CN116970053B

CN116970053B - Plant carotenoid synthesis related protein DcAPRR2, and coding gene and application thereof

Info

Publication number: CN116970053B
Application number: CN202311226749.3A
Authority: CN
Inventors: 徐志胜; 张凯; 熊爱生; 王璐
Original assignee: Sanya Research Institute Of Nanjing Agricultural University; Nanjing Agricultural University
Current assignee: Sanya Research Institute Of Nanjing Agricultural University; Nanjing Agricultural University
Priority date: 2023-09-22
Filing date: 2023-09-22
Publication date: 2024-01-30
Anticipated expiration: 2043-09-22
Also published as: CN116970053A

Abstract

The invention relates to the technical field of biology, in particular to a plant carotenoid synthesis related protein DcAPRR2, and a coding gene and application thereof. The protein of the present invention is any one of the following: a1 Protein with the amino acid sequence shown as SEQ ID No. 2; a2 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of A1) and having more than 80% of identity with the protein shown in A1) and regulating and controlling the synthesis of plant carotenoid; a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2). Experiments prove that the expression is over-expressedDcAPRR2The gene can promote carotenoid synthesis and knock outDcAPRR2The gene can reduce carotenoid synthesis, which shows that DcAPRR2 can regulate and control carrot carotenoid synthesis, and has important theoretical significance for carrot breeding.

Description

Plant carotenoid synthesis related protein DcAPRR2, and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a plant carotenoid synthesis related protein DcAPRR2, and a coding gene and application thereof.

Background

Carotenoids are a class of important pigments that are widely found in nature, more than 800 of which have been found. In plants, abundant carotenoids accumulate in flowers, fruits and root organs of many species, but the diversity of their colors is caused by obvious differences in species and content. Meanwhile, carotenoids play a vital role in the growth and development process of plants.

The carotenoid is used as an important source of provitamin A in human diet, and has effects of preventing night blindness, resisting oxidation, resisting cancer, and delaying aging. However, not every carotenoid is converted into vitamin A in the human body, and only a small fraction of it is possible, for example, to alpha-carotene and beta-carotene.

Carrot (Daucus carota L., chromosome number 2n=2x=18) contains abundant carotenoid, and the cultivation area of carrot per year in China is about 600 tens of thousands of acres, and the yield is the first world. Carotenoids are one of the important factors that determine the appearance and nutritional quality of carrots. Carrots with high carotenoid content are more expensive than carrots with lower carotenoid content. Therefore, the research develops the gene cloning and functional research on regulating and controlling the synthesis of carotenoid in the carrot, and the research result lays a foundation for regulating and controlling the content of carotenoid in the carrot, and has important significance for improving the economic benefit of the carrot.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control carotenoid synthesis of plants, and provides a protein, a coding gene and application thereof in order to solve the problems in the prior art.

The technical scheme adopted for solving the technical problems is as follows:

the protein provided by the invention can be any one of the following proteins:

a1 Protein with the amino acid sequence shown as SEQ ID No. 2;

a2 Protein obtained by substituting and/or deleting and/or adding amino acid residues of the protein of A1) and having more than 80% of identity with the protein shown in A1) and regulating and controlling the synthesis of plant carotenoid; for example, according to the amino acid sequence shown as SEQ ID No.2 and the conventional technical means in the art such as conservative substitution of amino acid, one or more amino acids can be substituted, deleted and/or added to obtain a protein mutant with the same function as the amino acid sequence shown as SEQ ID No.2 under the premise of not affecting the activity of the protein mutant;

a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2).

The protein described in A1) above is named DcAPRR2.

In order to facilitate purification or detection of the protein of A1), a tag protein may be attached to the amino-or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing.

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein, and the like.

The nucleotide sequence encoding the protein DcAPRR2 of the present invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein DcAPRR2 isolated according to the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein DcAPRR2 and have the function of the protein DcAPRR2.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Herein, the 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In the above, the protein is derived from carrot (Daucus carota l.).

