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A kind of 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: 2018-06-29
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 home and abroad is still far behind other plants, and the results are mostly concentrated on catechin synthesis. Cloning and functional characterization of enzymes, slow progress in transcriptional regulation

Method used

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  • A kind of tea tree csans promoter and application thereof
  • A kind of tea tree csans promoter and application thereof
  • A kind of tea tree csans promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: An isolated tea plant CsANS promoter and its preparation method

[0034] 1. Design of primers

[0035] According to the sequence of Camellia CsANS gene provided in NCBI, reverse amplification primers GSP1 and GSP2 were designed near 133bp downstream of the start codon.

[0036] 2. Obtaining the CsANS promoter

[0037] 2.1 Extraction of tea plant genomic DNA

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

[0039] 2.2 Separation and sequencing of CsAns promoter

[0040] The promoter sequence is separated using Genome-walking technology and TaKaRa Genome Walker kit (Clontech) is used to isolate the promoter region of the target gene. The principle and steps are as ( Figure 5 ).

[0041] 2.2.1 Restriction enzyme digestion of genomic DNA

[0042] According to the method of Genome Walker Universal Kit, the tea tree genomic DNA was digested with four restri...

Embodiment 2

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

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

[0059] CsANSpro / pBI101 is constructed based on the vector pBI101 and inserted into the promoter of the tea plant CsANS gene. Using BamHI and SalI double cut CsANSpro / pMD18T (forward) and pBI101, the small fragment of CsANSpro obtained by digestion and the large fragment of pBI101 were connected to obtain the plant expression vector CsANSpro / pBI101.

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

[0061] 2. Transformation of recombinant plasmid into Agrobacterium tumefaciens competent

[0062] Add 3μl of recombinant vector plasmid to 100ul Agrobacterium competent cells, mix gently and then ice bath for 30min. Quickly freeze in liquid nitrogen for 5 minutes, then water bath at 37°C for 5 minutes. Add 900μl YEB liquid medium, 28°C, 200rpm shaking cu...

Embodiment 3

[0072] Example 3: Promoter transcription regulation analysis

[0073] According to the tea tree ANS promoter sequence that the laboratory has obtained, through the PLACE database, comparative analysis.

[0074] 1 Tobacco transient transformation

[0075] 1.1 Construction of heterologous expression vector CsANSpro / pGreenII 0800-LUC

[0076] The plant expression vector CsANSpro / pGreenII 0800-LUC is constructed on the basis of the vector pGreenII 0800-LUC by inserting the promoter of the tea plant CsANS gene. The CsANSpro / pMD18T and pGreenII 0800-LUC were double-cut with BamH Ⅰ and Sal Ⅰ, and the small fragment of CsANSpro (forward) obtained by digestion was connected with the large fragment of pGreenII 0800-LUC to obtain the plant expression vector CsANSpro / pGreenII 0800-LUC.

[0077] The above-mentioned vector pGreenII 0800-LUC is as image 3 Shown.

[0078] 1.2 Construction of transcription factor expression vector

[0079] The transcription factor AtPAP1 was connected to the vector pCam...

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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 SEQ ID No.1, which has the basic transcription elements of a general promoter and conforms to the characteristics of eukaryotic gene promoters. The promoter can be expressed in heterologous systems (tobacco and Arabidopsis) after driving the reporter gene, and its activity is regulated by AtPAP, a key transcription factor in the plant anthocyanin synthesis pathway. Due to the great difficulties in tea tree whole genome sequencing and transgenic, the progress of tea tree gene promoter cloning and gene regulation mode analysis in tea tree is very slow. This is the first time that the promoters of genes related to catechin synthesis pathways have been isolated and identified from tea trees. The results of this invention can be used for in-depth research on the transcriptional regulation of CsANS genes and the catechin biosynthesis pathways involved in them, and can be applied to plant genetic engineering and Tea tree transgenic research.

Description

Technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a CsANS promoter, a key gene for the synthesis of tea 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 its super antioxidant activity can be used as a highly effective and multifunctional natural antioxidant and health medicine, which is of great benefit to human health. The development and utilization of catechins and the in-depth study of their metabolic pathways have increasingly highlighted their importance and attracted worldwide attention. [0003] In the biosynthesis of catechins, 4-coumarolyl-CoA produced in the pentose phosphate pathway (PPP) is used as a substrate to start the first step of the flavonoid pathway (FL pathway) reaction. Chalcone synthase (CH...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82C12N15/66A01H5/00A01H6/00
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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