阅读说明：本技术 一种长喙壳菌外源基因转化的方法 (Method for transforming exogenous gene of shell fungus longrostone ) 是由 李健强 张治萍 李迎宾 罗来鑫 于 2019-12-03 设计创作，主要内容包括：本发明公开了属于生物技术领域的一种长喙壳菌外源基因转化的方法。本发明的方法通过将长喙壳菌的原生质体与适量的含有筛选标记的外源DNA片段和含有向导RNA(SgRNA)的质粒载体进行混合,然后利用PEG介导的原生质体的转化及转化子筛选,从而将含有筛选标记的外源DNA片段转入长喙壳菌基因组中,实现了PEG介导的长喙壳菌原生质体转化,建立了一套完整的遗传转化、筛选方法,易获得表达潮霉素抗性蛋白且稳定遗传的转化菌株,转化效率较高。(The invention discloses a method for transforming exogenous genes of a shell bacterium longum, belonging to the technical field of biology. The method of the invention mixes the protoplast of the long coracoid shell bacterium with a proper amount of exogenous DNA segment containing a screening marker and a plasmid vector containing guide RNA (SgRNA), and then utilizes the PEG-mediated transformation of the protoplast and the screening of transformants, thereby transferring the exogenous DNA segment containing the screening marker into the genome of the long coracoid shell bacterium, realizing the PEG-mediated transformation of the protoplast of the long coracoid shell bacterium, establishing a set of complete genetic transformation and screening methods, easily obtaining a transformation strain expressing hygromycin resistance protein and stably inheriting, and having higher transformation efficiency.)

1. A method for transformation of chaetomium longum, comprising the steps of:

(1) preparing a protoplast suspension of the fusca longirostrata, then mixing the protoplast suspension, an exogenous DNA fragment or a plasmid thereof and a plasmid expressing CRISPR/Cas9-sgRNA, adding heparin sodium, standing, and centrifuging;

(2) centrifuging, removing supernatant, adding the precipitate into a culture medium for regeneration culture, transferring to a flat plate for growth culture, and screening to obtain a transformant.

2. The method as claimed in claim 1, wherein the exogenous DNA fragment comprises 1200bp upstream and downstream of the target gene to be knocked out.

3. The method of claim 2, wherein the exogenous DNA segment further comprises an exogenous gene segment to be inserted.

4. The method of claim 1, wherein the protoplast suspension is prepared by a method comprising: resuspending the protoplast in a buffer to prepare a protoplast suspension having a concentration of (1-10). times.10⁷one/mL.

5. The method according to claim 1, wherein the exogenous DNA fragment or the plasmid thereof and the CRISPR/Cas9-sgRNA expression plasmid are added in amounts of 0.1-10 μ g/100 μ L final concentration respectively. Preferably, it is 0.5 to 5. mu.g/100. mu.L, more preferably 2 to 5. mu.g/100. mu.L.

6. The method according to any one of claims 1 to 5, wherein the resting treatment comprises: after adding heparin sodium, standing on ice, adding PTC solution, standing, and resuspending with buffer solution.

7. The method according to claim 6, wherein the PTC solution is a 25mM Tris-HCl solution containing PEG4000 and calcium chloride, having a pH of 7.5, wherein the content of PEG4000 is 60-66%, and the concentration of calcium chloride is 50 mM; the adding amount of the PTC solution is as follows: the volume ratio of the protoplast suspension to the protoplast suspension is 25: 1-2;

the heparin sodium is a heparin sodium solution, the concentration is 5mg/mL, and the volume ratio of the addition amount to the protoplast suspension is 1: 20-40.

8. The method according to any one of claims 1 to 7, wherein the buffer is an STC buffer, the STC buffer is a 25mM Tris-HCl solution containing sorbitol and calcium chloride at a pH of 7.5, wherein the concentration of sorbitol is 1.0M and the concentration of calcium chloride is 50 mM.

9. The method of any one of claims 1 to 8, wherein the method of regenerative culture comprises: the culture medium is an OCM culture medium, and is cultured for 6-8h at room temperature, wherein the OCM culture medium comprises the following components: 5g/L of malt extract, 5g/L of yeast, 1.32g/L of ammonium phosphate and 0.6M of sucrose.

10. The method of any one of claims 1-9, wherein the method of growing culture comprises: coating on a Bottom Agar plate, culturing at room temperature for 1-2 days, covering an unset Top Agar culture medium on the Bottom Agar plate, and culturing until a transformant grows out;

the composition of the Bottom agar plate culture medium comprises: 5g/L of malt extract, 5g/L of yeast, 1.32g/L of ammonium phosphate, 16g/L of agar and 0.6M of cane sugar; the Top Agar plate culture medium comprises the following components: malt extract 20g/L, yeast 2g/L, agarose 10g/L and hygromycin 50 mg/L.

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a method for transforming exogenous genes of a shell bacterium longum.

Background

The long coracoid fungus is a pathogenic fungus which is widely distributed and seriously harmful, and can infect more than 14 woody and herbaceous plants of more than 30 species. The strain has been reported to harm crops such as sweet potatoes, pomegranate, eucalyptus, taro, loquat, rhizoma arisaematis, mango and the like in China. Wherein serious vascular bundle diseases are caused on pomegranate and mango, which are called cancer diseases in production.

At present, the research on the fungi is mostly concentrated on the aspect of traditional biology, the research on the aspect of molecular mechanism is rare, and relevant documents at home and abroad are searched without reports related to genetic transformation methods. In order to research the molecular biology of the coracoid fungus, an efficient genetic transformation system is required to be established, and the establishment of the efficient genetic transformation system is also a precondition for deeply researching the pathogenic molecular mechanism of the coracoid fungus on economic forest trees and herbaceous plants and the self growth and development of the strain. Therefore, establishing a simple, efficient and stable genetic transformation method has important significance for researching the pathogenic mechanism of pomegranate wilt, mango wilt and other diseases.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, the invention aims to provide a simple, efficient and stable genetic transformation method of the long coracoid fungus.

