A protein AeLCYE for improving plant carotenoid accumulation and salt tolerance, as well as its encoding gene and application

Abstract

The present invention discloses a protein AeLCYE for improving plant carotenoid accumulation and salt tolerance, as well as a coding gene and application thereof. The present invention provides a protein, which is (a) or (b): (a) a protein composed of an amino acid sequence shown in a sequence SEQ ID NO 2 in a sequence list; (b) a protein derived from a sequence SEQ ID NO 2 in a sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added to an amino acid residue sequence shown in a sequence SEQ ID NO 2 in a sequence list and the protein is related to plant stress resistance and is derived from the sequence SEQ ID NO 2. The present invention introduces the coding gene of the protein into Arabidopsis thaliana, and the carotenoid content in the transgenic Arabidopsis thaliana is significantly improved, while the salt tolerance of the transgenic Arabidopsis thaliana plant is improved. The protein AeLCYE for carotenoid biosynthesis and stress resistance and its coding gene are of great value in regulating plant carotenoid biosynthesis and salt tolerance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a protein AeLCYE for improving plant carotenoid accumulation and salt tolerance, and a coding gene and application thereof. Background Art

[0002] Okra is rich in protein, free amino acids, carotenoids, various vitamins, mineral elements such as phosphorus, iron, potassium, calcium, and viscous substances composed of pectin and polysaccharides. It has multiple health functions and is very popular among consumers. In animals, carotenoids also play a particularly important role, but animals cannot synthesize carotenoids themselves and can only take them from their daily diet. Studies have shown that carotenoids are closely related to human health and are indispensable nutrients in the human diet. They play a very important role in quenching free radicals, increasing human immunity, preventing cardiovascular and cerebrovascular diseases, preventing cancer, and protecting the eyes.

[0003] The synthesis of carotenoids in higher plants begins with the isoprenyl pyrophosphate (IPP) pathway. Three IPP molecules and one dimethylallyl pyrophosphate (DMAPP) molecule condense under the catalysis of geranylgeranyl pyrophosphate (GGPP) synthase (GGPS) to form C 20 GGPP. GGPP is a common precursor for the biosynthesis of many substances and is the most direct precursor for the formation of plant β-carotene. Two GGPP molecules condense under the action of phytoene synthase (PSY) to form a C 40 Phytoene. Phytoene is catalyzed by three enzymes: phytoene dehydrogenase (PDS), ζ-carotene dehydrogenase (ZDS), and β-carotene isomerase (CRTISO) to form lycopene. Lycopene undergoes two cyclization reactions by lycopene β-cyclase (LCYB) to form β-carotene; lycopene undergoes cyclization by LCYB and lycopene ε-cyclase (LCYE) to form α-carotene. α-carotene undergoes a one-step hydroxylation reaction by β-carotene hydroxylase (BCH) to form β-cryptoxanthin, and then undergoes another one-step hydroxylation reaction by BCH to form zeaxanthin. α-carotene undergoes a two-step hydroxylation reaction by BCH and ε-carotene hydroxylase (ECH) to form lutein.

[0004] There are large areas of salinized land in the world. According to statistics, there are 800 million hm2 of salinized land in the world. 2In irrigated areas, there are also secondary salinized lands that account for 33% of the cultivated land area. Soil salinization seriously affects the development of modern agriculture. Generally speaking, a salt concentration of 0.2%-0.5% will affect the growth of crops, but the salt content of saline-alkali land is mostly 0.6%-10%. The existence of large areas of salinized land has seriously affected food production and has become the main factor restricting agricultural production.

[0005] Therefore, cloning the key gene AeLCYE in okra carotenoid biosynthesis, using genetic engineering technology to increase the plant carotenoid content, while improving plant stress resistance, and breeding new okra varieties with high carotenoid content are important ways to improve the nutritional and health benefits of okra. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a protein AeLCYE related to improving plant carotenoid accumulation and salt tolerance, as well as its encoding gene and application.

[0007] Technical solution: The present invention first provides a protein, named AeLCYE protein or protein AeLCYE, derived from okra (Abelmoschus esculentus L.), as shown in any one of the following (a1) or (a2) or (a3):

[0008] (a1) a protein with an amino acid sequence as shown in SEQ ID NO.2;

[0009] (a2) a fusion protein obtained by connecting a protein tag to the N-terminus or / and the C-terminus of the amino acid sequence shown in SEQ ID NO.2;

[0010] (a3) A protein having more than 90% identity and the same function as the protein shown in a1) obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2.

[0011] Among them, SEQ ID NO.2 consists of 534 amino acid residues.

[0012] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0013] In the above proteins, a protein tag refers to a polypeptide or protein that is fused and expressed with a target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0014] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0015] In the above proteins, the above 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0016] The present invention also provides any of the following uses of the protein AeLCYE:

[0017] (b1) increasing the lutein content of plants;

[0018] (b2) preparing products with increased lutein content in plants;

[0019] (b3) increasing the zeaxanthin content of plants;

[0020] (b4) preparing products for increasing the zeaxanthin content of plants;

[0021] (b5) increasing the content of β-cryptoxanthin in plants;

[0022] (b6) preparing products with increased β-cryptoxanthin content in plants;

[0023] (b7) increasing the content of α-carotene in plants;

[0024] (b8) preparing products with increased α-carotene content in plants;

[0025] (b9) increasing the content of β-carotene in plants;

[0026] (b10) preparing a product with increased β-carotene content in plants;

[0027] (b11) increasing the total carotenoid content of plants;

[0028] (b12) preparing a product with increased total carotenoid content in plants;

[0029] (b13) improving plant salt tolerance;

[0030] (b14) preparing products for improving plant salt tolerance;

[0031] (b15) improving the rooting of plants under salt stress conditions;

[0032] (b16) preparing a product for improving rooting of plants under salt stress conditions;

[0033] (b17) improving the growth vigor of plants under salt stress conditions;

[0034] (b18) preparing a product for the growth of plants under salt stress conditions;

[0035] (b19) preparing the survival rate of plants under salt stress conditions;

[0036] (b20) preparing a product for improving the survival rate of plants under salt stress conditions;

[0037] (b21) increasing the soluble sugar content of plants under salt stress conditions;

[0038] (b22) preparing a product for increasing the soluble sugar content of plants under salt stress conditions;

[0039] (b23) increasing the relative water content of plants under salt stress conditions;

[0040] (b24) preparing a product for increasing the relative water content of plants under salt stress conditions;

