was used as a template, according to the above cloning conditions, and the upstream and downstream primers with attB linkers were used as primers to clone the target fragment. The target fragment is recovered from the gel.

[0084] 2. BP recombination reaction system and conditions

[0085] components Volume (µL) / tube attB-PCR recovery product with adapter 3 pDONR221 vector (150ng / µL) 2 BPase 2 TE Buffer, pH 8.0 to 10

[0086] Incubate at 25°C for 20 h, add 1 µl Proteinase K to each reaction, and place in a water bath at 37°C for 10 min. Transform Escherichia coli competent cells, pick a single colony, verify the bacterial liquid by PCR, and ext...

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Abstract

The invention discloses an expression vector of an arabidopsis gene REM16 and application to regulation and control of flowering stages of plants thereof. Experimental analysis shows that arabidopsis gene REM16 has a function of regulating and controlling the flowering stages; an overexpression vector and a gene silencing vector of the arabidopsis gene REM16 are obtained; the overexpression REM16 gene causes flowering relevant gene expression change in transgenic arabidopsis plants so as to accelerate arabidopsis to flower early; compared with the flowering time of wild plants, the expression is early flowering character; the silencing REM16 gene causes flowering relevant gene expression change in transgenic arabidopsis plants so as to delay flowering of arabidopsis; compared with the mild plants, the expression is late flowering character. The function of regulating and controlling flowering of the REM16 gene is determined by analyzing the expression of flowering relevant genes of the transgenic plant strains. According to the technical scheme, the expression vector of the arabidopsis gene REM16 has great significance for regulating and controlling the flowering stages of the plants.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to Arabidopsis gene REM16 The expression vector of and its application in regulating the flowering period of plants. Background technique [0002] Actin filament depolymerization factor (ADF / cofilin) ​​widely exists in eukaryotic cells and plays a very important role in maintaining cell morphology, cell transportation, energy conversion, information transmission, cell division and differentiation. Previous studies have shown that changes in the expression of Arabidopsis microfilament depolymerization factor ADF1 will affect the flowering period of plants, ADF1 Decreased expression results in delayed flowering. The wild-type Arabidopsis flowered in about 35±1 days and had 17 rosette leaves; while ADF1 The gene silenced plants flowered 2 weeks later than the wild type, and the number of rosette leaves increased to 26. Later, people analyzed in depth through gene knocko...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00
CPCC07K14/415C12N15/827
Inventor 董春海乔龙飞于延冲荣永恒崔宪奎赵宇航陈家才侯晓敏
Owner QINGDAO AGRI UNIV
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