The present invention also provides a biological material related to the above protein, which may be any one of the following:

b1 A nucleic acid molecule encoding a protein as described above;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

c1 A nucleic acid molecule that inhibits or reduces or silences the expression of a gene encoding a protein as described above;

c2 Expression of the gene encoding the nucleic acid molecule of C1);

c3 An expression cassette containing the coding gene of C2);

c4 A recombinant vector comprising the coding gene of C2) or a recombinant vector comprising the expression cassette of C3);

c5 A recombinant microorganism containing the gene encoding C2), or a recombinant microorganism containing the expression cassette of C3), or a recombinant microorganism containing the recombinant vector of C4);

c6 A transgenic plant cell line containing the coding gene of C2), or a transgenic plant cell line containing the expression cassette of C3), or a transgenic plant cell line containing the recombinant vector of C4);

c7 A transgenic plant tissue containing C2) said coding gene, or a transgenic plant tissue containing C3) said expression cassette, or a transgenic plant tissue containing C4) said recombinant vector;

c8 A transgenic plant organ containing the coding gene of C2), or a transgenic plant organ containing the expression cassette of C3), or a transgenic plant organ containing the recombinant vector of C4).

In the above biological material, the nucleic acid molecule of B1) is a gene represented by E1) or E2) as follows:

e1 A cDNA molecule with a coding sequence of SEQ ID No. 1;

e2 A DNA molecule with a nucleotide sequence of SEQ ID No. 1.

DNA molecule shown in SEQ ID No.1DcAPRR2Gene) encoding a protein DcAPRR2 whose amino acid sequence is SEQ ID No. 2.

The nucleotide sequence shown in SEQ ID NO.1 is the nucleotide sequence of a gene (CDS) encoding the protein DcAPRR2.

B1 The nucleic acid molecules may also comprise nucleic acid molecules which have been modified by codon preference on the basis of the nucleotide sequence indicated in SEQ ID No. 1.

B1 The nucleic acid molecule may also include a nucleic acid molecule having a nucleotide sequence identity of 95% or more to that shown in SEQ ID No.1 and being of the same species.

The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be an RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

Vectors described herein are well known to those of skill in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids or viral vectors, and in particular, the vector pCAMBIA1301.

Recombinant expression vectors containing DcAPRR2 genes can be constructed using existing plant expression vectors. Such plant expression vectors include, but are not limited to, vectors such as binary Agrobacterium vectors and vectors useful for microprojectile bombardment of plants, and the like. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to untranslated regions transcribed from the 3' end of plant genes including, but not limited to, agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase Nos genes), plant genes (e.g., soybean storage protein genes).

When the DcAPRR2 gene is used for constructing a recombinant plant expression vector, any one of enhanced promoters or constitutive promoters can be added before transcription initiation nucleotide thereof, including but not limited to, a cauliflower mosaic virus (CAMV) 35S promoter, a ubiquitin promoter (ubiquitin) of corn, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, such as by adding genes encoding enzymes or luminescent compounds that produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

As a specific example, the recombinant vector can be pCAMBIA1301-DcAPRR2, wherein the recombinant vector pCAMBIA1301-DcAPRR2 is a recombinant expression vector obtained by replacing a fragment (small fragment) between EcoRI and SalI recognition sites of the pCAMBIA1301 vector with a fragment with the nucleotide sequence of SEQ ID No.1 in a sequence table, and keeping other sequences of the pCAMBIA1301 vector unchanged.

The DcAPRR2 gene or the fragment of the gene provided by the invention is introduced into plant cells or receptor plants by using any vector capable of guiding the expression of exogenous genes in plants, and transgenic cell lines and transgenic plants with the altered carotenoid content of the plants can be obtained. Expression vectors carrying the DcAPRR2 gene can be used to transform plant cells or tissues by conventional biological methods using Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and the transformed plant tissues are cultivated into plants.

