阅读说明：本技术 烟草丝裂原活化蛋白激酶基因NtMAPK8及其应用 (Tobacco mitogen-activated protein kinase gene NtMAPK8 and application thereof ) 是由 杨文武 曾婉俐 高茜 向海英 宋春满 张建铎 邓乐乐 蒋佳芮 许力 贾凌 杨光宇 于 2020-06-11 设计创作，主要内容包括：本发明公开了一种烟草丝裂原活化蛋白激酶基因<I>NtMAPK8</I>及其应用,所述烟草丝裂原活化蛋白激酶<I>NtMAPK8</I>基因核苷酸序列如SEQ ID NO:1或SEQ ID NO:3所示,编码的氨基酸序列如SEQ ID NO:2或SEQ ID NO:4所示；本发明首次克隆烟草丝裂原活化蛋白激酶基因<I>NtMAPK8</I>,并构建得到了克隆载体及通过基因编辑载体构建NtMAPK8基因缺失的转基因突变株,从而使烟草叶片颜色由正常的绿色变成花白；因此可以利用基因编辑载体使NtMAPK8基因不表达,进行植物品种育种,获得植物花叶的观赏植物品种。(The invention discloses a tobacco mitogen activated protein kinase gene NtMAPK8 And application thereof, namely the tobacco mitogen-activated protein kinase NtMAPK8 The gene nucleotide sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 3, and the coded amino acid sequence is shown as SEQ ID NO. 2 or SEQ ID NO. 4; the invention clones the gene of the tobacco mitogen activated protein kinase for the first time NtMAPK8 Constructing a cloning vector and constructing a transgenic mutant strain with NtMAPK8 gene deletion by using a gene editing vector, so that the color of the tobacco leaves is changed from normal green to white; therefore, a gene editing vector can be usedThe NtMAPK8 gene is not expressed, and plant variety breeding is carried out to obtain ornamental plant varieties of plant mosaic.)

1. Tobacco mitogen activated protein kinase geneNtMAPK8The method is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

2. The tobacco mitogen-activated protein kinase gene according to claim 1NtMAPK8The method is characterized in that: the amino acid sequence of the gene code is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

3. A knock-out vector, comprising: the gene is obtained by designing a target site knockout sequence for the tobacco NtMAPK8 gene by using a CRISPR/Cas9 gene editing technology and then carrying out gene editing.

4. The knock-out vector according to claim 3, wherein: the nucleotide sequence of the knockout sequence of the target site is shown as SEQ ID NO. 7, after artificially synthesized linker sequences are added on two sides of the target site, primer pairs with the nucleotide sequences shown as SEQ ID NO. 8 and SEQ ID NO. 9 are obtained, and after the primer pairs are annealed, the primer pairs are connected to a Bsa I enzyme-digested vector pORE-Cas9, and the gene knockout vector pORE-Cas9/NtMAPK8 is obtained.

5. The tobacco mitogen-activated protein kinase gene of claim 1NtMAPK8Or the use of the gene knockout vector of claim 3 or 4 in the preparation of different color leaf plant varieties.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a tobacco mitogen-activated protein kinase geneNtMAPK8And applications thereof.

Background

Among plants, tobacco is an important commercial crop. The color of the tobacco leaves directly influences the color of the cut tobacco after the tobacco is baked, and the color is an important index for measuring the quality of the tobacco leaves.

The MAPKKK-MAPKK-MAPK cascade pathway is well conserved in plants, and plays an important role not only in the defense system of plants, but also in the growth and development of plants. However, no report about the color of leaves of MAPK gene is available.

Disclosure of Invention

Aiming at the existingThe technical deficiency of the invention is that the invention provides a tobacco mitogen activated protein kinase geneNtMAPK8It is shown as SEQ ID NO. 1 or SEQ ID NO. 2, and the amino acid sequence coded by the gene is shown as SEQ ID NO. 3 or SEQ ID NO. 4.

Another object of the present invention is to provide a tobacco mitogen-activated protein kinase gene containing the above-mentionedNtMAPK8The recombinant vector of (1).

The recombinant vector comprises a cloning vector and an expression vector; the preparation method of the cloning vector comprises the steps of carrying out PCR amplification by using cDNA of tobacco as a template and primers shown as SEQ ID NO. 5 and SEQ ID NO. 6; the obtained PCR product is connected to a T vector, and Escherichia coli is transformed to obtain a cloning vector.

