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A carrier for improving forest strawberry leaf regeneration efficiency and its preparation method and application

A technology for forest strawberry and regeneration efficiency, applied in the field of plant genetic engineering, can solve the problems of inability to obtain transgenic plants, infection, reduction of regeneration frequency, etc., and achieve the effects of enhanced regeneration ability, enhanced regeneration ability, and improved transformation efficiency.

Active Publication Date: 2022-06-21
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the gene transfer method applied to strawberries is mainly the Agrobacterium-mediated leaf disc method. Due to factors such as Agrobacterium infection in the culture, selective pressure in the selective regeneration culture, antibiotic action, and subculture, the regeneration frequency will be reduced. As a result, effective transgenic plants cannot be obtained, so whether the target gene is transferred to the recipient and the transgenic plants are obtained mainly depends on whether there is a stable and efficient in vitro regeneration system

Method used

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  • A carrier for improving forest strawberry leaf regeneration efficiency and its preparation method and application
  • A carrier for improving forest strawberry leaf regeneration efficiency and its preparation method and application
  • A carrier for improving forest strawberry leaf regeneration efficiency and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Obtaining the RNA interference fragment of the forest strawberry cytokinin receptor gene FveHK2

[0053] Cloning of the nucleotide forward sequence of the cytokinin receptor gene FveHK2:

[0054] According to the FveHK-2b sequence published in the NCBI database (http: / / www.ncbi.nlm.nih.gov) accession number: XM_004293409 as the template, the specific primer FveHK2-RNAi-F: 5'-TCAATGTCAGCAAGAACCAG-3' (SEQID No. 4) and FveHK2-RNAi-R: 5'-CAGGATGCTTGACCTATCAT-3' (SEQ ID No. 5).

[0055] Amplify the desired target fragment by PCR reaction, and the reaction system is shown in Table 1:

[0056] template cDNA 1μL (about 750ng) 5×Prime STAR GXL Buffer 10μL dNTP Mixture 4μL 0.2 μM forward primer 1μL 0.2 μM reverse primer 1μL Prime STAR GXL DNA Polymerase 1μL Sterilized distilled water up to 50μL

[0057] Reaction program: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 10 s, annealing at 55 °C fo...

Embodiment 2

[0062] Example 2: Construction of plant expression vector GN2300-FveHK2

[0063] Specific steps are as follows:

[0064] 1. Link the intron to the GN2300 vector to obtain the Intron-GN2300 vector containing the intron:

[0065] ① Using the sequence of NCBI accession number: LOC101294385 as a template, design specific primers Intron-F: 5'-GGACTCTGCAGGTCGACCATATTATTCAGGTACATTC-3' (SEQ ID No. 8) and Intron-R: 5'-TGTGATGCAGCTTGCAAAGCTCTAGAGATCGTTCAAA-3' (SEQ ID No. 8) .9), PCR amplification of the intron Intron fragment. The obtained Intron intron fragment sequence is shown in SEQ ID NO.3.

[0066] The reaction system is shown in Table 1.

[0067] Reaction program: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 10 s, annealing at 55 °C for 15 s, extension at 68 °C for 10 s, 32 cycles, and extension at 68 °C for 10 min.

[0068] ②GN2300 is a general carrier, the carrier diagram is as follows figure 1 The GN2300 universal vector was double digested with XbaI / Sal...

Embodiment 3

[0088] Example 3: Genetic Transformation of Forest Strawberry

[0089] (1) use the genetic transformation method mediated by Agrobacterium GV3101 to transform the vector obtained in Example 2 into forest strawberry, and the concrete steps are as follows:

[0090] ①Strain activation: streak the Agrobacterium GV3101 containing the inhibitory expression vector GN2300-FveHK2 on the solid medium, culture at 28°C for 2 days, inoculate it into the liquid LB medium, and culture at 28°C with shaking to an OD600 value of 0.4- 0.6, after centrifuging the bacterial liquid, pour off the supernatant, collect the bacterial pellet, resuspend the collected sediment with an equal volume of MS liquid medium, and put the resuspended bacterial liquid in the refrigerator for later use.

[0091] ②Material preparation: Take the leaves of the diploid forest strawberry ‘Hawaii 4’ tissue culture seedlings, and cut 3-4 wounds with a blade.

[0092] ③ Agrobacterium infection: transfer the cut strawberry ...

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Abstract

The invention belongs to the technical field of plant genetic engineering and specifically designs a carrier for improving regeneration efficiency of strawberry leaves and its application. The RNA interference fragment of forest strawberry cytokinin receptor gene FveHK2 was screened from forest strawberry, and the plant inhibitory expression vector GN2300‑FveHK2 of forest strawberry cytokinin receptor gene FveHK2 was constructed, and then transfected with genetically engineered bacteria, and transformed into two In the ploid forest strawberry 'Hawaii 4', the RNAi expression interference line of FveHK2 gene was obtained. Under the condition of isolated leaf tissue culture, the germination time of the FveHK2 gene suppressed expression line was earlier than that of the wild type, and the germination rate was significantly higher than that of the wild type. It indicated that the regenerative ability of the leaves of the transgenic line of strawberry transgenic line transformed with the FveHK2 interference vector was significantly improved. The invention has great significance to the regeneration of strawberry leaves and the improvement of transformation efficiency.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a carrier for improving the regeneration efficiency of forest strawberry leaves, a preparation method and application thereof. Background technique [0002] Strawberry (Fragaria×ananazsa Duch.) belongs to the genus Fragaria of the Rosaceae family, and is a perennial herb. The fruit is sweet and delicious, has high nutritional value, and has health benefits. In recent years, a large number of high-quality strawberry seedlings are urgently needed in production in various places. However, conventional seedlings are used for propagation, the reproduction coefficient is low and the cost is high. Now, tissue culture technology is used to propagate strawberry seedlings. The earliest research on in vitro culture of strawberries was carried out in the 1960s. Studies by Shimomura in Japan showed that the combination of heat treatment and growing point culture...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/12A01H6/74
Inventor 丁静马琳琳王永李春李义
Owner NANJING AGRICULTURAL UNIVERSITY
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