A salt-tolerant gene for Morchella mycelium and its use

Abstract

The present invention provides a Morchella mycelium salt-tolerance gene atg8, the nucleotide sequence of which is shown in the sequence table SEQ ID NO.1. The present invention also provides an amino acid sequence of a protein encoded by the above gene, the sequence of which is shown in SEQ ID NO.2. The present invention also provides the use of the above gene in improving the salt-tolerance growth of Morchella. The expression amount of atg8 in Morchella mycelium at different salt concentrations is obtained by fluorescent quantitative PCR, and then the role of the autophagy gene in the salt-tolerance stress of Morchella is simulated by adding an autophagy inhibitor. The experimental results show that the growth rate and mycelium diameter of Morchella mycelium with the addition of the autophagy inhibitor and under salt stress have obvious changes, and there are significant differences. It shows that the autophagy process plays an important role in the growth of Morchella mycelium in salt-tolerance stress, and also shows that the autophagy gene can improve the tolerance of Morchella mycelium to salt stress.

Description

Technical Field

[0001] The invention belongs to the field of biological genetic engineering and relates to Morchella, in particular to a Morchella mycelium salt-tolerance gene and application thereof. Background Art

[0002] Morchella is an important type of medicinal and edible fungi. It has attracted widespread attention from researchers around the world for its unique appearance, delicious taste and high economic value. In addition to being rich in amino acids, proteins, mineral elements and vitamins, Morchella also has various medicinal effects, such as antibacterial, antioxidant, anti-inflammatory, improving immunity, anti-allergic and lowering blood sugar. At the same time, researchers have been exploring the artificial cultivation of Morchella. At present, the artificial cultivation technology of Morchella has successfully achieved large-scale production. Six-sister Morchella is one of the main cultivated varieties in China. In previous studies, it was found that the hyphae of Six-sister Morchella has a certain salt tolerance. Therefore, this paper screened out the key autophagy gene atg8 from the salt stress tolerance of Morchella hyphae and verified its effect on the salt stress tolerance of Morchella hyphae. Summary of the invention

[0003] The invention provides a Morchella mycelium salt stress tolerance gene and application thereof. The Morchella mycelium salt stress tolerance gene and application thereof can be applied to the gene improvement of Morchella to make it adapt to land with high salt content.

[0004] The present invention provides a Morchella mycelium salt-tolerance gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the sequence length is 597 bp, specifically:

[0005] (SEQ ID NO.1).

[0006] The present invention also provides the amino acid sequence of the protein encoded by the above gene, as shown in SEQ ID NO. 2. The sequence length is 122, specifically:

[0007] MVHVSSFKNDHPLDKRREVAERIRSKYLDRIPVIVEKAPRSDAPDIDKKKYLVPADITVGKFVYEIRKHMTKVSAEKAIY LFVNNTIPPTAALISQIYERYKDEDGFLYITYSGENTFGSDL (SEQ ID NO. 2).

[0008] The present invention also provides application of the above gene in improving the salt-tolerant growth of Morchella.

[0009] The present invention also provides a method for promoting the growth of Morchella mycelium and the growth resistant to salt stress, wherein an autophagy inhibitor is added to the fertilizer, and the amino acid sequence of the autophagy inhibitor is shown in SEQ ID NO.2.

[0010] The present invention simulates the role of autophagy genes in the salt stress tolerance of Morchella by adding autophagy inhibitors. The experimental results show that the growth rate and mycelial diameter of Morchella hyphae with autophagy inhibitors and under salt stress have obvious changes, and there are significant differences. It shows that the autophagy process plays an important role in the growth of Morchella hyphae in salt stress tolerance, and also shows that autophagy genes can improve the tolerance of Morchella hyphae to salt stress.

[0011] Compared with the prior art, the present invention has a positive and obvious technical effect. The autophagy gene atg8 of the present invention is obtained based on the transcriptome experiment of salt stress tolerance of Morchella mycelium, and its expression level increases significantly under salt stress. Identifying its salt tolerance function and its function in improving the salt tolerance of Morchella will be of great significance for cultivating new varieties of Morchella. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 and Figure 2 The effects of autophagy inhibitors on mycelial growth of Morchella oleracea under salt stress are shown.

