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Application of cymbidium goeringii miR159b in enhancing cold sensitivity of plants

A sensitive, plant-based technology applied in the field of plant genetic engineering

Active Publication Date: 2020-03-24
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the role of miR159 in plant response to cold stress has not been reported. Therefore, using genetic engineering technology, it will be cloned from Chunlan miR159b 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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  • Application of cymbidium goeringii miR159b in enhancing cold sensitivity of plants
  • Application of cymbidium goeringii miR159b in enhancing cold sensitivity of plants
  • Application of cymbidium goeringii miR159b in enhancing cold sensitivity of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Functional Prediction of Genes

[0018] Material 1 used in this example is the roots, stems, and leaves of Chunlan "Songmei" in the vegetative growth period and reproductive growth period, and material 2 is Chunlan "Songmei" treated at 4°C for 0h, 2h, 6h, 12h, and 24h. The leaves and the two types of materials were quickly frozen in liquid nitrogen after harvest, 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,...

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 gro...

Embodiment 3

[0077] Example 3 Chunlan miR159b Phenotypic characterization of enhanced cold sensitivity in plants

[0078] 1) Obtainment and phenotype observation 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; After the confirmed transgenic plants are mature, harvest the T1 generation seeds and number them to obtain the T2 generation; same as the T1 generation, sterilize the T2 generation seeds and spread them on the selection medium containing antibiotics, place them at 22°C, and continuously Illumination; about 10 days, count the survival rate of T2 generation seeds with different numbers, select plants with a survival rate of 75%, transplant them and culture them in nutrient soil at 22°C, 16 h light / 8 h dark, and take leaves for positive detection; The positive T2 generation plants continue to be numbered, and the seeds are collected to obtain T...

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Abstract

The invention discloses cymbidium goeringii miR159b gene and application thereof in enhancing cold sensitivity of plants. The method comprises the following steps: obtaining a precursor sequence fragment encoding gene MIR159b of miR159b from a cymbidium goeringii cultivated variety 'Song mei', and performing expression analysis in cymbidium goeringii, constructing the precursor sequence fragment encoding gene MIR159b into an overexpression vector, introducing the overexpression vector into a target plant to verify the function, screening and culturing to obtain a T3-generation plant, wherein it is found that the root growth speed in the seedling stage is obviously higher than that of a wild type plant; after transplantation of the wild type and 35S: MIR159b transgenic plants into soil for60 days, carrying out low-temperature stress treatment at 4 DEG C, it is found that phenotypes such as main inflorescence sagging, non-standing, stem leaf sagging and curling, basal leaf yellowing andthe like of the transgenic plants are caused, so that the gene has wide application in production 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) miR159b Application in enhancing cold sensitivity of plants. 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. Chunlan has high requirements on the growth environment. During the growth process, it is easily affected by harsh environments such as high temperature, low temperature, and drought. Therefore, it is of great significance to study the molecular mechanism of plant response to abiotic stress and iden...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/20
CPCC12N15/113C12N15/8273C12N2310/141
Inventor 胡凤荣刘倩徐子涵李芳乐
Owner NANJING FORESTRY UNIV
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