阅读说明：本技术 获取含油菜黑胫病菌突变erg11基因的重组质粒的引物组合及方法 (Primer combination and method for obtaining recombinant plasmid containing phytophthora parasitica mutant ERG11 gene ) 是由 杨永青 李子钦 宋培玲 郝丽芬 燕孟娇 皇甫海燕 张福金 张欣昕 皇甫九茹 赵丽丽 于 2020-07-31 设计创作，主要内容包括：本发明提供了获取含油菜黑胫病菌突变ERG11基因的重组质粒的引物组合及方法,属于基因工程技术领域,所述引物组合包括第一引物对、第二引物对和第三引物对；所述第一引物对的上游引物的核苷酸序列如SEQ ID No.1所示,下游引物的核苷酸序列如SEQ IDNo.2所示；所述第二引物对的上游引物的核苷酸序列如SEQ ID No.3所示,下游引物的核苷酸序列如SEQ ID No.4所示；所述第三引物对的上游引物的核苷酸序列如SEQ ID No.5所示,下游引物的核苷酸序列如SEQ ID No.6所示。采用本发明提供的引物组合能够获得含油菜黑胫病菌突变ERG11基因的重组质粒。(The invention provides a primer combination and a method for obtaining recombinant plasmids containing phytophthora parasitica mutant ERG11 gene, belonging to the technical field of genetic engineering, wherein the primer combination comprises a first primer pair, a second primer pair and a third primer pair; the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4; the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 6. The primer combination provided by the invention can be used for obtaining the recombinant plasmid containing the phytophthora parasitica mutant ERG11 gene.)

1. Obtaining a primer combination of a recombinant plasmid containing a phytophthora parasitica mutant ERG11 gene, wherein the primer combination comprises a first primer pair, a second primer pair and a third primer pair;

the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2;

the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4;

the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 6.

2. A method for obtaining a recombinant plasmid containing a phytophthora parasitica mutant ERG11 gene is characterized by comprising the following steps:

1) extracting genome DNA of the phytophthora parasitica, and respectively amplifying by using the genome DNA as a template and using a first primer pair, a second primer pair and a third primer pair in the primer combination of claim 1 to obtain gene fragments of 1183bp, 242bp and 263 bp;

2) cloning the 1183bp, 242bp and 263bp gene fragments obtained in the step 1) into a pLAU2 vector through a Gibson reaction to obtain a recombinant plasmid.

3. The method according to claim 1, wherein the step 1) amplification procedure comprises: 3min at 95 ℃; 30s at 95 ℃, 30s at 58 ℃, 60s at 72 ℃ and 35 cycles; 5min at 72 ℃; storing at 4 ℃.

4. The method of obtaining as claimed in claim 1 or 3 wherein the 25 μ L amplification system is: 1 mu L of genome DNA,

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a primer combination and a method for obtaining a recombinant plasmid containing a phytophthora parasitica mutant ERG11 gene.

Background

The base mutation of the target gene ERG11 is an important way for plant pathogenic bacteria to generate drug resistance to DMI bactericide, and in the research of the target gene mutation, except for screening mutant strains naturally existing in the field, ultraviolet induced mutation is a more common method, random base mutation is generated by ultraviolet induction, and an indoor induced drug resistance mutant strain is finally obtained by combining domestication of indoor bactericide concentration gradient, and the research is reported in plant pathogenic bacteria such as fusarium graminearum, ustilago oryzae, sclerotinia sclerotiorum, rhizoctonia cerealis and the like. Although ultraviolet light can induce pathogenic bacteria to generate new genetic mutation, the genetic mutation frequency is relatively low, the period for screening drug-resistant mutant strains is relatively long, and the environment fitness of most mutant strains is possibly reduced.

Disclosure of Invention

In view of this, the present invention aims to provide a primer combination and a method for obtaining a recombinant plasmid containing a blackleg bacterium mutant ERG11 gene, and the primer combination provided by the present invention can obtain a recombinant plasmid containing a blackleg bacterium mutant ERG11 gene of an alkali mutation point at a fixed point.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a primer combination for obtaining a recombinant plasmid containing a phytophthora parasitica mutant ERG11 gene, which comprises a first primer pair, a second primer pair and a third primer pair;

the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2;

the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4;

the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 6.

The invention also provides a method for obtaining the recombinant plasmid containing the phytophthora parasitica mutant ERG11 gene, which comprises the following steps:

1) extracting genome DNA of the rape phytophthora parasitica, and respectively amplifying a first primer pair, a second primer pair and a third primer pair in the primer combination according to the technical scheme by using the genome DNA as a template to obtain gene fragments of 1183bp, 242bp and 263 bp;

2) cloning the 1183bp, 242bp and 263bp gene fragments obtained in the step 1) into a pLAU2 vector through a Gibson reaction to obtain a recombinant plasmid.

Preferably, the system amplified in step 1) is: 3min at 95 ℃; 30s at 95 ℃, 30s at 58 ℃, 60s at 72 ℃ and 35 cycles; 5min at 72 ℃; storing at 4 ℃.

Preferably, the amplification procedure is every 25. mu.lL comprises: 1. mu.L of genome DNA,

High-Fidelity 2 × Master Mix 12.5. mu.L, 10. mu.M upstream and downstream primers 1. mu.L each and ddH₂O9.5μL。

The invention provides a primer combination for obtaining a recombinant plasmid containing a phytophthora parasitica mutant ERG11 gene, which comprises a first primer pair, a second primer pair and a third primer pair; the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4; the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 6. The primer combination provided by the invention can be used for obtaining the recombinant plasmid containing the phytophthora parasitica mutant ERG11 gene.

