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Carrier for improving regeneration efficiency of strawberry leaves and application of carrier

A forest strawberry regeneration efficiency technology, applied in the field of plant genetic engineering, can solve problems such as infestation, inability to obtain transgenic plants, and reduced regeneration frequency, and achieve the effect of enhancing regeneration ability

Active Publication Date: 2019-05-10
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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  • Carrier for improving regeneration efficiency of strawberry leaves and application of carrier
  • Carrier for improving regeneration efficiency of strawberry leaves and application of carrier
  • Carrier for improving regeneration efficiency of strawberry leaves and application of carrier

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 NCBI database (http: / / www.ncbi.nlm.nih.gov) accession number: XM_004293409 published FveHK-2b sequence as template, design 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, 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

Sterile 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 5...

Embodiment 2

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

[0063] Specific steps are as follows:

[0064] 1. Connect 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. .9), PCR amplifies the intron Intron fragment. The sequence of the obtained Intron intron fragment 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 5min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 68°C for 10s, 32cycles, extension at 68°C for 10min.

[0068] ②GN2300 is a universal carrier, the carrier picture is as follows figure 1 As indicated, the GN2300 universal vector was digested with XbaI / SalI. ...

Embodiment 3

[0088] Example 3: Genetic transformation of Strawberry forest

[0089] (1) Using the genetic transformation method mediated by Agrobacterium GV3101, the vector obtained in Example 2 is transformed into forest strawberry, and the specific steps are as follows:

[0090] ① Strain activation: Agrobacterium GV3101 containing the expression vector GN2300-FveHK2 was streaked on a solid medium, cultured at 28°C for 2 days, inoculated into liquid LB medium, and cultured at 28°C until the OD600 value was 0.4- 0.6, after centrifuging the bacterial liquid, pour off the supernatant, collect the bacterial precipitate, resuspend the collected precipitate with an equal volume of MS liquid medium, and put the resuspended bacterial liquid into 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] ③Agrobacterium infection: Transfer the cut strawberry leaves t...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically designs a carrier for improving the regeneration efficiency of strawberry leaves and application of the carrier. An RNA interference fragments of a forest strawberry cytokinin receptor gene FveHK2 is screened from a forest strawberry, a plant inhibition expression vector GN2300-FveHK2 of the forest strawberry cytokinin receptor gene FveHK2 is constructed, then transfection is performed on transgenic engineering bacteria, the plant inhibition expression vector GN2300-FveHK2 is transferred into a diploidforest strawberry 'Hawaii 4' to obtain an RNAi expression interference strain line of the FveHK2 gene, under the condition of in-vitro leaf tissue culture, the germination time of the FveHK2 gene inhibition expression line is earlier than that of a wild type, and the budding rate of the FveHK2 gene inhibition expression line is significantly higher than that of the wild type, which indicates thatthe regeneration ability of the leaves of the forest strawberry transgenic line with the genetic transformation FveHK2 interference vector is significantly improved. The carrier for improving the regeneration efficiency of the strawberry leaves has important significance for the regeneration of strawberry leaves and the improvement of the 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 and its preparation method and application. Background technique [0002] Strawberry (Fragaria×ananazsa Duch.) belongs to the Rosaceae Fragaria genus and is a perennial herb. The fruit is sweet and delicious, with high nutritional value and health benefits. In recent years, there is an urgent need for a large number of high-quality strawberry seedlings in production in various places. However, conventional seedlings are used for propagation, with low reproduction coefficient and high cost. Now, tissue culture technology is mostly used to propagate strawberry seedlings. The earliest research on strawberry in vitro culture was in the 1960s. Research by Shimomura and others in Japan showed that the method of combining heat treatment and growth point culture to eliminate str...

Claims

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Application Information

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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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