CN116478259A - Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof - Google Patents

Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof Download PDF

Info

Publication number
CN116478259A
CN116478259A CN202211738140.XA CN202211738140A CN116478259A CN 116478259 A CN116478259 A CN 116478259A CN 202211738140 A CN202211738140 A CN 202211738140A CN 116478259 A CN116478259 A CN 116478259A
Authority
CN
China
Prior art keywords
protein
plants
plant
transgenic
aelcyb
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211738140.XA
Other languages
Chinese (zh)
Inventor
王飞兵
陈嘉敏
李佳男
徐海亮
叶玉秀
王尊欣
万陈中
张妍宁
李扬
张雨柔
程小萌
李纯
胡来宝
陈新红
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Huaiyin Institute of Technology
Original Assignee
Huaiyin Institute of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Huaiyin Institute of Technology filed Critical Huaiyin Institute of Technology
Priority to CN202211738140.XA priority Critical patent/CN116478259A/en
Publication of CN116478259A publication Critical patent/CN116478259A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nutrition Science (AREA)
  • Botany (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention discloses a plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and a coding gene and application thereof. Experiments prove that the coding gene of the protein is introduced into the arabidopsis, the carotenoid content in the transgenic arabidopsis is obviously improved, and the stress resistance of transgenic arabidopsis plants is improved. Therefore, the protein AeLCYB related to carotenoid biosynthesis and stress resistance and the encoding gene thereof have important theoretical significance and practical value in regulating and controlling plant carotenoid biosynthesis and stress resistance; the invention has wide application space and market prospect in the agricultural field.

