Construction method of fringed iris gene silencing system

A gene silencing and construction method technology, which is applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problems of low induction efficiency, difficulty in disinfection, low cost, etc., and achieve simple gene silencing system and difficult resistance Effects of infection and efficiency improvement

Pending Publication Date: 2021-12-03
ZHEJIANG UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

However, the genetic regeneration of pansies is relatively difficult, and the efficiency of callus induction using flower organs as explants is extremely low, the natural seed setting rate is low and the seeds are dormant, and it is impossible to use embryos as explants, and underground rhizomes as explants It is difficult to sterilize, and the induction efficiency is also...

Method used

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  • Construction method of fringed iris gene silencing system
  • Construction method of fringed iris gene silencing system
  • Construction method of fringed iris gene silencing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Utilizes TRV-VIGS technology to transfer green fluorescent protein (Green FluorescentProtein, GFP) gene into pansies

[0039] One-year-old plants of Phalaenopsis were purchased from Hangzhou Tianjing Aquatic Botanical Garden; pTRV1 and pTRV2 plasmids linked to GFP gene fragments were donated by the Department of Horticulture, China Agricultural University; Escherichia coli DH5α was purchased from Novizan Biotechnology Co., Ltd.; Earth Biotechnology Co., Ltd.

[0040] 1. Construction of Agrobacterium tumefaciens containing pTRV1 vector and pTRV2 vector with GFP reporter gene fragment respectively

[0041] Transform pTRV1 and the pTRV2 plasmid (pTRV2-GFP) connected with the GFP gene fragment (pTRV2-GFP) into Agrobacterium GV3101 by freeze-thawing method, culture in dark at 28°C for two days, pick a single clone, and use pTRV2-F and pTRV2-R primers for positive single-clonal PCR detection .

[0042] 2. Infection of pansies plants with TRV-GFP-containing Agro...

Embodiment 2

[0048] Example 2 Using TRV-VIGS technology to silence 1-year-old Phalaenopsis phytoene dehydrogenase ( Phytoenes Desaturase, PDS )Gene

[0049] The 1-year-old plants of Phalaenopsis were purchased from Hangzhou Tianjing Aquatic Botanical Garden; pTRV1 and pTRV2 plasmids were donated by the Horticulture Department of China Agricultural University; Escherichia coli DH5α was purchased from Novizan Biotechnology Co., Ltd.; Agrobacterium GV3101 was purchased from Shanghai Weidi Biotechnology Co., Ltd. .

[0050] 1. Cloning of pansy flower PDS gene fragment

[0051] Use the Tiangen polysaccharide polyphenol kit (cat.#RR047A) to extract the total RNA of the shoot tip of the pansies, and the operation steps are carried out according to the instructions of the kit. Using PrimeScript TM II. The 1st Strand cDNA Synthesis Kit (TAKARA, Cat#6210A; Lot#AK4601) reverse-transcribed the total RNA of pansies into cDNA, and the operation steps were carried out according to the instructions...

Embodiment 3

[0068] Example 3 Utilize TRV-VIGS technology to silence 1-month-old pansies PDS gene

[0069] One-month-old Phalaenopsis plants were purchased from Hangzhou Tianjing Aquatic Botanical Garden; pTRV1 and pTRV2 plasmids were donated by the Horticulture Department of China Agricultural University; Escherichia coli DH5α was purchased from Novizan Biotechnology Co., Ltd.; Ltd.

[0070] 1. Cloning of pansy flower PDS gene fragment

[0071] The content of this step is exactly the same as the operation in Example 2;

[0072] 2. Construction of Agrobacterium Containing Phalaenopsis PDS Gene TRV Silencing Vector

[0073] The content of this step is exactly the same as the operation in Example 2;

[0074] 3. Agrobacterium containing pTRV1 and pTRV2-PDS infected pansies plants

[0075] Add 50 μL of Agrobacterium GV3101 bacterial solution containing pTRV1 and pTRV2-PDS to 5 mL of LB culture solution containing 50 mg / L kanamycin and 50 mg / L rifampicin, and shake the bacteria at 28°C and ...

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Abstract

The invention relates to the technical field of biology, and aims to provide a construction method of a fringed iris gene silencing system. According to the construction method of the fringed iris gene silencing system, the TRV-VIGS system is applied to the gene function research of fringed iris for the first time. By establishing a rapid and efficient fringed iris virus-mediated gene silencing system, the optimal fringed iris infection receptor, the optimal bacterial liquid concentration and the optimal infection part are determined, fringed iris plants are infected by adopting a leaf back cross cutting injection method, so that the efficiency of agrobacterium infection and gene silencing is remarkably improved, and high-throughput and functional analysis of iris plant gene functions in monocotyledonous plants on a genome scale are realized. The fringed iris gene silencing system is simple, convenient, rapid and efficient, and can lay a foundation for analyzing a molecular mechanism of important characters of fringed iris.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a VIGS gene silencing system of pansies. Background technique [0002] Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing phenomenon, which can cause specific degradation of endogenous mRNA sequences, and corresponding phenotype changes, which can be passed on by plant phenotype or physiological indicators. Changes reflect the function of the gene. VIGS technology has the advantages of rapidity, high efficiency, and high throughput. It can conveniently and quickly study gene functions, and can even be used to study some gene functions that will cause death after mutation. Therefore, this technology is widely used in plant growth and development, resistance to diseases and insect pests, and metabolism. Regulation and other aspects of functional gene research. [0003] Iris japonica (Iris japonica) is a perennial flower of the famil...

Claims

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Application Information

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IPC IPC(8): C12N15/83C12N15/84C12N15/66C12N9/02A01H5/00A01H6/00
CPCC12N15/8218C12N15/8203C12N15/8205C12N15/66C12N9/001C12Y103/05005C12N2770/00043
Inventor 许曈张润龙邵灵梅王小斌李丹青张佳平夏宜平
Owner ZHEJIANG UNIV
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