Nicotiana alata gene NaD1 promoter and use thereof

A technology of promoters and tobacco flowers, applied in the field of genetic engineering, can solve the problems of unreported research on gene regulatory elements

Active Publication Date: 2019-08-23
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on NaD1 mainly involves protein function and mechanism of action, while the research on its gene regulatory elements has not been reported

Method used

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  • Nicotiana alata gene NaD1 promoter and use thereof
  • Nicotiana alata gene NaD1 promoter and use thereof
  • Nicotiana alata gene NaD1 promoter and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Cloning of the promoter According to the CTAB method in Wei Shenghua (2011), the DNA of Nicotiana tabacum (the plant material Nicotiana tabacum seeds were donated by Professor Chen Suiyun, School of Life Sciences, Yunnan University) DNA was extracted. Three specific primers were designed according to the instructions of Genome Walking Kit, and the upstream fragment of NaD1 gene was cloned by thermal asymmetric staggered PCR reaction. As shown in Table 1.

[0035] Table 1 Gene promoter amplification system

[0036]

[0037] Table 2 PCR amplification program

[0038]

[0039] Among them, the three specific primers were Na-sp1: 5'-AGCTTTTCTGCATGGTGGTTTGG-3', Na-sp2: 5'-GAGACAAACCATAGGCAACAAAGAGC-3': Na-sp3: 5'-GAAGCACAAGGAGCGAGCCATAGT-3'.

[0040] The third PCR product was separated on 1.2% agarose gel, and the target band was recovered and purified according to the instructions of the Gel Extraction Kit, and the pMD TM The method provided by the 19-T V...

Embodiment 2

[0041] Embodiment 2, identification and analysis of NaD1 gene promoter sequence

[0042] Design specific primers Na-p-644-F / R according to the sequencing results, and the sequence of Na-p-644-F is 5'- GTC GAC TCAGGTTATTTACTTTTTGGGG-3' (underlined as the SalI restriction site), Na-p-644-R sequence is 5'- GAATTC TGAAGCACAAGGAGCGAG-3' (underlined as Ecor1 restriction site).

[0043] Using Nicotiana japonica genomic DNA as a template Taq DNA Polymerase High FidelityKit amplifies its upstream fragment.

[0044] Among them, the PCR reaction system is: Platinum enzyme 1.0μl, 10×High Fidelity PCR Buffer 2.5μl, MgSO4 (50mM) 1.0μl, dNTP mix (10mM) 0.5μl, upstream and downstream primers 0.5μl, DNA 1.0μl, ddH 2 O 18.8μl, the final system is 25.0μl. The PCR reaction program was: 94°C pre-denaturation for 2min, (94°C 15sec, 60°C 30sec, 68°C 1min) 5 cycles, (94°C 15sec, 59°C 30sec, 68°C 1min) 5 cycles, (94°C 15sec, 58°C for 30sec, 68°C for 1min) for 25 cycles, 68°C for 10min, and 12°...

Embodiment 3

[0049] Example 3. Construction of pCAMBIA1391z-pNaD1-644::GUS and pCAMBIA1391z-pNaD1-310::GUS recombinant vectors

[0050] According to the obtained promoter sequence, synthesize pNaD1-644 and pNaD1-310 promoter fragments (310bp fragment upstream of ATG of NaD1 gene, SEQ ID NO.5), pNaD1-310 contains promoter basic elements TATA-box, CAAT-box and transcription factor binding elements. The aforementioned two fragments were connected to the front of the GUS reporter gene of the plant expression vector pCAMBIA1391z to drive its expression. The ligation product was transferred into DH5α strain for amplification, and the plasmid was extracted by alkaline lysis method for SalⅠ and EcoRI double enzyme digestion verification. It was detected by agarose gel. The electrophoresis results showed that the bands with expected sizes of 644bp and 310bp were obtained. The results are as follows: figure 2 As shown, it shows that the constructed vector is positive. The successfully constructed...

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Abstract

The invention discloses a Nicotiana alata gene NaD1 promoter and use thereof. The promoter has a nucleotide sequence shown in SEQ ID NO: 1. The invention further provides a vector containing the promoter and a host cell. The invention further relates to a nucleic acid constructor containing the promoter, a vector, a recombinant cell, a transgenic plant, an explant and a callus. The invention further discloses the use of the promoter. The Nicotiana alata gene NaD1 promoter and the use thereof have the advantages that the promoter contains a photoresponse element, the expression of the gene NaD1in flowers can be significantly changed under light stress treatment, and researching on regulation and control modes of the promoter has important use in more efficient utilization of protein NaD1.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter of Nicotiana tabacum NaD1 gene and its application. Background technique [0002] Promoter refers to the specific DNA sequence that RNA polymerase specifically recognizes and binds to guide the gene to start transcription. Eukaryotic promoters are divided into two regions, the core promoter region and the upstream sequence region. The core promoter region contains transcription initiation sites and TATA-box and other cis-acting elements; the upstream sequence region contains different regulatory expression elements, which are important for gene The efficiency, specificity and activity of transcription play a key role in ensuring the effectiveness and accuracy of gene transcription. According to the different modes of transcription, promoters can be divided into constitutive, tissue-specific and inducible. Understanding the component compositio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N1/21A01H5/00A01H6/82
CPCC07K14/415C12N15/8237
Inventor 赵懿琛赵德刚龚伟伟杨玲玲罗显麟
Owner GUIZHOU UNIV
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