Method for creating short and strong plant-type chrysanthemums

A technology of chrysanthemum and plant type, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc.

Active Publication Date: 2020-05-26
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The object of the present invention is to solve the problem of directional improvement of ch

Method used

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  • Method for creating short and strong plant-type chrysanthemums
  • Method for creating short and strong plant-type chrysanthemums
  • Method for creating short and strong plant-type chrysanthemums

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0085] Example 1. Cloning of CmYAB3.1

[0086] Take cut flower chrysanthemum'Shenma' as the material, take 0.15g leaves, extract the total RNA from the leaves 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 CmYAB3.1 according to the sequence information of the gene in the Chrysanthemum transcriptome database;

[0087] Upstream primer CmYAB3.1-F: 5'-ATGTCTACCATCACAGCCAC-3' (SEQ ID NO. 2),

[0088] Downstream primer CmYAB3.1-R: 5'-CTAATAATAAGGAGAGACACCAAC-3' (SEQ ID NO.3);

[0089] Use leaf cDNA as template to carry out PCR reaction, 50μL reaction system: 10×PCR Buffer 5.0μL, CmYAB3.1-F, CmYAB3.1-R primer each 1.0μL (20μmol·L -1 ), dNTP mix 4.0μL(2.5mmol·L -1 ), TaqDNA Polymerase 0.2μL, cDNA template 1μL, ddH 2 O 37.8μL; reaction procedure: 95°C pre-denaturation for 3min, then 94°C melting for 30sec,...

Example Embodiment

[0090] Example 2. Construction of plant expression vector pORE-R4-35sAA-CmYAB3.1

[0091] Primers were designed according to the full-length gene sequence of CmYAB3.1 for PCR reaction, and restriction sites Xho I and Sma I were introduced respectively upstream and downstream of the target gene CmYAB3.1. Using the plasmid containing SEQ ID NO.1 as a template, using high-fidelity enzyme (PrimeSTAR TM HS DNA Polymerase, TaKaRa) for PCR reaction, 50μL reaction system: 10×HS PCRBuffer 5.0μL, Cm YAB3.1-XhoI-F, Cm YAB3.1-SmaI-R primers 1.0μL each (20μmol·L -1 ), dNTPmix 4.0μL(2.5mmol·L -1 ), PrimeSTAR TM HS DNA Polymerase 0.4μL, cDNA template 1μL, ddH 2 O37.6μL; reaction procedure: 95°C pre-denaturation for 5min, then 94°C melting for 30sec, 55°C annealing for 30sec, 72°C extension for 1min, 35 cycles of reaction, 72°C extension for 10min; PCR product gel recovery kit (AXYGEN ,USA), then the recovered PCR product and plant overexpression vector pORE-R4-35sAA are double digested by ...

Example Embodiment

[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. Take 10μL of pORE-R4-35sAA-CmYAB3.1 vector plasmid, add 200μL of competent cells, ice bath for 30min, freeze in liquid nitrogen for 5min, 37℃ for 5min, add 800μL YEB liquid medium, pre-incubate at 28℃ for 4h at 200rpm, and coat Plate on YEB (50μg / mL rifampicin+50μg / mL kanamycin) solid medium, culture in the dark at 28°C for 2 days, pick out single clones for detection, and select positive clones of shaking bacteria to transform chrysanthemums.

[0096] The Agrobacterium used refers to the plant expression vector EHA105 conta...

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Abstract

The invention provides a method for creating a short, small and thick chrysanthemum through a transgenic technology, which belongs to the field of plant genetic engineering and transgenic breeding. The method comprises the following steps: cloning a CmYAB3.1 gene from chrysanthemum; constructing a plant expression vector of the CmYAB3.1 gene; transferring into chrysanthemum by adopting an agrobacterium-mediated method, and culturing to primarily obtain a resistant plant. The molecular detection on the transformed plant proves that the CmYAB3.1 gene is already integrated into the genome DNA ofthe transgenic plant and transcribed. Phenotypic observation and statistical analysis are carried out on transgenic plants, and compared with non-transgenic plants, the height of the transgenic plantsis obviously decreased, and stems are thickened. According to the method, the chrysanthemum plant type is regulated and controlled through the transgenic technology, the short and strong plants suitable for the chrysanthemum morifolium ramat are obtained, the novel and practical method is provided for regulating and controlling the important agronomic traits of the flowers through the genetic engineering technology and easily simplifying cultivation, and the method has high application value.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and transgenic breeding, and relates to a method for creating dwarf plant-type chrysanthemums through transgenic technology and an application thereof. Background technique [0002] Chrysanthemum (Chrysanthemum morifolium) originated in China and has a long history of cultivation. It is one of the top ten traditional famous flowers in my country and one of the four major cut flowers in the world. The chrysanthemum has the characteristics of rich flower colors and various flower poses, and is deeply loved by people. Plant type directly affects the ornamental quality of flowers, and is one of the main traits that breeders pay attention to. Chrysanthemums can be divided into ground cover chrysanthemums, cut chrysanthemums, potted chrysanthemums, and desk chrysanthemums according to different uses. Breeders regulate the plant type by constantly adjusting the cultivation method, so that it ca...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/14C12Q1/6895C12Q1/686C12N15/11
CPCC07K14/415C12N15/8261C12Q1/686C12Q1/6895C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 陈发棣张雪丁莲李松房伟民胡月姮
Owner NANJING AGRICULTURAL UNIVERSITY
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