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Chrysanthemum strigolactone synthetase gene CmCCD8 and application thereof in changing chrysanthemum flowering phase

A gene, chrysanthemum technology, applied in the field of plant genetic engineering and transgenic breeding

Inactive Publication Date: 2017-12-22
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The object of the present invention is to solve the problem of directional improvement of chrysanthemum ornamental traits, and to provide the genetic engineering application of strigolactone synthase gene CmCCD8 in regulating the flowering period of chrysanthemums

Method used

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  • Chrysanthemum strigolactone synthetase gene CmCCD8 and application thereof in changing chrysanthemum flowering phase
  • Chrysanthemum strigolactone synthetase gene CmCCD8 and application thereof in changing chrysanthemum flowering phase
  • Chrysanthemum strigolactone synthetase gene CmCCD8 and application thereof in changing chrysanthemum flowering phase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1. Cloning of CmCCD8 gene

[0086] Take cut chrysanthemum "Shenma" as material, take 0.15g of root system, extract the total RNA of root system according to the operation method of Trizol RNA extraction kit (TaKaRa), and perform reverse transcription according to M-MLV reverse transcription kit (TaKaRa) Obtain cDNA, and use primer 5 software to design specific primers to amplify CmCCD8 according to the sequence information of the gene in the chrysanthemum library;

[0087] Upstream primer CmCCD8-F: 5'-ATGGCTTCCTCCCTACTTTC-3' (SEQ ID NO. 2),

[0088] Downstream primer CmCCD8-R: 5'-TCAACATTTTGGAACCCAACAT-3' (SEQ ID NO.3);

[0089] Use root cDNA as template to carry out PCR reaction, 50μL reaction system: 10×PCR Buffer 5.0μL, CmCCD8-F, CmCCD8-R primers 1.0μL each (20μmol·L -1 ), dNTP mix 4.0μL(2.5mmol·L -1 ), Taq DNAPolymerase 0.2μL, cDNA template 1μL, ddH 2 O 37.8μL; reaction procedure: 95°C pre-denaturation for 5min, then 94°C melting for 45sec, 55°C annealing for 45sec,...

Embodiment 2

[0090] Example 2. Construction of plant expression vector pBIG-CmCCD8

[0091] Design primers based on the full-length gene sequence of CmCCD8 to perform PCR reaction, introduce restriction sites Xba I and BamH I upstream and downstream of the target gene CmCCD8, respectively, connect the PCR product to the pMD19-T vector, transform DH5α competent cells, and extract the positive plasmid The CmCCD8 fragment obtained by double digestion with Xba I and BamH I of the extracted positive plasmid was ligated with pBIG double digested with Xba I and BamH I, transformed, and the positive plasmid was extracted. Upstream primer CmCCD8-Xba I-F: 5′-GCTCTAGAGCATGGCTTCCTCCCTACTTTC-3′ (SEQ ID NO. 4), downstream primer CmCCD8-BamH I-R: 5′-CGGGATCCCGTCAACATTTTGGAACCCAACAT-3′ (SEQ ID NO. 5);

[0092] Using cut chrysanthemum'shenma' root cDNA as template, using high-fidelity enzyme (PrimeSTAR TM HS DNAPolymerase, TaKaRa) for PCR reaction, 50μL reaction system: 10×HS PCR Buffer 5.0μL, CmCCD8-XbaI-F,...

Embodiment 3

[0094] Example 3. Agrobacterium EHA105 mediates leaf disc method to transform chrysanthemum

[0095] Pick a single colony of EHA105 from YEB (50μg / mL rifampicin) plate, inoculate it in 50mL YEB liquid medium containing 50μg / mL rifampicin, culture at 200rpm and 28℃ to OD value 0.5, then ice bath the bacteria liquid Collect the bacteria by centrifugation for 30min, and suspend in 2mL pre-cooled 100mM CaCl 2 (20% glycerol) solution, 200μL / tube aliquot, set aside.

[0096] Take 10μL pBIG-CmCCD8 vector plasmid, add 200μL competent cells, ice bath for 30min, freeze in liquid nitrogen for 5min, 37℃5min, add 800μL YEB liquid medium, pre-incubate at 28℃200rpm for 4h, and coat the bacterial solution on YEB (50μg / mL rifampicin+50μg / mL kanamycin) solid medium, culture in the dark at 28°C for 2 days, pick a single clone for detection, select positive clones to OD = 0.5, centrifuge, discard the supernatant, and use MS (pH 5.8) The culture solution suspends the precipitate in an equal volume fo...

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Abstract

The invention belongs to the field of genetic engineering and transgenic breeding, and discloses a chrysanthemum strigolactone synthetase gene CmCCD8 and application thereof in changing a chrysanthemum flowering phase. The method for controlling the chrysanthemum flowering phase by virtue of a transgene CmCCD8 comprises the following steps: cloning a CmCCD8 gene in chrysanthemum morifolium 'Jinba';establishing a pBIG-CmCCD8 plant expression vector and transferring into a chrysanthemum leaf disc, and performing the subculture to primarily obtain a transgenic plant. The observation on a phenotype of the flowering phase of the transgenic plant discovers that compared with the non-transgenic plant, the transgenic plant can bloom normally under the sunlight for a long time. By transforming the endogenesis CmCCD8 gene, the flowering time of the chrysanthemum morifolium is controlled, and a novel and practical method can be provided for improving the ornamental quality of the chrysanthemum morifolium by utilizing the genetic engineering technology, so that the breeding process of the chrysanthemum biotechnology can be effectively promoted.

Description

Technical field [0001] The invention belongs to the field of plant genetic engineering and transgenic breeding, and relates to a method for regulating the flowering period of chrysanthemums by translating strigolactone synthase gene CmCCD8. Background technique [0002] Chrysanthemum morifolium (Chrysanthemum morifolium) is one of the top ten traditional flowers in my country and one of the four cut flowers in the world, and its consumption is second only to rose (Zheng et al. 2004). At present, most cut chrysanthemum varieties are short-day varieties, and the flowering period is mostly in autumn, which limits the annual production of chrysanthemums. At present, cultivation measures such as artificial shading, supplementary light, and heating are often used in production to adjust the flowering period to meet the needs of annual production, but it is easy to cause quality degradation and high cost and energy consumption. [0003] Strigolactone (SL) is a kind of terpenoid small mol...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/84A01H5/00
CPCC12N9/88C12N15/827
Inventor 陈素梅王银杰蒋甲福陈发棣房伟民管志勇廖园
Owner NANJING AGRICULTURAL UNIVERSITY
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