WO2024000237A1 - Iaa-po1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用 - Google Patents

Iaa-po1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用 Download PDF

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WO2024000237A1
WO2024000237A1 PCT/CN2022/102247 CN2022102247W WO2024000237A1 WO 2024000237 A1 WO2024000237 A1 WO 2024000237A1 CN 2022102247 W CN2022102247 W CN 2022102247W WO 2024000237 A1 WO2024000237 A1 WO 2024000237A1
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iaa
pleurotus ostreatus
gene
growth
strain
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PCT/CN2022/102247
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French (fr)
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崔筱
孔维丽
刘芹
张玉亭
张坐芳
王彦坡
胡素娟
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河南省农业科学院植物营养与资源环境研究所
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Priority to PCT/CN2022/102247 priority Critical patent/WO2024000237A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

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  • the present invention relates to the technical field of genetic engineering, and more specifically to the application of the IAA-PO1 gene in inducing the formation of primordia of Pleurotus ostreatus in the growth, development and stress resistance of Pleurotus ostreatus.
  • Pleurotus ostreatus is a wood-rotting fungus with wide adaptability. Compared with other edible fungi, oyster mushroom has strong vitality, easy cultivation, short cultivation cycle, wide substrate utilization, high biological efficiency, strong adaptability, and wide cultivation area. Rich in nutrients and delicious in taste, oyster mushrooms are increasingly favored by people. They are one of the most widely cultivated edible fungi in the world. They have also become the most widely cultivated species of fungi in my country, with an output of 6.8296 million tons, ranking first in the country. 3 (Statistics of China Edible Fungi Association, 2020).
  • the present invention provides the application of the IAA-PO1 gene in inducing the formation of Pleurotus ostreatus primordium in the growth, development and stress resistance of Pleurotus ostreatus.
  • IAA-PO1 gene in inducing the formation of Pleurotus ostreatus primordia and stress resistance in the growth and development of Pleurotus ostreatus.
  • the IAA-PO1 gene sequence is shown in SEQ ID NO.2.
  • the stress resistance is temperature stress, oxidative stress, acid-base stress.
  • the present invention discloses the application of the IAA-PO1 gene in inducing the formation of oyster mushroom primordium and the growth and development resistance of oyster mushroom.
  • the IAA-PO1 gene induces the oyster mushroom primordium in advance. Formation; IAA-PO1 gene is involved in regulating temperature stress, oxidative stress, acid-base stress during the growth of Pleurotus ostreatus, and is related to the integrity of the cell wall of Pleurotus ostreatus.
  • Figure 1 is a visual diagram of the growth conditions of the wild-type strain and the overexpression mutant strain of the present invention under different temperature conditions;
  • Figure 2 is a statistical diagram of the growth conditions of the wild-type strain and the overexpression mutant strain of the present invention under different temperature conditions;
  • Figure 3 is a diagram showing the relative expression levels of the IAA-PO1 gene of the wild-type strain and the overexpression mutant strain of the present invention under different temperature conditions;
  • Figure 4 is a diagram illustrating the primordium formation of the wild-type strain and the overexpression mutant strain of the present invention
  • A is WT; B is Mutant;
  • Figure 5 is a visual diagram of the growth conditions of the wild-type strain and the overexpression mutant strain of the present invention under different pH conditions;
  • Figure 6 is a statistical diagram of the growth conditions of the wild-type strain and the overexpression mutant strain of the present invention under different pH conditions;
  • Figure 7 is a visual diagram of the growth conditions of the wild-type strain and the overexpression mutant strain of the present invention under conditions of different concentrations of Congo red;
  • Figure 8 is a statistical diagram of the growth of the wild-type strain and the overexpression mutant strain of the present invention under conditions of different concentrations of Congo red;
  • Figure 9 is a visual diagram of the growth conditions of the wild-type strain and the overexpression mutant strain of the present invention under conditions of different concentrations of H 2 O 2 ;
  • Figure 10 is a statistical graph showing the growth of the wild-type strain and the overexpression mutant strain of the present invention under conditions of different concentrations of H 2 O 2 ;
  • Figure 11 is a standard curve
  • Figure 12 is a diagram showing the H 2 O 2 content in the wild-type strain and overexpression mutant strain of the present invention.