Description

Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an okra carotenoid biosynthesis and stress resistance related protein AeLCYB, a coding gene thereof and application thereof in improving the carotenoid content and stress resistance of plants.
Background
Okra contains abundant proteins, free amino acids, carotenoid, various vitamins, mineral elements such as phosphorus, iron, potassium, calcium and the like, and viscous substances composed of pectin, polysaccharide and the like, has various health care functions, and is welcomed by wide consumers. In animals, carotenoid substances also play an important role, but animals themselves cannot synthesize carotenoids and can only ingest them from the daily diet. Researches show that the carotenoid has close relation with human body health, is an indispensable nutrient substance in human diet structure, and has very important effects in quenching free radicals, increasing human immunity, preventing cardiovascular and cerebrovascular diseases, preventing cancers, protecting eyes and the like.
The synthesis of carotenoids in higher plants starts from the isoprene pyrophosphate (IPP) pathway. Three IPP molecules and one dimethyl propenyl pyrophosphate (DMAPP) molecule are condensed to form C under the catalysis of geranylgeranyl pyrophosphate (GGPP) synthase (GGPS) 20 GGPP. GGPP is a common precursor for biosynthesis of various substances, and is the most direct precursor for forming plant carotenoids. Condensing 2 GGPP molecules under the action of Phytoene Synthase (PSY) to form a C 40 Phytoene. Phytoene is co-catalyzed by Phytoene Dehydrogenase (PDS), zeta-carotene dehydrogenase (ZDS) and carotenoid isomerase (CRTISO) 3 enzymes to form lycopene. Lycopene undergoes twice lycopene beta-cyclase (LCYB) cyclization to produce beta-carotene; lycopene cyclizes to form alpha-carotene in LCYB and lycopene epsilon-cyclase (LCYE). Alpha-carotene is subjected to a one-step beta-carotene hydroxylase (BCH) hydroxylation reaction to produce beta-cryptoxanthin, and then is subjected to a one-step BCH hydroxylation reaction to produce zeaxanthin. Alpha-carotene is subjected to two-step hydroxylation reaction of BCH and epsilon-carotene hydroxylase (ECH) to generate lutein.
There is a large area of salinized land around the world. Statistically, 8 hundred million hm are shared worldwide 2 In the saline-alkali soil, secondary salinized soil accounting for 33% of the cultivated land area is also arranged in an irrigation area, and the salt ulcer of the soil seriously affects the development of modern agriculture. In China, nearly one tenth of the secondary salt crumbling land exists in 18 hundred million mu of cultivated land in China, and 2000 ten thousand hm of the secondary salt crumbling land exists in the land 2 Saline and alkaline wastelands. Generally, the salt concentration is 0.2% -0.5% which affects the growth of crops, but the salt content of saline-alkali soil is 0.6% -10%. The presence of large areas of salinized land severely affects grain production and becomes a major factor limiting agricultural production.
Therefore, cloning of the key gene AeLCYB for biosynthesis of okra carotenoid, improving plant carotenoid content by utilizing a genetic engineering technology, improving plant stress resistance, and cultivating novel okra varieties with high carotenoid content is an important way for improving nutrition and health care effects of okra.
Disclosure of Invention
The invention aims to solve the technical problems of effectively improving plant quality, especially improving carotenoid accumulation of plants and improving drought resistance and salt tolerance of the plants.
The present invention provides a protein, named AeLCYB protein or AeLCYB protein, derived from okra (Abelmoschus esculotus l.) as shown in any one of (a) or (b) or (c):
(a) A protein with an amino acid sequence shown as SEQ ID NO. 2;
(b) Fusion protein obtained by N-terminal or/and C-terminal connexin label of the amino acid sequence shown in SEQ ID NO. 2;
(c) The protein which has more than 90 percent of identity and identical functions with the protein shown in the (a) is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.
Wherein SEQ ID NO.2 consists of 503 amino acid residues.
The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.
Among the above proteins, a protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above proteins, the identity refers to the identity of amino acid sequences. 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.
In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
The protein AeLCYB related biological material also belongs to the protection scope of the invention, and the invention also provides a new application of the protein AeLCYB related biological material.
The related biological material of the protein is any one of the following (c 1) to (c 10):
(c1) A nucleic acid molecule encoding the protein of claim 1;
(c2) An expression cassette comprising the nucleic acid molecule of (c 1);
(c3) A recombinant vector comprising the nucleic acid molecule of (c 1) or a recombinant vector comprising the expression cassette of (c 2);
(c4) A recombinant microorganism comprising the nucleic acid molecule of (c 1), or a recombinant microorganism comprising the expression cassette of (c 2), or a recombinant microorganism comprising the recombinant vector of (c 3);
(c5) A transgenic plant cell line comprising the nucleic acid molecule of (c 1), or a transgenic plant cell line comprising the expression cassette of (c 2), or a transgenic plant cell line comprising the recombinant vector of (c 3);
(c6) Transgenic plant tissue comprising the nucleic acid molecule of (c 1), or transgenic plant tissue comprising the expression cassette of (c 2), or transgenic plant tissue comprising the recombinant vector of (c 3);
(c7) A transgenic plant organ comprising the nucleic acid molecule of (c 1), or a transgenic plant organ comprising the expression cassette of (c 2), or a transgenic plant organ comprising the recombinant vector of (c 3);
(c8) A transgenic plant comprising the nucleic acid molecule of (c 1), or a transgenic plant comprising the expression cassette of (c 2), or a transgenic plant comprising the recombinant vector of (c 3);
(c9) A tissue culture produced by regenerable cells of the transgenic plant of (c 8);
(c10) Protoplasts produced from the tissue culture of (c 9).
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
As a preferred technical scheme: the nucleic acid molecule encoding the above protein (i.e., the gene encoding the above protein) is a DNA molecule of at least one of the following (a 1) to (a 3);
(a1) A DNA molecule as shown in SEQ ID NO 1;
(a2) A DNA molecule which hybridizes under stringent conditions with the DNA molecule defined in (a 1) and which encodes a protein associated with plant carotenoid content and stress tolerance;
(a3) A DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA molecule defined in (a 1) and encoding a protein associated with carotenoid accumulation and stress resistance in plants.
Wherein, SEQ ID NO.1 is composed of 1512 nucleotides, the Open Reading Frame (ORF) is 1 st-1512 th from the 5' end, and the encoding amino acid sequence is protein shown as SEQ ID NO. 2.
The stringent conditions are hybridization and washing of the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing of the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time.
In the above-mentioned related biological material, the expression cassette of (c 2) refers to a DNA capable of expressing the protein AeLCYB in a host cell, and the DNA may include not only a promoter for initiating transcription of the AeLCYB gene but also a terminator for terminating transcription of the AeLCYB gene.
In the above related biological material, the recombinant vector of (c 3) may contain a DNA molecule shown in SEQ ID NO.2 for encoding the protein AeLCYB.
The recombinant vector containing the AeLCYB encoding gene expression cassette can be constructed by using a plant expression vector. The plant expression vector may be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa, pCAMBIA1391-Xb, etc. When the AeLCYB is used for constructing a recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like can be added before transcription initiation nucleotide thereof, and 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 a specific embodiment of the invention, the recombinant expression vector is obtained by inserting the coding gene between multiple cloning sites of a vector pCBGUS;
the vector pCBGUS is obtained by a method comprising the following steps:
(1) The pCAMBIA1301 vector is subjected to double enzyme digestion of HindIII and EcoRI, and a large vector fragment is recovered;
(2) The pBI121 vector was digested with HindIII and EcoRI, and a fragment containing the gusA gene was recovered;
(3) And (3) connecting the large vector fragment recovered in the step (1) with the fragment containing the gusA gene recovered in the step (2) to obtain the recombinant vector pCBGUS.
The pCAMBIA1301 vector was purchased from CAMBIA company; the pBI121 vector was purchased from Clontech.
Of the above-mentioned related biological materials, (c 4) the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.
In the above related biological material, the transgenic plant organ of (c 7) may be the root, stem, leaf, flower, fruit and seed of the transgenic plant.
In the above related biological material, the tissue culture of (c 9) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.
In the above related biological materials, none of the transgenic plant cell line, transgenic plant tissue and transgenic plant organ comprises propagation material.
Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant bacteria containing the genes encoding the proteins associated with plant stress tolerance or the nucleic acid molecules encoding the above proteins are also within the scope of the invention.
The use of the protein AeLCYB described above, or a related biological material of the protein AeLCYB described above, or the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line described above, in at least one of the following (b 1) - (b 4):
(b1) The application of the method in regulating and controlling the carotenoid content in plants; preferably, in increasing the carotenoid content in plants;
(b2) Use in the cultivation of transgenic plants with altered carotenoid content; preferably, in the cultivation of transgenic plants with increased carotenoid content;
(b3) Application in regulating plant stress resistance; preferably, in improving stress resistance of plants;
(b4) Use in the cultivation of transgenic plants with altered stress resistance; preferably in the cultivation of transgenic plants with increased stress resistance.
The stress resistance is at least one of salt resistance and drought resistance. The carotenoid content is at least one of lutein (lutein), zeaxanthin (zeaxanthin), beta-cryptoxanthin (beta-cryptoxantin), alpha-carotene (alpha-carotenes), beta-carotene (beta-carotenes) and total carotenoid (total carotenoids).
In detail, the application of the protein AeLCYB or any one of the following related biological materials of the protein AeLCYB is also within the scope of the present invention:
