CN116445503A - Cryptomeria fortunei CfNAC1 gene and expression protein and application thereof - Google Patents

Abstract

The invention discloses a cedar CfNAC1 gene and an expression protein and application thereof, belonging to the field of plant molecular biology. The nucleotide sequence of the Cryptomeria fortunei CfNAC1 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the invention takes the cryptoforming layer tissue of the cedar as a material, obtains the cedar CfNAC1 gene by cloning, and constructs an overexpression vector binary plant recombinant expression vector 35S on the basis: : the CfNAC1 is transferred into the lower epidermis of Nicotiana benthamiana to obtain transgenic plants, and the gene function identification result shows that the Cryptomeria fortunei CfNAC1 gene is an important transcription factor for promoting secondary xylem development and secondary wall thickening and improving lignin content, and has good application prospects in plant growth and development and breeding.

Description

Cryptomeria fortunei CfNAC1 gene and expression protein and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a Cryptomeria fortunei CfNAC1 gene, an expression protein and application thereof.

Background

The cedar (Cryptomeria fortunei) is a special tree species in China, also called China cedar, and is produced in the areas with the altitude below 1100 m such as Zhejiang Tianmu mountain, fujian nan-screen Sanqian eight hundred candles, jiangxi Lushan and the like. The plant is cultivated in Jiangsu south, zhejiang, anhui south, henan, hubei, hunan, sichuan, guizhou, yunnan, guangxi and Guangdong places, and has good growth. The young cedar can resist shadow slightly, and grow faster in warm and humid climates and mountain areas with acid soil, hypertrophic soil and good drainage; the side material is yellow and white, the core material is light and brown, the material is light and soft, the texture is straight, the structure is thin, the corrosion resistance is strong, and the side material is easy to process, thus being a good industrial building material. At present, the research on the Cryptomeria fortunei NAC transcription factor is less, and a reliable theoretical basis is lacked.

The common feature of NAC transcription factors in plants is that they contain a highly conserved and specific NAC domain, consisting of about 150 to 160 amino acids, at the N-terminus of the protein, which can bind DNA and other proteins, while the C-terminus has a highly mutated transcriptional regulatory region, which can activate or repress gene transcription. The entire N-terminal region of NAC transcription factor can be divided into 5 subdomains (A, B, C, D and E). Wherein subdomain a may be involved in the formation of a functional dimer; subdomains C and D are positively charged, highly conserved, are DNA binding sites, contain nuclear localization signals (nuclear localization signal, NLS), and a few NAC proteins have a Negative Regulatory Domain (NRD) in subdomain D, which can inhibit transcriptional activity; subdomains B and E may be involved in the functional diversity of the NAC gene. The C-terminal is a transcription regulatory region, which is a region with simpler sequence, often contains repeated sequences of serine-threonine, proline-glutamine or some acidic amino acids, which leads to inherent disorder of the C-terminal, so that the C-terminal lacks a stable three-dimensional structure. Although the C-terminal region of NAC transcription factors varies widely, NAC protein TRR of some different species have specific motifs that are conserved in certain subgroups of specific NAC subfamilies. The diversity of structures allows the NAC transcription factor to have a wide variety of functions, allowing the NAC transcription factor to exert important regulatory effects in nearly every growth and development stage of a plant and under various stress conditions, including biotic and abiotic stress responses (exogenous hormonal responses, pathogenic bacterial attack and external adverse environmental interference), anthocyanin synthesis, embryogenesis, auxin signal transduction, lignin synthesis, secondary xylem development, secondary wall formation, and the like.

Disclosure of Invention

Aiming at the problems existing in the prior art, the technical problem to be solved by the invention is to provide a Cryptomeria fortunei CfNAC1 gene. The invention aims to provide an expression protein of a cedar CfNAC1 gene and application thereof. The invention also solves the other technical problem of providing an application of the Cryptomeria fortunei CfNAC1 gene.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a Cryptomeria fortunei CfNAC1 gene has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expressed protein of the Cryptomeria fortunei CfNAC1 gene is shown as SEQ ID NO. 2.

A vector comprising the cedar CfNAC1 gene of claim 1.

The vector is a plant expression vector.