Drawings

FIG. 1 shows the construction scheme of mutant ERG11 gene and the primer positions (UMY-1, PM001, PM002, PM003, PM004, and UMY-2 are primers;

FIG. 2 shows that three segments of target gene fragments are amplified by three pairs of primers, and the lengths are 1183bp (1 and 2 in the figure), 242bp (3 and 4 in the figure) and 263bp (5 and 6 in the figure);

FIG. 3 is a schematic diagram of a recombinant plasmid structure;

FIG. 4 is an electrophoresis diagram showing the restriction enzyme Pst I digestion verification result for the recombinant plasmid, 1-10 are randomly selected recombinant plasmids, all obtain expected digestion fragments, the lengths are 8679bp, 2941bp and 429bp, and ck is an empty vector pLAU2 before recombination as a control;

FIG. 5 shows the sequencing result of recombinant plasmid (PM5 for example), wherein ATC in the sequence of the recombinant plasmid shown in FIG. A is mutated to GTC, TACGGA in the sequence of the recombinant plasmid shown in FIG. B is deleted, and TGG is mutated to GGG;

Detailed Description

The invention provides a primer combination for obtaining a recombinant plasmid containing a phytophthora parasitica mutant ERG11 gene, which comprises a first primer pair, a second primer pair and a third primer pair; the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4; the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 6.

In the invention, the rape phytophthora parasitica is preferably Leptosphaeria maculans.

In the present invention, the nucleotide sequence of the upstream primer (hereinafter referred to as UMY-1-F) of the first primer pair is shown as SEQ ID No.1, and specifically as follows:

GAAACCTAATCAATCAACATGGCTGTTCTTGCTACC；

the nucleotide sequence of the downstream primer (PM 002-R for short) is shown as SEQ ID No.2, and the nucleotide sequence is as follows:

GCATGATCGAGTGGACCGGGGCGTGG。

in the present invention, the nucleotide sequence of the upstream primer (hereinafter referred to as PM001-F) of the second primer pair is shown as seq id No.3, and specifically as follows:

CCACGCCCCGGTCCACTCGATCATGC；

the nucleotide sequence of the downstream primer (PM 004-R for short) is shown as SEQ ID No.4, and the nucleotide sequence is as follows:

CAACGCCCCCATCAATCTTCTCGTCGTCC。

in the present invention, the nucleotide sequence of the upstream primer (hereinafter referred to as PM003-F) of the third primer pair is shown as seq id No.5, and specifically as follows:

AGAAGATTGATGGGGGCGTTGTGTCCAAGG；

the nucleotide sequence of the downstream primer (hereinafter referred to as UMY-2-R) is shown as SEQ ID No.6, and specifically comprises the following steps:

gctcatagtcacatccctcactcgaccttctccctcctc。

in the invention, the design concept of the primers of the primer combination is as follows: according to the position of base mutation points to be introduced in ERG11 gene, the primers for introducing point mutation are designed in the range of 10-20bp extending from the left and right ends, the tail end of the primer is G or C, and the GC content is more than 40%. Primers PM001 and PM002 introduced ATC to GTC base mutation resulting in isoleucine (I) to valine (V); primers PM003 and PM004 introduce tyrosine (Y), glycine (G) deletion (TACGGA deletion) and tryptophan (W) mutation to glycine (G) (TGG mutation to GGG); wherein the primers PM001-F and PM002-R are base complementary sequences, and the lengths are both 26 bp; the PM003-F and the PM004-R comprise base complementary sequences with the length of 21bp, and the base complementary sequences have the function of amplifying homologous fragments while amplifying the base of the mutation point for the next step of target fragment recombination; primers UMY-1-F and UMY-2-R are located at the 5 'end and the 3' end of the target gene respectively, and comprise homologous fragments which can be amplified and are arranged at two sides of the cohesive end of the vector, the amplified fragments are connected with the vector, and the bold sequences can be modified correspondingly according to the actual expression vector.

The invention also provides a method for obtaining the recombinant plasmid containing the phytophthora parasitica mutant ERG11 gene, which comprises the following steps: 1) extracting genome DNA of the phytophthora parasitica, and respectively amplifying by using the genome DNA as a template and using a first primer pair, a second primer pair and a third primer pair in the primer combination of claim 1 to obtain gene fragments of 1183bp, 242bp and 263 bp; 2) cloning the 1183bp, 242bp and 263bp gene fragments obtained in the step 1) into a pLAU2 vector through a Gibson reaction to obtain a recombinant plasmid.

The method for extracting the genome DNA of the phytophthora parasitica is not particularly limited, and a conventional extraction method, such as a CTAB method, is adopted.

In the present invention, the procedure of amplification preferably comprises: 3min at 95 ℃; 30s at 95 ℃, 30s at 58 ℃, 60s at 72 ℃ and 35 cycles; 5min at 72 ℃; storing at 4 ℃. In the present invention, the amplification system is preferably 25. mu.L: 1. mu.L of genome DNA,High-Fidelity 2 × Master Mix 12.5. mu.L, 10. mu.M upstream and downstream primers 1. mu.L each and ddH₂O 9.5μL。

In the invention, the pLAU2 vector belongs to a gene expression vector used for agrobacterium-mediated genetic transformation in subsequent experiments, and ERG11 gene containing mutation sites is inserted into the genome of rape black shank strain under the action of agrobacterium T-DNA to obtain black shank strain transformation strain containing mutation ERG11 gene. In the present invention, the vector pLAU2 was constructed as follows: idnurm A, Urquhara.S., Vummadi D.R., etc. and CRISPR/Cas9-induced mutation of the osmosensor kinase of the methanopathogenic bacteria [ J ] Fungal Biol Biotechnol,2017,4:1-12.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.