Tea tree CsANS promoter and application thereof

A promoter and tea tree technology, applied in the field of plant genetic engineering, can solve the problems of lack of high-efficiency transgenic systems, slow progress in transcriptional regulation, etc., and achieve the effect of enhanced activity

Active Publication Date: 2016-05-04
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the limitations of various conditions, such as the lack of whole genome sequencing and high-efficiency transgenic systems, the molecular biology research on tea tree flavonoid pathways at...

Method used

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  • Tea tree CsANS promoter and application thereof
  • Tea tree CsANS promoter and application thereof
  • Tea tree CsANS promoter and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: A kind of isolated tea tree CsANS promoter and preparation method thereof

[0034] 1. Design of primers

[0035] According to the sequence of the tea tree CsANS gene provided by NCBI, reverse amplification primers GSP1 and GSP2 were designed around 133 bp downstream of the start codon.

[0036] 2. Acquisition of CsANS promoter

[0037] 2.1 Extraction of tea tree genomic DNA

[0038] The material used in the present invention is Longjing long-leaf tea, and the genomic DNA is extracted by using a TAKARA genomic DNA mini kit.

[0039] 2.2 Isolation and sequencing of CsAns promoter

[0040] The promoter sequence was isolated using Genome-walking technology, and the TaKaRaGenomeWalkerkit (Clontech Company) kit was used to isolate the promoter region of the target gene. Its principle and steps are as ( Figure 5 ).

[0041] 2.2.1 Restriction enzyme digestion of genomic DNA

[0042] According to the method of the GenomeWalkerUniversalKit kit, tea tree geno...

Embodiment 2

[0057] Example 2: Construction of CsANS promoter plant expression vector, its genetic transformation in Arabidopsis thaliana and screening of transgenic plants

[0058] 1. Construction of plant expression vector CsANSpro / pBI101

[0059] CsANSpro / pBI101 is constructed by inserting the promoter of tea tree CsANS gene on the basis of vector pBI101. CsANSpro / pMD18T (forward direction) and pBI101 were double cut with BamHI and SalI, and the small fragment of CsANSpro obtained by digestion was ligated with the large fragment of pBI101 to obtain the plant expression vector CsANSpro / pBI101.

[0060] The above-mentioned vector pBI101 such as figure 2 shown.

[0061] 2. Recombinant plasmid transformed into competent Agrobacterium tumefaciens

[0062] Add 3 μl of the recombinant vector plasmid to 100ul of Agrobacterium competent cells, mix gently and then ice-bath for 30min. Quick-frozen in liquid nitrogen for 5 minutes, followed by a water bath at 37°C for 5 minutes. Add 900 μl YEB ...

Embodiment 3

[0072] Example 3: Analysis of promoter transcription regulation

[0073] According to the tea tree ANS promoter sequence obtained in the laboratory, the PLACE database was used for comparative analysis.

[0074] 1 Transient transformation of tobacco

[0075] 1.1 Construction of heterologous expression vector CsANSpro / pGreenII0800-LUC

[0076] The plant expression vector CsANSpro / pGreenII0800-LUC is constructed on the basis of the vector pGreenII0800-LUC by inserting the promoter of the tea tree CsANS gene. CsANSpro / pMD18T and pGreenII0800-LUC were double cut with BamHI and SalI, and the small fragment of CsANSpro (forward) obtained by digestion was ligated with the large fragment of pGreenII0800-LUC to obtain the plant expression vector CsANSpro / pGreenII0800-LUC.

[0077] The above vector pGreenII0800-LUC such as image 3 shown.

[0078] 1.2 Construction of transcription factor expression vector

[0079] The transcription factor AtPAP1 was connected with the vector pCambi...

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Abstract

The invention provides a tea tree CsANS promoter and application thereof. The nucleotide sequence of the tea tree CsANS promoter is shown in the SEQ ID No.1. The tea tree CsANS promoter is provided with a basic transcription component of a common promoter and conforms to the characteristics of a eucaryon gene promoter. The promoter can be expressed in a heterogenetic source system (tobacco and Arabidopsis thaliana) after driving a reporter gene, and the activity of the promoter is controlled by a plant anthocyanin synthetic route key transcription factor AtPAP. Due to the fact that difficulty exists in tea tree whole genome sequencing and transgenosis, development of tea tree gene promoter cloning and analysis of a gene control mode in a tea tree is quite slow. The promoter of a catechinic acid synthetic route related gene is separated out and identified from a tea tree for the first time, and the tea tree CsANS promoter has the advantages that the promoter can be used for intensively studying CsANS gene transcriptional control and catechinic acid biosynthetic pathways in which the CsANS gene participates and applied to plant genetic engineering and tea tree transgenosis studying.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a CsANS promoter of a key gene for synthesis of tea tree catechins and an application thereof. Background technique [0002] Catechin compounds belong to polyphenols and are the most important secondary metabolites in tea. The synthesis and accumulation of catechins is not only closely related to the quality of tea, but also its super antioxidant activity can be used as an efficient and multifunctional natural antioxidant and health care medicine, which is of great benefit to human health. The development and utilization of catechin and the in-depth study of its metabolic pathway have increasingly highlighted its importance and attracted worldwide attention. [0003] In the biosynthesis of catechins, 4-coumaroyl-CoA produced in the pentose phosphate pathway (PPP) is firstly used as a substrate to start the first step of the flavonoid pathway (FL pathway) reaction. Chalcon...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/82C12N15/66A01H5/00
CPCC12N9/001C12N15/1003C12N15/8243C12Y103/01077
Inventor 洪高洁孙宗涛徐平李林颖
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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