阅读说明：本技术 一种笔筒树核外dna快速提取和分析鉴别的方法 (Method for quickly extracting, analyzing and identifying DNA outside pen container tree nucleus ) 是由 张君毅 周扬 蔡邦平 刘嘉 于 2019-12-06 设计创作，主要内容包括：本发明公开了一种笔筒树核外DNA快速提取和分析鉴别的方法,通过裂解液提取破除细胞壁后的笔筒树植物样品中的总DNA,再通过0.45μm的微孔径滤膜过滤实现去除细胞核基因组,得到包括mtDNA和cpDNA在内的核外DNA；设计笔筒树mtDNA中atp1基因的特异性引物,选择植物cpDNA-trnH基因序列引物,利用两对引物分别对核外DNA进行PCR扩增并对扩增产物进行测序分析。(The invention discloses a method for quickly extracting, analyzing and identifying DNA outside a pen container tree nucleus, which comprises the steps of extracting total DNA in a pen container tree plant sample with broken cell walls by lysate, and removing a nuclear genome by filtering through a micro-aperture filter membrane of 0.45 mu m to obtain the DNA outside the nucleus including mtDNA and cpDNA; designing specific primers of atp1 gene in mtDNA of the pen container tree, selecting plant cpDNA-trnH gene sequence primers, respectively carrying out PCR amplification on extranuclear DNA by using the two pairs of primers, and carrying out sequencing analysis on the amplified product.)

1. A method for quickly extracting, analyzing and identifying DNA outside a pen container tree nucleus is characterized by comprising the following steps:

1) extraction of extranuclear DNA

Extracting total DNA in the pen container tree plant sample with the broken cell wall by lysate, and filtering by a micro-aperture filter membrane of 0.45 mu m to remove the nuclear genome to obtain extra-nuclear DNA including mtDNA and cpDNA;

2) analytical identification of extranuclear DNA

Designing a specific primer of atp1 gene in mtDNA of the pen container tree, wherein the sequence of an upstream primer is SEQ ID NO: 01, the sequence of the downstream primer is SEQ ID NO: 02;

selecting plant trnH-psbA gene sequence primers, wherein the sequence of an upstream primer of the primers is SEQ ID NO: 03, the sequence of the downstream primer is SEQ ID NO: 04;

and respectively carrying out PCR amplification on the extranuclear DNA by using the two pairs of primers and carrying out sequencing analysis on the amplified products.

2. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 1, characterized in that: 1) extraction of extranuclear DNA

a) Grinding the plant sample of the penholder tree into sample powder in liquid nitrogen, and breaking cell walls; transferring sample powder to a centrifuge tube, adding a lysis solution preheated at 65 ℃, wherein the dosage ratio of the sample powder to the lysis solution is 1 g: 2 mL; water bath is carried out for 30 minutes at 65 ℃, the centrifuge tube is shaken up and down to be uniform every 5 minutes, and filtrate containing the pen container tree total DNA is obtained by filtration;

b) filtering the filtrate containing the pen container tree total DNA to a centrifuge tube by using a syringe filter through a micro-aperture filter membrane of 0.45 mu m, and freezing the filtrate in liquid nitrogen for 1 hour; then, after the filtrate is unfrozen at room temperature, inverting the centrifugal tube for 2-3 times, and centrifuging for 2 minutes at 8000g and 4 ℃; transferring the supernatant to a new centrifuge tube, adding two times of volume of-20 deg.C pre-cooled anhydrous ethanol, and centrifuging at 16800g and 4 deg.C for 5 min; discarding the supernatant, adding a washing solution, and resuspending; the suspension was centrifuged at 16800g for one minute, the supernatant was discarded and the precipitate was dissolved by adding TE buffer, and the pellet was frozen at-20 ℃ to obtain extranuclear DNA including mtDNA and cpDNA.

3. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 1 or 2, which is characterized in that: the pencil vase tree plant sample is fresh pencil vase tree leaves.

4. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 2, characterized in that: in the step 1), two layers of gauze are used for filtering during filtering, and the gauze is washed twice by using a lysate.

5. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 1 or 2, which is characterized in that: the pH of the lysis solution is 7.6, and the formulation is 350mM of mannitol, 30mM of 3-morpholine propanesulfonic acid, 1mM of EDTA ethylene diamine tetraacetic acid, 50 mu M of PVPP cross-linked polyvinylpyrrolidone and 11.2 mu M of cysteine.

6. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 2, characterized in that: in step 1) b), the pH of the washing solution was 7.2, and the formulation was 300mM mannitol, 20mM MOPS and 1mM EDTA.

7. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 2, characterized in that: the TE buffer had a pH of 8.0 and was formulated with 10mM Tris and 1mM EDTA.

8. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 1, characterized in that: in the step 2), the PCR amplification procedure is pre-denaturation at 90 ℃ for 4 minutes, then 35 cycles are carried out, and the cycle procedure is denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 1 minute for 35 seconds, extension at 72 ℃ for one minute, and finally extension at 72 ℃ for 10 minutes.

9. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 1, characterized in that: in the step 2), the amplification product of the PCR is electrophoresed on a 1% agarose gel.

10. The method for rapidly extracting, analyzing and identifying DNA outside a pen container tree nucleus as claimed in claim 1, characterized in that: the A260/A280 ratio of the extranuclear DNA is 1.032-1.042, and the yield is 172 ng/g-255 ng/g.

Technical Field

The invention relates to a method for quickly extracting, analyzing and identifying DNA outside a pen container tree nucleus.

Background

The existing methods for extracting mitochondrial DNA are classified into density gradient centrifugation, enzymatic digestion, column chromatography, cesium chloride ultracentrifugation, alkaline denaturation, DNase, Triton, and modified high-salt precipitation. The cesium chloride reagent is expensive, the requirements of an alkaline denaturation method and an enzyme digestion method on test conditions are strict, the yield of the Triton method is low, the degradation is easy, and the density gradient centrifugation method is complicated to operate. The improved high-salt precipitation method has the advantages of simplicity, convenience, economy, easy repetition and the like, is considered as a better extraction method by a plurality of literatures, and still requires higher operation environment.

The existing methods for extracting the DNA of the chloroplast of higher plants mainly comprise a DNase I method, a sucrose density gradient centrifugation method, a Percoll gradient method, an anhydrous method, a high-salt low-pH method and the like, and although the methods are applied in many plant researches, the problems of high requirements on instruments and equipment, complex steps, long time consumption and the like still exist.

The extranuclear DNA of the pen container tree mainly refers to mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA), and the rapid extraction technology of the extranuclear DNA is important for the construction of a gene bank of the pen container tree and the completion of full sequencing.

In-vitro DNA identification of plants often utilizes chloroplast or ribosomal gene sequences, such as matK, rbcL, ITS, and the like. Plant mitochondria have large variability and the design of related specific primers is laborious.

The rapid extraction and analysis of DNA outside the pen container tree nucleus is beneficial to the deep research of the scientific problems of plant system evolution, cytoplasm gene function and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a method for quickly extracting, analyzing and identifying DNA outside a pen container tree nucleus, and solves the problems of complicated extraction steps and long time consumption in the background technology.

The technical scheme adopted by the invention for solving the technical problems is as follows: the method for quickly extracting, analyzing and identifying the DNA outside the core of the pen container tree comprises the following steps:

1) extraction of extranuclear DNA

Extracting total DNA in the pen container tree plant sample with the broken cell wall by lysate, and filtering by a micro-aperture filter membrane of 0.45 mu m to remove the nuclear genome to obtain extra-nuclear DNA including mtDNA and cpDNA;

2) analytical identification of extranuclear DNA

Designing a specific primer of atp1 gene in mtDNA of the pen container tree, wherein

atp1(F)：TGTAGGAAAGGCCATGCCAG(SEQ ID NO：01)；

atp1(R)：TGCCCTATGCGTTGATTGGT(SEQ ID NO：02)；

Selecting a plant trnH-psbA gene sequence primer, wherein

psbA(F)：GTTATGCATGAACGTAATGCTC(SEQ ID NO：03)；

trnH(R)：CGCGCATGGTGGATTCACAATCC(SEQ ID NO：04)；

And respectively carrying out PCR amplification on the extranuclear DNA by using the two pairs of primers and carrying out sequencing analysis on the amplified products.