The microorganism described herein may be a yeast, bacterium, algae or fungus. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc. Specifically, agrobacterium tumefaciens GV3101 can be used.

The invention also provides the use of the protein dcapr 2 or the substance regulating the expression of a gene or the substance regulating the activity or content of said protein as described hereinbefore in any of the following:

u1) the use of the protein or the substance regulating the expression of a gene or the substance regulating the activity and/or the content of the protein in regulating the synthesis of plant carotenoids;

u2) the application of the protein or the expression substance of the regulatory gene or the substance for regulating the activity and/or the content of the protein in preparing products for regulating the content of plant carotenoid;

u3) the use of the protein or of a substance regulating the expression of a gene or of a substance regulating the activity and/or the content of the protein for cultivating plants with a high carotene content;

u4) the use of the protein or of a substance regulating the expression of a gene or of a substance regulating the activity and/or the content of said protein for the preparation of a product for cultivating plants having a carotenoid content;

u5) the use of the protein or of a substance regulating the expression of a gene or of a substance regulating the activity and/or the content of the protein in plant breeding.

Herein, the substance that regulates the activity and/or content of the protein may be a substance that regulates the expression of a gene encoding the protein DcAPRR2.

In the above application, the substance for regulating the expression of the gene or the substance for regulating the activity and/or content of the protein is a biological material related to the protein, and the biological material may be any of the following:

b1 A nucleic acid molecule encoding a protein as described above;

b2 An expression cassette comprising the nucleic acid molecule of B1);

c2 Expression of the gene encoding the nucleic acid molecule of C1);

c3 An expression cassette containing the coding gene of C2);

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

The invention also provides a method for regulating and controlling the synthesis of plant carotenoid, which comprises regulating and controlling the activity and/or content of the protein DcAPRR2 in target plants, or/and the expression level of the coding gene of the protein DcAPRR2, so as to regulate and control the content of the plant carotenoid.

In the above method, the controlling the activity and/or content of the protein DcAPRR2 in the target plant, or/and the expression level of the gene encoding the protein DcAPRR2 comprises introducing the gene encoding the protein into a recipient plantDcAPRR2Obtaining a target plant with altered carotenoid content; the saidDcAPRR2The coding gene codes for the protein DcAPRR2.

In the above applications and methods, the modulation may be enhancement, enhancement or upregulation.

In the above applications and methods, the modulation may be inhibition, reduction or silencing.

To facilitate identification and selection of transgenic cells or plants, the recombinant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color response, antibiotic markers or chemical resistance markers which are expressed in plants, etc. The transformed plants can also be screened directly in adversity without adding any selectable marker gene. The plants obtained by the above method may be transgenic plants, or plants obtained by conventional breeding techniques such as crossing. In the above methods, the transgenic plants are understood to include not only first to second generation transgenic plants but also their progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

The present invention also provides a method of growing plants having altered carotenoid content comprising:

1) Increasing, enhancing and/or upregulating the expression level of a gene encoding a protein as defined above in a plant of interest, or/and increasing, enhancing and/or upregulating the activity and/or content of a gene encoding a protein as defined above, to obtain a plant with increased carotenoid content;

2) Inhibiting or reducing or silencing the expression level of a gene encoding a protein as described above in a plant of interest, or/and inhibiting or reducing or silencing the activity and/or content of a gene encoding a protein as described above, to obtain a plant with reduced carotenoid content.

In a specific embodiment, a method of growing plants having reduced carotenoid content comprises the steps of: inhibiting expression of nucleic acid molecules encoding DcAPRR2 protein in the plant of interest to obtain transgenic plants having reduced carotenoid content.

