Rootstock improvement method and application

A rootstock and gene technology, applied in rootstock improvement methods and applications, in the field of long-distance transmission of fragments between rootstocks and ears to carry functional genes to regulate the agronomic traits of scions, which can solve the problems of inconspicuous regulation of phenotypes, low transfer efficiency, and inability to be applied industrially

Active Publication Date: 2021-04-02
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previously related technologies had problems such as low transfer efficiency, unclear regulatory phenotype, and inability to be applied in industry. The efficient transfer of mRNA fragments used in the present invention can increase the transfer efficiency to more than 90%.

Method used

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  • Rootstock improvement method and application
  • Rootstock improvement method and application
  • Rootstock improvement method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1: The process of obtaining transmissible gene sequences and constructing delivery vectors

[0127] 1. Transmissible gene sequence acquisition

[0128] tRNA Met , 3×CTCT, 3×(CTCT:tRNA Met ) were synthesized by Sangon Biotechnology Co., Ltd., and SalI and BglII enzyme cleavage sites were added at both ends of the sequence. See the sequence at the end of the article.

[0129] 2. Delivery vector construction (such as figure 1 shown)

[0130] tRNA Met , 3×CTCT, 3×(CTCT:tRNA Met ) and the pCAMBIA1305 vector are combined with SalI and BglII for double enzyme digestion, and the reaction system is shown in the following table:

[0131]

[0132] Perform agarose gel electrophoresis detection on the digested product, and recover target size fragments with the Axgen recovery kit. The steps are as follows:

[0133] (1) Put the cut gel into a 2mL centrifuge tube, add 500 μL of DE-A, sol at 65°C until the gel is completely dissolved, add 250 μL of DE-B, and put the ...

Embodiment 2

[0157] Example 2 Transferable Fragment Fusion Branching Functional Gene Regulates Branching Characters

[0158] 1. Tomato and Tobacco RNA Extraction

[0159] In order to amplify the full-length sequence of the SlZFP2 gene, the CTAB method was first used to extract RNA from tomato and tobacco leaves, and the specific steps were as follows:

[0160] (1) Preheat CTAB at 65°C, add 20 μL β-mercaptoethanol per 1 mL;

[0161] (2) Grind the sample in liquid nitrogen, take 0.5 g of the sample and put it into a 2 mL RNase-free centrifuge tube, add 1 mL of preheated CTAB, vortex for 30 seconds, and bathe in 65°C water for 10 minutes;

[0162] (3) Add 1mL CI (chloroform:isoamyl alcohol=24:1), and vortex;

[0163] (4) Place in a pre-cooled 4°C centrifuge at 13000rpm for 10min;

[0164] (5) Take the supernatant, add an equal volume of CI, and mix gently;

[0165] (6) 13000rpm in a centrifuge at 4°C for 10min;

[0166] (7) Take the supernatant, add 2 times the volume of isopropanol, and...

Embodiment 3

[0250] Example 3 Transmittable fragments fused with early flowering functional genes to regulate flowering traits (such as Image 6 shown)

[0251] In the previous study, we found that SlZFP2 can regulate tomato branching and also regulate tomato flowering period, so we further counted the flowering time of the above grafting system.

[0252] The results showed that: SlZFP2-tRNA Met , SlZFP2-3×CTCT, SlZFP2-3×(CTCT:tRNA Met ) tomato scion with transgenic tobacco as rootstock flowered earlier than the control SlZFP2, and SlZFP2-3×(CTCT:tRNA Met ) phenotype is the most obvious (such as Figure 5 shown). The above results indicated that the transmissible fragment fused the non-transmissible SlZFP2 early flowering functional gene into the scion, and finally translated into a functional protein in the scion to advance flowering.

[0253] The above examples illustrate: tRNA Met , 3×CTCT, 3×(CTCT:tRNA Met ) as a transmissible gene, can carry the non-transmissible functional gen...

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Abstract

The invention belongs to the technical field of biological grafting, and relates to a rootstock improvement method and application. The method includes step1, synthesizing a transfer gene; step 2, constructing the synthetic transfer gene and a functional gene on a vector; step 3, performing heterologous grafting with the tobacco transgenic in an agrobacterium infection mode as a stock and the wildtomatoes as scions; step 4, taking grafting openings and stem segments and flowers at different positions, extracting total RNA of plants, performing reverse transcription to obtain cDNA, and performing RT-PCR detection on transportation conditions of SlZFP2-tRNAMet, SlZFP2-3 * CTCT and the like in a heterologous grafting system; and step 5, investigating branching and flowering conditions of wild tomatoes on the transgenic rootstock. The method can be applied to fruit tree transgenic rootstock breeding, scion traits are efficiently improved in a fixed-point mode through rootstock transgenosis, rootstock genes are manually controlled, the scion traits are influenced, the regulation and control efficiency is improved, the breeding age limit is shortened, the manpower and material resourcecost is reduced, and the method is widely applied to various plants.

Description

technical field [0001] The invention belongs to the technical field of biological grafting, and relates to a rootstock improvement method and its application, in particular to a method for long-distance transfer between rootstocks and ears to carry functional genes to regulate the agronomic traits of scions, and more specifically to transferring mRNA (messenger ribonucleic acid) through rootstocks ) fragment fusion of important agronomic traits functional gene genetic transformation, and then regulate the technology of scion-related traits. Background technique [0002] Grafting is the most important method of asexual reproduction of horticultural crops; after grafting, the interaction between the rootstock and the scion will affect the growth and development, resistance, flowering, fruit yield and quality of the tree. In actual production, it is mainly the regulation of the rootstock to the scion, and selecting a suitable rootstock is the key to ensure the success of fruit ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00A01H6/82A01G2/30A01G22/05A01G22/45
CPCA01G2/30A01G22/05A01G22/45C12N15/8261C12N15/827
Inventor 李天忠王胜男王圣元郝理
Owner CHINA AGRI UNIV
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