(b1) The lutein content of the plant is improved;
(b2) Preparing a product for improving the lutein content of plants;
(b3) Increasing the zeaxanthin content of the plants;
(b4) Preparing a product for increasing the zeaxanthin content of the plant;
(b5) Increasing the beta-cryptoxanthin content of the plant;
(b6) Preparing a product for increasing the content of the plant beta-cryptoxanthin;
(b7) Increasing the alpha-carotene content of the plant;
(b8) Preparing a product for increasing the content of alpha-carotene in plants;
(b9) Increasing the beta-carotene content of the plant;
(b10) Preparing a product for increasing the content of plant beta-carotene;
(b11) Increasing the total carotenoid content of the plants;
(b12) Preparing a product for increasing the total carotenoid content of plants;
(b13) Improving the salt tolerance of plants;
(b14) Preparing a product for improving the salt tolerance of plants;
(b15) The drought resistance of the plants is improved;
(b16) Preparing a product for improving drought resistance of plants;
(b17) Improving rooting conditions of plants under drought and/or salt stress conditions;
(b18) Preparing a product for improving rooting conditions of plants under drought and/or salt stress conditions;
(b19) Improving the vigor of plants under drought and/or salt stress conditions;
(b20) Preparing a product of the vigor of a plant under drought and/or salt stress conditions;
(b21) Preparing the survival rate of the plant under drought and/or salt stress conditions;
(b22) Preparing a product that increases the survival rate of plants under drought and/or salt stress conditions;
(b23) Increasing plant abscisic acid content under drought and/or salt stress conditions;
(b24) Preparing a product for increasing the abscisic acid content of plants under drought and/or salt stress conditions;
(b25) Increasing the proline content of plants under drought and/or salt stress conditions;
(b26) Preparing a product for increasing the proline content of plants under drought and/or salt stress conditions;
(b27) Reducing plant H under drought and/or salt stress conditions 2 O 2 The content is as follows;
(b28) Preparation of plant H under drought and/or salt stress reducing conditions 2 O 2 Content of product
(b29) Reducing malondialdehyde content of plants under drought and/or salt stress conditions;
(b30) Preparing a product for reducing the malondialdehyde content of plants under drought and/or salt stress conditions;
(b31) Improving the SOD activity of plants under drought and/or salt stress conditions;
(b32) Preparing a product for improving the SOD activity of plants under drought and/or salt stress conditions;
(b33) Enhancing plant POD activity under drought and/or salt stress conditions;
(b34) Preparing a product for improving the POD activity of the plant under drought and/or salt stress conditions;
The application of the protein AeLCYB or related biological materials thereof in plant breeding is also within the protection scope of the invention.
The above application may specifically be the crossing of plants containing the protein AeLCYB or the related biological material (e.g. the gene AeLCYB encoding the protein AeLCYB) with other plants for plant breeding.
The invention further provides a method for cultivating transgenic plants with high carotenoid content, salt tolerance and drought resistance.
The method for cultivating the transgenic plant with high carotenoid content, drought resistance and salt tolerance comprises the steps of improving the expression quantity of a coding gene of protein AeLCYB in a target plant and/or the content of the protein AeLCYB and/or the activity of the protein AeLCYB to obtain the transgenic plant; the carotenoid content, drought resistance and salt tolerance of the transgenic plant are higher than those of the target plant.
In the above method, the method for increasing the expression level of the gene of the protein AeLCYB in the target plant and/or the content of the protein AeLCYB and/or the activity of the protein AeLCYB is to express or overexpress the protein AeLCYB in the target plant.
In the method, the method of expression or over-expression is to introduce the encoding gene of the protein AeLCYB into a target plant.
In the above method, the gene encoding the protein AeLCYB may be introduced into a target plant by a plant expression vector carrying the AeLCYB gene of the present invention. The plant expression vector carrying the gene AeLCYB of the present invention may be obtained by transforming plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and cultivating the transformed plant cells or tissues into plants.
In the method, the nucleotide sequence of the encoding gene of the protein AeLCYB is a DNA molecule shown in SEQ ID NO. 1.
In a specific embodiment of the present invention, the plant expression vector carrying the gene AeLCYB of the present invention may be pCAMBIA1301-AeLCYB. Specifically, pCAMBIA1301-AeLCYB was obtained by inserting the DNA molecule shown in SEQ ID NO.1 into pCAMBIA1301 vector using restriction enzymes HindIII and EcoRI.
In the above method, the improvement of plant quality is mainly characterized by at least one of increasing lutein content, increasing zeaxanthin content, increasing β -cryptoxanthin content, increasing α -carotene content, increasing β -carotene content, and increasing total carotenoid content of the plant.
In the method, the drought resistance and the salt tolerance are mainly reflected in improving the survival rate of plants, increasing the ABA content, increasing the proline content, improving the SOD activity, improving the POD activity and reducing H 2 O 2 At least one of the content and the reduced content of malondialdehyde.
In the present invention, the plant is any one of the following (e 1) to (e 4):
(e1) Dicotyledonous plants;
(e2) Monocotyledonous plants;
(e3) Cruciferous plants;
(e4) Arabidopsis thaliana.
Compared with the prior art, the invention has the beneficial effects that: the protein encoded by the AeLCYB gene provided by the invention can improve the accumulation and stress resistance of the total carotenoid of plants: the over-expression of the AeLCYB gene can improve the accumulation of total carotenoid in plants, drought resistance and salt tolerance. The carotenoid content in the transgenic plant is measured, and the result shows that the lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene and total carotenoid content of the transgenic arabidopsis plant are obviously improved compared with that of the wild arabidopsis plant; lutein content is 1.10 times and 1.04 times of wild type plants respectively; the zeaxanthin content is 1.06 times and 1.03 times that of wild type plants respectively; the beta-cryptoxanthin content is 1.06 times and 1.02 times that of wild type plants respectively; the alpha-carotene content is 2.28 times and 2.50 times that of wild plants respectively; beta-carotene content was 1.84 times and 1.75 times that of wild type plants, respectively; the total carotenoid content was 1.33-fold and 1.26-fold, respectively, compared to wild type plants. Under NaCl stress, the transgenic plant shows a good growth state, and the root length and fresh weight of the over-expressed transgenic arabidopsis material are respectively improved by 262-275% and 55-64% compared with the wild type WT material; under the stress of PEG6000, the root length and fresh weight of the over-expressed transgenic arabidopsis thaliana material are respectively increased by 244-287 percent and 108-118 percent compared with the wild type WT material; the survival rate of the over-expressed transgenic arabidopsis is obviously higher than that of a wild plant, and compared with the wild plant, the survival rate of the over-expressed transgenic arabidopsis is respectively improved by 6730 to 6992 percent and 2302 to 2402 percent, and the over-expressed transgenic arabidopsis has very strong salt tolerance and drought resistance; the specific expression is that the over-expression transgenic arabidopsis material increases the ABA content, the proline content, the SOD activity and the POD activity and reduces H 2 O 2 Content and malondialdehyde content. The result shows that the AeLCYB gene and the protein encoded by the AeLCYB gene play an important role in improving carotenoid content of plants and resisting high-salt and drought processes. The AeLCYB protein and the coding gene thereof provided by the invention have important application value in the research of improving the carotenoid content and stress resistance of plants. The invention is to be used in agricultureThe industrial field has wide application space and market prospect.
Drawings
FIG. 1 is a schematic diagram of an expression vector of an Abelmoschus esculentus AeLCYB gene plant of the invention.
FIG. 2 is a graph showing the results of PCR detection of an AeLCYB transgenic Arabidopsis plant of the present invention.
FIG. 3 expression of the AeLCYB gene of the invention in overexpressing Arabidopsis lines and wild type Arabidopsis plants.
FIG. 4 is a graph showing the standard curves of lutein (lutein), zeaxanthin (zeaxanthin), β -cryptoxanthin (β -cryptoxantin), α -carotene (α -carotene) and β -carotene (β -carotene) in the present invention.
FIG. 5 growth and rooting of the AeLCYB transgenic Arabidopsis plants of the invention on MS medium with 200mM NaCl and 25% PEG6000, where WT is wild Arabidopsis plant and L3 and L4 are transgenic Arabidopsis plant.
FIG. 6A salt tolerance and drought resistance pot identification of an AeLCYB transgenic Arabidopsis plant of the invention, wherein WT is a wild Arabidopsis plant, and L3 and L4 are transgenic Arabidopsis plants.
FIG. 7A stress-resistant physiological and biochemical index measurement of an AeLCYB transgenic Arabidopsis plant of the present invention, wherein WT is a wild type Arabidopsis plant, and L3 and L4 are transgenic Arabidopsis plants.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
In the following examples, the test materials used and their sources include:
okra (Abelmoschus esculentus) is preserved in laboratory at the plant production and processing practice education center of Jiangsu province of the national institute of sciences and food engineering.
Coli DH 5. Alpha. Was maintained by laboratory at the plant production and processing practice education center of Jiangsu province, the institute of Kogyo life sciences and food engineering. Cloning vectors PMD-18-Simple T, various restriction enzymes, taq polymerase, ligase, dNTPs, 10 XPCR buffer and DNA marker were purchased from Takara Bio-engineering Co., ltd. All chemicals were purchased from sigma chemical company in the united states and from Shanghai national pharmaceutical chemical reagent company.
The general Molecular biology procedures in the present invention are described in detail in Molecular cloning, 2nd ed. Cold Spring Harbor Laboratory Press, 1989.
Conventional genetic manipulation in the examples described below is carried out in reference to Molecular cloning literature [ Sambook J, frets EF, mannsdes Tet al. In: molecular cloning.2nd ed. Cold Spring Harbor Laboratory Press,1989 ].