The inhibition of expression of the nucleic acid molecule encoding the DcAPRR2 protein in the plant of interest can be achieved specifically by introducing into the plant of interest a gene editing vector targeting the nucleic acid molecule encoding the DcAPRR2 protein. The inhibition of expression of the nucleic acid molecule encoding the DcAPRR2 protein in the plant of interest can be achieved specifically by introducing into the plant of interest a gene editing vector targeting the nucleic acid molecule encoding the DcAPRR2 protein. Specifically, the gene editing vector is a vector based on Cas9 gene editing technology. Specifically, the gene editing vector expresses sgRNA and Cas9 protein. The sgrnas target nucleic acid molecules encoding DcAPRR2 proteins. Specifically, the targets of the sgrnas are: 5'-CCATTTCCTGAACAGAGCTG-3' and 5'-AGCAGGAAGTGACAAGTACC-3'.

The invention also provides a method for cultivating plants with increased carotenoid content, comprising the following steps: increasing the content of DcAPRR2 protein in the plant to obtain the plant with increased carotenoid content.

In the above application or method, the plant is any one of the following:

c1 Dicotyledonous plants;

c2 Plant of the order umbelliferae;

c3 An Umbelliferae plant;

c4 Plants of the genus Dauci Sativae;

c5 Carrot (Dauci Sativae).

The present invention identifies a gene DcAPRR2 which regulates carotenoid synthesis and codes a transcription factor. The transgenic knockout experiment proves that the gene positively regulates and controls the synthesis of carrot carotenoid. The acquisition of the protein DcAPRR2 related to the synthesis of the carrot carotenoid can provide important candidate genes and breeding means for expanding the planting area of the carrot variety, provide guiding basis for realizing the precise regulation and control of the synthesis of the carrot carotenoid, and have important theoretical significance for carrot breeding.

Drawings

FIG. 1. Ji Touhuang expression level of DcAPRR2 in fleshy root, petiole and leaf of carrot.

FIG. 2 shows the carotenoid content change pattern obtained by overexpressing DcAPRR2 in Ji Touhuang carrot.

FIG. 3 shows the sequence mutation information of target sites of gRNA positions and different DcAPRR2 knockout lines.

FIG. 4 shows carotenoid content change patterns obtained by knocking out DcAPRR2 in Ji Touhuang carrots.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, carriers and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated. The obtained data were processed using Excel 2013 and SPSS21.0 statistical software, the experimental results were expressed as mean ± standard deviation, with significant differences using One-way ANOVA test, P < 0.05 (x), P < 0.01 (x).

Example 1 carrotDcAPRR2Cloning of Gene full-Length cDNA

According to carrot genome data, primer premier 5.0 is used for designing a Primer, ji Touhuang carrot fleshy root cDNA is used as a template for carrying out PCR amplification on the full length, and one carrot is screenedDcAPRR2And (3) a gene. The detailed steps are as follows:

the research material Ji Touhuang carrot is planted in a phytotron of a genetic and germplasm innovation utilization laboratory of Nanjing agricultural university, and the seedling age is 90d. Selecting the fleshy roots of the yellow carrots with the same head and immediately quick-freezing the fleshy roots with liquid nitrogen. RNA extraction was performed by reference to the manual of the polysaccharide polyphenol plant total RNA extraction kit (shanghai Pu Di, china). After the completion of the extraction, the quality of the extracted RNA was checked by agarose gel electrophoresis of 1.2%, and the concentration and quality of the RNA were checked by a NanoDrop 2000 spectrophotometer. The synthesis of the first strand of cDNA was performed with reference to the operating manual of HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wind) (Nanjinouzan, china). The first strand cDNA obtained was used forDcAPRR2Amplification of genes. PCR amplification is carried out by using the cDNA as a template and a specific forward primer DcAPRR2-F1 and a specific reverse primer DcAPRR2-R1 to obtain the gene for regulating and controlling carotenoid synthesisDcAPRR2. In the invention, the sequence of the forward primer F1 is shown as SEQ ID No. 3, and is specificallyIs DcAPRR2-F1: 5'-ATGGTTTTGAATGCTAATGATTTATTGGA-3'; the sequence of the reverse primer R1 is shown as SEQ ID No. 4, specifically DcAPRR2-R1: 5'-CTATGTCAGTGGAGATGTGTGCC-3'. The PCR system amounted to 20. Mu.L: primeSTAR Max Premix (2X) 10. Mu.L, template 1. Mu.L, forward and reverse primers 1. Mu. L, ddH each ₂ O7. Mu.L. PCR was performed as follows: pre-denaturation at 98 ℃ of 10 s;35 amplification cycles including denaturation at 98℃for 10s, annealing at 55℃for 10s, and extension at 72℃for 30s; after the cycle was completed, the extension was carried out at 72℃for 1 min, followed by incubation at 4 ℃. After amplification, the PCR product of a single target band was detected by 1.2% agarose gel electrophoresis, and specific target bands were recovered according to the gel recovery kit (Nanjinouzan, china) instructions for extraction.

