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Fraxinus mandshurica U6 gene promoter proFmU6.6 and cloning and application thereof

A promoter, pnc-121-pro technology, applied in the field of biology and plant transgenic, can solve the problems of limited application, lack of application, lack of research on U6 promoter, etc.

Active Publication Date: 2022-07-05
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • 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 proFmU6.6 and cloning and application thereof
  • Fraxinus mandshurica U6 gene promoter proFmU6.6 and cloning and application thereof
  • Fraxinus mandshurica U6 gene promoter proFmU6.6 and cloning and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The acquisition of the ash U6 gene promoter proFmU6.6, the specific operation is as follows:

[0046] (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(GTCCC TTCGGGGACATCCGATAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATG ACACGCATAAATCGAGAAATGGTCCAAATTTT)及大豆GmU6启动子的snRNA序列(GTCCCTT CGGGGACATCCGATAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCACAAATCGAGAAATGGTCCAAATTTT)与水曲柳基因组序列进行比 Right (BLAST). The alignment results were checked, and the positions with sequence homology higher than or equal to 99% were selected, and the upstream 1800 bp sequence was analyzed by the cis-acting element of the promoter using the Plant CARE online analysis software. The analysis results showed that USE (Upstream SequenceElement) and 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 proFmU6.6.

[0047] (2) Design specific primers up...

Embodiment 2

[0064] Detection of promoter activity of ash U6 gene promoter , the specific operations are as follows:

[0065] (1) Construction of the detection vector for the promoter activity of the Fraxinus mandshurica U6 gene:

[0066] 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:

[0067] pNC-proFmU6.6-F: CAGTGGTCTCTGTCCAGTCCT ACATCAACTCCAACACCGCC

[0068] pNC-proFmU6.6-R: CGGTCTCAGCAGACCACAAGT GACGAGAGGAACGACGGAAA

[0069] pNC-proFmU6.6.1-F: CAGTGGTCTCTGTCCAGTCCT CCAACCACCAACCGCATGT

[0070] pNC-proFmU6.6.2-F: CAGTGGTCTCTGTCCAGTCCT ACAGTTCGATTGAACTGTGACTTC

[0071] pNC-proFmU6.6.3-F: CAGTGGTCTCTGTCCAGTCCT GGCAATCCATTAGACTTTTGAG

[0072] pNC-proFmU6.6.4-F: CAGTGGTCTCTGTCCAGTCCT GAAGGTGTGGCGAGAAATCTTAT

[0073] pNC-proFmU6.6.1 / 2 / 3 / 4-R: CGGTCTCAGCAGACCACAAGT AATTTTATCGGATGTCCCCG (underlined sequence homologous ...

Embodiment 3

[0085] The construction of the truncated promoter proFmU6.6.4 gene editing recombinant vector of the ash U6 gene, the specific operation is as follows:

[0086] (1) The pSC1-GmU6-GmUbi3 vector was digested with Asc I and Lgu I double enzymes, the soybean GmU6 promoter on the vector was removed, and a large fragment of the 15472bp vector backbone was recovered.

[0087] (2) Design primers containing homology arms for homologous recombination cloning:

[0088] pSC1-proFmU6.6.4-F: ATCTTTCACTGGCGCGCC GAAGGTGTGGCGAGAAATCTTAT

[0089] pSC1-proFmU6.6.4-R: TCTAGCTCTAAAACAGAAGAGC AATTTTATCGGATGTCCCCG (underlined sequence homologous to pSC1-GmU6-GmUbi3 vector)

[0090] (3) Using the positive recombinant monoclonal plasmid of the truncated promoter FmU6.6.4 of the ash U6 gene as a template, the DNA fragment of the truncated promoter FmU6.6.4 of the ash U6 gene containing homology arms at both ends was obtained by PCR amplification , using 2×MasterAssembly Mix to homologously recomb...

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Abstract

The invention relates to a fraxinus mandshurica U6 gene promoter proFmU6.6 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 proFmU6.6, is successfully cloned for the first time, the fraxinus mandshurica U6 gene promoter activity detection vector is successfully constructed, and through transient transformation of fraxinus mandshurica seedlings, GUS dyeing and gus gene transcription expression verification, the promoter is proved to have efficient transcriptional activity. Experiments prove that the four types of fraxinus mandshurica U6 gene truncated promoters, namely, the proFmU6.6.1, the proFmU6.6.2, the proFmU6.6.3 and the proFmU6.6.4, all have the starting activity, and are 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 proFmU6.6 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 proFmU6.6, 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, the tree generation cycle is long, the genetic ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/70C12N15/84A01H5/00A01H6/00C12Q1/6897
CPCC12N9/1247C12N15/10C12N15/70C12N15/8205C12N15/8212C12N15/8216C12Q1/6897C12Y207/07006C12Q2531/113Y02A50/30
Inventor 曾凡锁高尚珠齐凤慧詹亚光辛颖关欣张桂芹卢晗
Owner NORTHEAST FORESTRY UNIVERSITY
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