Example 1 acquisition of Abelmoschus esculentus AeLCYB protein and its coding Gene
1. Experimental materials
Reference Wang Xu et al (2014) [ Wang Xu, han Chunle, zhou Yanan, wang Chunguo, song Wenqin, chen Chengbin. Cloning and expression analysis of okra chalcone synthase gene aehs. Plant genetic resources journal, 2014, 15 (3): 561-567), removing leaf material of okra plant of variety five-Fu, quick freezing with liquid nitrogen, and preserving at-80deg.C.
2. Leaf total RNA extraction and purification
About 2.0g of five-leaf blades are taken, ground into powder in liquid nitrogen, added into a 10mL centrifuge tube, and leaf blade total RNA is extracted by using an Applygen plant RNA extraction kit (Applygen Technologies Inc, beijin), wherein the kit comprises: plant RNAreagent, plant tissue lysis, isolation of RNA, removal of Plant polysaccharides and polyphenols; extraction Reagent, removing proteins, DNA, polysaccharides and polyphenols by organic extraction; plant RNAAid, removing Plant polysaccharide polyphenols and secondary metabolites. mRNA was purified from total RNA using QIAGEN Oligotex Mini mRNAKit (QIAGEN, gmbH, germany). Finally, 1. Mu.L of the sample was subjected to 1.2% agarose gel electrophoresis to determine the integrity, 2. Mu.L of the sample was diluted to 500. Mu.L, and the mass (OD 260) and purity (OD) of the sample were determined by an ultraviolet spectrophotometer 260 /OD 280 ) The total RNA of five-leaf blades is extracted, and detected by non-denaturing gel agarose gel electrophoresis, 28S and 18S bands are clear, and the brightness ratio of the two bands is 1.5-2:1, which indicates that the total RNA is not degraded, and the purified mRNA meets the experimental requirements, and can be used for cloning the full length of Abelmoschus esculentus AeLCYB protein cDNA.
3. Full-length cloning of Abelmoschus esculentus Aelcyb protein cDNA
The full-length cloning of the AeLCYB protein cDNA is carried out by designing a primer by using the sequence of the AeLCYB gene cDNA.
The primer sequences were as follows:
AeLCYB-GC-F：5’-CCATGGATACTTTACTTAGAAC-3’
AeLCYB-GC-R：5’-CTTATTCTCTATCCTGCACT-3’
reverse transcription of five-leaf total RNA with Oligo (dT) as template, PCR amplification with high-fidelity Fastpfu enzyme at 95 deg.C for 1min, subsequent cycles of 95 deg.C for 20s,53 deg.C for 20s and 72 deg.C for 1min, 40 cycles, and final extension at 72 deg.C for 10min. And detecting the PCR amplification product by agarose gel electrophoresis to obtain an amplified fragment with the length of 1155 bp.
The result of the steps is combined to obtain the target cDNA sequence, and the nucleotide sequence of the target cDNA sequence is shown as a sequence SEQ ID NO 1 in a sequence table. The sequence SEQ ID NO 1 in the sequence table consists of 1512 bases, and the 1 st to 1512 th bases from the 5' end are taken as open reading frames thereof, so as to code protein with an amino acid residue sequence shown in the sequence SEQ ID NO 2 in the sequence table. The sequence SEQ ID NO 2 of the sequence Listing consists of 503 amino acid residues. The gene is named as AeLCYB, and the protein encoded by the gene is named as AeLCYB.
EXAMPLE 2 construction of AeLCYB Gene overexpression vector
The DNA fragment containing the nucleotide shown in SEQ ID NO 1 of the sequence Listing, which was identified as correct by sequencing in example 1, was digested with BamH I and Sac I, and the DNA fragment was recovered by 1% agarose gel and passed through T 4 The DNA ligase connects the recovered AeLCYB gene fragment with a pYPx245 plasmid containing double 35S promoters, and the recombinant plasmid AH128 containing the grape AeLCYB gene is obtained by enzyme digestion identification and sequence analysis and determination. The expression vector also contained a gusA reporter gene and a kanamycin resistance marker gene with introns, and the vector is shown in FIG. 1.
EXAMPLE 3AeLCYB Gene-transformed Arabidopsis thaliana
The plant expression vectors pCAMBIA1301-AeLCYB of the okra AeLCYB gene constructed in example 2 are used for transforming Arabidopsis thaliana by a dipping method, and the specific method is as follows:
1. preparation of Agrobacterium
(1) The pCAMBIA1301-AeLCYB was transformed into Agrobacterium tumefaciens EHA105 strain (Biovector Co., LTD) by electric shock to obtain recombinant Agrobacterium containing pCAMBIA1301-AeLCYB, and the recombinant Agrobacterium was plated on kanamycin-resistant plates to screen transformants.
(2) The Agrobacterium single bacteria were inoculated into 5mL LB liquid medium (rifampicin 50. Mu.g/mL, chloramphenicol 100. Mu.g/mL), and cultured at 28℃for 20h at 250 rpm.
(3) 1mL of the bacterial liquid is transferred into 20-30mL of LB liquid medium (rifampicin 50. Mu.g/mL, chloramphenicol 100. Mu.g/mL), and cultured at 28 ℃ for about 12 hours at 250rpm, and OD 600 is measured to be approximately equal to 1.5.
(4) The cells were collected by centrifugation at 8000rpm,4℃for 10min, resuspended in Agrobacterium transformation permeate (5% (g/100 ml) sucrose, 0.05% (v/v) Silwet L-77) and diluted to OD 600. Apprxeq.0.8.
2. Transformation of Arabidopsis thaliana by flower dipping
(1) Immersing the flower bolts of the arabidopsis thaliana in the dyeing liquid, slightly stirring for about 10s, taking out, covering the arabidopsis thaliana with a fresh-keeping bag after all transformation is finished, keeping a moist environment, horizontally placing, culturing at 22 ℃ in a dark place, and removing the fresh-keeping bag for vertical culture after 24 hours.
(2) After the primary transformation is carried out for 4 days, the transformation can be carried out again, the transformation is repeated twice, and the total transformation is carried out for three times, so that the transformation can be carried out on the flower buds in different periods developed on inflorescences, and the transformation efficiency is improved.
(3) After growing for about two months, seeds are collected and stored in a refrigerator at 4 ℃ for later use.
After the arabidopsis transformed by the dip-flower method grows for about two months, the arabidopsis flowers and the knots normally bloom.
Example 4 molecular detection of AeLCYB Gene transgenic Arabidopsis plants
1. Screening of transgenic Arabidopsis seeds
(1) 25-30mg of seeds were weighed into a 1.5mL centrifuge tube.
(2) 1mL of 75% ethanol was sterilized for 1min (shaking continuously), centrifuged at 8000rpm for 5s, and the supernatant was removed.
(3) 1mL of the filtered bleach powder (2.5%) was added and sterilized for 15min (shaking, sufficient sterilization), centrifuged at 8000rpm for 5s, and the supernatant removed.
(4) Washing with sterile water for 3-4 times.
(5) Seeds were evenly spread on 1/2MS plates (hygromycin 50. Mu.g/mL), sealed with Parafilm, placed in a refrigerator at 4℃for two days, and incubated at 22℃for 10 days with 16h of light.
(6) Transplanting the resistant plants into a pot for culture, detecting GUS activity after the seedlings are slightly bigger, and selecting positive plants (T) 1 ) Culturing until flowers and fruits come out, collecting T 1 T tied up on plants 2 Seeds are further screened to obtain T 3 Seed.
2. PCR detection of transgenic Arabidopsis plants
(1) Test method
Extraction of T by CTAB method 3 Genomic DNA of arabidopsis transgenic plants and wild type plants. PCR detection was performed by conventional methods using the following primers for the AeLCYB gene: primer 1:5'-ACAGCGTCTCCGACCTGATGCA-3' and Primer2:5'-AGTCAATGACCGCTGTTATGCG-3'.
(2) Test results
The result of electrophoresis detection and amplification is shown in FIG. 2 (in FIG. 2, lane M is Maker; lane W is water; lane P is positive control (recombinant plasmid pCAMBIA 1301-AeLCYB), lane WT is wild type Arabidopsis plant, and lanes L1-L10 are Arabidopsis transgenic plants transformed with pCAMBIA 1301-AeLCYB). From the figure, the arabidopsis transgenic plants transformed with pCAMBIA1301-AeLCYB and the positive control amplify a 591bp target band, which shows that the AeLCYB gene is integrated into the genome of arabidopsis, and proves that the regenerated plants are transgenic plants; wild type Arabidopsis plants did not amplify the 591bp band of interest. Transgenic plants were subsequently analyzed for function.
3. qRT-PCR detection of transgenic arabidopsis plant
(1) Test method
Will be positive T 3 RNA is extracted from the strain of the Arabidopsis thaliana transformed into AeLCYB, cDNA is obtained through reverse transcription, qRT-PCR is carried out, and untransformed wild type is used as a control. AtActin gene is an internal reference: atActin-F:5'-GCACCCTGTTCTTCTTACCGA-3' and AtActin-R:5'-AGTAAGGTCACGTCCAGCAAGG-3'; the sequence of the AeLCYB primer is as follows: aeLCYB-F:5'-GAACTCTTCCTTTAGCCAACA-3'And AeLCYB-R:5'-AATGAAACCCAGCCACAA-3'.
(2) Test results
As shown in FIG. 3, WT is a wild type Arabidopsis plant, and L1-L7 are positive T 3 The substitution of the AeLCYB arabidopsis thaliana shows that the AeLCYB has different degrees of expression in transgenic arabidopsis thaliana plants.
Example 5 high Performance liquid chromatography determination of the carotenoid content of leaves of AeLCYB Gene transgenic Arabidopsis plants
1. Preparation of a Standard sample
(a) Beta-carotene (beta-carotenes), zeaxanthin and lutein (lutein) standards were purchased from sigma company under the trade numbers C4582-10MG, 14681-1, 95507, respectively; the β -cryptoxanthin standard was purchased from the beijing huaman reciprocal biochemistry company under the trade designation 0317S.
(b) A-carotene (a-carotenee) standard: since the α -carotene standard is extremely degradable, no commercial standard is made and must be extracted by itself. The specific method comprises the following steps:
(1) And (5) putting the diced carrots into a food processor for breaking.
(2) The crushed carrot slurry is introduced into a large mortar containing 5g of diatomite and mixed uniformly.
(3) Adding proper amount of precooled acetone, grinding, and extracting carotene from radix Dauci Sativae into acetone.
(4) Pouring the grinding liquid into a grinding funnel for vacuumizing, pumping the yellow liquid into a triangular flask, taking out the dry substance in the grinding funnel, putting the dry substance into a large mortar, adding a proper amount of precooled acetone, and grinding with force again. Repeating for 5-6 times until the color of the grinding fluid becomes colorless.
(5) Pouring golden yellow liquid (carotene extract liquid) in a triangular flask into a separating funnel for several times, and pouring 300mL ddH after pouring the carotene extract liquid once 2 O, standing for a little, and discharging the lower transparent liquid layer into a waste liquid bottle.
(6) The carotene extract liquid is subjected to ddH for a plurality of times 2 After O-washing, the emulsion formed is removed thoroughly by the final washing with saturated salineAnd a layer for discharging the transparent waste liquid of the lower layer.
(7) The water outlet hole of the separating funnel is wiped by absorbent paper. Discharging the golden petroleum ether layer (high purity carotene extract) into a dry conical flask; adding proper amount of anhydrous sodium sulfate (Na) 2 SO 4 ) Drying (to anhydrous Na 2 SO 4 Crystals are in a dispersed state), and shaking the mixture to suck residual moisture in the extract.