The plant expression vector is a binary plant recombinant expression vector 35S: : cfNAC1.

Use of the cedar CfNAC1 gene for increasing lignin content in tobacco plants and/or for promoting secondary xylem development in tobacco plants and/or for promoting secondary wall thickening in tobacco plants.

The application of the cedar CfNAC1 gene in improving lignin content in tobacco plants and/or promoting secondary xylem development in tobacco plants and/or promoting secondary wall thickening in tobacco plants comprises the following steps:

(1) Constructing a vector of a cedar CfNAC1 gene;

(2) Transforming the constructed vector of the cedar CfNAC1 gene into the lower epidermis of Nicotiana benthamiana;

(3) Cultivation and screening to obtain tobacco plants with increased lignin content and/or secondary xylem development and/or secondary wall thickening

Compared with the prior art, the invention has the beneficial effects that:

the cedar CfNAC1 gene disclosed by the invention has the effects of promoting secondary xylem development and secondary wall thickening and improving lignin content, and compared with a wild type, the tobacco plant lignin content of the cedar CfNAC1 gene is obviously increased; histochemical staining results showed that: compared with the wild type, the secondary xylem area of the CfNAC 1-transformed plant is obviously thickened, and xylem cells are closely arranged; further, secondary wall thickness observations showed that: the secondary cell walls of CfNAC 1-transformed plants were significantly thickened compared to the wild type. Cryptomeria fortunei CfNAC1 has effects of promoting secondary xylem development and secondary wall thickening, and increasing lignin content.

Drawings

FIG. 1 is a diagram of pCambia1302 and PBI121 overexpression vectors;

FIG. 2 is a subcellular map of the Cryptomeria fortunei CfNAC1 gene;

FIG. 3 is a wild-type and Cryptomeria fortunei transcription factor 35S: : cfNAC1 transgenic plant lignin content map, WT represents wild-type lignin content, 35S: : cfNAC1 denotes turn 35S: : lignin content of CfNAC1 gene plants;

FIG. 4 is a wild-type and Cryptomeria fortunei transcription factor 35S: : a secondary xylem development map of a CfNAC1 transgenic plant; WT represents wild type, 35S: : cfNAC1 denotes turn 35S: : cfNAC1 gene plants;

FIG. 5 is a wild-type and Cryptomeria fortunei transcription factor 35S: : secondary wall thickness plot of CfNAC1 transgenic plants, left panel for wild type and right panel for transgenic 35S: : cfNAC1 plants.

Detailed Description

The invention is further described below in connection with specific embodiments. In the following examples, the procedures not described in detail are all routine biological experimental procedures, and can be performed with reference to molecular biology laboratory manuals, journal literature published in the prior art, and the like, or according to the kit and product instructions. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1: cloning of the Cryptomeria fortunei transcription factor CfNAC1 Gene

1. RNA extraction

The experimental material used in the invention is the tissue of the cambium of the cedar: the plant with good growth vigor is collected from a flower house of university of Nanjing forestry in 2022, and RNA is extracted by using a centrifugal column type RNA extraction kit of Baitaike biotechnology company, and then quality and concentration detection is carried out.

2. Synthesis and reverse transcription PCR of first strand of cDNA

3. Cryptomeria fortunei CfNAC1 Gene clone

The primers CfNAC1-F and CfNAC1-P, which are the upstream and downstream primers of the CfNAC1 gene, were designed as primers for the amplification reaction, and the sequences of the primers were as follows:

CfNAC1-F：5′-ATGACTCTGTCAGTTAACGG-3′，

CfNAC1-R：5′-TTACTTGCCAAAATTCCAC-3′。

after the reaction, the PCR product was separated by 1.5% agarose gel electrophoresis to give a length of about 1107bp. Sequencing after purification and recovery, wherein the sequencing result is shown as SEQ ID NO.1, the nucleotide sequence length is 1107bp, the coded protein sequence is shown as SEQ ID NO.2, and the protein consists of 375 amino acids.