The invention also aims to provide a gene knockout vector, which is obtained by gene editing after designing a target site for a tobacco NtMAPK8 gene by using a CRISPR/Cas9 gene editing technology, wherein the nucleotide sequence of a knockout sequence of the target site is shown as SEQID NO. 7; the nucleotide sequences of the primer pair obtained by adding artificially synthesized linker sequences on both sides of the target site are shown as SEQ ID NO. 8 and SEQ ID NO. 9; and (3) annealing the primer pair, and connecting the primer pair to a BsaI digested vector pORE-Cas9 to obtain a gene knockout vector pORE-Cas9/NtMAPK 8.

Another object of the present invention is to provide a transgenic plant prepared by a method comprising: transforming agrobacterium-infected cells by using the gene knockout vector pORE-Cas9/NtMAPK8, and then transforming infected plants to obtain the gene knockout vector.

The transgenic plant is transgenic tobacco; when the transgenic tobacco is transformed and infected, the vector pORE-Cas9/NtMAPK8 is adopted to transform the agrobacterium-induced competence, and the agrobacterium-mediated tobacco leaf disc transformation method is utilized to carry out genetic transformation on the tobacco to obtain tobacco plants with different colors. Specifically, the color of the leaf changes from green to light green.

Another object of the present invention is to provide the above-mentioned gene of a tobacco mitogen-activated protein kinaseNtMAPK8The method is applied to the preparation of plant varieties with different leaf colors.

Compared with the prior art, the invention has the following advantages and effects:

the invention clones the gene of the tobacco mitogen activated protein kinase for the first timeNtMAPK8And constructing a cloning vector and a knockout vector; the NtMAPK8 protein participates in MAPK signal transmission and can be constructed by a gene editing vectorNtMAPK8The transgenic mutant strain with the deleted gene can change the color of the tobacco leaves from normal green to white, and the specific reason is still unclear.

Obtained by the inventionNtMAPK8The leaf color of the knockout mutant strain is partially changed to white, and thus can be made to white by using a gene editing vectorNtMAPK8The gene is not expressed, and the plant variety breeding is carried out to obtain the ornamental plant variety of the plant mosaic.

Drawings

FIG. 1 shows the experimental results of the present inventionNtMAPK8Expression level of genes in different tissues, panel a being the result of transcript MAPK8 a; panel B is the result of transcript MAPK 8B;

FIG. 2 is a schematic diagram of the target site design of NtMAPK8 gene knockout in the experiment of the present invention;

FIG. 3 is a phenotype diagram of T0 generation transgenic line NtMAPK8 mutant in the experiment of the present invention.

Detailed Description

The present invention is further illustrated in detail below with reference to the drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples are, unless otherwise specified, conventional reagents and conventional methods.

The conditions of the biological materials, the experimental reagents, the consumables and the like related to the embodiment are as follows:

tobacco variety: the seeds of the Honghuadajinyuan are provided by national key laboratories of silkworm genome biology at southwest university;

carrier: the CRISPR/Cas9 basic knockout vector is provided by national emphasis laboratories of silkworm genome biology at southwest university; pEASY-Blunt Zero Cloning Kit vectors purchased from Beijing all-purpose gold Biotechnology Co., Ltd;

the strain is as follows: trans1-T1 chemically competent cells and LBA4404 Agrobacterium chemically competent cells, all purchased from Beijing Quanji Biotech, Inc.;

primers and sequencing: the primer synthesis and the DNA sequencing are both completed by Beijing Hua large gene;

experimental reagent: the Plant DNA extraction Kit EasyPure Plant Genomic DNA Kit (containing RNase A) and the DNA Purification Kit EasyPure PCR Purification Kit PCR are purchased from Beijing all-purpose gold company; trizol reagent (Invitrogen, USA) for RNA extraction; the reverse transcription kit comprises TransScript One-Step gDNAremoval and cDNA Synthesis SuperMix purchased from Beijing all-purpose gold company; t4 ligase, restriction enzyme BsaI, DNA amplificate, available from NEB; PrimeSTAR Max DNA polymerase was purchased from Takara.

Experimental equipment: PCR amplification apparatus Veriti (Gene Co., Ltd.); gel imaging system ultraviolet gel imager (BIO-RAD); quantitative PCR instrument, ABI7500 fast real-time PCR system (Saimeri fly).