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Chunlan mir390a Application in controlling plant root development

A technology of spring orchids and plants, applied in the field of plant genetic engineering

Active Publication Date: 2020-12-25
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how miR390 affects the development of the root system of Chunlan has not yet been described. Therefore, using genetic engineering technology, it will be cloned from Chunlan. miR390a The transfer of precursor genes into other plants is of great significance for the study of their functions and has great application prospects

Method used

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  • Chunlan <i>mir390a</i> Application in controlling plant root development
  • Chunlan <i>mir390a</i> Application in controlling plant root development
  • Chunlan <i>mir390a</i> Application in controlling plant root development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Functional Prediction of Genes

[0018] The materials used in this example are the roots, stems, and leaves of Chunlan "Songmei" in the vegetative growth period and reproductive growth period. After harvesting, they were quickly frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator (-80°C).

[0019] 1) Extraction of Total Small RNA from Chunlan tissues

[0020] According to the instructions of the TaKaRa plant total RNA extraction kit, the specific operation is as follows:

[0021] The ultra-low temperature cryopreserved Chunlan tissues were quickly transferred to a mortar pre-cooled with liquid nitrogen, and the tissues were ground with a pestle, during which liquid nitrogen was continuously added until they were ground into powders; the powdered samples were added to the In a 1.5 mL sterile tube containing 450 μl Buffer PE, pipette repeatedly until there is no obvious precipitation in the lysate; centrifuge the lysate at 12,000 rpm a...

Embodiment 2

[0032] Example 2 Cloning and Transformation of Genes

[0033] The plant material used in this embodiment is the fresh leaf of Chunlan "Song Mei", Arabidopsis thaliana ( Arabidopsis thaliana ) is of the "Columbia" type. The Escherichia coli strain used is Trans5α, which is used for gene cloning and overexpression vector construction. The vector construction is as follows: figure 2 Shown; the Agrobacterium strain is GV3101, which is used to transform Arabidopsis; the plant expression vector used in the experiment is pBI121. The strains used were purchased from Qingke Biotechnology Co., Ltd. and Price Biotechnology Co., Ltd., respectively.

[0034] 1) Extraction of gDNA from Chunlan leaves

[0035] Follow the instructions of the TaKaRa Plant Genomic DNA Extraction Kit, the specific operations are:

[0036] Transfer the fresh leaves of Chunlan "Songmei" to a liquid nitrogen precooled mortar, grind the tissue with a pestle, and add liquid nitrogen continuously until it is g...

Embodiment 3

[0069] Example 3 Chunlan miR390a Characterization of functions controlling plant root development

[0070] 1) Acquisition of transgenic homozygous plants: Harvested transgenic T1 generation seeds were sterilized, screened and cultivated, then transplanted in nutrient soil, cultured at 22°C, 16 h light / 8 h dark; the preliminary confirmed transgenic plants were retained after testing , harvest the seeds of the T1 generation after maturity, number them, and obtain the T2 generation; same as the T1 generation, sterilize the T2 generation seeds and spread them on the screening medium containing antibiotics, place them at 22°C, and continuously light them; 10 days Left and right, the survival rate of T2 generation seeds with different numbers was counted, and the plants with a survival ratio of 75% were selected for transplantation and cultured in nutrient soil at 22°C, 16 h light / 8 h dark, and the leaves were taken for positive detection; positive T2 generation plants Continue num...

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Abstract

The invention discloses a cymbidium goeringii miR390a gene and application of the cymbidium goeringii miR390a gene for controlling plant root system development. The precursor sequence segment encoding gene of the miR390a is obtained from a cymbidium goeringii cultivated variety "Song Mei", expression analysis is carried out in the cymbidium goeringii, then, the precursor sequence segment encodinggene is constructed to an overexpression vector, the overexpression vector is imported into an objective plant to verify the function of the overexpression vector so as to discover the phenotypes ofan arabidopsis thaliana plant that an overexpression miR390a gene has a high root system growth speed in a seedling stage and a euphylla formation speed is quickened, and therefore, the gene has a wide purpose in the production and the seed breeding of orchids and other horticultural plants.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a kind of Chunlan non-coding RNA (ribonucleic acid) miR390a Application in controlling plant root development. Background technique [0002] Orchidaceae (Orchidaceae) is one of the largest families of flowering plants, with more than 25,000 species in the world, accounting for about 10% of all flowering plants. Chunlan ( Cymbidium goeringii ) belongs to the small-flowered ground orchid species in the Orchidaceae genus. It has a peculiar flower shape, elegant flower color, quiet flower fragrance, beautiful leaf appearance, and high ornamental and economic value. However, it takes a long time for Chunlan to bloom from seedlings. Wild Chunlan seedlings need about ten years to bloom, and artificial tissue culture seedlings need at least three years to bloom under the condition of proper maintenance. An old saying goes: "The roots are deep and the leaves are luxu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/06A01H6/20
CPCC12N15/8218C12N15/8261
Inventor 胡凤荣刘倩徐子涵丁彦芬
Owner NANJING FORESTRY UNIV
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