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Cucumis sativus endogenous promoter and application thereof

A promoter and molecular technology, applied in the field of cucumber endogenous promoters, can solve the problems of gene silencing, less selectivity, less research work on isolation of cucumber endogenous efficient promoters, etc.

Active Publication Date: 2021-12-24
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In genetic engineering operations, simultaneous use of the same type of promoter to drive multiple foreign genes may lead to gene silencing or co-suppression, so different promoter elements are required. At present, the promoters commonly used in cucumber genetic engineering are mainly cauliflower flowers and leaves The virus CaMV35S promoter has less selectivity, and there is still relatively little research on the isolation of cucumber endogenous efficient promoters. Therefore, the isolation of cucumber endogenous promoters with excellent activity is of great significance for the research of cucumber functional genes and the improvement of genetic breeding.

Method used

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  • Cucumis sativus endogenous promoter and application thereof
  • Cucumis sativus endogenous promoter and application thereof
  • Cucumis sativus endogenous promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Mining and Analysis of Cucumber Candidate Promoters

[0041] The RNA-seq (3 biological replicates) and ATAC-seq libraries (3 biological replicates) were constructed using the cotyledon of cucumber 7-day seedlings. In order to enrich the gene expression in different periods and tissues of cucumber, another 12 sets of RNA-seq data were downloaded from the NCBI public database. Firstly, the RNA-seq data is analyzed. After quality control to remove low-quality reads and sequence connectors, the original data is compared to the cucumber reference genome to evaluate the expression of each gene and calculate the expression of each gene in each sample. The average value of expression in , and the 30 genes with the smallest variance among the top 100 expression values ​​were taken out, and 26 of them were selected as the key research objects in the next step. To further analyze the ATAC-seq data, first perform quality control on the ATAC-seq library, including removin...

Embodiment 2

[0042] Example 2 Comparative Analysis of Transcriptional Activity of Cucumber Candidate Promoters

[0043] In order to verify the transcriptional activities of the above 11 cucumber candidate promoter elements, the fluorescent reporter vector pTX1101 was selected (this carrier is a GFP fluorescent reporter vector, and the GFP gene transcription is initiated by the 35S promoter derived from cauliflower mosaic virus (CaMV). The terminator of the ketose-1,5-bisphosphate carboxylase small subunit (rbcS) E9 gene to terminate GFP gene transcription) as a backbone vector (such as figure 1 shown). The CsCRE01-CsCRE11 candidate promoter fragments were respectively amplified from cucumber genomic DNA using specific primers containing homologous sequences to the backbone vector, and the fragments recovered from the gel and the fragments of the backbone vector pTX1101 digested with AscI and SbfI were subjected to Gibson analysis. After assembling and replacing the CaMV 35S promoter on th...

Embodiment 3

[0061] Example 3 Transformation of Cucumber Hairy Roots Verifies the Transcriptional Activity of Candidate Promoters

[0062] In the above examples, the transcriptional functional activities of 11 candidate promoters were successfully verified at the cellular level. In order to further verify the functions of the candidate promoters in plants, CsCRE02, CsCRE10, CsCRE10, and The CsCRE11 promoter element, considering that the expressions of 4, 10, and 11 in cucumber are not much different, so 4 was removed when verifying the activity of hairy root transformation. By replacing the CaMV 35S promoter element on the constructed plant expression vector pLB42, the plant binary expression of the β-glucuronidase gene (GUS) and the green fluorescent protein gene (GFP) driven by the CsCRE02, CsCRE10, and CsCRE11 promoters were respectively obtained. Expression vector.

[0063] The specific operation is to use cucumber genomic DNA as a template to amplify the CsCRE02, CsCRE10, and CsCRE11...

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Abstract

The invention relates to the technical field of plant biology, in particular to a cucumis sativus endogenous promoter and application. The invention aims to provide a cucumis sativus endogenous high-activity promoter element. The invention provides a DNA (Deoxyribose Nucleic Acid) molecule which is named as CsCRE02. The promoter CsCRE02 not only can efficiently and stably drive gene expression in cucumis sativus, but also can efficiently and stably drive gene expression in other plant species such as rice and the like, and a good tool is provided for genetic oriented improvement of cucumis sativus by using modern biological technologies such as a transgenic technology or a genome editing technology. The promoter has great value in biological research of cucumis sativus.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a cucumber endogenous promoter and application thereof. Background technique [0002] Cucumber (Cucumis sativus) is a plant of the genus Cucumis (Cucurbitaceae) in the Cucurbitaceae family. It is widely distributed all over the world. However, due to the narrow genetic basis and long breeding years of cucumber itself, it is difficult to make breakthroughs in conventional genetic manipulation techniques in cucumber, which greatly affects the basic research of cucumber functional genome and the practice of molecular breeding. [0003] With the rapid development of genome engineering technologies such as transgenic and genome editing, cucumber functional genome research and germplasm innovation practices have ushered in new opportunities and challenges. Transgenic technology introduces pre-designed exogenous DNA into recipient cells through biotechnological means and integrates i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/34
CPCC07K14/415C12N15/8222
Inventor 张勇唐旭张韬郑雪莲周建平
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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