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A kind of endogenous strong promoter phsp of smut smut and its expression vector and application

An endogenous promoter and strong promoter technology, applied in the field of genetic engineering, can solve the problems of low expression level and unstable fluorescence, and achieve the effect of improving the expression intensity

Active Publication Date: 2022-03-18
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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  • A kind of endogenous strong promoter phsp of smut smut and its expression vector and application
  • A kind of endogenous strong promoter phsp of smut smut and its expression vector and application
  • A kind of endogenous strong promoter phsp of smut smut and its expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning and sequencing of the endogenous pHSP 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 genomic DNA bands of Ustilago sativa were clear and complete, which could meet the needs of PCR amplification. Amplified with specific primers Hs-F: 5'-CATCGGGCCGCATGGCTGAG-3' (as shown in SEQ ID NO.2); Hs-R: 5'-GATGACGATTCTAAGGCTGTTGC-3' (as shown in SEQ ID NO.3) The full sequence of pHSP is 928bp (as shown in SEQ ID NO.1), cloned into the pMD 19-T vector, and verified by sequencing, the plasmid pMD19-pHSP containing the pHSP sequence of Ustilago smut was obtained.

Embodiment 2

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

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

[0034] According to the pHSP promoter sequence obtained in Example 1, design specific primers H-F: 5'- CGAAATTCGAGCTC G GTACC CATCGGGCCGCATGGCTGAG-3' (as shown in SEQ ID NO.4), wherein italics GGTACC Is the recognition site of the restriction endonuclease KpnI; H-R: 5'- CTCGCCCTTGCTCA CCATGG GATGACGATTCTAAGGCTGTTGC-3' (as shown in SEQID 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 promoter Otef, go through 1% agarose gel electrop...

Embodiment 3

[0040] Example 3: Evaluation of the Strength and Stability of the Endogenous Promoter pHSP 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 ) transformed the transformants obtained by transforming Ustilago smut, and extracted RNA according to the instructions. All operations were RNase free operations. After the extraction was completed, the concentration and quality of the extracted RNA were determined by UV analysis. Vazyme’s HiScript II Q RT SuperMix was used to The forqPCR (+gDNA wiper) kit was reverse-transcribed according to the operation instructions of the kit 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-TGATGCC...

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Abstract

The invention discloses an endogenous strong promoter pHSP of smut smut and its expression vector and application, belonging to the technical field of genetic engineering. The present invention includes: a strong endogenous promoter pHSP of Ustilago smut, the nucleotide sequence of which is shown in SEQ ID NO.1; an expression vector containing strong endogenous promoter pHSP of Ustilago smut; Driving the transcription and expression of the eGFP gene, constructing a stable expression system and obtaining the application of engineering smut smut has improved eGFP expression intensity and fluorescence stability. The present invention combines endogenous pHSP promoter and strong terminator nosT of Ustilago smut with eGFP The genes are connected to construct an effective plasmid vector pUe-cbx-Hsp, which improves eGFP The expression intensity of the start eGFP Compared with otef and TFIID2, the gene transcription level was enhanced by more than 9 times and 11 times respectively.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an endogenous strong promoter pHSP of smut fungus 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 com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/80C12N15/65C12N15/64C12N15/67C12N1/15C12R1/645
CPCC07K14/375C12N15/10C12N15/80C12N15/65C12Q2531/113
Inventor 张雅芬卞加慧叶子弘夏文强汤近天崔海峰俞晓平
Owner CHINA JILIANG UNIV
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