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Birch spl2 gene and its protein involved in plant morphogenesis and flower development

A technology of plant morphology and birch, applied in plant genetic improvement, genetic engineering, botanical equipment and methods, etc., can solve problems such as not involving birch SPL2 gene sequence, unclear function of SPL gene, few reports on AtSPL gene function, etc. To achieve the effect of regulating flower fertility

Active Publication Date: 2020-01-14
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the function of the SPL gene of birch with a relatively long juvenile period is not clear, and there is no relevant patent report involving the sequence and function of the birch SPL2 gene
[0003] In addition, in Arabidopsis, most of the SPL gene functions have been identified, while the AtSPL gene functions in the G1 region are rarely reported

Method used

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  • Birch spl2 gene and its protein involved in plant morphogenesis and flower development
  • Birch spl2 gene and its protein involved in plant morphogenesis and flower development
  • Birch spl2 gene and its protein involved in plant morphogenesis and flower development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning of the birch BpSPL2 gene

[0030] (1) Acquisition of plant material

[0031] The experimental material Birch male flowers were collected from the birch forest of Northeast Forestry University. Immediately after collection, refrigerate to -80°C refrigerator for future use. (2) Extraction of birch RNA

[0032] Birch total RNA was performed using the improved CTAB method, and the purity and concentration of total RNA were measured using agarose gel electrophoresis and ultraviolet spectrophotometer. Use the ThermoScriptTM RT-PCR System Reverse Transcription Kit to synthesize the first strand of cDNA, and then use BpSPL2-F and BpSPL2-R for PCR amplification. PCR reaction system: cDNA template 1 μL, 10×Buffer 5 μL, dNTP (2.5 mM) 4 μL, Forward and reverse primers each 2 μL (10 μM), Taq Plus DNA polymerase 1 μL, sterilized distilled water to make up 50 μL. The PCR amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for ...

Embodiment 2

[0037] Embodiment 2: The plant expression vector construction of birch BpSPL2 gene

[0038] (1) Using gateway technology to construct plant expression vectors, the specific steps are as follows:

[0039] a. Compared with the strains carrying the entry vector pENTR-BpSPL2 and the Gateway vector pGWB5, they were cultured in LB liquid medium containing 50 mg / L kanamycin and different concentrations of hygromycin (hyg) to screen for tide Practical concentration of mycin.

[0040] b. Take 2 μL of plasmid pENTR-BpSPL2 and 2 μL of plasmid pGWB5 in a 200 μL PCR tube and mix well.

[0041] c. Place the LR ClonaseTMEnzyme II mix on ice for 2 minutes and shake it twice, about 2 seconds each time.

[0042] d. Add 1 μL LR ClonaseTMEnzyme II mix to the mixed plasmid system, mix well, and centrifuge briefly.

[0043] e. In a PCR machine, keep warm at 25°C for 6h.

[0044] f. Add 0.5 μL of proteinase K to the reaction system to terminate the reaction, mix well and keep warm at 37°C for 10...

Embodiment 3

[0047] Example 3: Functional analysis of birch BpSPL2 gene overexpression in Arabidopsis

[0048] (1) Cultivation of Arabidopsis thaliana sterile seedlings:

[0049] a) Take an appropriate amount of seeds in a centrifuge tube, add an appropriate amount of sterile distilled water, vortex fully, centrifuge for a short time, and suck out the floating seeds and sterile water together.

[0050] b) Add 1mL of 70% ethanol, vortex for 10s, fully suspend the seeds in the ethanol solution, suck out the ethanol (the ethanol disinfection time should not be too long, avoid ethanol penetration, reduce the seed activity), add sterile ddH2O, shake and wash twice .

[0051] c) Add 1mL of 10% sodium hypochlorite, vortex for 5min, centrifuge at 12000rpm for 1min, wash with 1mL of sterile ddH2O for 3-5 times in the ultra-clean workbench until the solution in the centrifuge tube becomes colorless, and reduce the impact of residual sodium hypochlorite on the seeds. influences.

[0052] d) Spread...

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Abstract

The invention relates to a white birch SPL2 gene participating plant morphogenesis and floral development and a coding protein thereof and belongs to the gene technical field of molecular biology. A nucleotide sequence of a related gene BpSPL2 is as shown in a sequence table SEQ ID NO; 1. The amino acid sequence is as shown in SEQ ID NO: 2. The invention provides the white birch SPL2 gene and a plant expression vector thereof. The gene is over-expressed by means of an agrobacterium-mediated method in Arabidopsis, the obtained transgenetic Arabidopsis purified strain has phenotypes that petals are unfolded and pistils and stamens are withered and wizened, another part of transgenetic strain is dwarf, and the quantity of primary stems or side branches on the main stem is increased. The BpSPL2 gene can affect floral development and floral development of Arabidopsis. The invention provides a gene resource and a theoretical foundation for regulating floral abortion and plant morphogenesis of forests by means of transgenic technology.

Description

[0001] Technical field: The present invention relates to a birch SPL2 gene and its protein involved in plant morphogenesis and flower development, and belongs to the field of gene technology in molecular biology. Background technique: [0002] SPL (SQUAMOSA promoter-binding protein-like) is a plant-specific transcription factor that widely exists in green plants. Initially, two SPL genes were screened from the snapdragon inflorescence cDNA library, named SBP1 and SBP2, respectively. It is named after being able to recognize and bind to the promoter of SQUAMOSA. SPL proteins constitute a diverse family of transcription factors, all of which have a highly conserved segment of 76 amino acids, which binds to the promoter region of the downstream target gene to regulate the expression of the target gene, and is named the SBP domain. The SBP domain contains a novel zinc finger structure and a nuclear localization signal, which is involved in the entry of transcription factors into t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415
CPCC07K14/415
Inventor 刘雪梅胡晓晴张淇王晟宇张正一
Owner NORTHEAST FORESTRY UNIVERSITY
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