Application of Citrus citpitp1 Gene and Its Nucleotide Sequence in Transformation of Prokaryotic Vector Promoter

A technology of nucleotide sequence and promoter, applied in the field of genetic engineering

Active Publication Date: 2022-05-06
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, usually the object of promoter mining is concentrated on the upstream DNA sequence at the 5' end of the gene, and there is no research to confirm that the gene sequence itself can also be used as a promoter

Method used

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  • Application of Citrus citpitp1 Gene and Its Nucleotide Sequence in Transformation of Prokaryotic Vector Promoter
  • Application of Citrus citpitp1 Gene and Its Nucleotide Sequence in Transformation of Prokaryotic Vector Promoter
  • Application of Citrus citpitp1 Gene and Its Nucleotide Sequence in Transformation of Prokaryotic Vector Promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Acquisition of Citrus CitPITP1 Gene Sequence and CDS

[0029] Mature leaves of Guanxi honey pomelo without diseases and insect pests were picked, quick-frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature freezer, and the total DNA of the pulp was extracted by CTAB method. According to the citrus genome sequence, Primer5 was used to design amplification primers for the CitPITP1 gene for PCR, and the PCR product was subjected to TA cloning and sequencing to obtain its gene sequence, as shown in SEQ ID NO: 1, the amplification primer sequence, PCR system and reaction program as follows:

[0030] Its primer sequence is:

[0031] Forward primer CitPITP1 gene F: 5'-CACAACAAAGTCCAGCGTCG-3';

[0032] Reverse primer CitPITP1 gene R: 5'-ACAGCAACAGACAGTAGTTTCACC-3'.

[0033] The PCR system is as follows:

[0034]

[0035] The PCR reaction procedure is as follows:

[0036]

[0037] The fruit of Guanxi honey pomelo was picked 150 days after flowering, t...

Embodiment 2

[0062] Analysis of promoter activity of CitPITP1 CDS sequence in prokaryotes

[0063] Select eGFP as the reporter gene. First, in order to obtain the CDS sequence of the eGFP reporter gene, any vector containing eGFP can be used as a template for amplifying the eGFP CDS. According to the sequence of the plasmid pHSE401-35S-eGFP, the plasmid pHSE401-35S-eGFP was used as Template, using Primer5 to design specific amplification primers for PCR, the amplification primer sequences and PCR system and reaction procedures are as follows:

[0064] Forward primer eGFP extension fragment F: 5'-GCACATACAAATGGACGAACG-3'; reverse primer eGFP extension fragment R: 5'-GCCTGAATGGCGAATGCTA-3'.

[0065] The PCR system is as follows:

[0066]

[0067] The PCR reaction procedure is as follows:

[0068]

[0069] The PCR reaction product was separated by agarose gel electrophoresis and purified by Biomiga's DNA Gel / PCR Purification Miniprep Kit to obtain the eGFP extension fragment.

[0070...

Embodiment 3

[0089] Example 3 CitPITP1 promoter key sequence mining and application

[0090] Submit the CitPITP1 gene sequence and its CDS sequence to BPROM (http: / / www.softberry.com / berry.phtml?topic=bprom&group=programs&subgroup=gfindb) and BDGP (http: / / www.fruitfly.org / seq_tools / promoter .html) website for prokaryotic promoter prediction, and the results show that the sequence contains -10element (TGCTATAAT) and -35element (TTGGTG), which are located at positions 396-404 and 376-381 of SEQ ID NO: 2, respectively. Further use the method of promoter truncation analysis to obtain 5 nucleotide sequences of different lengths, which are respectively located in the full-length CitPITP1 CDS of 1-1008 in SEQ ID NO: 2, CitPITP1NO in 6-1008, and CitPITP1NO in 357-1008 of SEQ ID NO: 2. The gene sequences of CitPITP1Narrow1, CitPITP1Narrow2 of No. 376-1008, and CitPITP1Narrow3 of No. 396-1008 are shown in 2, 4, 5, 6, and 7 respectively. Primer5 was used to design corresponding specific amplification...

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Abstract

The invention discloses the application of a citrus CitPITP1 gene and its nucleotide sequence in prokaryotic vector promoter transformation. The nucleotide sequence of the CitPITP1 gene is shown in SEQ ID NO:1. The present invention screens the CitPITP1 gene from the genome of citrus, and finds from the CitPITP1 gene that a 64bp nucleotide sequence on exon 4 encoding the CRAL_TRIO domain of the CitPITP1 gene can drive gene expression in prokaryotes; therefore, the sequence containing CitPITP1_key64 The method can be applied to the transformation of a vector promoter in the field of genetic engineering.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the application of a citrus CitPITP1 gene and its nucleotide sequence in prokaryotic vector promoter transformation. Background technique [0002] During the development of organisms, transcription is one of the fundamental processes of molecular biology, which converts the genetic code in DNA into RNA molecules. Transcription initiation is a key step, which determines the position of the transcription start site (TSS) and the spatiotemporal expression pattern and intensity of transcription. The transcription initiation apparatus (RNA polymerase) needs to recognize and bind a specific DNA sequence to initiate transcription. This sequence is called a promoter, and it is usually a non-coding DNA sequence located upstream of the 5' end of the gene. [0003] In the fields of genetic engineering and synthetic biology, the promoter is one of the important elements to regulate the e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N15/113
CPCC07K14/415C12N15/70C12N2830/34
Inventor 徐强邬庆江付佳玲孙娟
Owner HUAZHONG AGRI UNIV
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