(8) The treated golden yellow liquid was poured into a dry, spherical bottle, and 100mL of 10% (g/100 mL) KOH in methanol was added to saponify and 0.1% (g/100 mL) dibutylhydroxytoluene.
(9) The spherical bottle is connected with a rotary evaporator, the other interface of the rotary evaporator is connected with a vacuum pump, liquid phase petroleum ether is evaporated in vacuum, the organic phase is concentrated to about 5mL, then petroleum ether (< 5 mL) is sucked by a rubber head dropper to clean orange solid matters on the wall of the spherical bottle, the rubber head dropper repeatedly blows the orange liquid phase, so that the solid matters are fully dissolved, and the carotene extract with higher purity is obtained, and the working time is about 50min.
(10) The column packing diatomite and magnesium oxide (1:1, w:w) are activated for 4 hours at 110 ℃, cooled and dried in a dryer. An iron stand is prepared, and a glass chromatographic column is placed on the iron stand and fixed. And a triangle bottle with a port is connected below the glass chromatographic column, and the triangle bottle is connected with a vacuum pump through a leather hose.
(11) Filling the column: carefully pouring the diatomite and magnesia mixture activated at high temperature into a glass chromatographic column, and carefully compacting by using a plunger rod from time to time, filling the column by about 20cm, and ensuring the cylindrical surface level; and adding a refined anhydrous sodium sulfate layer 1cm above the cylindrical surface, and then plugging a cotton wool layer about 1.5cm, so as to ensure the flatness of the sodium sulfate layer and the cotton wool layer. After compacting again, the vacuum pump is turned on and the vacuum is pumped for 1h.
(12) Petroleum ether is passed through a column: after vacuumizing for 1 hour, the vacuum pump is not required to be closed, the petroleum ether is added along the column wall for moistening the column, the vacuum pump is adjusted to enable the flow rate to be 2-3 drops per second, the plunger rod is used for keeping the level of the solid surface, and finally the petroleum ether is used for moistening the whole chromatographic column.
(13) Extracting with dropperCarefully transferring the substance into a chromatographic column until the sample layer enters near anhydrous Na 2 SO 4 When in layer, the petroleum ether phase of the round bottom flask is transferred into a chromatographic column, and the petroleum ether phase is always higher than anhydrous Na in the column passing process 2 SO 4 A layer.
(14) In the chromatographic working process, the existence of petroleum ether liquid phase above the solid phase is ensured, and the petroleum ether must be immediately replenished once insufficient (the chromatographic column needs to be protected from light and can be wrapped by using tinfoil paper).
(15) The alpha-carotene is the first substance to flow out of the chromatographic column, so when the layered alpha-carotene layer is about to flow out of the chromatographic column, the vacuum pump is temporarily turned off, a new triangular flask with a mouth is replaced to contain the alpha-carotene, after the connection is made, the vacuum pump is turned on, and golden yellow alpha-carotene flows out into the triangular flask (the triangular flask also needs to be protected from light).
(16) Pouring the obtained golden yellow liquid into a glass screw centrifuge tube for preservation, and noting the extraction time of a standard sample in detail, sealing the Parafilm strictly, keeping the tinfoil paper away from light, and preserving the golden yellow liquid upright in a refrigerator at the temperature of minus 80 ℃.
(17) Take out a small amount of N 2 Blow drying (sealing the rest of the extract, keeping away from light at-70deg.C for use), and dissolving with solvent of 1mLV acetonitrile/V methanol/V dichloromethane=45:20:35.
(18) After complete dissolution of the solute using a 1mL disposable syringe, transfer through a 0.22 μl filter into a 2mL brown sample vial, and 50 μl sample is used to detect the purity of the extracted sample.
2. Configuration of standard substance and drawing of standard curve
Directly using the extracted and detected alpha-carotene (alpha-carotenes) for preparing a mixed standard sample; lutein (Lutein) standard sample is dissolved by absolute ethyl alcohol and diethyl ether; zeaxanthin (Zeaxanthin) is dissolved in acetone; dissolving a beta-cryptoxanthin (beta-cryptoxantine) standard sample with diethyl ether and petroleum ether; beta-carotene (beta-catote) standard was dissolved in petroleum ether. After the standard sample is dissolved, 100 mu l of each standard sample is taken to a small volumetric flask (5 mL) to fix the volume, and the absorbance of each standard sample is measured by an ultraviolet-visible spectrophotometer under the specific wavelength of each component. And (3) calculating the concentration according to the formula (1), checking the concentration calculated by the formula (1) by the formula (2), and configuring the concentration into 50mL mixed standard samples by the formula (3). After the mixed standard sample is concentrated by nitrogen, petroleum ether is used for volume fixing. And respectively taking 1mL, 2mL, 3mL and 5mL of mixed standard samples, repeating 3 times for 45mL to establish a standard curve, and drawing the standard curve when the measured value of the standard sample meets the concentration range of each component listed in Table 1. The standard curves for lutein (lutein), zeaxanthin (zeaxanthin), beta-cryptoxanthin (beta-cryptoxantin), alpha-carotene (alpha-carotenes) and beta-carotene (beta-carotenes) in the mixed standard are shown in figure 4.
Wherein OD is the absorbance;is the absorption coefficient;
calibration concentration (μg/mL) =concentration c×purity (%) (2)
Purity (%) = standard HPLC peak area/HPLC peak total area x 100
a＝(50×b)/c (3)
Wherein 50 is 50mL of total volume of the mixed standard sample; a is the amount of the added standard sample (mug/mL)
b is the median concentration range (μg/mL); c is the calibration concentration (μg/mL)
TABLE 1 absorption coefficient and concentration ranges for standard solutions
3. Transgenic arabidopsis plant carotenoid extraction
Arabidopsis leaves: taking 2w of positive T transplanted into nutrient soil 3 Replacing leaves of the AeLCYB arabidopsis plants and wild arabidopsis plants, quick-freezing with liquid nitrogen, grinding into powder, weighing about 0.6g of ground samples, and extracting carotenoid. The method comprises the following steps:
(1) For positive T 3 Grinding the plant samples of the substituted AeLCYB Arabidopsis and wild ArabidopsisSampling;
(2) Weighing 0.6g of ground sample into a 25mL screw glass centrifuge tube, adding 6mL of 0.1% (g/100 mL) BHT absolute ethyl alcohol, and swirling for 20s;
(3) Taking out and adding 120 mu L of 80% (g/100 ml) potassium hydroxide solution;
(4) Vortex for 20s, then put into water bath of 85 ℃ for 5min;
(5) Taking out, swirling for 20s, and putting into water bath at 85 ℃ for 5min;
(6) Immediately placing the mixture on ice after taking out, and immediately adding 3mL of precooled ddH2O;
(7) 3mL of n-hexane was added and vortexed for 20s;
(8) Centrifuging at 2700rpm for 5min, and sucking the supernatant into another new screw glass centrifuge tube by using a pipetting gun;
(9) Repeating the steps 7 and 8 for 3 times, and adding the supernatant into another new screw glass centrifuge tube, wherein the final volume reaches about 12mL;
(10) 3mL of precooled ddH was added to a new screw glass centrifuge tube with supernatant 2 O, vortex, 2700rpm centrifugal 5min;
(11) Sucking the upper layer solution (n-hexane layer) into a new sharp-bottomed glass centrifuge tube by using a pipette;
(12) Adding 3mL of n-hexane into the water phase screw pipe, swirling, centrifuging for 5min, sucking the upper n-hexane layer into a new sharp bottom glass pipe, and repeating for 2 times;
(13) Vacuum centrifugal drying total n-hexane;
(14) 1mL of mobile phase was added to the dried, sharp-bottomed glass centrifuge tube, and after pipetting and mixing, the liquid was carefully added to a brown sample bottle through a 0.22 μm filter and measured by High Performance Liquid Chromatography (HPLC).
4. Transgenic arabidopsis plant carotenoid assay
(1) Test method
The measurement was performed by high performance liquid chromatography (high performance liquid chromatography: model 1200 of Agilent company, usa) as follows:
(1) Collecting the carotenoid solution in rotary evaporator, and collecting the carotenoid solution with N 2 And (5) drying.
(2) Immediately dissolving carotenoid in 1mL acetonitrile, methanol, dichloromethane (V, V) =45:20:35.
(3) After filtering the sample to be tested through a 0.22 μl syringe, 10 μl sample is added directly to the column.
(4) By YMC 30 Chromatographic column (250 mm×4.6mm,5 nm) with acetonitrile/methanol/dichloromethane (V/V) =75:20:5 as mobile phase, and detecting change of absorption peak at 450nm with flow rate of 1.8 mL/min.
(5) The assay was repeated 3 times for each sample tested.
(2) Test results
The carotenoid content of transgenic arabidopsis leaves, transgenic empty vector control arabidopsis leaves and wild type control arabidopsis leaves were determined according to the standard curves established above for lutein (lutein), zeaxanthin (zeaxanthin), β -cryptoxanthin (β -cryptoxantin), α -carotene (α -carotene) and β -carotene (β -carotene) (fig. 4). Wherein the total carotenoid content is the sum of the five carotenoid contents. The results are shown in Table 2, wherein L3 and L4 in Table 2 respectively represent samples to be tested of 2 transgenic Arabidopsis plant leaves; CK represents the leaves of the transgenic empty vector control arabidopsis plant; WT represents wild type arabidopsis plant leaves. The carotenoid content in the transgenic empty vector control Arabidopsis plant leaves (CK) and the wild control Arabidopsis plant leaves (WT) is not significantly different; the lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene and total carotenoid content of the transgenic arabidopsis plant are obviously improved compared with that of the wild arabidopsis plant; lutein content is 1.10 times and 1.04 times of wild type plants respectively; the zeaxanthin content is 1.06 times and 1.03 times that of wild type plants respectively; the beta-cryptoxanthin content is 1.06 times and 1.02 times that of wild type plants respectively; the alpha-carotene content is 2.28 times and 2.50 times that of wild plants respectively; beta-carotene content was 1.84 times and 1.75 times that of wild type plants, respectively; the total carotenoid content was 1.33-fold and 1.26-fold, respectively, compared to wild type plants.
The carotenoid content results show that the over-expression of the AeLCYB gene remarkably improves the carotenoid accumulation of plants, and the AeLCYB gene plays an important role in improving plant quality.
TABLE 2 carotenoid content in leaves of AeLCYB transgenic Arabidopsis plants