Example 2: construction and function verification of Cryptomeria fortunei CfNAC1 gene expression vector

1. The subcellular vector used in the invention is pCambia1302, and the over-expression vector is PBI 121. The endonucleases used in the present invention are BamH I enzyme, ecoR I and Xba I enzyme (subcellular localization endonucleases are BamH I enzyme and EcoR I; over-expressed endonucleases are BamH I enzyme and Xba I enzyme). The primer sequences for designing the localization of homologous recombination subcellular are as follows:

CfNAC1-F：5′-tatgaccatgattacgaattcATGACTCTGTCAGTTAACGG-3′，

CfNAC1-R：5′-caggtcgactctagaggatccTTACTTGCCAAAATTCCAC-3′。

the sequence of the over-expressed primer is as follows:

CfNAC1-F：5′-gagaacacgggggactctagaATGACTCTGTCAGTTAACGG-3′，

CfNAC1-R：5′-ggactgaccacccggggatccTTACTTGCCAAAATTCCAC-3′。

the double cleavage reaction is as follows:

the recombination reaction is as follows:

after sequencing verification, recovering and purifying the target vector after the recombination reaction to obtain a binary plant recombination expression vector 35S of the target gene: : cfNAC1 (fig. 1).

2. Transformation of Agrobacterium GV3101

(1) Taking agrobacterium tumefaciens competence preserved at-80 ℃ to be melted to an ice water mixed state at room temperature or palm, and then inserting the melted agrobacterium tumefaciens competence into ice;

(2) Adding 0.01-1 μg plasmid DNA (high conversion efficiency, preferably pre-experiment to determine the amount of plasmid added before first use) into 100 μl competent cells, and stirring with a flick;

(3) Sequentially placing in ice bath for 5min, quick-freezing with liquid nitrogen for 5min, water bath at 37deg.C for 5min, and ice bath for 5min;

(4) Adding 700 μl of LB liquid medium (without any antibiotics), shaking at 28deg.C and 200rpm for 3 hr;

(5) Centrifuging at 6000rpm for 1min;

(6) Discarding part of the supernatant, taking about 100-120 mu L of bacterial liquid, gently blowing and mixing to re-suspend bacterial blocks, coating on LB (Kan+) solid plates containing corresponding antibiotics, standing for 15min, and airing the bacterial liquid on the surface;

(7) Placing the mixture in a 28 ℃ incubator to perform dark culture for 42-48 hours;

(8) Positive colonies were detected by PCR and stored at 4 ℃ for subsequent transgenic infestation of tobacco.

3. Agrobacterium activation and infestation

(1) Activating and propagating the agrobacterium tumefaciens bacterial liquid with correct positive detection, putting the agrobacterium tumefaciens bacterial liquid into a 100mL sterile conical flask, diluting the agrobacterium tumefaciens bacterial liquid into 50mL LB liquid culture medium (Kan+Rif) according to the ratio of 1:50, and culturing the agrobacterium tumefaciens bacterial liquid at 28 ℃ for 36-48h at 200rpm until the bacterial liquid is turbid in color, wherein the OD value A600 is about 1.0;

(2) Transferring the bacterial liquid into a sterile centrifuge tube, centrifuging at 5000rpm for 10min at room temperature, discarding supernatant, re-suspending the bacterial liquid with 1/2MS or MS liquid culture medium, and diluting to A600=0.6 for transformation;

(3) The diluted bacterial liquid is put into a constant temperature shaking table for 30min at 28 ℃ and 200rpm for infection.

4. Subcellular localization observations

(1) Sucking the agrobacterium heavy suspension by using a 1mL syringe without a needle, injecting the lower epidermis of the Nicotiana benthamiana, and slightly pushing a piston to slowly infiltrate the agrobacterium liquid into the whole tobacco leaves;

(2) Dark cultures 1d, light cultures 2d after infestation and then observations were made using a laser confocal microscope.

(3) The results are shown in FIG. 2, where it can be seen that there is a distinct green fluorescent signal in the nucleus and no signal in other regions, consistent with the description of NAC transcription factors functioning biologically in the nucleus.