For analysis in Ji Touhuang carrotDcAPRR2Expression patterns of genes at different tissue sites were determined using Real-time qPCR technologyDcAPRR2The expression level of the gene was analyzed. According toDcAPRR2The sequence of the coding region of the gene is designed into the upstream and downstream PCR primers for amplifying the whole coding region of the gene by using Primer 5.0 software according to the principle of general Primer design. RNA was extracted from the fleshy root, petiole and leaf of the carrot of period Ji Touhuang d, and the first strand cDNA was synthesized using the kit, and the synthesis was performed by referring to the manual of HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wind). The 20. Mu.L reaction system comprises: 10. Mu.L SYBR Green (Nanjinouzan, china), 5. Mu.L sterilized ultrapure water, 1. Mu.L cDNA, 2. Mu.L forward primer, dcAPRR2-F2: 5'-AGGACCGCAACTATGTCTCGC-3' (SEQ ID No. 5), 2. Mu.L reverse primer, dcAPRR2-R2:5'-ATCCAACCTTGATAACCCTGCC-3' (SEQ ID No. 6). (DcActin 1 is used as an internal reference, and the sequence is shown as follows: dcActin 1-F5'-CCTGAGGAGCACCCTGTACTTCTT-3' (SEQ ID No. 7), and DcActin 1-R5'-AACATACATGGCAGGGACATTGAA-3' (SEQ ID No. 8).

The procedure for RT-qPCR was as follows: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s, annealing at 60℃for 20 s,40 cycles; then the melting curve is obtained by gradually heating from 65 ℃ to 95 ℃ and preserving the temperature at 4 ℃ for 5 min.

The RT-qPCR experimental result shows that the Ji Touhuang carrot different tissues are used as materialsDcAPRR2Gene meat qualityThe roots, the petioles and the leaves are expressed. As shown in FIG. 1, dcAPRR2 was most expressed in the leaf.

1、DcAPRR2Obtaining of Gene overexpression Ji Touhuang carrot callus

1.1 construction of recombinant vector comprising the DcAPRR2 DNA molecule shown in SEQ ID No.2

ConstructionDcAPRR2Gene overexpression vector pCAMBIA1301-DcAPRR 2: will beDcAPRR2The coding sequence of the gene (SEQ ID NO.2 was constructed on vector pCAMBIA1301 (CAMBIA Co.) to give overexpressionDcAPRR2The recombinant vector of the gene is named pCAMBIA1301-DcAPRR2.

The recombinant vector pCAMBIA1301-DcAPRR2 is a recombinant expression vector obtained by replacing a fragment (small fragment) between EcoRI and SalI recognition sites of the pCAMBIA1301 vector with a DNA fragment with a nucleotide sequence of SEQ ID No.2 in a sequence table, and keeping other sequences of the pCAMBIA1301 vector unchanged.