Example 6 identification of salt tolerance and drought resistance of AeLCYB Gene transgenic Arabidopsis plants
1. In vitro identification of salt tolerance and drought resistance of transgenic plants
(1) Test method
Transgenic arabidopsis and wild type seeds are sown and subcultured on a 1/2MS medium of 200mM NaCl and 25% (g/100 ml) PEG6000 after sterilization, and after stress culture for 2 weeks, the growth state and rooting condition of arabidopsis plants are observed.
(2) Test results
The results show that under the conditions of salt stress and PEG6000 treatment, the results are shown in figure 5, and the over-expression arabidopsis material and the wild type material are both due to the existence of the conditions of salt stress and PEG6000 stress, so that the plant becomes smaller; however, compared with the wild type WT, the over-expression Arabidopsis material has relatively good growth state, and the growth potential data statistics shows that under the salt stress, the root length and fresh weight of the over-expression Arabidopsis material are respectively improved by 262-275% and 55-64% compared with the wild type WT material; under the stress of PEG6000, the root length and fresh weight of the over-expressed Arabidopsis thaliana material are respectively increased by 244-287 percent and 108-118 percent compared with the wild WT material; the method shows that the over-expression of the AeLCYB gene obviously improves the salt tolerance and drought resistance of transgenic Arabidopsis plants.
2. Potted plant identification of salt tolerance and drought resistance of transgenic plant
(1) Test method
After 2 weeks of culture of transgenic arabidopsis and wild seeds on a 1/2MS medium, the plants were transplanted into pots for 2 weeks of culture, and then subjected to salt and drought stress treatment. Irrigating 1 time every 2 days with 1/2 Hoagland nutrient solution containing 300mM NaCl, 200mL each time, treating for 4 weeks, observing plant growth condition and counting survival rate; after 6 weeks of drought treatment, plants were observed for growth, photographed and their survival investigated. The following calculation methods related to the improvement of survival rate are: (over-expressed plant survival-wild-type plant survival) ×100%/wild-type plant survival.
(2) Test results
The results show that the results of salt tolerance and drought resistance potted plant identification are shown in figure 6, the growth state of the transgenic plant is obviously better than that of a wild plant after 4 weeks of salt treatment or 6 weeks of drought treatment, the survival rate of the transgenic plant is obviously higher than that of the wild plant, and the survival rate of the transgenic plant is respectively improved by 6730-6992% and 2302-2402% compared with that of the wild plant; the method shows that the over-expression of the AeLCYB gene obviously improves the salt tolerance and drought resistance of transgenic Arabidopsis plants.
Example 7 determination of physiological and Biochemical index of resistance of AeLCYB Gene transgenic Arabidopsis plants
Aba assay
(1) Test method
ABA plays an important role in plant stress response. ABA can improve the salt tolerance of plants, relieve osmotic stress and ion stress caused by excessive salt, maintain water balance, induce plant osmotic regulator substances proline to accumulate in large quantity, maintain the stability of cell membrane structures and improve the activity of protective enzymes. During drought damage stress, ABA can obviously reduce leaf water evaporation, reduce leaf cell membrane permeability, increase leaf cell soluble protein content, induce biomembrane system protective enzyme formation, reduce membrane lipid peroxidation degree, enhance antioxidant capacity and improve drought resistance of plants.
Assay methods reference [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thiana. Molecular Genetics and Genomics,2016,291:1545-1559 ], the ABA content of Arabidopsis plants was examined. The Arabidopsis plants were Arabidopsis plants which were not subjected to stress treatment for 2 weeks in the above pot culture assays, arabidopsis plants which were subjected to salt treatment for 1 week in the above pot culture assays, and Arabidopsis plants which were subjected to dry drought treatment for 2 weeks in the above pot culture assays. The experiment was repeated three times and the results averaged.
(2) Test results
The experimental results are shown in FIG. 7A (Normal is a blank control, salt stress is Salt stress, and Drright stress is Drought stress). The results show that the ABA content of the transgenic arabidopsis L3 plants and L4 plants is significantly higher than that of the wild arabidopsis plants.
2. Proline content determination
(1) Test method
Under normal conditions, plants have very low free proline content, but when subjected to salt, drought and other stresses, free amino acids accumulate in large quantities, and the accumulation index is related to stress resistance of the plants. Thus, proline can be used as a biochemical indicator of stress resistance in plants.
Assay methods reference [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thiana. Molecular Genetics and Genomics,2016,291:1545-1559 ], the proline content of Arabidopsis plants was examined. The Arabidopsis plants were Arabidopsis plants which were not subjected to stress treatment for 2 weeks in the above pot culture assays, arabidopsis plants which were subjected to salt treatment for 1 week in the above pot culture assays, and Arabidopsis plants which were subjected to dry drought treatment for 2 weeks in the above pot culture assays. The experiment was repeated three times and the results averaged.
(2) Test results
The experimental results are shown in FIG. 7B (Normal is a blank control, salt stress is Salt stress, and Drright stress is Drought stress). The results show that the proline content of the transgenic arabidopsis thaliana L3 plants and L4 plants is significantly higher than that of the wild arabidopsis thaliana plants.
3.H 2 O 2 Content determination
(1) Test method
H due to enhanced in vivo active oxygen metabolism in plants under adverse conditions or aging 2 O 2 Accumulation occurs. H 2 O 2 Can oxidize biomacromolecules such as nucleic acid, protein and the like in cells directly or indirectly and damage cell membranes, thereby accelerating aging and disintegration of cells. Thus H 2 O 2 The more the content of (2)The higher the level of stress injury a plant is subjected to.
Assay methods reference [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thiana. Molecular Genetics and Genomics,2016,291:1545-1559 ], the MDA content of Arabidopsis plants was examined. The Arabidopsis plants were Arabidopsis plants which were not subjected to stress treatment for 2 weeks in the above pot culture assays, arabidopsis plants which were subjected to salt treatment for 1 week in the above pot culture assays, and Arabidopsis plants which were subjected to dry drought treatment for 2 weeks in the above pot culture assays. The experiment was repeated three times and the results averaged.
(2) Test results
The experimental results are shown in FIG. 7C (Normal is a blank control, salt stress is Salt stress, and Drright stress is Drought stress). The results show that the H of the transgenic Arabidopsis L3 plants and L4 plants 2 O 2 The content is significantly lower than that of wild type Arabidopsis plants.
MDA content determination
(1) Test method
Plant organ aging or injury in adverse circumstances often occurs, and Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of adverse circumstances injury of plants, i.e. the higher the MDA content, the greater the degree of adverse circumstances injury of plants.
Assay methods reference [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thiana. Molecular Genetics and Genomics,2016,291:1545-1559 ], the MDA content of Arabidopsis plants was examined. The Arabidopsis plants were Arabidopsis plants which were not subjected to stress treatment for 2 weeks in the above pot culture assays, arabidopsis plants which were subjected to salt treatment for 1 week in the above pot culture assays, and Arabidopsis plants which were subjected to dry drought treatment for 2 weeks in the above pot culture assays. The experiment was repeated three times and the results averaged.
(2) Test results
The experimental results are shown in FIG. 7D (Normal is a blank control, salt stress is Salt stress, and Drright stress is Drought stress). The results show that the MDA content of the transgenic Arabidopsis L3 plants and L4 plants is significantly lower than that of the wild type Arabidopsis plants.
SOD Activity assay
(1) Test method
Superoxide dismutase (SOD) activity can be used as a physiological and biochemical index of plant stress resistance. The lower the activity of SOD, the greater the extent to which the plant suffers from stress injury.
Assay methods reference [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thiana. Molecular Genetics and Genomics,2016,291:1545-1559 ], the SOD activity of Arabidopsis plants was tested. The Arabidopsis plants were Arabidopsis plants which were not subjected to stress treatment for 2 weeks in the above pot culture assays, arabidopsis plants which were subjected to salt treatment for 1 week in the above pot culture assays, and Arabidopsis plants which were subjected to dry drought treatment for 2 weeks in the above pot culture assays. The experiment was repeated three times and the results averaged.
(2) Test results
The experimental results are shown in FIG. 7E (Normal is a blank control, salt stress is Salt stress, and Drright stress is Drought stress). The results show that the SOD activity of the transgenic Arabidopsis L3 plants and L4 plants is obviously higher than that of wild Arabidopsis plants.
POD Activity assay
(1) Test method
Peroxidase (POD) activity can be used as a physiological and biochemical indicator of plant stress resistance. The lower the activity of POD, the greater the extent to which the plant suffers from stress injury.
Assay methods reference [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thiana. Molecular Genetics and Genomics,2016,291:1545-1559 ], POD activity of Arabidopsis plants was examined. The Arabidopsis plants were Arabidopsis plants which were not subjected to stress treatment for 2 weeks in the above pot culture assays, arabidopsis plants which were subjected to salt treatment for 1 week in the above pot culture assays, and Arabidopsis plants which were subjected to dry drought treatment for 2 weeks in the above pot culture assays. The experiment was repeated three times and the results averaged.
(2) Test results
The experimental results are shown in FIG. 7, wherein F (Normal is a blank control, salt stress is Salt stress, and Drright stress is Drought stress). The results show that the POD activity of the transgenic Arabidopsis L3 plants and L4 plants is significantly higher than that of the wild Arabidopsis plants.
The measurement result of the physiological and biochemical index shows that the over-expression of the AeLCYB gene obviously improves the salt tolerance and drought resistance of the transgenic Arabidopsis plant.