5. Tobacco leaf disc mediated infection

(1) Pre-culturing: cutting wild type non-resistant tobacco leaves into leaf discs of about 1cm multiplied by 1cm in an ultra clean bench, and culturing in dark for 2-3d until leaf edges curl;

(2) Co-cultivation: placing the leaf disc into diluted bacterial liquid, soaking, vibrating and infecting for 10-15min, taking out the leaf disc, placing the leaf disc on sterile filter paper to suck away excessive bacterial liquid, placing the infected leaf disc on MS solid culture medium (a layer of sterile filter paper is laid on the culture medium) without any antibiotics, and carrying out dark culture at 28 ℃ for 2-3d;

(3) And (3) differentiation culture: transfer the dark co-cultured leaf discs to differentiation medium (MS medium, 2.0 mg/L6-BA, 0.05mg/L NAA,50mg/L Kan,100mg/L TMT) for differentiation culture;

(4) Screening and culturing: re-inoculating the callus differentiated for about 20-25 days into a screening culture medium (MS culture medium, 2.0 mg/L6-BA, 0.05mg/L NAA,50mg/L Kan,150mg/L TMT), and continuously differentiating adventitious buds;

(5) And (3) strong seedling cultivation: cutting off after about 2-3 weeks and transferring to strong seedling culture medium (MS culture medium, 1.0 mg/L6-BA, 0.1mg/L NAA,50mg/L Kan,200mg/L TMT) for strong seedling culture when adventitious bud length reaches about 1 cm;

(6) Rooting culture: when the length of the adventitious bud reaches 1-2cm, placing the adventitious bud into a rooting culture medium (1/2 MS culture medium, 200mg/L TMT) for about 10-15d to grow adventitious roots, hardening off the aseptic seedlings with good root system growth for 4-5d, and transferring the aseptic seedlings into soil for culture;

(7) Wild tobacco leaf disks not infected with agrobacterium were also set as negative controls.

6. Histochemical staining of transgenic plants

(1) Collecting tobacco stems of mature wild-type and transgenic plants near the basal region, and cutting into thin slices using a Leica microtome;

(2) A phloroglucinol formulation; preparing a 5% phloroglucinol solution dissolved in ethanol, and then mixing with concentrated hydrochloric acid and water (v/v/v=1:1:1);

(3) The mixed liquid is dripped on a slice, kept stand for 30s, and then placed on a microscope stage for observation and photographing.

7. Phenotype observation and index determination of transgenic plants

And detecting lignin content of the positive clone plants, performing secondary xylem histochemical staining and performing secondary wall environment scanning electron microscope observation. The results are shown in FIG. 3, where the lignin content of CfNAC 1-transformed plants was significantly increased compared to the wild type; the results are shown in fig. 4-5, which show that the secondary xylem region of CfNAC 1-transformed plants is significantly thickened and xylem cells are closely aligned compared to wild type; the secondary cell walls of CfNAC 1-transformed plants were significantly thickened compared to the wild type. To sum up: the results of histochemical staining observation, lignin content and secondary wall thickness measurement show that the cedar CfNAC1 has the effects of promoting secondary xylem development and secondary wall thickening and improving lignin content.

Claims (7)

1. A Cryptomeria fortunei CfNAC1 gene has a nucleotide sequence shown in SEQ ID NO. 1.

2. The expressed protein of the cedar CfNAC1 gene as claimed in claim 1, wherein the amino acid sequence is shown in SEQ ID No. 2.

3. A vector comprising the cedar CfNAC1 gene of claim 1.

4. A vector comprising the cryptomeria CfNAC1 gene of claim 3, wherein the vector is a plant expression vector.

5. The vector containing the cedar CfNAC1 gene of claim 4, wherein said plant expression vector is a binary plant recombinant expression vector 35S: : cfNAC1.

6. Use of the cedar CfNAC1 gene of claim 1 for increasing lignin content in tobacco plants and/or promoting secondary xylem development in tobacco plants and/or promoting secondary wall thickening in tobacco plants.

7. Use of the cedar CfNAC1 gene according to claim 6 for increasing lignin content in tobacco plants and/or promoting secondary xylem development in tobacco plants and/or promoting secondary wall thickening in tobacco plants, comprising the steps of:

(1) Constructing a vector of a cedar CfNAC1 gene;

(2) Transforming the constructed vector of the cedar CfNAC1 gene into the lower epidermis of Nicotiana benthamiana;

(3) And culturing and screening to obtain tobacco plants with increased lignin content and/or secondary xylem development and/or secondary wall thickening.