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Construction method and application of fluorescence marker for pollen tube vacuole of plant

A technology of plant pollen and construction method, applied in the field of genetic engineering, can solve problems such as lack of molecular means

Inactive Publication Date: 2021-05-28
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to enrich germplasm resources, it is imperative to screen male sterile lines in wild species in nature, but currently there are relatively few molecular methods for detecting pollen viability

Method used

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  • Construction method and application of fluorescence marker for pollen tube vacuole of plant
  • Construction method and application of fluorescence marker for pollen tube vacuole of plant
  • Construction method and application of fluorescence marker for pollen tube vacuole of plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Obtaining of Arabidopsis thaliana inositol transporter INT1 gene sequence and construction of plant expression vector

[0019] Log in to the Arabidopsis Information Resource Database website

[0020] https: / / www.arabidopsis.org / servlets / TairObject? type=sequence&id=125264, find the sequence (SEQ ID No.1) of the inositol transporter INT1 gene, design PCR amplification primers according to the CDS of INT1,

[0021] INT1 CDS-F:5'-CACCatgacattgacgatcccaaac-3'

[0022] INT1 CDS-R:5'-ttaagattgagatccctgctcg-3'

[0023] The wild-type Arabidopsis Col-0 was cultivated, and the seedlings of Arabidopsis thaliana grown for 4 days in long-day sunlight were taken as materials, and the total plant RNA was extracted with the kit of Kangwei Century Company, and the cDNA was obtained by reverse transcription PCR, and then the cDNA was used as a template to amplify Increment sequence.

[0024] The specific steps for extracting RNA are as follows:

[0025] (1) Take 30-50 mg o...

Embodiment 2

[0047] The acquisition of embodiment 2 transgenic Arabidopsis plants

[0048] Firstly, the plants are cultivated to the flowering stage, and then transformed with the Agrobacterium dipping method, and the transgenic plants are screened after the seeds are harvested.

[0049] The surface disinfection and cultivation methods of Arabidopsis seeds are as follows:

[0050](1) Use a sieve to remove excess impurities and only keep the seeds, and divide into 1.5mL centrifuge tubes. Treat with 70% ethanol (prepared with sterilized Tween water) for 5 minutes in an ultra-clean bench, then treat with 2.6% NaClO (prepared with sterilized Tween water) for 10 minutes, then rinse with sterilized Tween water four times, Then spread the seeds evenly on 1 / 2MS solid medium.

[0051] (2) Vernalize in a refrigerator at 4°C for 3 days, and then place them in a long-day light incubator for more than 4 days. After the seeds germinate and grow larger true leaves, the seedlings can be transplanted int...

Embodiment 3

[0068] Example 3 Fluorescence Observation of Transgenic Arabidopsis

[0069] After the transgenic plants flowered, pollen was collected for pollen tube germination and fluorescence observation.

[0070] (1) Take 10 mL of freshly prepared liquid pollen germination medium in a new graduated centrifuge tube.

[0071] (2) Weigh 0.1 g of special agar powder for pollen germination with low melting point, heat it in a microwave oven to melt it completely, and be careful not to boil the culture medium and overflow the centrifuge tube.

[0072] (3) Pipette 1.9 mL into a petri dish with a diameter of 5.5 cm, spread it evenly on the bottom of the petri dish, open the lid and cool for a few minutes to make it solidify.

[0073] (4) Use special tweezers for pollination to take the best flower that blooms that day, remove the pistil, and gently apply pollen to the surface of the medium, pay attention to spreading evenly, and do not puncture the medium.

[0074] (5) Take a petri dish with ...

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Abstract

The invention relates to a construction method of a fluorescence marker for a pollen tube vacuole of a plant. The construction method comprises the following steps: cloning a CDS sequence of an inositol transport protein gene AT2G43330 INT1, wherein the sequence of the CDS sequence is represented by SEQ ID No. 1 or is a nucleotide sequence having the same function and formed by replacing, deleting or adding one or more basic groups to the CDS sequence of the inositol transport protein gene AT2G43330 INT1; linking INT1 to a Gateway expression vector of a red fluorescent protein (RFP) started by a pollen tube-specific promoter, and expressing fusion protein RFP-INT1 in plant pollen through the vector, wherein RFP is the red fluorescent protein, and INT1 is an amino acid sequence coded by arabidopsis AT2G43330; and transforming the plant, and conducting screening and identifying to obtain a transgenic plant with a fluorescence signal. The red marker constructed by the invention can be applied to scientific research and is used for determining subcellular localization of unknown proteins; and meanwhile, through instantaneous transformation, the dynamic state of the vacuole of the plant pollen can be rapidly displayed, pollen viability is indirectly reflected, and excellent germplasm and genetic materials are convenient to screen.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a construction method and application of plant pollen tube vacuole fluorescent markers. Background technique [0002] The fertilization of plants is crucial for fruiting, and the function of pollen is particularly critical in the process of double fertilization. Many breeding techniques are carried out using male sterility, such as three-line hybrid rice, photosensitive male sterile line rice, etc. In order to enrich germplasm resources, it is imperative to screen male sterile lines in wild species in nature, but currently there are relatively few molecular methods for detecting pollen viability. The pollen of many plants can germinate and grow into pollen tubes in vitro. Pollen tubes are polar cells that can grow rapidly. During the growth process, along with the dynamic changes of organelles, the vacuoles of pollen tubes often have a network structure, and their i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/65C12N5/10A01H5/02A01H6/20A01H6/82A01H6/56
CPCC07K14/415C12N15/8231C12N15/65
Inventor 万智远
Owner XUZHOU NORMAL UNIVERSITY
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