1.2 transferring the recombinant vector constructed in the step 1.1 into Ji Touhuang carrot

The plasmids (recombinant vectors pCAMBIA1301-DcAPRR 2) and pCAMBIA1301 which are constructed in the step 1.1 are respectively transferred into agrobacterium tumefaciens GV3101 in an empty load (as a control), and after being cultured for 72 hours at 28 ℃, single clones are selected, positive clones are screened by colony PCR, the positive clones are subjected to genetic transformation on hypocotyls of conventional seed of the carrot with the same head yellow by using an agrobacterium-mediated method, and the grown transgenic DcAPRR2 gene positive calli (35S: dcAPRR2#1, 35S: dcAPRR2#2, 35S: dcAPRR2#3) and the empty calli (EV) of the transgenic pCAMBIA1301 are screened and used for subsequent experiments.

2. Transgenic plantsDcAPRR2Determination of carotenoid content in carrot callus of gene Ji Touhuang

The method for extracting and measuring carotenoid comprises the following steps: the sample was ground to a powder in liquid nitrogen and accurately weighed 50 mg as the extraction material. The extract material was transferred into acetone 1mL, mixed extracted twice and the supernatants combined. The extracted supernatant was filtered through a 0.45 μm filter. The carotenoid content is measured by a Woltth ACQUITY ultra-high performance liquid chromatography systemAnd (5) setting. The detection wavelength is 450nm, the mobile phase is methanol, acetonitrile=90:10, and the flow rate is 1mLmin ^-1 20. Mu.L of the extract was injected into an HPLC Hedera ODS-2C 18 analytical column (inner diameter 250 mm. Times.4.6 mm, particle size 5 μm) for chromatographic separation and measurement, and the column temperature was 30 ℃. The content was calculated from the standard curve of the standard and expressed in μg/g fresh weight (μg/g FW).

As shown in FIG. 2, the Ji Touhuang carrot carotenoid content was greatly increased after the DcAPRR2 was overexpressed as compared with the control. The above results indicate that DcAPRR2 positively regulates carrot carotenoid synthesis.

Example 4 knockoutDcAPRR2Gene validation

1. Construction of knockout vectors

1. CRISPR-Cas9 gene editing target sequence selection. After inputting the cDNA sequence of DcAPRR2 into CRISP-GE website, the most reliable 2 sites were selected and determined as target sites, all locatedDcAPRR2Is located in the 6 th exon region of (2)DcAPRR2Is defined in the specification. The first target sequence is 5'-CCATTTCCTGAACAGAGCTG-3', which is positioned at 475-494 of SEQ ID No. 1; the second target sequence is 5'-AGCAGGAAGTGACAAGTACC-3', which is located at positions 555-574 of SEQ ID No. 1.

2. Constructing a gene editing vector. The sgRNA sequences are designed according to the two target site sequences, and are respectively placed in AtU-29 and AtU d and then are connected in series, bsaI enzyme digestion site sequences are added at two ends to form the sgRNA expression cassette sequence (shown as SEQ ID No. 9), and the sgRNA expression cassette sequence is sent to a company for synthesis (Nanjing Jinsri, china). And (3) cutting the sgRNA expression cassette sequence by BsaI, recovering a short band by agarose gel electrophoresis, and inserting the short band into the BsaI-cut pYLCRISPR-Cas9Pubi-H to keep other sequences of the pYLCRISPR-Cas9Pubi-H vector unchanged to obtain the recombinant plasmid pYLCRISPR-Cas9Pubi-H-sgRNA.

2. Obtaining of knockout Material

1. The recombinant plasmid pYLCRISPR-Cas9Pubi-H-sgRNA is introduced into the agrobacterium tumefaciens GV3101 to obtain recombinant agrobacterium GV3101/pYLCRISPR-Cas9Pubi-H-sgRNA.

