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Fusion protein and encoding gene for labeling microtubule skeleton, method for constructing transgenic leguminous plants

A technology of fusion gene and coding gene, which is applied in the field of genetic engineering, can solve the problems that the transient expression technology cannot be realized, and the microtubule skeleton cannot be labeled, and achieve the effect of clear labeling, strong specificity, and strong green fluorescent signal

Active Publication Date: 2021-09-07
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] There are certain differences in the sequence of tubulin in different plants. In Arabidopsis, the main function of the microtubule skeleton is to participate in the elongation and growth of various tissues and organs. In legumes, the microtubule skeleton is mainly in the It plays a role in the process of symbiotic nitrogen fixation. Previous studies have shown that the mutation of a protein MtDREPP that regulates both microfilaments and microtubules does not affect the elongation and growth of alfalfa root hairs but affects the infection efficiency of rhizobia and the number of nodules. It is speculated that It is possible that the microtubule skeleton in alfalfa is not fully represented by genes in Arabidopsis
In addition, the 35S strong promoter currently used in most plants to mark the microtubule skeleton will lead to chimeric expression after being transferred into plants, and cannot mark the microtubule skeleton in roots and root hair cells
Although transient expression can label microtubules in alfalfa root and root hair cells, microtubule labeling in other parts cannot be achieved by transient expression technology

Method used

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  • Fusion protein and encoding gene for labeling microtubule skeleton, method for constructing transgenic leguminous plants
  • Fusion protein and encoding gene for labeling microtubule skeleton, method for constructing transgenic leguminous plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A plant expression vector

[0029] Its construction method includes the following steps:

[0030] A DNA molecule named TUA6Pro::GFP-MtTUA6-GFP-PZP211 shown in SEQ ID NO.1 was artificially synthesized. In SEQ ID NO.1, the 1-2519th position is the promoter of TUA6 gene; the 2520-5303rd position of SEQ ID NO.1 is the GFP-MtTUA6-GFP fusion gene, and the coding sequence of the fusion gene encodes SEQ ID NO .2 The fusion protein GFP-MtTUA6-GFP shown in 2; the 2520-3236th position of SEQ ID NO.1 is the coding gene of N-terminal GFP, which encodes the GFP shown in the 1st-239th position of SEQ ID NO.2; No. 3257-4586 of SEQ ID NO.1 is the coding gene of alfalfa tubulin MtTUA6, which encodes the alfalfa TUA6 protein shown in No. 240-689 of SEQ ID NO.2; No. 4587 of SEQ ID NO.1 Position -5303 is the coding gene of C-terminal GFP, which encodes the GFP shown in the 690-928 positions of SEQ ID NO.2.

[0031] Specific steps are as follows:

[0032] (1) Construction of intermediate...

Embodiment 2

[0041] A recombinant Agrobacterium

[0042] Its construction method is as follows:

[0043] Mix Agrobacterium tumefaciens EHA105 competent cells with PZP211-TUA6Pro::GFP-MtTUA6-GF P plasmid, incubate on ice for 30 minutes, freeze in liquid nitrogen for 2 minutes, recover at 37°C for 3-5 minutes, add 1 mL of liquid YEB medium, Shake culture at 180 rpm at 28°C for 4 hours, collect the bacterial cells, and culture on YEB solid medium containing a final concentration of 50 mg / L spectinomycin and 100 mg / L rifampicin at 28°C for 36 hours. Pick a single clone shake, use PCR and enzyme digestion to identify positive clones, obtain recombinant Agrobacterium containing PZP211-TUA6Pro::GFP-MtTUA6-GFP expression vector, named PZP211-TUA6Pro::GFP-MtTUA6-GFP / EHA105, to obtain the infecting bacteria used for genetic transformation of alfalfa.

Embodiment 3

[0045] A kind of transgenic leguminous plant-transgenic alfalfa

[0046] Its construction method is as follows:

[0047] The genetic transformation of alfalfa adopts leaf disc transformation method. The activated PZP211-TUA6Pro::GFP-MtTU A6-GFP / EHA105 was cultured in shake flasks overnight at 30°C (200rpm) in a final concentration of 50mg / L spectinomycin and a final concentration of 100mg / L rifampicin in 2mLYE B liquid medium; 1-2mL of the overnight bacterial solution was transferred to a 200ml Erlenmeyer flask containing 30mL of YEB liquid medium for amplification and culture, OD 600 The nm should reach 0.6. The bacteria were collected by centrifugation at 3000g for 2min, and resuspended with 50mL sterile SH3a medium. For transformation, leaves from 4 to 6 week old plants grown in vitro or in the greenhouse were selected. The leaves grown in the greenhouse are put into a 50mL centrifuge tube (20-30 leaves per tube). The leaves are washed for the first time with water added...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a fusion protein for labeling a microtubule skeleton, an encoding gene, and a method for constructing a transgenic leguminous plant. The amino acid sequence of the fusion protein is shown in SEQ ID NO. 2, and at the same time, the present invention also relates to the nucleotide sequence encoding the fusion protein, a plant expression vector containing the sequence, recombinant Agrobacterium and transgenic leguminous plants According to the construction method of the invention, the recombinant Agrobacterium is transformed into legumes, and the transgenic legumes that mark the microtubule skeleton in the legume can be obtained by screening, and at the same time, the double GFP tag can make the green fluorescence signal stronger.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing fusion proteins and coding genes for marking microtubule skeletons and transgenic leguminous plants. Background technique [0002] The plant microtubule skeleton is formed by the combination of α-tubulin and β-tubulin, which has highly dynamic characteristics. When the cells are stimulated by external environmental signals, the microtubule array quickly reorganizes, and the plant cell growth, cell division, and vesicle transport , organelle transport, cell wall synthesis, and biotic and abiotic stress responses. Some plant-specific microtubule arrays are formed in plant cells, such as periplasmic microtubules (Cortical Microtubules, CMTs), early prophase band (Preprophase Band, PPB) and membrane-forming body (Phragmoplast). PPB, centrosome, spindle microtubules and membranous bodies participate in cell mitosis, and periplasmic microtubules a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/84C12N15/65C12N1/21A01H5/00A01H6/54
CPCC07K14/415C07K2319/60C12N15/65C12N15/8212
Inventor 王显玲邱天麒曹启江郭南南岳剑茹苏媛媛杨卉张少斌
Owner SHENYANG AGRI UNIV
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