In a preferred embodiment of the present invention, 1) the extraction of extranuclear DNA comprises the steps of:

a) grinding the plant sample of the penholder tree into sample powder in liquid nitrogen, and breaking cell walls; transferring sample powder to a centrifuge tube, adding a lysis solution preheated at 65 ℃, wherein the dosage ratio of the sample powder to the lysis solution is 1 g: 2 mL; water bath is carried out for 30 minutes at 65 ℃, the centrifuge tube is shaken up and down to be uniform every 5 minutes, and filtrate containing the pen container tree total DNA is obtained by filtration;

b) filtering the filtrate containing the pen container tree total DNA to a centrifuge tube by using a syringe filter through a micro-aperture filter membrane of 0.45 mu m, and freezing the filtrate in liquid nitrogen for 1 hour; then, after the filtrate is unfrozen at room temperature, the centrifugal tube is inverted from top to bottom for 2-3 times, and the centrifugal tube is centrifuged for 2 minutes at 8000g and 4 ℃; transferring the supernatant to a new centrifuge tube, adding two times of volume of-20 deg.C pre-cooled anhydrous ethanol, and centrifuging at 16800g and 4 deg.C for 5 min; discarding the supernatant, adding a washing solution, and resuspending; the suspension was centrifuged at 16800g for one minute, the supernatant was discarded and the precipitate was dissolved by adding TE buffer, and the pellet was frozen at-20 ℃ to obtain extranuclear DNA including mtDNA and cpDNA.

In a preferred embodiment of the present invention, the pencil vase tree plant sample is fresh pencil vase tree leaves.

In a preferred embodiment of the present invention, in the step 1), two layers of gauze are used for filtration, and the gauze is washed twice with the lysate.

In a preferred embodiment of the present invention, the pH of the lysis solution is 7.6, and the formulation is 350mM of mannitol, 30mM of 3-morpholine propanesulfonic acid, 1mM of EDTA-EDTA, 50. mu.M of PVPP cross-linked polyvinylpyrrolidone and 11.2. mu.M of cysteine.

In a preferred embodiment of the present invention, in step 1) b), the pH of the washing solution is 7.2, and the formulation is 300mM mannitol, 20mM MOPS and 1mM EDTA.

In a preferred embodiment of the invention, the TE buffer has a pH of 8.0 and is formulated with 10mM Tris and 1mM EDTA.

In a preferred embodiment of the present invention, in the step 2), the PCR amplification procedure is pre-denaturation at 90 ℃ for 4 minutes, followed by 35 cycles, wherein the cycle procedure comprises denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 1 minute for 35 seconds, extension at 72 ℃ for one minute, and final extension at 72 ℃ for 10 minutes.

In a preferred embodiment of the present invention, in the step 2), the amplification product of the PCR is electrophoresed on a 1% agarose gel.

In a preferred embodiment of the present invention, the A260/A280 ratio of the extranuclear DNA is 1.032-1.042, and the yield is 172 ng/g-255 ng/g.

Compared with the background technology, the technical scheme has the following advantages:

1. the invention designs a specific primer aiming at the mitochondrial ATP1 gene of the pen container tree, the gene codes ATP synthase α subunit protein ATP1, the extracted extranuclear DNA sample can be respectively subjected to the PCR amplification of mtDNA and cpDNA, and the sequencing efficiency is high;

2. compared with the existing method for extracting the DNA outside the plant nucleus, the method firstly extracts the total DNA of the pen container tree material, and then uses a syringe filter to enable the extracting solution to pass through a micro-pore diameter membrane of 0.45 mu m so as to achieve the purposes of removing the nuclear genome and reserving the DNA outside the nucleus, thereby simplifying the extraction steps and shortening the operation time;

3. compared with the existing method for extracting the DNA outside the plant nucleus, the extraction scheme of the invention does not use toxic reagents with strong irritation, such as β -mercaptoethanol and the like, and is safe and environment-friendly.

Drawings

FIG. 1 is an electrophoresis photograph of a mitochondrial atp1 target gene fragment;

FIG. 2 is a photograph of an electrophoresis of a trnH-psbA target gene fragment;

FIG. 3 is a diagram showing the sequencing peaks of the PCR amplification products.

Detailed Description