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Method and related biological material for cultivating transgenic plant of fluorescent protein mCherry labeled microtubulin

A technology of transgenic plants and fluorescent proteins, which is applied in the field of cultivating transgenic plants labeled with fluorescent protein mCherry-labeled tubulin, can solve the problems of chimeric expression, non-expression of cell expression, unstable expression of fusion protein, etc., and achieve the effect of stable expression.

Inactive Publication Date: 2015-11-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

However, due to the use of 35S as a strong promoter, gene silencing often occurs in the transgenic progeny lines, showing that the expression of the fusion protein is unstable, and there is a chimeric expression phenomenon in which some cells express and some do not express, which seriously affects the expression of the fusion protein. micro observation
Especially as a fluorescent label strain, it is generally observed under another (or more) mutant or transgenic background. This gene silencing phenomenon is even more serious, often resulting in no expression of fluorescent proteins, which severely limits the ability of fluorescent proteins to express. Application of tagged strains

Method used

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  • Method and related biological material for cultivating transgenic plant of fluorescent protein mCherry labeled microtubulin
  • Method and related biological material for cultivating transgenic plant of fluorescent protein mCherry labeled microtubulin
  • Method and related biological material for cultivating transgenic plant of fluorescent protein mCherry labeled microtubulin

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Embodiment 1

[0053] Embodiment 1, the expression vector pEarleyGate302-P used in the method for cultivating the transgenic plant of fluorescent protein mCherry labeling tubulin TUB6 :: Preparation of mCherry-TUB6 and its related biomaterials

[0054] The name shown in the artificially synthesized SEQ ID No.1 is P TUB6 :: DNA molecule of mCherry-TUB6. In SEQ ID No.1, positions 1-3004 are promoter P TUB6 ; The 3005-5074th position of SEQ ID No.1 is the mCherry-TUB6 fusion gene, the coding sequence of the mCherry-TUB6 fusion gene is the 3005-5074th position of SEQ ID No.1, encoding the fusion shown in SEQ ID No.2 Protein mCherry-TUB6; No. 3005-3712 of SEQ ID No.1 is the coding gene of mCherry, which encodes mCherry shown in No. 1 to No. 236 of SEQ ID No.2; No. 3713-3713 of SEQ ID No.2 Position 3724 is the coding gene of GA-Linker, which encodes the GA-Linker shown in the 237-240th position of SEQ ID No.2; the 3725-5074th position of SEQ ID No.2 is the coding gene of plant tubulin TUB6 , w...

Embodiment 2

[0060] Embodiment 2, the method for cultivating the transgenic plant of fluorescent protein mCherry labeling tubulin

[0061] 1.P TUB6Transfected pEarleyGate302-P that initiates the expression of mCherry-TUB6 fusion protein TUB6 :: Preparation of mCherry-TUB6 Arabidopsis (using pEarleyGate302-P TUB6 :: mCherry-TUB6 to cultivate transgenic plants with fluorescent protein mCherry-labeled tubulin)

[0062] Arabidopsis transformation was done by flower dipping method. Activated GV3101 / pEarleyGate302-P TUB6 :: mCherry-TUB6 was cultured in shake flask at 28°C for 24 hours in a final concentration of 50mg / mL kanamycin and 14mg / mL gentamicin in 5mL YEP liquid medium; after 200mL expansion culture, centrifuge at 4°C at 3600rpm for 15min to collect Bacterial cells were resuspended with 5% sucrose solution, and silwet L-77 with a volume ratio of 0.05% was added to obtain a bacterial suspension; the Colombian ecotype Arabidopsis thaliana inflorescences that had grown to the full flowe...

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Abstract

The invention discloses a method and a related biological material for cultivating a transgenic plant of fluorescent protein mCherry labeled microtubulin. The method comprises the step of importing PTUB6:mCherry-TUB6 to a receiver plant to obtain the transgenic plant of the fluorescent protein mCherry labeled microtubulin, wherein PTUB6:mCherry-TUB6 is formed by connecting mCherry-TUB6 fusion genes with PTUB6 for starting transcription of the mCherry-TUB6 fusion genes, the mCherry-TUB6 fusion genes are genes for encoding fusion protein mCherry-TUB6, the fusion protein mCherry-TUB6 is protein obtained by fusing fluorescent protein mCherry with plant microtubulin, and PTUB6 is a DNA molecule shown in 1st-3004th positions of a nucleotide sequence shown in SEQ ID No.1.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for cultivating transgenic plants marked with fluorescent protein mCherry and tubulin and related biological materials. Background technique [0002] The plant microtubule cytoskeleton is an organizational form of the plant cytoskeleton that plays a vital role in plant cell growth, cell division, 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 are closely related to cell morphology. [0003] In many studies, there is a need to link microtubules to biological processes, so visualization of ...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N1/21C12N5/10A01H5/00G01N21/64
Inventor 孔照胜柳婷田娟于艳军
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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