Regulation and application of transcription factor lfc1 on fruiting body development of Flammulina velutipes

A technology of transcription factor and Flammulina velutipes, applied in the field of genetic engineering, can solve the problems of blind screening, labor-consuming and time-consuming

Active Publication Date: 2021-07-06
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
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Problems solved by technology

Although the above three breeding methods can breed strains with excellent traits, they are labor-intensive and time-consuming, and the screening is relatively blind

Method used

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  • Regulation and application of transcription factor lfc1 on fruiting body development of Flammulina velutipes
  • Regulation and application of transcription factor lfc1 on fruiting body development of Flammulina velutipes
  • Regulation and application of transcription factor lfc1 on fruiting body development of Flammulina velutipes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Nucleotide and amino acid sequence analysis of LFC1

[0046] 1. Nucleotide sequence analysis of lfc1

[0047] The upstream and downstream sequences of the lfc1 gene were each extended by 2000 bp as the reference sequence, and the Zillions of OligosMapped (ZOOM) software was used to map the transcriptome reads to the reference sequence.

[0048] The analysis showed that the lfc1 gene was 1172bp in length from start codon to stop codon, including 6 introns with sizes of 53bp, 54bp, 55bp, 48bp, 52bp and 64bp, respectively.

[0049] The sequence is shown in SEQ ID NO.1.

[0050] 2. Analysis of the amino acid sequence of lfc1

[0051] with SMART ( http: / / smart.embl-heidelberg.de / ) to analyze the amino acid sequence of LFC1, the results show that the protein LFC1 encodes a protein with a GAL4-like Zn(II)2Cys6 domain, the structure is as follows figure 1 The molecular weight was 31802.0 Da and the isoelectric point was 9.32 when analyzed by the software DNAMAN.

[0052]...

Embodiment 2

[0054] Construction of lfc1 overexpression mutant strains and lfc1 knockdown mutant strains.

[0055] The pBHg-BCA1 plasmid used in the present invention is a binary expression vector that can be used for Agrobacterium transformation, and the used Escherichia coli is DH5α strain.

[0056] 1. Construction of lfc1 overexpression vector

[0057] (1) Use primers Pgpd-lfc1OE-F and Pgpd-lfc1OE-R to carry out PCR amplification with Flammulina velutipes genomic DNA as a template to obtain Flammulina velutipes gpd promoter fragment (Pgpd-lfc1OE), the gpd promoter contains the first inner part of the gpd gene Introns and exons, the target sequence is 920bp.

[0058] The primer sequences are as follows: Pgpd-lfc1OE-F: 5'-CAGATCCCCCGAATTAGTCGTGGGTCCAGCATTTTG-3';

[0059] Pgpd-lfc1OE-R: 5'-ACTACGACATGACCTGTAAAATGGTGAGCAAGA-3'.

[0060] The PCR reaction program was: 98°C for 30sec; 98°C for 10sec, 65°C for 90sec, 72°C for 60sec (25 cycles); 72°C for 10min; 4°C.

[0061] (2) PCR amplific...

Embodiment 3

[0151] The mycelial growth observation experiment was performed on the lfc1 mutant strain.

[0152] (1) The origin strains of Flammulina velutipes FL19 and lfc1 overexpressed mutant strains (lfc1 OE#15 ,lfc1 OE#23 and lfc1 OE#24 ) and lfc1 knockdown mutant strains (lfc1 RNAi#35 ,lfc1 RNAi#232 and lfc1 RNAi#309 ) were used to make bacterial blocks of the same size (d=5mm) with a hole punch, and were respectively inoculated on CYM plates without drugs, and photographs were taken after culturing at 25°C for 7 days.

[0153] (2) In the process of culturing, measure the growth length of mycelium every 24 hours, make records, and calculate the average growth rate of each strain.

[0154] The results of the analysis of the growth conditions and growth rates of different strains are as follows: on the CYM plate, the growth of the hyphae of the lfc1 overexpression mutant strain and the lfc1 knockdown expression strain was similar to that of the starting strain FL19, and both produ...

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Abstract

The invention belongs to the technical field of genetic engineering. The invention discloses a transcription factor LFC1 involved in regulating the development of the sporocarp of Flammulina velutipes, its encoding gene and its application. The nucleotide sequence of the gene encoding the transcription factor LFC1 is shown in SEQ ID NO.1 in the sequence listing; the amino acid sequence of the transcription factor LFC1 is shown in SEQ ID NO.2 in the sequence listing. The present invention finds through overexpression and RNAi experiments on lfc1 in Flammulina velutipes that lfc1 is an important negative regulation factor for the development of fruiting bodies of Flammulina velutipes, and affects the generation of primordia, length of stipe and cap shape of Flammulina velutipes. Knockdown expression of lfc1 on the one hand promotes the growth of Flammulina velutipes stipe and increases the commodity value of Flammulina velutipes, on the other hand shortens the fruiting cycle, reduces energy consumption, and reduces production costs. However, the high expression of lfc1 leads to the deformity of the fruiting bodies of Flammulina velutipes. Therefore, the specific expression level of lfc1 in Flammulina velutipes strains is an important factor for the development of the fruiting bodies of Flammulina velutipes, and lfc1 can be used in the breeding of fine strains of Flammulina velutipes, which is convenient for screening Flammulina velutipes with long stipe and small canopy.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. Background technique [0002] Flammulina velutipes, also known as shiitake mushrooms, oyster mushrooms, shiitake mushrooms, etc. (Yu Rongli, Qin Xusheng et al. 2004), is a traditional cultivated edible mushroom in my country. Flammulina velutipes fruiting body is slender and crisp, and the cap is smooth and tender. At the same time, it has rich nutrition and delicious taste. It is one of the important commercial edible fungi and is widely cultivated all over the world, especially in Asian countries (Chang and Buswell 1996). . The dry weight of Flammulina velutipes accounts for 31.2% of protein and 5.8% of fat. It is a good high-protein and low-fat food. In addition, Flammulina velutipes is also rich in vitamins B1, B2, B3, C, D, E, unsaturated fatty acids and 18 kinds of amino acids (Zhou Ping 2014, Sun Chuanbo, Jiang Ming et al. 2015). As an economical edible fungus, Flammulina ve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/375C12N15/31C12N15/80C12N15/66C12Q1/6895C12Q1/04C12R1/645
CPCC07K14/375C12N15/66C12N15/80C12Q1/6895C12Q2600/158
Inventor 李少杰吴塔菊孙宪昀张振颖
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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