Therefore, the technical scheme of the invention is as follows:

a method for transformation of chaetomium longum comprising the steps of:

(1) preparing a protoplast suspension of the fusca longirostrata, then mixing the protoplast suspension, an exogenous DNA fragment or a plasmid thereof and a plasmid expressing CRISPR/Cas9-sgRNA, adding heparin sodium, standing, and centrifuging;

(2) centrifuging, removing supernatant, adding the precipitate into a culture medium for regeneration culture, transferring to a flat plate for growth culture, and screening to obtain a transformant.

In the above method, the exogenous DNA fragment comprises 800bp upstream and downstream of the target gene to be knocked out, such as 800bp, 900bp, 1000bp, 1100bp or 1200bp upstream and downstream of the target gene.

In the above method, the exogenous DNA fragment further comprises a selection or marker gene, for example, a hygromycin gene.

In the above method, the screening or marker gene may be a double screening or double marker gene.

In the above method, further, the foreign DNA fragment may further comprise a foreign gene fragment to be inserted.

In the above method, optionally, the exogenous DNA fragment comprises the target gene to be knocked out with upstream 800-.

In the above method, the plasmid containing the exogenous DNA fragment, the original backbone thereof, optionally, is a PSK (-) plasmid.

In the above method, the method for preparing the protoplast suspension comprises: resuspending the protoplast in a buffer to prepare a protoplast suspension having a concentration of (1-10). times.10⁷one/mL.

In the method, the adding amount of the exogenous DNA fragment or the plasmid thereof and the plasmid for expressing CRISPR/Cas9-sgRNA are 0.1-10 mug/100 mug respectively. Optionally, the amount added is 0.5-5 μ g/100 μ L, or the amount added is 2-5 μ g/100 μ L, respectively.

In the above method, the CRISPR/Cas9-sgRNA expression plasmid, optionally, the original plasmid thereof is pCRISPR/Cas-U6-1, and the construction method refers to the article "Tailor-Made CRISPR/Cas System for high efficiency engineered Gene Replacement in the Rice Blast funus". Wherein the sgRNA is designed and prepared according to a target gene.

In the above method, the standing treatment comprises: after adding heparin sodium, standing on ice, adding PTC solution, standing, and resuspending with buffer solution.

In the method, the PTC solution is 25mM Tris-HCl solution containing PEG4000 and calcium chloride, the pH value is 7.5, the content of the PEG4000 is 60-66%, and the concentration of the calcium chloride is 50 mM; the adding amount of the PTC solution is as follows: the volume ratio of the protoplast suspension to the protoplast suspension is 25: 1-2;

the heparin sodium is a heparin sodium solution, the concentration is 5mg/mL, and the volume ratio of the addition amount to the protoplast suspension is 1: 20-40.

In the above method, the buffer is STC buffer, and the STC buffer is 25mM Tris-HCl solution containing sorbitol and calcium chloride, and has a pH value of 7.5, wherein the concentration of the sorbitol is 1.0M, and the concentration of the calcium chloride is 50 mM.

In the above method, the method for regenerative culture comprises: the culture medium is an OCM culture medium, and is cultured for 6-8h at room temperature, wherein the OCM culture medium comprises the following components: 5g/L of malt extract, 5g/L of yeast, 1.32g/L of ammonium phosphate and 0.6M of sucrose.

In the above method, the method for growth culture comprises: spread on a Bottom Agar plate, and after 1-2 days of culture at room temperature, the unset Top Agar medium is overlaid on the Bottom Agar plate and cultured until the transformants grow out.

In the above method, the composition of the Bottom agar plate medium comprises: 5g/L of malt extract, 5g/L of yeast, 1.32g/L of ammonium phosphate, 16g/L of agar and 0.6M of cane sugar; the Top Agar plate culture medium comprises the following components: malt extract 20g/L, yeast 2g/L, agarose 10g/L and hygromycin 50 mg/L.

The invention has the beneficial effects that:

the invention mixes the protoplast of the long coracoid shell bacterium with a proper amount of exogenous DNA segment containing a screening marker and a plasmid vector containing guide RNA (SgRNA), and then utilizes the PEG-mediated transformation of the protoplast and the screening of transformants, thereby transferring the exogenous DNA segment containing the screening marker into the genome of the long coracoid shell bacterium, realizing the PEG-mediated transformation of the long coracoid shell bacterium protoplast, establishing a set of complete genetic transformation and screening methods, easily obtaining a transformation strain which expresses hygromycin resistance protein and is stable in heredity, and having higher transformation efficiency. The method of the invention can be applied to the molecular biology research of the coracoid fungus, such as the research of the pathogenic molecular mechanism of the fungus to economic forest and herbaceous plants, the growth and development of the fungus, and the like.

Drawings

FIG. 1 is a colony morphology of transformants positive for the strain MG-1-7 of Corynoruslongus.

FIG. 2 is an agarose electrophoresis picture of a partial transformant of the strain MG-1-7 of Corynorum longirostratum; in the figure, M: DNA marker, 1: amplification result of MG-1-7 wild-type strain, 2-3: positive control for hygromycin resistance gene, 4-15: amplification result of partial transformant of MG-1-7 strain, N: negative control (ddH)₂O)。

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The materials and devices used in the present invention are commercially available unless otherwise specified.

The invention is further described below with reference to the drawings and examples of the specification, but the invention is not limited thereto.