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Fraxinus mandshurica U6 gene promoter proFmU6.7 and cloning and application thereof

A promoter and gene technology, applied in application, angiosperm/flowering plants, introduction of foreign genetic material using vectors, etc., can solve the problems of lack of research, lack of application, short transcription activity of U6 promoter, etc., and achieve efficient and accurate germplasm The effect of innovative, efficient and precise variety genetic improvement and efficient activation of activity

Active Publication Date: 2022-07-05
NORTHEAST FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there is still a lack of research on the U6 promoter in Mandshurica mandshurica, and the lack of an applicable, as short as possible, endogenous U6 promoter with high transcriptional activity has become the basis for the construction of the Mandshurica mandshurica CRISPR / Cas9 gene editing system. Restriction factors also limit the application of CRISPR / Cas9 system in genetic breeding and germplasm innovation of Mandshurica mandshurica

Method used

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  • Fraxinus mandshurica U6 gene promoter proFmU6.7 and cloning and application thereof
  • Fraxinus mandshurica U6 gene promoter proFmU6.7 and cloning and application thereof
  • Fraxinus mandshurica U6 gene promoter proFmU6.7 and cloning and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The acquisition of the ash U6 gene promoter proFmU6.7, the specific operation is as follows:

[0038] (1)根据U6 snRNA序列在不同物种间的保守性,利用拟南芥AtU6启动子的snRNA序列(GTCCCTTCGGGGACATCCGATAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCATAAATCGAGAAATGGTCCAAATTTT)及大豆GmU6启动子的snRNA序列(GTCCCTTCGGGGACATCCGATAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCACAAATCGAGAAATGGTCCAAATTTT)与水曲柳基因组序列进行比对(BLAST ). Check the alignment results, select the positions with sequence homology higher than or equal to 99%, and use the Plant CARE online analysis software to analyze the cis-acting element of the promoter of the upstream 1800bp sequence. The analysis results showed that USE (Upstream Sequence Element) and the basic transcription-related TATA-like Box were located at 60bp and 30bp upstream of the transcription initiation site of these U6 genes, respectively. One of the promoter sequences was selected and named proFmU6.7.

[0039] (2) Design specific primers upstream and downstream of ...

Embodiment 2

[0052] The detection of the promoter activity of the Fraxinus mandshurica U6 gene promoter is as follows:

[0053] (1) Construction of the detection vector for the promoter activity of the Fraxinus mandshurica U6 gene:

[0054] Specific primers containing homology arms for homologous recombination clone with pNC-121-pro (pBI121 frame, GUS reporter gene, NC clone frame replaced by 35S promoter of pBI121) were designed as follows:

[0055] pNC-proFmU6.7-F: CAGTGGTCTCTGTCCAGTCCT TATAACCAACCATTCCTTCTCACTG

[0056] pNC-proFmU6.7-R: CGGTCTCAGCAGACCACAAGT TTCTGGACAAAACCCTGGC

[0057] pNC-proFmU6.7.4-F: CAGTGGTCTCTGTCCAGTCCT TAACATCGTTGGGTAAATG

[0058] pNC-proFmU6.7.4-R: CGGTCTCAGCAGACCACAAGT AATTTTATCGGATGTCCCCG (underlined sequence homologous to pNC-121-pro vector)

[0059] Using the ash U6 gene promoter FmU6.7 and the ash U6 gene truncated promoter proFmU6.7.4 positive recombinant monoclonal plasmid as templates, the ash U6 gene promoter containing homologous arms at bo...

Embodiment 3

[0065] The construction of the truncated promoter proFmU6.7.4 gene editing recombinant vector of the ash U6 gene, the specific operation is as follows:

[0066] (1) Construction of the truncated promoter proFmU6.7.4 gene editing vector of Fraxinus mandshurica U6 gene

[0067] The pSC1-GmU6-GmUbi3 vector was digested with Asc I and Lgu I, and the soybean GmU6 promoter was removed from the vector, and a large 15472bp vector backbone fragment was recovered.

[0068] Design primers containing homology arms for homologous recombination cloning:

[0069] pSC1-proFmU6.7.4-F: ATCTTTCACTGGCGCGCC TAACATCGTTGGGTAAATG

[0070] pSC1-proFmU6.7.4-R: TCTAGCTCTAAAACAGAAGAGC AATTTTATCGGATGTCCCCG (underlined sequence homologous to pSC1-GmU6-GmUbi3 vector)

[0071] Using ash U6 gene truncated promoter FmU6.7.4 positive recombinant monoclonal plasmid as template, PCR amplification obtained the ash U6 gene truncated promoter FmU6.7.4 DNA fragment containing homology arms at both ends, using 2...

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Abstract

The invention relates to a fraxinus mandshurica U6 gene promoter proFmU6.7 as well as cloning and application thereof. According to the invention, the fraxinus mandshurica RNA polymerase III type promoter, namely the fraxinus mandshurica endogenous U6 gene promoter proFmU6.7, is successfully cloned for the first time, an activity detection carrier of the fraxinus mandshurica U6 gene promoter is successfully constructed, and through instantaneous transformation of fraxinus mandshurica seedlings and GUS dyeing verification, the promoter is proved to have efficient transcriptional activity. Experiments prove that the fraxinus mandshurica U6 gene truncated promoter proFmU6.7.4 has starting activity and is successfully applied to a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9) system. According to cloning of the fraxinus mandshurica U6 gene promoter proFmU6.7 and expression analysis of promoter activity, an efficient promoter sequence is provided for research on transformation of fraxinus mandshurica and related plants, and efficient and accurate germplasm innovation and variety genetic improvement of fraxinus mandshurica can be achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of plant transgenic technology, and specifically relates to a ash RNA polymerase type III promoter, more particularly to a ash U6 gene promoter proFmU6.7, and further discloses its clone methods and applications. Background technique [0002] Fraxinus mandshurica is a large deciduous tree of the genus Oleaceae, and it is listed as a national second-class protected endangered species. Since the first acquisition of transgenic tobacco plants in 1983, the genetic transformation of woody plants has also received attention. As an important part of forest genetic engineering, genetic transformation plays a role in disease resistance, insect resistance, improved traits and genetic engineering breeding of woody plants. important role. In the field of life sciences, mutants play a crucial role in the study of gene function. However, due to the long generation cycle, high genetic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/10A01H5/00A01H6/00
CPCC07K14/415C12N15/8216C12N2830/34Y02A50/30
Inventor 曾凡锁高尚珠陈晓慧齐凤慧王希刚刘海涛张振峰韩士君马铭浩
Owner NORTHEAST FORESTRY UNIVERSITY
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