Claims (10)

1. A protein which is at least one of the following (a) - (c):
(a) A protein with an amino acid sequence shown as SEQ ID NO. 2;
(b) Fusion protein obtained by N-terminal or/and C-terminal connexin label of amino acid sequence shown as SEQ ID NO. 2;
(c) The protein which has more than 90 percent of identity and identical functions with the protein shown in the (a) is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.
2. The protein-related biomaterial of claim 1, which is any one of the following (c 1) - (c 10):
(c1) A nucleic acid molecule encoding the protein of claim 1;
(c2) An expression cassette comprising the nucleic acid molecule of (c 1);
(c3) A recombinant vector comprising the nucleic acid molecule of (c 1) or a recombinant vector comprising the expression cassette of (c 2);
(c4) A recombinant microorganism comprising the nucleic acid molecule of (c 1), or a recombinant microorganism comprising the expression cassette of (c 2), or a recombinant microorganism comprising the recombinant vector of (c 3);
(c5) A transgenic plant cell line comprising the nucleic acid molecule of (c 1), or a transgenic plant cell line comprising the expression cassette of (c 2), or a transgenic plant cell line comprising the recombinant vector of (c 3);
(c6) Transgenic plant tissue comprising the nucleic acid molecule of (c 1), or transgenic plant tissue comprising the expression cassette of (c 2), or transgenic plant tissue comprising the recombinant vector of (c 3);
(c7) A transgenic plant organ comprising the nucleic acid molecule of (c 1), or a transgenic plant organ comprising the expression cassette of (c 2), or a transgenic plant organ comprising the recombinant vector of (c 3);
(c8) A transgenic plant comprising the nucleic acid molecule of (c 1), or a transgenic plant comprising the expression cassette of (c 2), or a transgenic plant comprising the recombinant vector of (c 3);
(c9) A tissue culture produced by regenerable cells of the transgenic plant of (c 8);
(c10) Protoplasts produced from the tissue culture of (c 9).
3. The related biological material according to claim 2, wherein the nucleic acid molecule encoding the protein of claim 1 is a DNA molecule of at least one of the following (a 1) to (a 3);
(a1) A DNA molecule as shown in SEQ ID NO 1;
(a2) A DNA molecule which hybridizes under stringent conditions with the DNA molecule defined in (a 1) and which encodes a protein associated with plant carotenoid content and stress tolerance;
(a3) A DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA molecule defined in (a 1) and encoding a protein associated with carotenoid accumulation and stress resistance in plants.
4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 3.
5. Use of a protein as claimed in claim 1, or a related biological material as claimed in claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line as claimed in claim 4, in at least one of the following (b 1) to (b 4):
(b1) The application of the method in regulating and controlling the carotenoid content in plants;
(b2) Use in the cultivation of transgenic plants with altered carotenoid content;
(b3) Application in regulating plant stress resistance;
(b4) Use in the cultivation of transgenic plants with altered stress resistance.
6. A method for growing a transgenic plant having a high carotenoid content and/or stress resistance, which method is characterized in that the transgenic plant is obtained by increasing the content or activity of the protein of claim 1 in a plant of interest or by increasing the expression level of a nucleic acid molecule encoding the protein of claim 1 in a plant of interest; at least one of carotenoid content and stress resistance of the transgenic plant is higher than that of the target plant.
7. A method for cultivating plants having a high carotenoid content and/or stress resistance, which comprises cultivating transgenic plants obtained by the method according to claim 6.
8. The protein as claimed in claim 1, or the related biological material as claimed in claim 2 or 3, or the use as claimed in claim 5, or the method as claimed in claim 6 or 7, characterized in that: wherein the stress resistance is at least one of salt tolerance and drought resistance.
9. The protein as claimed in claim 1, or the related biological material as claimed in claim 2 or 3, or the use as claimed in claim 5, or the method as claimed in claim 6 or 7, characterized in that: wherein the carotenoid content is at least one of lutein (lutein), zeaxanthin (zeaxanthin), beta-cryptoxanthin (beta-cryptoxantin), alpha-carotene (alpha-carotenes), beta-carotene (beta-carotenes) and total carotenoids (total carotenoids).
10. The protein as claimed in claim 1, or the related biological material as claimed in claim 2 or 3, or the use as claimed in claim 5, or the method as claimed in claim 6 or 7, characterized in that: the plant is any one of the following (c 1) to (c 4):
(c1) Dicotyledonous plants;
(c2) Monocotyledonous plants;
(c3) Cruciferous plants;
(c4) Arabidopsis thaliana.
CN202211738140.XA 2022-12-31 2022-12-31 Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof Pending CN116478259A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211738140.XA CN116478259A (en) 2022-12-31 2022-12-31 Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211738140.XA CN116478259A (en) 2022-12-31 2022-12-31 Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof

Publications (1)

Publication Number Publication Date
CN116478259A true CN116478259A (en) 2023-07-25

Family

ID=87223821

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211738140.XA Pending CN116478259A (en) 2022-12-31 2022-12-31 Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof

Country Status (1)

Country Link
CN (1) CN116478259A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116970053A (en) * 2023-09-22 2023-10-31 南京农业大学三亚研究院 Plant carotenoid synthesis related protein DcAPRR2, and coding gene and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116970053A (en) * 2023-09-22 2023-10-31 南京农业大学三亚研究院 Plant carotenoid synthesis related protein DcAPRR2, and coding gene and application thereof
CN116970053B (en) * 2023-09-22 2024-01-30 南京农业大学三亚研究院 Plant carotenoid synthesis related protein DcAPRR2, and coding gene and application thereof

Similar Documents

Publication Publication Date Title
CN108864267B (en) Sweet potato carotenoid synthesis and salt-tolerant drought-resistant related protein IbARF5 as well as encoding gene and application thereof
US11512322B2 (en) Transgenic plant and the method for producing the same
CN109678943B (en) Protein AeZDS and coding gene and application thereof
US20160068860A1 (en) Transgenic plants
CN111218470B (en) Method for regulating and controlling stress resistance of plants
CN107475263B (en) Betula platyphylla SPL2 gene participating in plant morphogenesis and flower development and protein thereof
CN113621040B (en) Du pear salt-tolerant gene PbHSF3A and application thereof in plant salt-tolerant genetic improvement
CN109593738B (en) Okra carotenoid biosynthesis and stress resistance related protein AePDS, and coding gene and application thereof
CN108047320A (en) The application of Protein G mMYB12 and encoding gene in isoflavones accumulation and salt tolerance is improved
KR100896487B1 (en) OsRDCP1 gene increasing plant stress resistance and transgenic plants transformed by OsRDCP1 gene
CN111218455B (en) IbAITR5 gene from sweet potato, and protein coded by same and application thereof
CN116478259A (en) Plant carotenoid bio-enhancement and stress resistance related protein AeLCYB, and coding gene and application thereof
Qin et al. Genetic transformation of lipid transfer protein encoding gene in Phalaenopsis amabilis to enhance cold resistance
CN116445464A (en) Related protein AeLCYE for improving carotenoid accumulation and salt tolerance of plants, and coding gene and application thereof
CN111153979A (en) Drought-resistant related protein IbBT4, and coding gene and application thereof
CN106591324B (en) Millet SiASR4 gene and application
CN116536283B (en) Abelmoschus esculentus carotenoid biosynthesis and stress resistance related protein AePSY, and encoding gene and application thereof
CN107663232B (en) Plant anti-adversity associated protein OsIAA18 and its encoding gene and application
CN115851660A (en) Application of protein OsPIS and coding gene in improving plant stress resistance
CN111171124B (en) Plant stress resistance related protein VvIAA18, coding gene and application
AU758346B2 (en) Nucleic acid comprising the sequence of a promoter inductible by stress and a gene sequence coding for stilbene synthase
CN115948456A (en) Fusion gene capable of increasing synthesis amount of patchouli alcohol and method
CN112391406A (en) Method for promoting growth of strawberries and biological material used by same
WO2007003346A2 (en) Fungicidal agents
CN113881689B (en) Transgenic plant of apple ion transporter MdCAX2L-1 and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination