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Recognition method for transporting mRNA (messenger ribonucleic acid) of GAPC1 gene among cucurbitaceae rootstocks and ears

A technology of genes and gene fragments, applied in the field of plant molecular biology, can solve problems such as many requirements, cumbersome processes, and difficult operations

Pending Publication Date: 2020-08-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can accurately detect the transmissibility of genes in the phloem, the process is cumbersome, requires many requirements, and is not easy to operate, which adds a lot of difficulty to our experimental process.

Method used

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  • Recognition method for transporting mRNA (messenger ribonucleic acid) of GAPC1 gene among cucurbitaceae rootstocks and ears
  • Recognition method for transporting mRNA (messenger ribonucleic acid) of GAPC1 gene among cucurbitaceae rootstocks and ears
  • Recognition method for transporting mRNA (messenger ribonucleic acid) of GAPC1 gene among cucurbitaceae rootstocks and ears

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: cucumber and pumpkin grafting

[0042] (1) Cultivation of scion and rootstock: The rootstock and scion seeds are respectively raised for seedlings, until the scion grows to one leaf and one heart, the cotyledons of the stock are fully expanded, and the thickness of the hypocotyls of the stock and the scion remains consistent.

[0043] (2) Hypocotyl grafting: use blade to cut obliquely downward 30 ° of slopes at 1 cm below the cotyledons of the scion, remove all the cotyledons of the stock, obliquely cut 1 knife at 1 cm below the cotyledons of the stock with a blade, and the length of the section is consistent with the scion, and then Align the scion with the cutting surface of the rootstock and fix it tightly with grafting clips; then graft the cotyledons of the rootstock to the cutting surface of the original scion, and fix the grafting interface with grafting clips.

[0044] (3) Management after grafting: the grafted seedlings are placed in the seedling ...

Embodiment 2

[0045] Example 2: total RNA extraction

[0046] Total RNA was extracted using the CTAB method, and the method steps were as follows:

[0047] (1) Take 2 g of blade material, grind it into fine powder in liquid nitrogen.

[0048] (2) Add 10ml 65°C preheated CTAB extract and 500μl β-mercaptoethanol to each tube, vigorously vortex homogenate, and incubate in a 65°C water bath for 30min.

[0049] (3) Add 10ml of chloroform:isoamyl alcohol (24:1) to each tube, vortex to mix, and ice-bath for 10min. Centrifuge at 12000rpm for 10min at 4°C.

[0050] (4) Take the supernatant, add 1 / 3 volume of 8M LiCl and 500 μl β-mercaptoethanol, and precipitate overnight at -20°C.

[0051] (5) Centrifuge at 12000 rpm for 20 min at 4°C.

[0052](6) Discard the supernatant, dissolve the precipitate with 4ml guanidine isothiocyanate denaturing solution, add 120μl β-mercaptoethanol and 880μl 2M NaAc (pH4.0), mix well and add 5ml of phenol: chloroform: isoamyl alcohol (25:24 : 1), vortex mix, ice ba...

Embodiment 3

[0058] Example 3: RNA reverse transcription into cDNA

[0059] Using TaKaRa Bio reagents, PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time), according to the company instructions:

[0060] (1) Genomic DNA removal reaction

[0061] Prepare the reaction mixture on ice according to the following components. In order to ensure the accuracy of the preparation of the reaction solution, when performing each reaction, prepare the Master Mix according to the number of reactions + 2, and then distribute it into each reaction tube. Add the RNA sample last.

[0062]

[0063] 2min at 42°C (or 5min at room temperature)

[0064] 4°C

[0065] (2) Reverse transcription reaction

[0066] Please prepare the reaction solution on ice. In order to ensure the accuracy of the preparation of the reaction solution, when performing various reactions, the Master Mix should be prepared according to the number of reactions + 2, and then 10 μl should be dispensed into each reaction...

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Abstract

The invention provides a molecular recognition method for transmission of GAPC1 gene mRNA between cucurbitaceae rootstocks and scions. The molecular recognition method comprises the following steps: 1) grafting by taking cucumbers as scions and taking pumpkins as rootstocks; 2) designing specific primers of the GAPC1 genes of the cucumbers and the pumpkins; 3) performing RT-PCR on cucumbers, pumpkins and grafted cucumbers and pumpkins by using the primers, and comparing RT-PCR product conditions before and after grafting, and 4) constructing a cloning vector of GAPC1. The method provided by the invention is quick, sensitive, high in accuracy, simple and convenient, and can be applied to other cucurbitaceae plants.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to a molecular identification method for long-distance transmission of GAPC1 gene mRNA molecules between rootstocks and scions of Cucurbitaceae plants. Background technique [0002] Grafting is the technique of connecting the buds or branches of a plant to the appropriate part of another plant, so that the two join to form a new plant. In the field of horticulture, grafting is often used to obtain traits such as resistance, dwarfing, and high quality, and improve product quality. A large number of studies have shown that the use of resistant rootstocks for grafting can enhance the root absorption capacity of vegetable grafted seedlings and improve the stress resistance of grafted plants. [0003] A large number of previous studies have found that RNA, as an information molecule, is transported between grafted plant roots and ears by intercellular and long-distance information...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12Q1/6895
CPCC07K14/415C12Q1/6895
Inventor 张文娜刘梓溪
Owner CHINA AGRI UNIV
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