White birch SPL2 gene participating plant morphogenesis and floral development and protein thereof

A plant morphology and genetic technology, applied in plant genetic improvement, genetic engineering, botanical equipment and methods, etc., can solve problems such as no birch SPL2 gene sequence, unclear SPL gene function, and few AtSPL gene functions. To achieve the effect of regulating flower fertility

Active Publication Date: 2017-12-15
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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  • White birch SPL2 gene participating plant morphogenesis and floral development and protein thereof
  • White birch SPL2 gene participating plant morphogenesis and floral development and protein thereof
  • White birch SPL2 gene participating plant morphogenesis and floral development and protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning of Betula platyphylla BpSPL2 gene

[0030] (1) Obtaining plant materials

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

[0032] The total RNA of Betula platyphylla was carried out using a modified CTAB method, and the purity and concentration of the total RNA were measured by agarose gel electrophoresis and UV spectrophotometer. Use ThermoScript TM 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 1μL, 10×Buffer 5μL, dNTP (2.5mM) 4μL, positive and negative Make up 2μL (10μM) of each primer, 1μL of Taq Plus DNA polymerase, and 50μL of sterilized distilled water. PCR amplification conditions were: 94°C pre-denaturation for 5 minutes; 9...

Embodiment 2

[0037] Example 2: Construction of plant expression vector of Betula platyphylla BpSPL2 gene

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

[0039] a. Compare the strains carrying the entry vector pENTR-BpSPL2 and the Gateway vector pGWB5, and culture them in LB liquid medium containing 50 mg / L kanamycin and different concentrations of hygomycin (hyg) to screen the tide Practical concentration of mycin.

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

[0041] c. Put the LR ClonaseTM Enzyme II mix on ice for 2 minutes and then shake it quickly twice, each time about 2 seconds.

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

[0043] e. Incubate at 25°C for 6h in the PCR machine.

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

Embodiment 3

[0047] Example 3: Functional analysis of the overexpression of Betula platyphylla BpSPL2 in Arabidopsis

[0048] (1) Cultivation of aseptic seedlings of Arabidopsis:

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

[0050] b) Add 1 mL of 70% ethanol, vortex for 10 seconds to fully suspend the seeds in the ethanol solution, suck out the ethanol (the sterilization time of ethanol should not be too long, avoid the infiltration of ethanol and reduce the seed activity), add sterile ddH 2 O, shake and clean twice.

[0051] c) Add 1mL 10% sodium hypochlorite, vortex fully for 5min, centrifuge at 12000rpm for 1min, in the ultra-clean workbench, use 1mL sterile ddH 2 O, shake and wash 3-5 times until the solution in the centrifuge tube becomes colorless to reduce the impact of residual sodium hypochlorite on ...

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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 technical field of gene technology in molecular biology. Background technique: [0002] SPL (SQUAMOSA promoter-binding protein-like) is a type of plant-specific transcription factor, which is widely present 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 highly conserved segments containing 76 amino acids, which bind to the promoter region of downstream target genes to regulate the expression of target genes, and are named SBP domains. The SBP domain contains a new zinc finger structure and a nuclear localization signal. It participates in the entry of transcription...

Claims

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Application Information

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Patent Type & Authority Applications(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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