Ustilago esculenta endogenous promoter pEF as well as expression vector and application thereof

A technology of endogenous promoters and expression vectors, applied in the field of genetic engineering, can solve the problems of unstable fluorescence and low expression levels

Active Publication Date: 2021-04-20
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the expression system constructed by the heterologous promoter Otef is mainly used, although the expression system driven by it eGFP The protein can be normally expressed in the Ustilago smut strain, but the expression level is not high and the fluorescence is unstable. Therefore, it is necessary to screen out a strong promoter with efficient and stable expression for the functional gene research of Ustilago smut and its interaction with Zizania. foundation for mechanism research

Method used

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  • Ustilago esculenta endogenous promoter pEF as well as expression vector and application thereof
  • Ustilago esculenta endogenous promoter pEF as well as expression vector and application thereof
  • Ustilago esculenta endogenous promoter pEF as well as expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning and sequencing of the endogenous pEF promoter of Ustilago smut

[0031] The total DNA template of Ustilago sativa UET1 strain was extracted by CTAB method, and detected by 0.8% agarose electrophoresis, the extracted genome DNA bands of Ustilago sativa were clear and complete, which could meet the needs of PCR amplification. Amplified with specific primers EF-F: 5'-GTGGTGAGCAAGGCACTTTGA-3' (as shown in SEQ ID NO.2); EF-R: 5'-CCATACCCAAAAAACATCATTCAAA-3' (as shown in SEQ ID NO.3) The full sequence of pEF is 1314bp (as shown in SEQ ID NO.1), cloned into the pMD 19-T vector, and verified by sequencing, the plasmid pMD19-pEF containing the pEF sequence of Ustilago smut was obtained.

Embodiment 2

[0032] Embodiment 2: Ustilago smut endogenous pEF promoter drives eGFP Gene expression in Ustilago smut

[0033] 1. Construction of endogenous pEF promoter and strong terminator nosT and eGFP gene-linked plasmid vector

[0034] According to the pEF promoter sequence obtained in Example 1, design specific primers E-F: 5'- TCGAAATTCGAGCTC G GTACC GTGGTGAGCAAGGCACTTTGA-3' (as shown in SEQ ID NO.4), wherein italics GGTACC Is the recognition site of the restriction endonuclease KpnI; E-R: 5'- CTCGCCCTTGCTCA CCATGG TTTGAATGATGTTTTTGGTGTATGG-3' (as shown in SEQ ID NO.5), where italics CCATGG Is the recognition site of the restriction endonuclease NcoI. The underlined orthopedic letters are fragments homologous to the ends of the vector required for the seamless junction construction of the vector.

[0035] Using the pUMa932 plasmid as a template, use restriction enzymes KpnI and NcoI to double-digest it, cut off the eGFP upstream promoter Otef, go through 1% agar...

Embodiment 3

[0040] Example 3: Evaluation of the Strength and Stability of the Endogenous Promoter pEF of Ustilago smut

[0041] 1. Evaluation of eGFP expression level

[0042] The transformant obtained in Example 2 and the expression vector (heterologous promoter Otef and endogenous promoter TFIID2) used by S. eGFP ) transformants obtained by transforming Ustilago smut, RNA extraction was carried out according to the instructions, and all operations were RNase free operations. After the extraction was completed, UV analysis was used to measure the concentration and quality of the extracted RNA, and Vazyme’s HiScript II Q RT SuperMix was used. The forqPCR (+gDNA wiper) kit was reverse-transcribed according to the kit’s operating instructions to obtain the reverse-transcribed product, which was detected by ChamQUniversal SYBR qPCR Master Mix, and the detection primers were:

[0043] Primer sequences for eGFP:

[0044] qF-AACCGCATCGAGCTGAAG (as shown in SEQ ID NO.8);

[0045] qR-TGATGCCGT...

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Abstract

The invention discloses an ustilago esculenta endogenous promoter pEF as well as an expression vector and application thereof, and belongs to the technical field of gene engineering. The nucleotide sequence of the ustilago esculenta endogenous promoter pEF is shown as SEQ ID NO.1. The invention discloses the expression vector containing the ustilago esculenta endogenous promoter pEF, an application of the ustilago esculenta endogenous promoter pEF to driving transcription and expression of an eGFP gene, constructing a stable expression system and obtaining engineering ustilago esculenta, and an application of the ustilago esculenta endogenous promoter pEF to improving expression intensity and fluorescence stability of eGFP. The ustilago esculenta endogenous pEF promoter and a strong terminator nosT are connected with the eGFP gene, an effective plasmid vector pUe-cbx-EF is constructed, more choices are provided for construction of an ustilago esculenta genetic transformation vector, and a foundation is laid for functional gene research of the ustilago esculenta.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an endogenous promoter pEF of smut smut and its expression vector and application. Background technique [0002] Ustilago is an important endophytic fungus in wild rice. After infecting Zizania zizania plants, Ustilago can inhibit heading and flowering and induce the base of the stem to expand, gradually forming a spindle-shaped edible fleshy stem, that is, " Zizania ". Wild wild rice is rich in nutrition, not only contains sugar, organic nitrogen, fat, protein, fiber, vitamins, but also has high medicinal value, and has various functions such as dispelling heat, promoting body fluid, quenching thirst, removing yellow eyes, and hangover poison , so its economic value is high. [0003] At present, the interaction mechanism between Zizania smut and Zizania plants is not clear, leading to the production, quality control and breeding of Zizania completely re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/80C12N15/65C12N15/66C12N1/15C12R1/645
Inventor 叶子弘张雅芬卞加慧夏文强汤近天崔海峰俞晓平
Owner CHINA JILIANG UNIV
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