  • Figure 13 is a diagram showing the relative expression of the IAA-PO1 gene of the wild-type strain and the overexpression mutant strain of the present invention under different concentrations of H 2 O 2 ;
  • Example 1 Obtaining genes related to IAA synthesis during the development of Pleurotus ostreatus fruiting body
  • the primordial stage samples of Pleurotus ostreatus treated with 10 -3 mol ⁇ L -1 and 10 -8 mol ⁇ L -1 IAA were used as materials, and the untreated primordial stage samples were used as controls to study the genes in the ALDH gene family of Pleurotus ostreatus.
  • the expression of gene No. 2762552025 was found to have the lowest expression level under the treatment condition of 10 -3 mol ⁇ L -1 and the highest expression level under the treatment condition of 10 -8 mol ⁇ L -1 .
  • the gene was named is IAA-PO1.
  • the promoter sequence of the IAA-PO1 gene is shown in SEQ ID NO.1.
  • the gene sequence of IAA-PO1 is shown in SEQ ID NO.2.
  • the genomic DNA of P99 strain of Pleurotus ostreatus was extracted and the promoter sequence of IAA-PO1 gene was amplified.
  • the amplification primers are as follows:
  • ALDHIAA-QDZ-up 5'-GC TCTAGA ACACATATCAATTCATGGC-3';Xba1; SEQ ID NO.3;
  • ALDHIAA-QDZ-down 5'-TCC CCCGGG CGTGCTCGTAGATAAGAG-3';Sma1; SEQ ID NO. 4.
  • PCR reaction system PrimeSTAR HS (Premix) 12.5 ⁇ L, upstream primer (10 ⁇ M) 1 ⁇ L, downstream primer (10 ⁇ M) 1 ⁇ L, genomic DNA 1 ⁇ L, ddH 2 O 9.5 ⁇ L.
  • PCR reaction program 98°C 10s, 58°C 15s, 72°C 2min, 30 cycles.
  • T-IAA-QDZ was digested by Xba1/Sma1, it was connected to the pCB1532 plasmid (Xiao Cui, Yi Wei, Xiang-Li Xie, et al.
  • Mitochondrial and peroxisomal Lon proteases play opposing roles in reproduction and growth but co-function in the normal development, stress resistance and longevity of Thermomyces lanuginosus[J], Fungal genetic and biology, 2017,103: 42-54.), the connected vector is named p-QDZ vector.
  • the enzyme digestion system is:
  • T-IAA-QDZ vector plasmid 5 ⁇ L, 10 ⁇ buffer 1 ⁇ L, Xba1 0.5 ⁇ L, Sma1 0.5 ⁇ L, RNase 0.15 ⁇ L, ddH 2 O 2.85 ⁇ L;
  • the extracted genomic DNA of Pleurotus ostreatus strain P99 was used as a template to amplify the gene sequence of IAA-PO1.
  • the amplification primers are as follows:
  • ALDHIAA-PO1-up 5'-TCC CCCGGG ATGGCGCAGAGCACAGTG-3';Sma1; SEQ ID NO.5;
  • ALDHIAA-PO1-down 5'-AA CTGCAG TCAAAAGGCCACGCCA-3';Pst1; SEQ ID NO. 6.
  • PCR reaction system PrimeSTAR HS (Premix) 12.5 ⁇ L, upstream primer (10 ⁇ M) 1 ⁇ L, downstream primer (10 ⁇ M) 1 ⁇ L, genomic DNA 1 ⁇ L, ddH 2 O 9.5 ⁇ L.
  • PCR reaction program 98°C 10s, 58°C 15s, 72°C 3min, 30 cycles.
  • T-IAA-PO1 was digested by Sma1/Pst1 and then connected to the p-QDZ vector.
  • the enzyme digestion system is:
  • p-QDZ carrier 5 ⁇ L, 10Xbuffer 1 ⁇ L, Sma1 0.5 ⁇ L, Pst1 0.5 ⁇ L, RNase 0.15 ⁇ L, ddH 2 O 2.85 ⁇ L;
  • T-IAA-PO1 carrier 5 ⁇ L, 10Xbuffer 1 ⁇ L, Sma1 0.5 ⁇ L, Pst1 0.5 ⁇ L, RNase 0.15 ⁇ L, ddH 2 O 2.85 ⁇ L;
  • the overexpression vector that was verified to be correct was named p-IAA-PO1.
