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Carrier for delaying fragaria X ananassa Duch. leaf senescence, and preparation method and application thereof

A forest strawberry and leaf aging technology, applied in the field of plant genetic engineering, to achieve the effect of delaying strawberry leaf aging, prolonging the functional period, and delaying leaf aging

Active Publication Date: 2020-07-31
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the urgent need to delay the senescence of strawberry leaves in the prior art, the object of the present invention is to provide a plant suppression expression vector of the forest strawberry cytokinin receptor gene FveHK4

Method used

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  • Carrier for delaying fragaria X ananassa Duch. leaf senescence, and preparation method and application thereof
  • Carrier for delaying fragaria X ananassa Duch. leaf senescence, and preparation method and application thereof
  • Carrier for delaying fragaria X ananassa Duch. leaf senescence, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Obtaining the RNA interference fragment of the forest strawberry cytokinin receptor gene FveHK4

[0053] Cloning of Sense and Antisense Strands of Cytokinin Receptor Gene FveHK4 Nucleotides:

[0054] According to the FveHK4 sequence published in NCBI database (http: / / www.ncbi.nlm.nih.gov) accession number: XM_004297360 as a template, design specific primers

[0055] FveHK4-RNAi-F: 5'-AGAAGCAAAGAGGTAGTTGAAG-3' (SEQ ID No.4) and

[0056] FveHK4-RNAi-R: 5'-CACTTTGTTATCAGCATCCATG-3' (SEQ ID No.5), amplified by PCR reaction to obtain the positive sense of the RNA interference fragment of the forest strawberry cytokinin receptor gene FveHK4 shown in SEQ ID NO.1 Chain, the length is 225bp;

[0057] Also use the above template to design specific primers

[0058] FveHK4-RNAi-F2: 5'-CACTTTGTTATCAGCATCCATG-3'(SEQ ID No.6) and

[0059] FveHK4-RNAi-R2: 5'-AGAAGCAAAGAGGTAGTTGAAG-3' (SEQ ID No. 7), the antisense strand of the RNA interference fragment of the FveHK4 cyto...

Embodiment 2

[0065] Embodiment 2: Construction of plant expression vector GN2300-FveHK4

[0066] Specific steps are as follows:

[0067] 1. Connect the intron to the GN2300 vector to obtain the Intron-GN2300 vector containing the intron:

[0068] ① Obtaining the intron (Intron) fragment: 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 amplified Intron fragment. The sequence of the obtained Intron intron fragment is shown in SEQ ID NO.3.

[0069] The PCR reaction system is shown in Table 1.

[0070] Reaction conditions: 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.

[0071] ② Digest the GN2300 universal vector with XbaI / SalI double enzymes.

[0072] The double enzyme diges...

Embodiment 3

[0091] Example 3: Genetic Transformation of Strawberry Forest

[0092] (1) using the genetic transformation method mediated by Agrobacterium to transform the forest strawberry with the carrier obtained in Example 2, the specific steps are as follows:

[0093] ①Activation of strains: Streak the GV3101 Agrobacterium containing the expression vector GN2300-FveHK4 on the LB solid medium containing 50mg / L Kan and 50mg / L Rif, place it upside down in a constant temperature incubator at 28°C, and culture in the dark 2d. After a single colony grows, pick a moderately sized, plump and round single colony and inoculate it into an 800 μl LB liquid culture-based 2ml centrifuge tube containing the same concentration of antibiotics, and incubate at 200 rpm at 28°C for 16 hours with shaking. Take 100μl of the bacterial solution and add it to a new 100ml LB medium, shake and culture at 200rpm at 28°C for about 15h to make the OD600 value 0.6-0.8. Centrifuge at 5000rpm at 4°C for 5min, and res...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a carrier for delaying fragaria X ananassa Duch. leaf senescence and an application thereof. An RNA interference fragment of a fragaria X ananassa Duch. cytokinin receptor gene FveHK4 is screened from fragaria X ananassa Duch.; a plant inhibition expression vector GN2300-FveHK4 of the gene is constructed and is transferred into diploid fragaria X ananassa Duch. 'Hawaii 4', and an RNAi expression interference strain of the FveHK4 gene is obtained, wherein vegetative growth of the strain is notobviously different from vegetative growth of a wild type. Under a dark condition, compared with a wild type, senescence of the FveHK4-RNAi in-vitro leaves is obviously delayed, which shows that thesenescence speed of the leaves of the fragaria X ananassa Duch. transgenic line of the genetic transformation FveHK4 interference carrier is slowed down, and the functional period is prolonged. The carrier has important significance in delaying strawberry leaf senescence, increasing the photosynthetic production of leaves and further increasing the strawberry yield.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a carrier for delaying the senescence of strawberry leaves and an application thereof. Background technique [0002] Crop leaves are an important place for plants to carry out photosynthesis, and they are also one of the most sensitive organs of plant aging. Leaf senescence is a process of decline in life activities that leads to natural death of leaves, which is manifested as yellowing in form. Changes in the color of plant leaves can reflect the senescence of the leaves and the loss of chlorophyll. The gradual loss of chlorophyll to yellow is the most obvious feature of leaf senescence. Leaf senescence has an important impact on crop yield and quality. Studies have reported that spraying appropriate concentrations of cytokinins on the leaves of corn, rice and other crops can delay leaf senescence, improve crop quality, and have a significant effe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29C12N1/21A01H5/12A01H6/74
CPCC12N15/8205C12N15/8218C12N15/8261C12N15/8295C07K14/415Y02A40/146
Inventor 丁静咸志慧王永李义
Owner NANJING AGRICULTURAL UNIVERSITY
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