Method for regulating and controlling growth of chrysanthemum petals through conversion of CmTCP20 gene

A technology of cmtcp20-gate-f and gene, which is applied in the field of regulating the growth of chrysanthemum petals by transfecting CmTCP20 gene, can solve the problem of lack of systematic and in-depth research on TCP regulation of petals

Active Publication Date: 2016-06-15
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The above studies have shown that the TCP gene family is involved in the regulation of the growth and development of plant floral organs, but there are differences in the me...

Method used

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  • Method for regulating and controlling growth of chrysanthemum petals through conversion of CmTCP20 gene
  • Method for regulating and controlling growth of chrysanthemum petals through conversion of CmTCP20 gene
  • Method for regulating and controlling growth of chrysanthemum petals through conversion of CmTCP20 gene

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Experimental program
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Effect test

Embodiment 1

[0109] Cloning of embodiment 1.CmTCP20 gene

[0110] The cut flower chrysanthemum 'Shenma' was used as material, and 0.15 g of petals were taken, and the total RNA of the petals was extracted according to the operation method of the Trizol RNA extraction kit (TaKaRa), and obtained by reverse transcription according to the M-MLV reverse transcription kit (TaKaRa). cDNA, according to the sequence information of the gene in the chrysanthemum library, use primer5 software to design specific primers to amplify CmTCP20;

[0111] Upstream primer CmTCP20-F: 5'-ATGACTGATCCCAACACC-3' (SEQ ID NO.2),

[0112] Downstream primer CmTCP20-R: 5'-CTGACCCTCTGAACCTCG-3' (SEQ ID NO.3);

[0113] Use petal cDNA as a template to carry out PCR reaction, 50 μL reaction system: 10×PCRBuffer 5.0 μL, CmTCP20-F, CmTCP20-R primers 1.0 μL each (20 μmol L -1 ), dNTPmix4.0μL (2.5mmol L -1 ), TaqDNAPolymerase 0.2μL, cDNA template 1μL, ddH 2 O37.8 μL; reaction program: pre-denaturation at 95°C for 5 min, the...

Embodiment 2

[0114] Example 2. Construction of plant expression vector pMDC43-CmTCP20

[0115] Primers were designed according to the full-length gene sequence of CmTCP20 for PCR reaction, restriction sites BamHI and NotI were introduced into the upstream and downstream of the target gene CmTCP20 respectively, the PCR product was connected to the pMD19-T vector, and DH5α competent cells were transformed, positive plasmids were extracted, and The extracted positive plasmid was digested with BamHI and NotI to obtain the CmTCP20 fragment and the pENTR obtained by BamHI and NotI double digestion TM 1A ligation, transformation, the extracted positive plasmid was linearized by PvuI single enzyme digestion, and then carried out LR recombination reaction (Invitrogen, USA) with the plant expression vector pMDC43 plasmid, transformed, and the positive plasmid was extracted.

[0116] Upstream primer CmTCP20-GATE-F: 5'-CGCGGATCCGGATGACTGATCCCAACACC-3' (SEQ ID NO.4), downstream primer CmTCP20-GATE-R: 5...

Embodiment 3

[0120] Embodiment 3. Agrobacterium EHA105 mediates leaf disk method to transform chrysanthemum

[0121] Pick a single colony of EHA105 from a YEB (50 μg / mL rifampicin) plate, inoculate it in 50 mL of YEB liquid medium containing 50 μg / mL rifampicin, cultivate it at 200 rpm, 28°C until the OD value is 0.5, and then bathe the bacteria in ice 30min, centrifuge to collect the bacteria, suspend in 2mL pre-cooled 100mM CaCl 2 (20% glycerol) solution, 200 μL / tube aliquoted for use. Take 10 μL of pMDC43-CmTCP20 vector plasmid, add 200 μL of competent cells, ice-bath for 30 minutes, freeze in liquid nitrogen for 5 minutes, 37°C for 5 minutes, add 800 μL of YEB liquid medium, pre-culture for 4 hours at 28°C and 200 rpm, and spread the bacterial solution on YEB (50 μg / mL Li Fuping + 50 μg / mL kanamycin) solid medium, cultured in the dark at 28°C for 2 days, picked a single clone for detection, and selected positive clones to transform chrysanthemums.

[0122] The Agrobacterium used refe...

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Abstract

The invention belongs to the field of plant genetic engineering and transgenic breeding and relates to a method for regulating and controlling growth of chrysanthemum petals through conversion of the CmTCP20 gene.The method includes the following steps that the CmTCP20 gene is cloned from florist's chrysanthemum 'ShenMa'; a plant expression vector of the CmTCP20 gene is constructed; the plant expression vector is transferred into chrysanthemum through an agrobacterium-mediated method, culturing is conducted, and a resistant plant is preliminarily obtained; the converted plant is subjected to PCR and quantitative RT-PCR detection to verify that the CmTCP20 endogenous gene is integrated to a genome DNA of the transgenic plant and subjected to transcription; the transgenic plant is subjected to phenotypic observation and statistic analysis, and it is found that compared with a plant not subjected to transgenosis, the petal growth amount of the transgenic plant is obviously increased.According to the method, the endogenous CmTCP20 transcription factors are converted and subjected to normal transcriptional expression, so that growth of the chrysanthemum petals is regulated and controlled, a novel and practical method is provided for improving ornamental quality of chrysanthemum through a genetic engineering technology, and the chrysanthemum biotechnology breeding process 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 growth of chrysanthemum petals by transfecting CmTCP20 gene. Background technique [0002] Chrysanthemum (Chrysanthemum morifolium) is one of the top ten famous flowers in my country and one of the four major cut flowers in the world. It has a long history of cultivation. Chrysanthemum has characteristics such as rich flower color, various flower positions, is deeply liked by people, and is widely cultivated in the world. The TCP family is a class of plant-specific transcription factors discovered in the last ten years, and has received extensive attention because it plays an important role in plant morphological development and evolution. More and more studies have shown that the TCP gene family is involved in the morphogenesis of floral organs, for example, the PsCYCs gene has an asymmetric expression pattern in pea flo...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C12N15/82C12N15/66C12Q1/68A01H5/00
CPCC07K14/415C12N15/66C12N15/8205C12N15/827C12Q1/6895C12Q2600/13C12Q2600/158
Inventor 陈发棣王晶晶王海滨宋爱萍蒋甲福陈素梅
Owner NANJING AGRICULTURAL UNIVERSITY
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