  • step (2) Aspirate the protoplasts (containing the target DNA) in step (1) into the center of the 50ml EP tube, add 500 ⁇ L PEG transformation solution, turn the centrifuge tube to mix gently, then add 500 ⁇ L PEG transformation solution, and place at 28°C Place in the incubator for 10 minutes, then place at room temperature for 20 minutes;
  • step (3) Pipette 200 ⁇ L of the mixed solution in step (2) into a TB3 (sucrose 200g/L, yeast extract 3g/L, acid hydrolyzed casein 3g/L, agar 7.5g/L) (without any antibiotics) plate, Rotate the plate so that the mixture covers the entire plate, place it in a 28°C incubator for 14 hours, and then pour about 10 ml of TB3 culture medium containing 200 ⁇ g/ml chlorsulfuron-methyl. After solidification, place it in a 28°C incubator to avoid light culture;
  • TB3 sucrose 200g/L, yeast extract 3g/L, acid hydrolyzed casein 3g/L, agar 7.5g/L
  • Protoplast transformation method The linearized p-IAA-PO1 vector digested by /SUR-R is the amplification primer, and the genomic DNA of each resistant transformant is used as the template for PCR amplification.
  • the primer sequence of SUR-F/SUR-R is as follows:
  • PCR reaction system 1 ⁇ L of DNA template, 12.5 ⁇ L of Taq PCR Master Mix polymerase, 1 ⁇ L of upstream and downstream primers (10 ⁇ molL -1 ), and 4.5 ⁇ L of ultrapure water.
  • PCR amplification program 94°C 2min; 94°C 30s, 55°C 30s, 72°C 1min, 30 cycles; 72°C 10min. After verification by PCR amplification and sequencing, positive overexpression mutant strains can be obtained.
  • IAA-PO1 gene is involved in the regulation of basic growth conditions of Pleurotus ostreatus
  • IAA-PO1 gene is involved in high temperature/low temperature stress of Pleurotus ostreatus
  • CDS sequence of IAA-PO1 is shown in SEQ ID NO.9.
  • primer sequences of qALDHIAA-PO1-up/qALDHIAA-PO1-down are as follows:
  • qALDHIAA-PO1-up 5’-GCTATTATGCTGGATGGGCT-3’; SEQ ID NO.10;
  • the primer sequence of the internal reference gene is as follows:
  • the qRT-PCR reaction system is: 1 ⁇ L cDNA template, 5 ⁇ L 5 ⁇ SYBR Green Mix, 0.5 ⁇ L each of upstream and downstream primers (10 ⁇ mol/L), and 3 ⁇ L ultrapure water.
  • the reaction program was: pre-denaturation at 95°C for 1 min, 95°C for 10 s, 55°C for 30 s, and 72°C for 30 s, 40 cycles.
  • the qRT-PCR results are shown in Figure 3.
  • the results in Figure 3 show that as the culture temperature continues to increase, the expression of the IAA-PO1 gene increases.
  • the gene expression of the wild-type strain is the optimal culture temperature. (25°C), and the gene expression level of the overexpression mutant strain was 1.41 times that of the wild strain.
  • the IAA-PO1 gene is involved in regulating the acidic/alkaline environment required for the growth of Pleurotus ostreatus mycelium.
  • P99 strains and overexpression mutants with a diameter of 5 mm were inoculated onto PDA culture medium with pH values of 4, 5, 6, 7, 8, and 9, and cultured in a 25°C incubator for 6 days. Photographs were taken and the colony diameters were measured. , the results are shown in Figure 5- Figure 6. The results showed that compared with the wild-type strain, the overexpression mutant strain had no significant difference when cultured on the PDA medium with an optimal pH of 6 (P>0.05). The growth rate on the PDA medium with pH 9 was significantly faster, and it was more suitable to grow on the PDA medium with pH 9, indicating that the IAA-PO1 gene was more resistant to alkaline environments.
  • IAA-PO1 gene is involved in cell wall integrity and oxidative stress response of Pleurotus ostreatus
  • the IAA-PO1 gene affects the cell wall integrity of Pleurotus ostreatus
  • Congo red is a chemical reagent that binds to beta-1,4 glucan in cell walls and is often used to test the integrity of cell walls.