2. Taking recombinant agrobacterium GV3101/pYLCRISPR-Cas9Pubi-H-sgRNA obtained in the step 1, and carrying out genetic transformation on the hypocotyl of the conventional radix Dauci Sativae seed gomphostema niveum by adopting an agrobacterium-mediated method to obtain a transgenic plant.

3. And (5) screening plants with variant target sequences from the transgenic plants.

The specific method comprises the following steps: and taking plant leaves, extracting genome DNA, and carrying out PCR amplification by adopting a primer pair consisting of DcAPRR2-F3 and DcAPRR 2-R3.

DcAPRR2-F3:5'-TGGCAGCATGTACTTCACAAG-3' (as shown in SEQ ID No. 10);

DcAPRR2-R3: 5'-TGCCTTTTTCCGACTAGCTTTG-3' (SEQ ID No. 11).

And (3) recovering PCR amplified products, sequencing, analyzing a website DSDecode (http:// dsDecode. Scgene. Com /) by a CRISPR target editing mode after sequencing success, comparing the website DSDecode with a gene standard sequence by combining a manual peak reading mode, and analyzing the editing modes of each target sequence and upstream and downstream sequences thereof, wherein the result is shown in figure 3.

Sequencing results showed that, compared to control material WT, in transgenic plantsDcAPRR2The nucleotide sequence was mutated. Based on the results of the sequence alignment, a double chromosome was obtained in the Ji Touhuang carrot backgroundDcAPRR2Positive lines #1, #2 and #3 (i.e., a pair of plants with mutations in homologous chromosomes) were knocked out.

In transgenic carrot plants, positive lines #1, #2 and #3 with knockout of the double chromosome DcAPRR2 produced the following mutations near the target site, leading to premature termination of protein translation (FIG. 3).

Mutant plant #1 has coding gene of DcAPRR2 protein in two homologous chromosomes compared with control material(DcAPRR2Genes) were mutated as follows: a base T is inserted between the 571 th site and the 572 th site of the chromosome SEQ ID No.1, and other nucleotides are unchanged. The other chromosome SEQ ID No.1 has one base A inserted between the 571-572 th sites and the other nucleotides unchanged. These two mutations result in incomplete domain of the translated protein and loss of function, thus knocking out DcAPRR2 gene.

Mutant plant #2 was compared to the control material, and the gene encoding the DcAPRR2 protein (DcAPRR 2 gene) was mutated as follows in two homologous chromosomes: one of the chromosome SEQ ID No.1 has 478-571 site base deleted and other nucleotides unchanged. One base C is inserted between the 478 th site and the 479 th site of the other chromosome SEQ ID No.1, the 563 th site and the 571 th site are deleted, and other nucleotides are unchanged. These two mutations result in incomplete domain of the translated protein and loss of function, thus knocking out DcAPRR2 gene.

Mutant plant #3 was mutated in the coding gene for DcAPRR2 protein (DcAPRR 2 gene) in two homologous chromosomes as compared to the control material as follows: a base A is inserted between 477-478 sites of a chromosome SEQ ID No.1, and other nucleotides are unchanged. One base T is inserted between 477 th and 478 th sites of the other chromosome SEQ ID No.1, one base G is inserted between 571 th and 572 th sites, and other nucleotides are unchanged. These two mutations result in incomplete domain of the translated protein and loss of function, thus knocking out DcAPRR2 gene.

The DcAPRR2 transgenic lines (DcAPRR 2-KO#1, dcAPRR 2-KO#2 and DcAPRR 2-KO#3) and the control material (QTH) control material were double-chromosome knocked out in the context of Ji Touhuang carrot obtained in example 5 above.

Carrot plant carotenoid content determination is carried out in a laboratory by crop inheritance and germplasm innovation of Nanjing agricultural university. Double chromosome knockout DcAPRR2 transgenic lines (DcAPRR 2-KO#1, dcAPRR 2-KO#2 and DcAPRR 2-KO#3) were selected from 3 plants per line, and the experiment was repeated three times.