[0013] Figure 3 The expression level of the autophagy gene atg8 in Morchella mycelium at different salt concentrations is shown. DETAILED DESCRIPTION

[0014] Example 1 Discovery of the autophagy gene atg8

[0015] The present invention studies Morchella Liumei No. 8 (the product can also be purchased commercially) of the Edible Fungi Institute of Shanghai Academy of Agricultural Sciences, performs transcriptome analysis on Morchella mycelium with different salt concentrations (0mM, 20mM, 40mM, 60mM), screens out the key differential gene atg8, as shown in SEQ ID NO.1, and finds that it is significantly expressed under different salt concentrations.

[0016] Example 2 Fluorescence quantitative PCR of autophagy gene atg8

[0017] The test tube culture of Morchella liumei 8 (this product can also be purchased commercially) stored in the Edible Fungi Institute of Shanghai Academy of Agricultural Sciences was inoculated on a PDA flat culture medium covered with cellophane, cultured at 22° C. in the dark for 8 days, and then the hyphae were collected.

[0018] RNA was extracted using an RNA extraction kit (RNAiso Plus, TAKARA), and the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Evo MM-MLV RT Mix Kit with gDNA Clean for qPCR, ACCURATE BIOLOGY). The detailed operation steps refer to the respective instructions.

[0019] Morchella ACT1 is the internal reference gene, and the qRT-PCR primers are shown in the table below. Green ProTaq HS qPCR Kit, ACCURATE BIOLOGY) was used to perform qRT-PCR on a StepOnePlus fluorescent quantitative PCR instrument (Applied Biosystems, USA) to analyze the expression of atg8 gene in Morchella salt stress. The detailed operation steps refer to the respective instructions. Three biological replicates were set for each sample, and 2 -ΔΔCT The relative gene expression was calculated by the method and converted into log2FC. The results are shown in the table below.

[0020] The expression level of the autophagy gene atg8 in Morchella mycelium at different salt concentrations was obtained by qRT-PCR. The results showed that the expression level of atg8 increased significantly with the increase of salt concentration.

[0021] Gene Primer sequences ACT1 GTACCCTGGTATTGCCGACCGGACGATGGAAGGACCACTC(SEQ ID NO.3) ATG8 CCATCCGCATCCCTACTCTCGTGGCCTACCTGCTGCATC(SEQ ID NO.4)

[0022] Example 3

[0023] like Figure 1 As shown, the present invention also observes the effect of autophagy inhibitors on the growth of Morchella mycelium under salt stress. Nacl solutions of different concentrations (0mM, 20mM, 40mM, 60mM) were added to the Morchella plate culture medium, which was used as a control group for comparison. The experimental group was based on the control group and each group added with equal doses of autophagy inhibitors. The culture was carried out at 22°C in the dark for 8 days, and the growth of mycelium was observed. The results of the test on the tolerance of Morchella mycelium to the addition of autophagy inhibitors and to salt stress showed that: with the deepening of salt stress, the growth rate and mycelium diameter of Morchella mycelium showed a downward trend. At the same time, there were obvious differences in the growth rate and mycelium diameter of Morchella mycelium with autophagy inhibitors and those under salt stress. The control group had better resistance to salt stress, as shown in Figure 2. Figure 1 , 2 .

[0024] This indicates that autophagy inhibitors delay the growth of Morchella mycelium, and also indirectly indicates that autophagy genes have the ability to improve Morchella's resistance to salt stress and promote mycelium growth.

[0025]

Claims

1. An application of a salt-tolerant gene of Morchella mycelium in the salt-tolerant growth of Morchella, characterized in that: The Morchella is Morchella lilii; the nucleotide sequence of the Morchella mycelium salt tolerance gene is shown in SEQ ID NO.1; The application method comprises the following steps: adding Nacl solutions of different concentrations, 0 mM, 20 mM, 40 mM and 60 mM, to a Morchella plate culture medium, and adding an equal dose of an autophagy inhibitor to each group on the basis of the aforementioned addition of the Nacl solution; the amino acid sequence of the autophagy inhibitor is shown in SEQ ID NO.2; culturing at 22° C. in the dark for 8 days; as the salt stress deepens, the growth rate and the diameter of the hyphae of Morchella lilii are both on a downward trend, and at the same time, the growth rate and the diameter of the hyphae of the Morchella with the autophagy inhibitor added are significantly different from those under salt stress.

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