  • the activated P99 strain with a diameter of 5 mm and the overexpression mutant strain were inoculated onto PDA culture medium containing 0 ppm, 100 ppm, 200 ppm, and 300 ppm Congo red respectively, and cultured in a 25°C incubator for 6 days. Photographs were taken and the growth status of the colonies was recorded. , the results are shown in Figures 7-8. The results showed that compared with the wild-type strain, the overexpression mutant strain grew significantly faster on PDA medium with 100 ppm, 200 ppm, and 300 ppm Congo red (P ⁇ 0.01), and the hyphae became dense and resistant to Congo red. Therefore, the IAA-PO1 gene is involved in the cell wall integrity response of Pleurotus ostreatus.
  • the IAA-PO1 gene is involved in regulating oxidative stress in Pleurotus ostreatus.
  • Hydrogen peroxide content C ⁇ Vt/FW ⁇ V1 (C: Check the sample on the standard curve ( Figure 11) Hydrogen peroxide content, Vt: total volume of sample extraction solution, V1: volume of sample extraction solution used for measurement, FW: tissue fresh weight), calculate the hydrogen peroxide content, and the results are shown in Figure 12.
  • the results show that the H 2 O 2 content of the wild-type strain is 12.5 ⁇ mol/g, and the H 2 O 2 content of the over-expression mutant strain is 8.4 ⁇ mol/g. Compared with the wild-type strain, the H 2 O 2 content of the over-expression mutant strain is 12.5 ⁇ mol/g. Very significant reduction.
  • the qRT-PCR reaction system is: 1 ⁇ L cDNA template, 5 ⁇ L 5 ⁇ SYBR Green Mix, 0.5 ⁇ L each of upstream and downstream primers (10 ⁇ mol/L), and 3 ⁇ L ultrapure water.
  • the reaction program was: pre-denaturation at 95°C for 1 min, 95°C for 10 s, 55°C for 30 s, and 72°C for 30 s, 40 cycles.
  • the qRT-PCR results are shown in Figure 13.

Abstract

本发明公开了IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用，属于基因工程技术领域。本发明公开的IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用，IAA-PO1基因诱导平菇原基提前形成；IAA-PO1基因参与调控平菇生长过程中温度胁迫、氧化性胁迫、酸碱胁迫，并与平菇细胞壁完整性有关。

Description

IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用 技术领域
本发明涉及基因工程技术领域，更具体的说是涉及IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用。
背景技术
平菇是一种适应性广的木腐真菌，与其他食用菌相比，平菇具有生命力强，栽培容易，栽培周期短，基质利用广，生物学效率高，适应性强，栽培区域广阔，营养丰富，口味鲜美的特点，平菇越来越受到人们的青睐，是世界上广泛栽培的食用菌之一，也成为我国菌类栽培面最广的种类且产量682.96万吨，位居全国第三(中国食用菌协会统计，2020)。但是，在平菇的生产过程中，存在的主要问题是外界环境因素的变化严重影响平菇的产量和质量，影响经济效益；另外，平菇原基有效利用率也是影响平菇高产的主要因素之一。基于此，选育出原基利用率高、抗逆性强的平菇菌株成为平菇生产中亟待解决的问题。
因此，提供IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用。
为了实现上述目的，本发明采用如下技术方案：
IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用，所述IAA-PO1基因序列如SEQ ID NO.2所示。
进一步，所述抗逆的逆境为温度胁迫、氧化性胁迫、酸碱胁迫。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用，IAA-PO1基因诱导平菇原基提前形成；IAA-PO1基因参与调控平菇生长过程中温度胁迫、氧化性胁迫、酸碱胁迫，并与平菇细胞壁完整性有关。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明野生型菌株和过表达突变株在不同温度条件下的生长情况直观图；
图2附图为本发明野生型菌株和过表达突变株在不同温度条件下的生长情况统计图；
图3附图为本发明野生型菌株和过表达突变株在不同温度条件下IAA-PO1基因的相对表达量；
图4附图为本发明野生型菌株和过表达突变株的原基形成情况；
其中，A为WT；B为Mutant；
图5附图为本发明野生型菌株和过表达突变株在不同pH条件下的生长情况直观图；
图6附图为本发明野生型菌株和过表达突变株在不同pH条件下的生长情况统计图；
图7附图为本发明野生型菌株和过表达突变株在不同浓度刚果红条件下的生长情况直观图；
图8附图为本发明野生型菌株和过表达突变株在不同浓度刚果红条件下的生长情况统计图；
图9附图为本发明野生型菌株和过表达突变株在不同浓度H 2O 2条件下的生长情况直观图；
图10附图为本发明野生型菌株和过表达突变株在不同浓度H 2O 2条件下的生长情况统计图；
图11附图为标准曲线；
图12附图为本发明野生型菌株和过表达突变株中H 2O 2含量；
图13附图为本发明野生型菌株和过表达突变株在不同浓度H 2O 2条件下IAA-PO1基因的相对表达量；
图2、3、6、8、10、12、13中，大写字母表示在P＜0.01条件下呈极显著性差异，小写字母表示在P＜0.05条件下呈显著性差异；误差线为标准差。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1平菇子实体发育过程中IAA合成相关基因的获得
以经10 -3mol·L -1和10 -8mol·L -1IAA处理的平菇原基期样品为材料，以不经处理的原基期样品为对照，研究平菇ALDH基因家族中各基因的表达情况，发现2762552025号基因在10 -3mol·L -1处理条件下表达量最低，在10 -8mol·L -1处理条件下表达量最高，作为后续研究的候选基因，该基因命名为IAA-PO1。
IAA-PO1基因的启动子序列如SEQ ID NO.1所示。
Figure PCTCN2022102247-appb-000001
Figure PCTCN2022102247-appb-000002
Figure PCTCN2022102247-appb-000003
IAA-PO1的基因序列如SEQ ID NO.2所示。
Figure PCTCN2022102247-appb-000004
Figure PCTCN2022102247-appb-000005
Figure PCTCN2022102247-appb-000006
实施例2 IAA-PO1基因过表达突变株的获得
1)构建p-QDZ载体
(1)扩增启动子基因片段
提取平菇P99菌株基因组DNA，扩增IAA-PO1基因的启动子序列。扩增引物如下：
ALDHIAA-QDZ-up：5’-GC TCTAGAACACATATCAATTCATGGC-3’；Xba1；SEQ ID NO.3；
ALDHIAA-QDZ-down：5’-TCC CCCGGGCGTGCTCGTAGATAAGAG-3’；Sma1；SEQ ID NO.4。
PCR反应体系：PrimeSTAR HS(Premix)12.5μL，上游引物(10μM)1μL，下游引物(10μM)1μL，基因组DNA 1μL，ddH 2O 9.5μL。
PCR反应程序：98℃ 10s，58℃ 15s，72℃ 2min，30个循环。
进行琼脂糖凝胶电泳后，切胶回收，获得启动子基因片段。将经测序正确的IAA-QDZ启动子片段连接到pMD-18T载体上，命名为T-IAA-QDZ载体，T-IAA-QDZ经Xba1/Sma1酶切后，连接到pCB1532质粒(Xiao Cui,Yi Wei,Xiang-Li Xie,etal.Mitochondrial and peroxisomal Lon proteases play opposing roles in reproduction and growth but co-function in the normal development,stress resistance and longevity of Thermomyces lanuginosus[J],Fungal genetic and biology,2017,103:42-54.)，连接后的载体命名为p-QDZ载体。
(2)酶切
酶切体系为：
pCB1532质粒5μL，10×buffer 1μL，Xba1 0.5μL，Sma1 0.5μL，RNA酶0.15μL，ddH 2O 2.85μL；
T-IAA-QDZ载体质粒5μL，10×buffer 1μL，Xba1 0.5μL，Sma1 0.5μL，RNA酶0.15μL，ddH 2O 2.85μL；
(3)连接
连接体系：
酶切后的启动子基因片段4.5μL，酶切后的pCB1532质粒0.5μL，10×T4 buffer1μL，T4 DNA连接酶1μL，ddH 2O 3μL。
获得p-QDZ载体。
2)构建p-IAA-PO1载体
(1)扩增IAA-PO1基因片段
以提取的平菇P99菌株基因组DNA为模板，扩增IAA-PO1的基因序列。扩增引物如下：
ALDHIAA-PO1-up：5’-TCC CCCGGGATGGCGCAGAGCACAGTG-3’；Sma1；SEQ ID NO.5；
ALDHIAA-PO1-down：5’-AA CTGCAGTCACAAAGGCCACGCCA-3’；Pst1；SEQ ID NO.6。
PCR反应体系：PrimeSTAR HS(Premix)12.5μL，上游引物(10μM)1μL，下游引物(10μM)1μL，基因组DNA 1μL，ddH 2O 9.5μL。
PCR反应程序：98℃ 10s，58℃ 15s，72℃ 3min，30个循环。
进行琼脂糖凝胶电泳后，切胶回收，获得IAA-PO1基因片段。
将经测序正确的IAA-PO1基因片段连接到pMD-18T载体上，命名为T-IAA-PO1，T-IAA-PO1经Sma1/Pst1酶切后，连接到p-QDZ载体上。
(2)酶切
酶切体系为：
p-QDZ载体5μL，10Xbuffer1μL，Sma1 0.5μL，Pst1 0.5μL，RNA酶0.15μL，ddH 2O 2.85μL；
T-IAA-PO1载体5μL，10Xbuffer1μL，Sma1 0.5μL，Pst1 0.5μL，RNA酶0.15μL，ddH 2O 2.85μL；
(3)连接
连接体系：
酶切后的IAA-PO1基因片段4.5μL，酶切后的p-QDZ载体0.5μL，10×T4 buffer1μL，T4 DNA连接酶1μL，ddH 2O 3μL。
将检验正确的过表达载体命名为p-IAA-PO1。
3)原生质体转化
P99原生质体制备方法：
①5mm平菇P99菌块接于150ml PD液体培养基中培养5d，无菌打碎机打碎后接到新鲜的150ml PD液体培养基中培养1d。
②在超净工作台中，将菌丝经铺有两层无菌滤膜的无菌漏斗中过滤，用无菌去离子水漂洗，然后用protoplast buffer漂洗并过滤；
③称取0.3g的融壁酶溶解于3mL Novozyme buffer中，并用0.25μm的滤器过滤除菌；
④将过滤收集到的菌丝加入到过滤除菌的融壁酶溶液及17mL的protoplast buffer的150ml三角瓶中，于28℃，80rpm振荡孵化4.5h，1h后开始境检原生质体的形成情况，当大多数菌丝被消化后停止振荡孵化；
⑤通过六层擦镜纸把原生质体过滤到一个新的无菌的50ml离心管中，加入30mL 0.6M的KCl溶液，充分混匀；于3000×g，4℃，离心10min；
⑥弃上清液，用10mL STC溶液充分悬浮，于3000×g，4℃，离心10min，此步骤重复一次；