The method for extracting and measuring carotenoid comprises the following steps: carrot fleshy root was ground into powder in liquid nitrogen and accurately weighed 50 mg as an extraction material. The extract material was transferred into acetone 1mL, mixed extracted twice and the supernatants combined. The extracted supernatant was filtered through a 0.45 μm filter. The carotenoid content was determined by means of a Shimadzu LC-20A high performance liquid chromatography system (Shimadzu co., kyoto, japan). The detection wavelength was 450nm, the mobile phase was methanol/acetonitrile=90:10, the flow rate was 1mLmin-1, and 20. Mu.L of the extract was injected into an HPLC Hedera ODS-2C 18 analytical column (inner diameter 250 mm. Times.4.6 mm, particle size 5 μm) for chromatographic separation and measurement, and the column temperature was 30 ℃. The content was calculated from the standard curve of the standard and expressed in μg/g fresh weight (μg/g FW).

Data were processed using Excel 2013 and SPSS21.0 statistical software, experimental results were expressed as mean ± standard deviation, with significant differences using One-way ANOVA test, P < 0.05 (x), and P < 0.01 (x).

As shown in FIG. 2, after DcAPRR2 knockout, the carrot carotenoid content is greatly reduced. The results show that DcAPRR2 positively regulates carrot carotenoid synthesis.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims

1. The DcAPRR2 protein and its related biomaterials are used in any one of the following U1) -U5):

u1) use in regulating plant carotenoid synthesis;

u2) use in the preparation of products for controlling plant carotenoids;

u3) use in cultivating plants with a high carotenoid content;

u4) use in the production of a product for growing plants with a high carotenoid content;

u5) use in plant breeding of high carotenoid content;

wherein the amino acid sequence of the DcAPRR2 protein is shown as SEQ ID No. 2;

the biomaterial is any one of the following B1) to B7):

b1 Nucleic acid molecules encoding a protein having an amino acid sequence as shown in SEQ ID No. 2;

b2 An expression cassette comprising the nucleic acid molecule of B1);

the modulation is selected from (C1) or (C2):

(C1) Increasing the content of the carotenoids by up-regulating the expression level of the coding gene of the DcAPRR2 protein or up-regulating the activity and/or content of the DcAPRR2 protein;

(C2) Lowering carotenoid content by inhibiting the expression level of the coding gene of DcAPRR2 protein or inhibiting the activity and/or content of DcAPRR2 protein;

the plant is carrot.

2. A method of up-regulating plant carotenoid synthesis, characterized by: up-regulating activity and/or content of protein with amino acid sequence shown as SEQ ID No.2 or expression level of coding gene of protein with amino acid sequence shown as SEQ ID No.2 in target plant to up-regulate carotenoid synthesis.

3. The method according to claim 2, characterized in that: the up-regulating of the activity and/or content of the protein with the amino acid sequence shown as SEQ ID No.2 in the target plant, or the expression level of the coding gene of the protein with the amino acid sequence shown as SEQ ID No.2 comprises the steps of introducing the coding gene of the protein into a receptor plant to obtain the target plant with the carotenoid content higher than that of the receptor plant; the coding gene codes protein with the amino acid sequence shown as SEQ ID No. 2.

4. A method of growing plants having altered carotenoid content in the plants, comprising:

1) Up-regulating the expression level of the coding gene of the protein with the amino acid sequence shown as SEQ ID No.2 in the target plant, or up-regulating the activity and/or content of the protein with the amino acid sequence shown as SEQ ID No.2 to obtain the plant with increased carotenoid content;

2) Inhibiting the expression level of the coding gene of the protein with the amino acid sequence shown as SEQ ID No.2 in the target plant or inhibiting the activity and/or content of the protein with the amino acid sequence shown as SEQ ID No.2 to obtain the plant with reduced carotenoid content;

the plant is carrot.