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Fluorescent identification method for transfer of mRNA molecules between stock and scion of plants

An identification method and fluorescence technology, which is applied in the field of plant molecular biology, can solve the problems of inability to observe mRNA transfer intuitively, and achieve the effect of low cost, high transgenic efficiency and simple process

Active Publication Date: 2018-07-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the problem in the prior art that the transfer of mRNA cannot be observed intuitively after transgene grafting by constructing a carrier with a specific tag for the gene, and to provide a fluorescent identification method for the transfer of mRNA molecules between plant stock and ear

Method used

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  • Fluorescent identification method for transfer of mRNA molecules between stock and scion of plants
  • Fluorescent identification method for transfer of mRNA molecules between stock and scion of plants
  • Fluorescent identification method for transfer of mRNA molecules between stock and scion of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Gene RNA Extraction

[0038] The total RNA from the phloem of Du pear was extracted by the CTAB method (Zhang Yugang et al., 2005), dissolved in 30 μL DEPC water, detected by electrophoresis, and stored in a -80°C refrigerator for later use.

[0039] (1) Remove DNA from RNA:

[0040] (2) Treat at 37°C for 30 minutes; add 550 μL RNase-free water (treated with 0.1% DEPC), then add an equal volume of 600 μL CI and mix well;

[0041] (3) Centrifuge at 10000rpm for 10min at 4°C, absorb the supernatant, then add an equal volume of CI and mix gently; centrifuge at 10000rpm for 10min at 4°C;

[0042] (4) Aspirate the supernatant, add 2 times the volume of absolute ethanol to the tube, and precipitate at -20°C for 1 hour;

[0043] (5) Centrifuge at 12000rpm for 20min at 4°C; discard the supernatant, then add 1mL of 75% ethanol to rinse and precipitate, centrifuge at 12000rpm for 5min, rinse twice and suck out excess ethanol with a pipette;

[0044] (6) Put it in an ...

Embodiment 2

[0047] Example 2 Construction of overexpressed GFP-PbHMGR1 vector and reverse transcription of RNA into cDNA

[0048] The RNA obtained in Example 1 was reverse-transcribed into cDNA according to conventional methods, and the obtained cDNA was used as a template for the following PCR amplification.

[0049] According to the plant HMGR1 gene sequence information of Pyrus betulaefolia Bunge, referring to the restriction site on the pCAMBIA1305 vector, PbHMGR1 CDS selects BglII and SpeI as the gene insertion site, the underline indicates the restriction site, and the primers are designed as follows:

[0050] Upstream primer: 5'- AGATCT ATGGACGTCCGAAGGC-3', (SEQ ID NO.2)

[0051] Downstream primer: 5'- ACTAGT TTAAGCGGACGCAACAG-3' (SEQ ID NO. 3).

[0052] The above primers were synthesized by Zhongmei Taihe Biotechnology Co., Ltd.

[0053] PCR reaction system: 2×Es Taq MasterMix (Dye) was purchased from (Beijing Kangwei Century Biotechnology Co., Ltd., CW0682S)

[0054]

...

Embodiment 3

[0061] Preparation and transformation of embodiment 3 Agrobacterium tumefaciens competent

[0062] 1. Preparation of competent cells of Agrobacterium tumefaciens

[0063] (1) Pick a single colony, add it to 20mL YEP liquid medium containing 20mg / L Rif, shake culture (28°C, 200rpm, under dark conditions) for 24-48h, shake the Agrobacterium until the appropriate OD600 value is 0.5 .

[0064] (2) Ice bath for 30 minutes;

[0065] (3) Centrifuge at 5000rpm for 10min at 4°C;

[0066] (4) Operate in an ultra-clean bench, discard the supernatant, add 0.15M NaCl, and resuspend;

[0067] (5) Centrifuge at 6000rpm for 5min at 4°C after 20min in ice bath;

[0068] (6) discard the supernatant, add 20mM CaCl 2 , resuspended, dispensed, 200 μL per tube, added an equal volume of 50% glycerol, and stored at -80°C.

[0069] 2. Transformation of Agrobacterium tumefaciens competent cells

[0070] (1) Take 100 μL of competent cells, put them on ice, and gently suspend the cells after compl...

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Abstract

The invention discloses a fluorescent identification method for transfer of mRNA molecules between stock and scion of plants and relates to the field of plant molecular biology. In the invention, a carrier, in which a gene carrying a fluorescent protein tag, is constructed for performing transgenosis, thus performing micro-grafting to a transgenic stock and a wild-type scion in a sterile glass bottle. Because an adventitious root is easy to grow on the scion existing in a damp environment, existence of fluorescence sent form the adventitious root on the scion can be detected by a laser scanning confocal microscope, thus determining whether long-distance transfer of genes between the stock and scion occurs or not; or whether the long-distance transfer of genes between the stock and scion occurs or not is determined by using nested-PCR for identifying existence of specific stripes on both the stock and scion. The method finally achieves intuitive observation of the long-distance transferof genes between the stock and scion. The method is intuitive, quick, sensitive and accurate, can be applied to various plant genes without limitation of species, and has extensive application prospect in the field of plant molecular biology.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to a fluorescence identification method for transferring mRNA molecules between plant stock and ear. Background technique [0002] In the practice of horticultural crop production, the application of grafting technology can improve plant resistance, increase yield, and improve fruit quality. used widely. In the production of fruit trees, it is found that different rootstocks can have different effects on the physiological and biochemical characteristics of the scion, flowering and fruiting, environmental adaptability, and tree growth and development, and these effects will eventually affect the formation of fruit and the later economic value. to a crucial role. [0003] So far, many endogenous mRNA molecules that can be transmitted long-distance by plant phloem have been found, such as Arabidopsis Aux / IAA18 and Aux / IAA28 (Notaguchi et al., 2012), squash CmNACP (Ruiz-Medrano ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6848C12N15/82C12N15/65
CPCC12N15/65C12N15/8212C12Q1/6848C12Q2531/113
Inventor 李天忠郝理王胜男王圣元陈佳琪
Owner CHINA AGRI UNIV
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