⑦显微镜下计数，并调整原生质体在STC中的终浓度达到1×10 6/mL，并始终置于冰上；
如需储存，加入7％DMSO，每管分装200μL于2ml EP管中，储存于-80℃备用。
原生质体转化法的具体步骤如下：
(1)将200μL 1×10 6/mL的p99原生质体与1-5μg线性化的p-IAA-PO1载体DNA充分混匀，置于冰上孵化30min；
(2)将步骤(1)中的原生质体(含有目的DNA)吸取到50ml EP管中央，加入500μL PEG转化液中，转动离心管轻轻混匀，再加入500μL PEG转化液，置于28℃培养箱中10min，然后于室温放置20min；
(3)吸取200μL步骤(2)中的混合液于TB3(蔗糖200g/L，酵母浸粉3g/L，酸水解酪蛋白3g/L，琼脂7.5g/L)(无任何抗生素)平板中，转动平板使混合液铺满整个平板，置于28℃培养箱中培养14h，再倒入约10ml加有200μg/ml氯嘧磺隆的TB3培养基，待凝固后置于28℃培养箱中避光培养；
(4)待TB3上层培养基中出现单菌落时(约铺氯嘧磺隆上层板后两天，注意每天观察)，挑转化子于PDA固体培养基中培养，提取DNA进行验证。
原生质体转化法将经Xba1酶切的线性化的p-IAA-PO1载体转入到P99原生质体中，挑取抗氯嘧磺隆转化子，提取各抗性转化子基因组DNA，以SUR-F/SUR-R为扩增引物，各抗性转化子基因组DNA为模板，进行PCR扩增。
其中，SUR-F/SUR-R的引物序列如下：
SUR-F：5’-CTCCCATGGCCGACGCTCTTG-3’；SEQ ID NO.7；
SUR-R：5’-CCACTACGCTCGGCCCTCTCATAA-3’；SEQ ID NO.8；
PCR反应体系：DNA模板1μL，Taq PCR Master Mix聚合酶12.5μL，上下游引物(10μmolL -1)各1μL，超纯水4.5μL。
PCR扩增程序：94℃ 2min；94℃ 30s，55℃ 30s，72℃ 1min，30个循环；72℃ 10min。经PCR扩增及测序验证，可得到阳性过表达突变株。
实施例3 IAA-PO1基因功能验证
(一)IAA-PO1基因参与平菇基本生长条件的调控
1)IAA-PO1基因参与平菇高温/低温胁迫
(1)将直径5mm的P99菌株(WT)及过表达突变株(Mutant)接种到PDA培养基上，分别置于10℃、15℃、20℃、25℃、30℃、35℃培养箱中培养6天，照相并记录其菌落直径，结果见图1-图2。结果表明：与野生型菌株相比，过表达突变株在20℃、30℃、35℃培养条件下生长速度极显著性增快，且在15℃培养条件下，与野生型菌株相比，菌丝变得致密。
(2)提取不同温度培养条件下野生型菌株及过表达突变株的RNA，反转录为cDNA，以P99菌株及过表达突变株cDNA为模板，以qALDHIAA-PO1-up/qALDHIAA-PO1-down为扩增引物，进行qRT-PCR扩增，Actin为内参。
其中，IAA-PO1的CDS序列如SEQ ID NO.9所示。
Figure PCTCN2022102247-appb-000007
Figure PCTCN2022102247-appb-000008
qALDHIAA-PO1-up/qALDHIAA-PO1-down的引物序列如下：
qALDHIAA-PO1-up：5’-GCTATTATGCTGGATGGGCT-3’；SEQ ID NO.10；
qALDHIAA-PO1-down：5’-TTCACAAAGTTTCAGGGCAGT-3’；SEQ ID NO.11；
内参基因的引物序列如下：
Actin-F：5’-CCGTCCCCATCTATGAAGGT-3’；SEQ ID NO.12；
Actin-R：5’-GGTATCCTCGCTCCATCAAAT-3’；SEQ ID NO.13；
qRT-PCR反应体系为：cDNA模板1μL，5×SYBR Green Mix 5μL，上下游引物(10μmol/L)各0.5μL，超纯水3μL。反应程序为：95℃预变性1min，95℃ 10s，55℃ 30s，72℃ 30s，40个循环。
qRT-PCR结果见图3，图3结果表明，随着培养温度的不断升高，IAA-PO1基因的表达量随之提高，35℃培养条件下，野生型菌株基因表达量是最适培养温度(25℃)下的4.3倍，过表达突变株基因表达量是野生菌株的1.41倍。
(3)将25℃培养7d至满板的P99野生型菌株及过表达突变株放置于15℃培养箱中培养10天后，发现过表达突变株原基提前形成，见图4。
2)IAA-PO1基因参与调控平菇菌丝生长所需酸性/碱性环境
将直径5mm的P99菌株及过表达突变株接种到pH分别为4、5、6、7、8、9的PDA培养基上，置于25℃培养箱中培养6天，照相并测量其菌落直径，结果见图5-图6。结果表明：与野生型菌株相比，过表达突变株除了在最适pH为6的PDA培养基上培养无显著性差异之外(P＞0.05)，在pH为4、5、7、8、9的PDA培养基上生长速度均极显著性变快，且更适宜在pH为9的PDA培养基上生长，表明IAA-PO1基因更耐碱性环境。
(二)IAA-PO1基因参与平菇细胞壁完整性及氧化性胁迫反应
以平菇P99IAA-PO1过表达突变株为研究对象，测定平菇菌丝体在不同浓度的刚果红(0ppm、100ppm、200ppm、300ppm)、H 2O 2(0μM、5μM、 10μM)胁迫条件下，P99菌株的过表达突变株菌落生长速度的变化情况，确定IAA-PO1是否参与了平菇细胞壁完整性及氧化性胁迫的调控。
1)IAA-PO1基因影响平菇细胞壁完整性
刚果红，是一种能够与细胞壁中的β-1,4葡聚糖相结合的化学试剂，通常用来检测细胞壁的完整性。
将经活化后直径5mm的P99菌株及过表达突变株分别接种到含有0ppm、100ppm、200ppm、300ppm刚果红的PDA培养基上，置于25℃培养箱中培养6天，照相并记录菌落生长状况，结果见图7-图8。结果表明：相对于野生型菌株，过表达突变株在100ppm、200ppm、300ppm刚果红的PDA培养基上生长速度极显著性增快(P＜0.01)，且菌丝变得致密，对刚果红抗性增强，因此，IAA-PO1基因参与平菇细胞壁完整性应答反应。
2)IAA-PO1基因参与调控平菇氧化性胁迫
(1)将经活化后直径5mm的P99菌株及过表达突变株分别接种到含有0μM、5μM、10μM H 2O 2的PDA固体培养基上，置于25℃培养箱中避光培养6天，照相并记录菌落生长状况，结果见图9-图10。结果表明：与野生型菌株相比，添加不同浓度外源H 2O 2对菌丝生长速度影响差异显著，当添加5μM、10μM的H 2O 2时，过表达突变株菌丝生长速度极显著变快(P＜0.01)。
(2)将经活化后直径为5mm的P99菌株及过表达突变株分别接种到PD液体培养基中，置于25℃摇床中150rpm避光培养6天，分别称取3g经摇床培养6天且在吸水纸上吸干的野生型及过表达突变株的菌丝于研体内，加入3ml 4℃预冷的丙酮研磨成匀浆后，转入15ml离心管中4000r/min离心15min，将上清液转至新的15ml离心管中，获得样品提取液。用1ml移液器吸取各样品提取液1ml，各管中分别加入0.1ml 5％硫酸钛和0.2ml浓氨水，3000r/min离心10min，弃去上清液，留下沉淀，于各管中加入5ml的2M硫酸，待沉淀完全溶解后于415nm波长下测各管中的吸光值，按照公式：过氧化氢含量＝C×Vt/FW×V1(C：标准曲线(图11)上查得样品中过氧化氢含量，Vt：样品提取液总体积，V1：测定时用样品提取液体积，FW：组织鲜重)，计算过氧化氢含量，结果见图12。结果显示：野生型菌株H 2O 2的含量为12.5 μmol/g，过表达突变株H 2O 2的含量为8.4μmol/g，与野生型菌株相比，过表达突变株H 2O 2含量极显著性降低。
(3)将经活化后直径为5mm的P99菌株及过表达突变株分别接种到分别含有0μM、2μM、3μM、4μM、5μM H 2O 2的铺有玻璃纸的PDA固体培养基上，于25℃培养箱中避光培养6天，6天后，从玻璃纸上刮取各菌株的菌丝，迅速放置到液氮中并提取野生型菌株和过表达突变株的RNA，反转录成cDNA后，以qALDHIAA-PO1-up/qALDHIAA-PO1-down为扩增引物，进行qRT-PCR扩增，Actin为内参。
qRT-PCR反应体系为：cDNA模板1μL，5×SYBR Green Mix 5μL，上下游引物(10μmol/L)各0.5μL，超纯水3μL。
反应程序为：95℃预变性1min，95℃ 10s，55℃ 30s，72℃ 30s，40个循环。qRT-PCR结果见图13。
结果表明：经H 2O 2处理后，野生型菌株及过表达突变株的IAA-PO1基因表达量均上调表达，且过表达突变株IAA-PO1基因表达量极显著高于野生型(P＜0.01)，分别是野生型的1.29、1.24、1.21、1.24倍。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (2)

  1. IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用，其特征在于，所述IAA-PO1基因序列如SEQ ID NO.2所示。
  2. 根据权利要求1所述的IAA-PO1基因在诱导平菇原基形成和平菇生长发育抗逆中的应用，其特征在于，所述抗逆的逆境为温度胁迫、氧化性胁迫、酸碱胁迫。
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