[0041] (b25) reducing the H2O2 content of plants under dry-salt stress conditions;

[0042] (b26) Preparation of products for reducing H2O2 content in plants under salt stress conditions

[0043] (b27) reducing the malondialdehyde content of plants under salt stress conditions;

[0044] (b28) preparing a product for reducing the malondialdehyde content of plants under salt stress conditions;

[0045] (b29) Increase the activity of plant SOD under salt stress conditions;

[0046] (b30) preparing a product for increasing the SOD activity of plants under salt stress conditions;

[0047] (b31) increasing plant CAT activity under salt stress conditions;

[0048] (b32) preparing a product for increasing CAT activity in plants under salt stress conditions;

[0049] (b33) increasing plant APX activity under salt stress conditions;

[0050] (b34) preparing a product for increasing the APX activity of plants under salt stress conditions;

[0051] (b35) increasing plant POD activity under salt stress conditions;

[0052] (b36) preparing a product for increasing the POD activity of plants under salt stress conditions;

[0053] The above-mentioned protein AeLCYE-related biological materials also belong to the protection scope of the present invention. The present invention also provides new uses of the protein AeLCYE-related biological materials.

[0054] The application of the protein AeLCYE-related biological materials of the present invention in any of the following is also within the protection scope of the present invention:

[0055] (b1) increasing the lutein content of plants;

[0056] (b2) preparing products with increased lutein content in plants;

[0057] (b3) increasing the zeaxanthin content of plants;

[0058] (b4) preparing products for increasing the zeaxanthin content of plants;

[0059] (b5) increasing the content of β-cryptoxanthin in plants;

[0060] (b6) preparing products with increased β-cryptoxanthin content in plants;

[0061] (b7) increasing the content of α-carotene in plants;

[0062] (b8) preparing products with increased α-carotene content in plants;

[0063] (b9) increasing the content of β-carotene in plants;

[0064] (b10) preparing a product with increased β-carotene content in plants;

[0065] (b11) increasing the total carotenoid content of plants;

[0066] (b12) preparing a product with increased total carotenoid content in plants;

[0067] (b13) improving plant salt tolerance;

[0068] (b14) preparing products for improving plant salt tolerance;

[0069] (b15) improving the rooting of plants under salt stress conditions;

[0070] (b16) preparing a product for improving rooting of plants under salt stress conditions;

[0071] (b17) improving the growth vigor of plants under salt stress conditions;

[0072] (b18) preparing a product for the growth of plants under salt stress conditions;

[0073] (b19) preparing the survival rate of plants under salt stress conditions;

[0074] (b20) preparing a product for improving the survival rate of plants under salt stress conditions;

[0075] (b21) increasing the soluble sugar content of plants under salt stress conditions;

[0076] (b22) preparing a product for increasing the soluble sugar content of plants under salt stress conditions;

[0077] (b23) increasing the relative water content of plants under salt stress conditions;

[0078] (b24) preparing a product for increasing the relative water content of plants under salt stress conditions;

[0079] (b25) reducing the H2O2 content of plants under dry-salt stress conditions;

[0080] (b26) Preparation of products for reducing H2O2 content in plants under salt stress conditions

[0081] (b27) reducing the malondialdehyde content of plants under salt stress conditions;

[0082] (b28) preparing a product for reducing the malondialdehyde content of plants under salt stress conditions;

[0083] (b29) Increase the activity of plant SOD under salt stress conditions;

[0084] (b30) preparing a product for increasing the SOD activity of plants under salt stress conditions;

[0085] (b31) increasing plant CAT activity under salt stress conditions;

[0086] (b32) preparing a product for increasing CAT activity in plants under salt stress conditions;

[0087] (b33) increasing plant APX activity under salt stress conditions;

[0088] (b34) preparing a product for increasing the APX activity of plants under salt stress conditions;

[0089] (b35) increasing plant POD activity under salt stress conditions;

[0090] (b36) preparing a product for increasing the POD activity of plants under salt stress conditions;

[0091] In the above application, the relevant biological material is any one of the following (c1)-(c10):

[0092] (c1) a nucleic acid molecule encoding the protein AeLCYE;

[0093] (c2) an expression cassette containing the nucleic acid molecule described in (c1);

[0094] (c3) a recombinant vector containing the nucleic acid molecule described in (c1), or a recombinant vector containing the expression cassette described in (c2);

[0095] (c4) a recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3);

[0096] (c5) a transgenic plant cell line containing the nucleic acid molecule described in (c1), or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3);

[0097] (c6) transgenic plant tissue containing the nucleic acid molecule described in (c1), or transgenic plant tissue containing the expression cassette described in (c2), or transgenic plant tissue containing the recombinant vector described in (c3);

[0098] (c7) a transgenic plant organ containing the nucleic acid molecule described in (c1), or a transgenic plant organ containing the expression cassette described in (c2), or a transgenic plant organ containing the recombinant vector described in (c3);

[0099] (c8) a transgenic plant containing the nucleic acid molecule described in (c1), or a transgenic plant containing the expression cassette described in (c2), or a transgenic plant containing the recombinant vector described in (c3);

[0100] (c9) tissue cultures produced from regenerable cells of the transgenic plant described in (c8);

[0101] (c10) Protoplasts produced from the tissue culture described in (c9).

[0102] 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.

[0103] In the above-mentioned related biological materials, (c1) the nucleic acid molecule encoding the protein AeLCYE can be specifically any one of the following (d1) or (d2) or (d3):

[0104] (d1) DNA molecule shown in SEQ ID NO.1;

[0105] (d2) the coding sequence is the DNA molecule shown in SEQ ID NO.1;

[0106] (d3) A DNA molecule that hybridizes with the DNA molecule defined in (d1) or (d2) under stringent conditions and encodes the protein AeLCYE.

[0107] Among them, SEQ ID NO.1 consists of 1605 nucleotides, and its open reading frame (ORF) is from position 1 to position 1605 from the 5′ end, encoding a protein with an amino acid sequence as shown in SEQ ID NO.2.

[0108] The stringent conditions are hybridization and membrane washing twice at 68°C in a 2×SSC, 0.1% SDS solution, each for 5 min, and hybridization and membrane washing twice at 68°C in a 0.5×SSC, 0.1% SDS solution, each for 15 min.

[0109] The expression cassette, recombinant expression vector, transgenic cell line or recombinant bacteria containing the coding gene of the protein related to plant stress resistance also fall within the protection scope of the present invention.

[0110] In the above-mentioned related biological materials, the expression cassette described in (c2) refers to a DNA capable of expressing the protein AeLCYE in a host cell, and the DNA may include not only a promoter for initiating the transcription of the AeLCYE gene, but also a terminator for terminating the transcription of the AeLCYE gene.

[0111] In the above-mentioned related biological materials, the recombinant vector (c3) may contain a DNA molecule encoding protein AeLCYE as shown in SEQ ID NO.2.

[0112] A recombinant vector containing the AeLCYE encoding gene expression cassette can be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBl121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When AeLCYE is used to construct a recombinant vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., 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 can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene.

[0113] In a specific embodiment of the present invention, the recombinant expression vector is a recombinant expression vector obtained by inserting the encoding gene into the multiple cloning site of the vector pCBGUS;

[0114] The vector pCBGUS is obtained by a method comprising the following steps:

[0115] (1) The pCAMBIA1301 vector was double-digested with HindIII and EcoR I to recover the large vector fragment;

[0116] (2) The pBI121 vector was double-digested with HindIII and EcoR I to recover the fragment containing the gusA gene;

[0117] (3) The large vector fragment recovered in step (1) is connected with the fragment containing the gusA gene recovered in step (2) to obtain the recombinant vector pCBGUS.

[0118] The pCAMBIA1301 vector was purchased from CAMBIA; the pBI121 vector was purchased from Clontech.

[0119] Among the above-mentioned related biological materials, the recombinant microorganism (c4) can specifically be yeast, bacteria, algae and fungi.

[0120] Among the above-mentioned related biological materials, the transgenic plant organ described in (c7) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0121] Among the above-mentioned related biological materials, the tissue culture described in (c9) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0122] Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

[0123] The application of the above protein AeLCYE or its related biological materials in plant breeding also falls within the protection scope of the present invention.

[0124] Among the above applications, the application in plant breeding can specifically be hybridizing plants containing the protein AeLCYE or the related biological materials (such as the protein AeLCYE encoding gene AeLCYE) with other plants to carry out plant breeding.

[0125] The present invention further provides a method for cultivating transgenic plants with high carotenoid content and high salt tolerance.

[0126] The method for cultivating transgenic plants with high carotenoid content and high salt tolerance of the present invention comprises increasing the expression amount of the gene of protein AeLCYE and / or the content of the protein AeLCYE and / or the activity of the protein AeLCYE in the target plant to obtain the transgenic plant; the carotenoid content and salt tolerance of the transgenic plant are higher than those of the target plant.

[0127] In the above method, the method for increasing the expression level of the gene of protein AeLCYE and / or the content of protein AeLCYE and / or the activity of protein AeLCYE in the target plant is to express or overexpress protein AeLCYE in the target plant.

[0128] In the above method, the expression or overexpression method is to introduce the gene encoding the protein AeLCYE into the target plant.

[0129] In the above method, the gene encoding the protein AeLCYE can be introduced into the target plant through a plant expression vector carrying the AeLCYE gene of the present invention. The plant expression vector carrying the gene AeLCYE of the present invention can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant cells or tissues can be cultivated into plants.

[0130] In the above method, the nucleotide sequence of the gene encoding the protein AeLCYE is the DNA molecule shown in SEQ ID NO.1.

[0131] In a specific embodiment of the present invention, the plant expression vector carrying the gene AeLCYE of the present invention can be pCAMBIA1301-AeLCYE. Specifically, pCAMBIA1301-AeLCYE is obtained by inserting the DNA molecule shown in SEQ ID NO.1 into the pCAMBIA1301 vector using restriction endonucleases Hind III and EcoR I.

[0132] In the above method, the improvement of plant quality is mainly reflected in increasing the lutein content, zeaxanthin content, β-cryptoxanthin content, α-carotene content, β-carotene content and total carotenoid content of the plant.

[0133] In the above method, the high salt tolerance is mainly reflected in increasing the survival rate of plants, increasing the soluble sugar content, increasing the relative water content, increasing the SOD activity, increasing the CAT activity, increasing the APX activity, increasing the POD activity, reducing the H2O2 content and reducing the malondialdehyde content.

[0134] In the present invention, the plant is any one of the following (e1) to (e4):

[0135] (e1) dicotyledons;

[0136] (e2) monocots;

[0137] (e3) Cruciferae;

[0138] (e4) Arabidopsis thaliana.

[0139] The protein encoded by the AeLCYE gene provided by the present invention can improve the accumulation of carotenoids and stress resistance of plants: overexpression of the AeLCYE gene can improve the accumulation of total carotenoids and salt tolerance of plants. The carotenoid content in the transgenic plants was measured, and the results showed that the content of lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and total carotenoids in the transgenic Arabidopsis plants was significantly increased compared with that in the wild-type Arabidopsis plants; the lutein content was 1.08 to 1.28 times that of the wild-type plants; the zeaxanthin content was 1.03 to 1.11 times that of the wild-type plants; the β-cryptoxanthin content was 1.15 to 1.29 times that of the wild-type plants; the α-carotene content was 1.54 to 1.84 times that of the wild-type plants; the β-carotene content was 1.23 to 1.39 times that of the wild-type plants; and the total carotenoid content was 1.13 to 1.32 times that of the wild-type plants. Under NaCl stress, transgenic plants showed good growth status. The root length and fresh weight of overexpressed transgenic Arabidopsis materials increased by 78-172% and 19-38% respectively compared with wild-type WT materials; the survival rate and fresh weight of overexpressed transgenic Arabidopsis were significantly higher than those of wild-type plants, and increased by 14731-17008% and 153-188% respectively compared with wild-type plants, expressing strong salt tolerance; specifically, overexpressed transgenic Arabidopsis materials increased soluble sugar content, relative water content, SOD activity, CAT activity, APX activity, POD activity, and reduced H202 content and malondialdehyde content. The results showed that the AeLCYE gene and the protein it encoded played an important role in increasing the content of carotenoids and resisting high salt in plants.

[0140] Beneficial effects: Compared with the prior art, the invention has the following advantages: the AeLCYE protein and its encoding gene provided by the invention have important application value in the research of increasing the carotenoid content and stress resistance of plants. The invention will have broad application space and market prospects in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1 A schematic diagram of the plant expression vector of the okra AeLCYE gene of the present invention;

[0142] Figure 2 Figure 1 shows the PCR test results of the AeLCYE transgenic Arabidopsis thaliana plant of the present invention;

[0143] Figure 3 Expression of the AeLCYE gene of the present invention in overexpressing Arabidopsis strains and wild-type Arabidopsis plants;

[0144] Figure 4The standard curve diagram of lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene in the present invention;

[0145] Figure 5 Growth and rooting of AeLCYE transgenic Arabidopsis plants of the present invention on MS medium containing 200 mM NaCl and 25% PEG6000 (A) and potted identification (B), WT is a wild-type Arabidopsis plant, and L1, L2, L3 and L4 are transgenic Arabidopsis plants;

[0146] Figure 6 In the present invention, the physiological and biochemical indexes of stress resistance of the AeLCYE transgenic Arabidopsis plant are measured, WT is a wild-type Arabidopsis plant, and L1, L2, L3 and L4 are transgenic Arabidopsis plants. DETAILED DESCRIPTION

[0147] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

[0148] In the following examples, the test materials used and their sources include:

[0149] Okra (Abelmoschus esculentus) variety Wufu, Taiwan, China, is preserved by the laboratory of Jiangsu Plant Production and Processing Practice Education Center, College of Life Science and Food Engineering, Huaiyin Institute of Technology.

[0150] Escherichia coli DH5α was stored in the laboratory of Jiangsu Plant Production and Processing Practice Education Center, School of Life Science and Food Engineering, Huaiyin Institute of Technology. Cloning vector PMD-18-Simp1e T, various restriction endonucleases, Taq polymerase, ligase, dNTP, 10×PCR buffer and DNA marker were purchased from Bao Bioengineering Dalian Co., Ltd. All chemical reagents were purchased from Sigma Chemical Company and Shanghai Sinopharm Chemical Reagent Company.

[0151] For details of conventional molecular biological operations in the present invention, please refer to Molecular Cloning. 2nd ed. Cold Spring Harbor Laboratory Press, 1989.

[0152] Conventional gene manipulations in the following examples were performed with reference to molecular cloning literature [Sambook J, Frets EF, Mannsdes T et al. In: Molecular Cloning. 2nd ed. Cold Spring Harbor Laboratory Press, 1989].

[0153] Example 1 Obtaining okra AeLCYE protein and its encoding gene

[0154] 1. Experimental Materials

[0155] Referring to the method of 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 AeCHS. Journal of Plant Genetic Resources, 2014, 15(3): 561-567], the leaf material of okra variety Wufu plant in Taiwan, China was removed, quick-frozen in liquid nitrogen, and stored at -80°C.

[0156] 2. Extraction and purification of total RNA from leaves

[0157] About 2.0 g of leaves of Taiwan Wufu were taken, ground into powder in liquid nitrogen, added to a 10 mL centrifuge tube, and the total RNA of the leaves was extracted using the Applygen Plant RNA Extraction Kit (Applygen Technologies Inc, Beijing). The kit includes: Plant RNA Reagent, for plant tissue lysis, RNA isolation, and removal of plant polysaccharides and polyphenols; Extraction Reagent, for organic extraction to remove proteins, DNA, polysaccharides and polyphenols; Plant RNA Aid, for removal of plant polysaccharides, polyphenols and secondary metabolites. mRNA was purified from total RNA using QIAGEN Oligotex Mini mRNA Kit (QIAGEN, GmbH, Germany). Finally, 1 μL was taken and tested for integrity by 1.2% agarose gel electrophoresis, and another 2 μL was taken and diluted to 500 μL, and its quality (OD 260 ) and purity (OD 260 / OD 280 ), total RNA was extracted from leaves of Taiwania wufu, China, and detected by non-denaturing agarose gel electrophoresis. The 28S and 18S bands were clear, and the brightness ratio between the two was 1.5-2:1, indicating that the total RNA was not degraded. The purified mRNA met the experimental requirements and could be used for cloning the full-length cDNA of okra AeLCYE protein.

[0158] 3. Full-length cloning of okra AeLCYE protein cDNA

[0159] Primers were designed based on the AeLCYE gene cDNA sequence for full-length cloning of AeLCYE protein cDNA.

[0160] The primer sequences are as follows:

[0161] AeLCYE-GC-F: 5'-ATGTTTTTGTTAGCTCCGAAGG-3'

[0162] AeLCYE-GC-R: 5'-CTAGATTGTG AGATATGTTCT-3'

[0163] The total RNA of Wufu leaves in Taiwan, China was reverse transcribed with Oligo (dT) as a template, and PCR amplification was performed using the high-fidelity FastPfu enzyme. The PCR conditions were 95°C for 1 min, followed by 95°C for 20 s, 53°C for 20 s, and 72°C for 1 min, for 40 cycles, and finally extended at 72°C for 10 min. The PCR amplification product was detected by agarose gel electrophoresis, and an amplified fragment of 1605 bp in length was obtained.

[0164] Based on the results of the above steps, the target cDNA sequence was obtained, and its nucleotide sequence is shown in the sequence SEQ ID NO 1 in the sequence list. The sequence SEQ ID NO 1 in the sequence list consists of 1605 bases, and the 1st to 1605th bases from the 5' end are its open reading frame, encoding a protein having the amino acid residue sequence shown in the sequence SEQ ID NO 2 in the sequence list. The sequence SEQ ID NO 2 in the sequence list consists of 534 amino acid residues. The gene was named AeLCYE, and the protein encoded by it was named AeLCYE.

[0165] Example 2 Construction of AeLCYE gene overexpression vector

[0166] The DNA fragment containing the nucleotides shown in the sequence table SEQ ID NO 1 that was correctly identified by sequencing in Example 1 was double-digested with BamH I and Sac I, and the DNA fragment was recovered by 1% agarose gel. The recovered AeLCYE gene fragment was ligated with the pYPx245 plasmid containing a double 35S promoter by T4 DNA ligase, and the recombinant plasmid AH128 containing the grape AeLCYE gene was obtained by enzyme digestion identification and sequence analysis. The expression vector also contains a gusA reporter gene and a kanamycin resistance marker gene with an intron. The vector is as shown in Figure 1 shown.

[0167] Example 3 AeLCYE gene transformation into Arabidopsis thaliana

[0168] The plant expression vector pCAMBIA1301-AeLCYE of the okra AeLCYE gene constructed in Example 2 was transformed into Arabidopsis thaliana by the floral dipping method, and the specific method is as follows:

[0169] 1. Preparation of Agrobacterium

[0170] (1) pCAMBIA1301-AeLCYE was transformed into Agrobacterium tumefaciens EHA105 strain (Biovector Co., LTD) by electroporation to obtain recombinant Agrobacterium containing pCAMBIA1301-AeLCYE, and the recombinant Agrobacterium was spread on a plate containing kanamycin resistance to select transformants.

[0171] (2) A single Agrobacterium bacterium was selected and inoculated into 5 mL of LB liquid culture medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL) and cultured at 28°C and 250 rpm for 20 h.

[0172] (3) Take 1 mL of bacterial solution and transfer it into 20-30 mL of LB liquid medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL), culture at 28°C, 250 rpm for about 12 h, and measure OD600≈1.5.

[0173] (4) The cells were collected by centrifugation at 8000 rpm and 4°C for 10 min, resuspended in Agrobacterium transformation infiltration medium (5% sucrose, 0.05% Silwet L-77) and diluted to OD600≈0.8.

[0174] 2. Arabidopsis floral dip transformation

[0175] (1) Immerse the inflorescence of Arabidopsis thaliana in the above infection solution, gently stir for about 10 seconds and then take it out. After all the transformation is completed, cover the Arabidopsis thaliana with a fresh-keeping bag to maintain a moist environment, place it horizontally, and culture it in the dark at 22°C. After 24 hours, remove the fresh-keeping bag and culture it upright.

[0176] (2) Four days after the initial transformation, another transformation can be performed, repeated twice, for a total of three transformations. This allows the flower buds at different developmental stages on the inflorescence to be transformed, thus improving the transformation efficiency.

[0177] (3) After about two months of growth, the seeds were collected and stored in a refrigerator at 4°C until use.

[0178] The Arabidopsis transformed by the floral dipping method grew for about two months and then flowered and produced fruit normally.

[0179] Example 4 Molecular Detection of AeLCYE Gene Transgenic Arabidopsis Plants

[0180] 1. Screening of transgenic Arabidopsis seeds

[0181] (1) Weigh 25-30 mg of seeds and place them in a 1.5 mL centrifuge tube.

[0182] (2) Disinfect with 1 mL of 75% ethanol for 1 min (shaking continuously), centrifuge at 8000 rpm for 5 s, and remove the supernatant.

[0183] (3) Add 1 mL of filtered bleach (2.5%) and disinfect for 15 min (shake continuously to ensure thorough disinfection), centrifuge at 8000 rpm for 5 s, and remove the supernatant.

[0184] (4) Wash with sterile water 3-4 times.

[0185] (5) The seeds were evenly spread on a 1 / 2 MS plate (hygromycin 50 μg / mL), sealed with a Parafilm film, placed in a 4°C refrigerator for two days, and cultured at 22°C with 16 h light for 10 days.

[0186] (6) The resistant plants were transplanted into pots for cultivation. When the seedlings grew larger, GUS activity was tested and positive plants (T1) were selected and cultured until flowering and fruiting. T2 seeds produced on the T1 plants were collected and further screened to obtain T3 seeds.

[0187] 2. PCR detection of transgenic Arabidopsis plants

[0188] (1) Test method

[0189] The genomic DNA of T3 Arabidopsis transgenic plants and wild-type plants were extracted by CTAB method. PCR detection was performed by conventional methods, and the primers of AeLCYE gene used were: Primer 1: 5'-ACAGCGTCTCCGACCTGATGCA-3' and Primer 2: 5'-AGTCAATGACCGCTGTTATGCG-3'.

[0190] (2) Test results

[0191] The results of electrophoresis detection amplification are shown in Figure 2 ( Figure 2 In the figure, lane M is Maker; lane w: water; lane P: positive control (recombinant plasmid pCAMBIA1301-AeLCYE); lane WT: wild-type Arabidopsis plants; lanes L1-L10: transgenic Arabidopsis plants transformed with pCAMBIA1301-AeLCYE). As can be seen from the figure, the transgenic Arabidopsis plants transformed with pCAMBIA1301-AeLCYE and the positive control amplified a 591bp target band, indicating that the AeLCYE gene has been integrated into the Arabidopsis genome and proving that these regenerated plants are transgenic plants; the wild-type Arabidopsis plants did not amplify the 591bp target band. The transgenic plants are for subsequent functional analysis.

[0192] 3. qRT-PCR detection of transgenic Arabidopsis plants

[0193] (1) Test method

[0194] RNA was extracted from the positive T3 generation AeLCYE-transformed Arabidopsis lines, and cDNA was obtained by reverse transcription for qRT-PCR, with the untransformed wild type as the control. The AtActin gene was used as the internal reference: AtActin-F: 5'-GCACCCTGTTCTTCTTACCGA-3' and AtActin-R: 5'-AGTAAGGTCACGTCCAGCAAGG-3'; the AeLCYE primer sequences were: AeLCYE-F: 5'-TGGTTCATCCTGCTACTGG-3' and AeLCYE-R: 5'-CACATCCAACTCGGCAAA-3'.

[0195] (2) Test results

[0196] The results are as follows Figure 3 As shown, WT is a wild-type Arabidopsis plant, and L1-L7 are all positive T3 generation AeLCYE-transfected Arabidopsis plants, indicating that AeLCYE is expressed to different degrees in transgenic Arabidopsis plants.

[0197] Example 5 Determination of carotenoid content in leaves of AeLCYE transgenic Arabidopsis plants by high performance liquid chromatography

[0198] 1. Preparation of Standard Samples

[0199] (a) β-carotene, zeaxanthin, and lutein were purchased from Sigma with product numbers C4582-10MG, 14681-1, and 95507, respectively. β-cryptoxanthin was purchased from Beijing Huameihuli Biochemical Company with product number 0317S.

[0200] (b) α-carotene standard: Since a-carotene standard is very easy to degrade, there is no commercial standard and it must be extracted by yourself. The specific method is as follows:

[0201] (1) Chop the diced carrots in a food processor.

[0202] (2) Pour the crushed carrot pulp into a large mortar containing 5 g of diatomaceous earth and mix well.

[0203] (3) Add an appropriate amount of pre-cooled acetone and grind vigorously to extract the carotene in the carrots into the acetone.

[0204] (4) Pour the grinding liquid into the frosted funnel and evacuate it. The yellow liquid will be pumped into the conical flask. Take out the dry material in the frosted funnel with a spoon and put it into a large mortar. Add an appropriate amount of pre-cooled acetone and grind it vigorously again. Repeat 5-6 times until the color of the grinding liquid becomes colorless.

[0205] (5) Pour the golden liquid (carotene extract) in the conical flask into the separatory funnel several times. After each pouring of the carotene extract, pour in 300 mL of ddH2O, let it stand for a while, and drain the lower transparent liquid layer into the waste liquid bottle.

[0206] (6) After the carotene extract is washed with ddH2O several times, it is washed with saturated brine for the last time to completely remove the formed milky layer, and the transparent waste liquid at the lower layer is also discharged.

[0207] (7) Wipe the outlet of the separatory funnel with absorbent paper. Drain the golden petroleum ether layer (high-purity carotene extract) and pour it into a dry conical flask; add an appropriate amount of anhydrous sodium sulfate (Na2SO4) to dry (until the anhydrous Na2SO4 crystals are dispersed), shake, and absorb the residual water in the extract.

[0208] (8) Pour the treated golden liquid into a dry spherical bottle, and then add 100 mL of 10% KOH methanol solution for saponification and 0.1% butylated hydroxytoluene.

[0209] (9) Connect the spherical bottle to a rotary evaporator, and connect the other port of the rotary evaporator to a vacuum pump. Vacuum evaporate the liquid petroleum ether and concentrate the organic phase to about 5 mL. Then use a rubber-tipped dropper to absorb the petroleum ether (<5 mL) to wash the orange solid substance on the wall of the spherical bottle. Repeatedly blow the orange liquid phase with the rubber-tipped dropper to fully dissolve the solid substance to obtain a higher purity carotene extract. The working time is about 50 min.

[0210] (10) Activate the column filler with diatomaceous earth and magnesium oxide (1:1) at 110°C for 4 hours, cool and dry in a desiccator. Prepare an iron stand, place the glass chromatography column on the stand and fix it. Connect a conical flask with a mouth under the glass chromatography column, and connect the conical flask to a vacuum pump through a leather hose.

[0211] (11) Filling the column: Carefully pour the high temperature activated diatomaceous earth and magnesium oxide mixture into the glass chromatography column, and compact it carefully with a plunger rod from time to time. Fill the column about 20 cm to ensure that the column surface is level; then add a 1 cm layer of refined anhydrous sodium sulfate above the column surface, and then insert a layer of about 1.5 cm of absorbent cotton to ensure that the sodium sulfate layer and the absorbent cotton layer are level. After compacting again, turn on the vacuum pump and evacuate for 1 hour.

[0212] (12) Petroleum ether column: After evacuating the column for 1 hour, do not turn off the vacuum pump. Add petroleum ether along the column wall to lubricate the column. Adjust the vacuum pump to a flow rate of 2-3 drops per second and use a plunger rod to keep the solid surface level. Finally, allow the petroleum ether to rinse the entire column.

[0213] (13) Use a dropper to carefully transfer the concentrated extract into the chromatography column. When the sample layer is close to the anhydrous Na2SO4 layer, transfer the petroleum ether phase used to rinse the round-bottom flask into the chromatography column. During the column flow, ensure that the petroleum ether phase is always higher than the anhydrous Na2SO4 layer.

[0214] (14) During the chromatography process, the presence of a liquid phase of petroleum ether above the solid phase must be ensured. If the petroleum ether is insufficient, it must be replenished immediately (the chromatography column needs to be protected from light and can be wrapped in tin foil).

[0215] (15) α-carotene is the first substance to flow out of the chromatography column. Therefore, when the α-carotene layer is about to flow out of the chromatography column, temporarily turn off the vacuum pump and replace it with a new Erlenmeyer flask with a mouth to hold the α-carotene. After the connection is completed, turn on the vacuum pump and the golden α-carotene will flow out into the Erlenmeyer flask (the Erlenmeyer flask also needs to be protected from light).

[0216] (16) The obtained golden liquid was poured into a glass screw-cap centrifuge tube for storage. The standard sample extraction time was marked in detail. The tube was tightly sealed with parafilm, protected from light with tin foil, and stored upright in a -80°C refrigerator.

[0217] (17) A small amount was taken out and dried with N2 gas (the remaining large amount of extract was sealed and stored in the dark at -70°C for future use), and then dissolved in 1 mL of a solvent of V acetonitrile: V methanol: V dichloromethane = 45:20:35.

[0218] (18) After the solute was completely dissolved using a 1-mL disposable syringe, it was transferred to a 2-mL brown injection vial through a 0.22-μL filter. The purity of the extracted sample was tested by injection of 50 μL.

[0219] 2. Configuration of standard products and drawing of standard curve

[0220] The extracted and detected α-carotene was directly used for the preparation of mixed standard samples; the lutein standard was dissolved in anhydrous ethanol and ether; the zeaxanthin standard was dissolved in acetone; the β-cryptoxanthin standard was dissolved in ether and petroleum ether; and the β-carotene standard was dissolved in petroleum ether. After the standard samples were dissolved, 100 μl of each was taken to a small volume bottle (5 mL) and the absorbance value of each component was measured at a specific wavelength using a UV-visible spectrophotometer. The concentration was calculated according to formula (1), and the concentration calculated by formula (1) was corrected by formula (2). The mixed standard was prepared into 50 mL using formula (3). The mixed standard was concentrated with nitrogen and then made up to volume with petroleum ether. Take 1mL, 2mL, 3mL and 5mL of the mixed standard sample and repeat 3 times for a total of 45mL to establish a standard curve. The standard curve can be drawn if the measured value of the standard sample meets the concentration range of each component listed in Table 1. The standard curves of lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene in the mixed standard sample are as follows: Figure 4 shown.

[0221]

[0222] Where OD is the absorbance value; is the absorption coefficient;

[0223] Calibration concentration (μg / mL) = concentration c × purity (%) (2)

[0224] Purity (%) = standard sample HPLC peak area / HPLC peak total area × 100

[0225] a=(50×b) / c (3)

[0226] Where 50 is the total volume of the mixed standard sample, 50 mL; a is the amount of standard sample added (μg / mL)

[0227] b is the median of the concentration range (μg / mL); c is the calibration concentration (μg / mL)

[0228] Table 1 Absorption coefficient and concentration range of standard solutions

[0229]

[0230] 3. Carotenoid Extraction from Transgenic Arabidopsis Plants

[0231] Arabidopsis leaves: Take the leaves of positive T3 transgenic AeLCYE Arabidopsis plants and wild-type Arabidopsis plants that have been transplanted into nutrient soil for 2 weeks, freeze them in liquid nitrogen, and then grind them into powder. Weigh about 0.6g of each ground sample for β-carotene extraction. The method is as follows:

[0232] (1) Grinding positive T3 generation AeLCYE transgenic Arabidopsis and wild-type Arabidopsis plant samples;

[0233] (2) Weigh 0.6 g of the ground sample into a 25 mL screw-cap glass centrifuge tube, add 6 mL of 0.1% BHT anhydrous ethanol, and vortex for 20 seconds;

[0234] (3) Take out and add 120 μL of 80% potassium hydroxide solution;

[0235] (4) Vortex for 20 seconds and place in an 85°C water bath for 5 minutes;

[0236] (5) Take out, vortex again for 20 seconds, and place in 85°C water bath for 5 minutes;

[0237] (6) Place on ice immediately after taking out and add 3 mL of pre-cooled ddH2O;

[0238] (7) Add 3 mL of n-hexane and vortex for 20 seconds;

[0239] (8) Centrifuge at 2700 rpm for 5 min and transfer the supernatant to another new screw-capped glass centrifuge tube using a pipette;

[0240] (9) Repeat steps 7 and 8 three times and transfer the supernatant to another new screw-cap glass centrifuge tube to a final volume of about 12 mE;

[0241] (10) Add 3 mL of pre-cooled ddH20 to a new screw-cap glass centrifuge tube containing the supernatant, vortex, and centrifuge at 2700 rpm for 5 min;

[0242] (11) Use a pipette to transfer the upper solution (hexane layer) into a new conical bottom glass centrifuge tube;

[0243] (12) Add 3 mL of n-hexane to the screw-cap tube of the aqueous phase, vortex, centrifuge for 5 min, and then pipette the upper n-hexane layer into a new conical bottom glass tube. Repeat twice.

[0244] (13) vacuum centrifugal drying of total n-hexane;

[0245] (14) Add 1 mL of mobile phase to a dried conical-bottom glass centrifuge tube. Mix by pipetting and then carefully add the liquid to a brown sample bottle through a 0.22 μm filter and measure by high performance liquid chromatography (HPLC).

[0246] 4. Carotenoid Determination in Transgenic Arabidopsis Plants

[0247] (1) Test method

[0248] The method is as follows:

[0249] (1) The carotenoid solution obtained above was collected in a rotary evaporator and dried with N2.

[0250] (2) Carotenoids were immediately dissolved in 1 mL of a solvent of acetonitrile:methanol:dichloromethane (V:V:V) = 45:20:35.

[0251] (3) After filtering the sample through a 0.22 μL syringe, add 10 μL of the sample directly to the chromatographic column.

[0252] (4) Using YMC C 30 A chromatographic column (250 mm×4.6 mm, 5 nm) was used, with acetonitrile:methanol:dichloromethane (V:V:V)=75:20:5 as the mobile phase and a flow rate of 1.8 mL / min. Changes in the absorption peak at a wavelength of 450 nm were detected.

[0253] (5) Repeat the measurement three times for each sample.

[0254] (2) Test results

[0255] According to the above-established standard curves of lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene ( Figure 4), and the carotenoid content of transgenic Arabidopsis leaves, empty vector control Arabidopsis leaves and wild-type control Arabidopsis leaves was measured respectively. The total β-carotene content is the sum of the contents of the five carotenoids. The results are shown in Table 2, where L3 and L4 represent the test samples of leaves of two transgenic Arabidopsis plants, respectively; CK represents the leaves of empty vector control Arabidopsis plants; and WT represents the leaves of wild-type Arabidopsis plants. There was no significant difference in the β-carotene content in the leaves of the empty vector control Arabidopsis plants (CK) and the wild-type control Arabidopsis plants (WT); the contents of lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and total carotenoids in the transgenic Arabidopsis plants were significantly increased compared with those in the wild-type Arabidopsis plants; the lutein content was 1.08 to 1.28 times that of the wild-type plants, respectively; the zeaxanthin content was 1.03 to 1.11 times that of the wild-type plants, respectively; the β-cryptoxanthin content was 1.15 to 1.29 times that of the wild-type plants, respectively; the α-carotene content was 1.54 to 1.84 times that of the wild-type plants, respectively; the β-carotene content was 1.23 to 1.39 times that of the wild-type plants, respectively; and the total carotenoid content was 1.13 to 1.32 times that of the wild-type plants, respectively.

[0256] The results of carotenoid content showed that overexpression of the AeLCYE gene significantly increased the accumulation of carotenoids in plants, and the AeLCYE gene played an important role in improving plant quality.

[0257] Table 2 Carotenoid content in leaves of AeLCYE transgenic Arabidopsis plants

[0258]

[0259] Example 6 Identification of salt tolerance of transgenic Arabidopsis thaliana plants with AeLCYE gene

[0260] 1. In vitro identification of salt tolerance of transgenic plants

[0261] (1) Test method

[0262] Transgenic Arabidopsis thaliana and wild-type seeds were sterilized and sown and subcultured on 200 mM NaCl 1 / 2MS medium. After 2 weeks of stress culture, the growth status and rooting of Arabidopsis thaliana plants were observed.

[0263] (2) Test results

[0264] The results showed that under salt stress conditions, Figure 5A. Both the overexpression Arabidopsis material and the wild-type material became smaller due to the presence of salt stress conditions; however, the overexpression Arabidopsis material had a relatively good growth state compared with the wild-type WT. Growth potential data statistics showed that under salt stress, the root length and fresh weight of the overexpression Arabidopsis material increased by 78-172% and 19-38% respectively compared with the wild-type WT material; indicating that overexpression of the AeLCYE gene significantly improved the salt tolerance of transgenic Arabidopsis plants.

[0265] 2. Potted identification of salt tolerance of transgenic plants

[0266] (1) Test method

[0267] After transgenic Arabidopsis and wild-type seeds were cultured on 1 / 2MS medium for 2 weeks, the plants were transplanted into pots and cultured for 2 weeks before being subjected to salt stress. The plants were irrigated with 1 / 2 Hoagland nutrient solution containing 300mM NaCl once every 2 days, 200mL each time, for 4 weeks, and the growth of the plants was observed and the survival rate was calculated. The following calculation method for the improvement of the survival rate is: (overexpression plant survival rate-wild-type plant survival rate)*100% / wild-type plant survival rate.

[0268] (2) Test results

[0269] The results showed that the salt tolerance potted plants were identified. Figure 5 B. After 4 weeks of salt treatment, the growth state of transgenic plants was significantly better than that of wild-type plants, and the survival rate of transgenic plants was significantly higher than that of wild-type plants, which was increased by 14731-17008% compared with wild-type plants; the fresh weight of transgenic plants was significantly higher than that of wild-type plants, which was increased by 153-188% compared with wild-type plants; indicating that overexpression of the AeLCYE gene significantly improved the salt tolerance of transgenic Arabidopsis plants.

[0270] Example 7 Determination of Physiological and Biochemical Resistance Indexes of AeLCYE Gene Transgenic Arabidopsis Plants

[0271] 1. Determination of soluble sugar content

[0272] (1) Test method

[0273] Soluble sugar is an organic osmotic regulator used by plants to respond to saline-alkali stress, such as glucose and sucrose, which play a role similar to protective enzymes in cells. At the same time, as carbohydrates, they provide sufficient material and energy supply for the synthesis of organic matter. Under adverse stress, the higher the soluble sugar content in plants, the less likely the plant cells will lose water under adverse conditions, the greater the possibility of survival, and the higher the plant resistance.

[0274] The determination method refers to the literature [Hong Zhai, Feibing Wang, Zengzhi Si, Jinxi Huo, Lei Xing, Yanyan An, Shaozhen He, Qingchang Liu. A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato. Plant Biotechnology Journal, 2016, 14: 592-602], and the soluble sugar content of Arabidopsis plants is detected. The Arabidopsis plants are Arabidopsis plants without stress treatment for 2 weeks in the above potted identification and Arabidopsis plants treated with salt for 1 week in the above potted identification. The experiment needs to be repeated three times, and the results are averaged.

[0275] (2) Test results

[0276] The experimental results are shown in Figure 6 A (Norma1 is the blank control, Salt stress is salt stress). The results showed that the soluble sugar content of transgenic Arabidopsis thaliana L1 plants, L2 plants, L3 plants and L4 plants was significantly higher than that of wild-type Arabidopsis thaliana plants.

[0277] 2. Determination of relative water content

[0278] (1) Test method

[0279] Relative water content is an important indicator reflecting the water status of plants. The relative water content of plant tissues is positively correlated with plant stress resistance, and the relative water content of cells reflects the degree of stress on cells.

[0280] Determination method: Reference [Yufeng Yang, Shikai Guan, Hong Zhai, Shaozhen He, Qingchang Liu. Development and evaluation of a storage root-bearing sweet potato somatic hybrid between Ipomoea batatas (L.) Lam. and I. triloba L. Plant Cell, Tissue and Organ Culture, 2009, 99: 83-89], to detect the relative water content of Arabidopsis plants. The Arabidopsis plants were Arabidopsis plants without stress treatment for 2 weeks in the above potted plant identification and Arabidopsis plants treated with salt for 1 week in the above potted plant identification. The experiment was repeated three times, and the results were averaged.

[0281] (2) Test results

[0282] The experimental results are shown in Figure 6 Middle B (Normal is blank control, Salt stress is salt stress). The results showed that the relative water content of transgenic Arabidopsis thaliana L1 plants, L2 plants, L3 plants and L4 plants was significantly higher than that of wild-type Arabidopsis thaliana plants.

[0283] 3. Determination of H2O2 content

[0284] (1) Test method

[0285] When plants are in adverse conditions or aging, H2O2 accumulates due to the enhanced metabolism of active oxygen in the body. H2O2 can directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells and damage cell membranes, thereby accelerating cell aging and disintegration. Therefore, the higher the H2O2 content, the greater the degree of damage to plants caused by adverse conditions.

[0286] The determination method refers to the literature [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana. Molecular Genetics and Genomics, 2016, 291: 1545-1559], and the MDA content of Arabidopsis plants is detected. The Arabidopsis plants are Arabidopsis plants without stress treatment for 2 weeks in the above potted identification and Arabidopsis plants treated with salt for 1 week in the above potted identification. The experiment needs to be repeated three times, and the results are averaged.

[0287] (2) Test results

[0288] The experimental results are shown in Figure 6 Middle C (Normal is blank control, Salt stress is salt stress). The results showed that the H2O2 content of transgenic Arabidopsis L1 plants, L2 plants, L3 plants and L4 plants was significantly lower than that of wild-type Arabidopsis plants.

[0289] 4. Determination of MDA content

[0290] (1) Test method

[0291] When plant organs age or suffer damage under adverse conditions, membrane lipid peroxidation often occurs. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of damage suffered by the plant under adverse conditions. That is, the higher the MDA content, the greater the degree of damage suffered by the plant under adverse conditions.

[0292] The determination method refers to the literature [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana. Molecular Genetics and Genomics, 2016, 291: 1545-1559], and the MDA content of Arabidopsis plants is detected. The Arabidopsis plants are Arabidopsis plants without stress treatment for 2 weeks in the above potted identification and Arabidopsis plants treated with salt for 1 week in the above potted identification. The experiment needs to be repeated three times, and the results are averaged.

[0293] (2) Test results

[0294] The experimental results are shown in Figure 6 The results showed that the MDA content of transgenic Arabidopsis thaliana L1, L2, L3 and L4 plants was significantly lower than that of wild-type Arabidopsis thaliana plants.

[0295] 5. SOD activity assay

[0296] (1) Test method

[0297] Superoxide dismutase (SOD) activity can be used as a physiological and biochemical indicator of plant stress resistance. The lower the SOD activity, the greater the degree of damage to the plant caused by adversity.

[0298] The determination method refers to the literature [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates fiavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana. Molecular Genetics and Genomics, 2016, 291: 1545-1559], and the SOD activity of Arabidopsis plants was detected. The Arabidopsis plants were Arabidopsis plants without stress treatment for 2 weeks in the above potted identification and Arabidopsis plants treated with salt for 1 week in the above potted identification. The experiment was repeated three times, and the results were averaged.

[0299] (2) Test results

[0300] The experimental results are shown in Figure 6 Middle E (Normal is blank control, Salt stress is salt stress). The results showed that the SOD activity of transgenic Arabidopsis thaliana L1 plants, L2 plants, L3 plants and L4 plants was significantly higher than that of wild-type Arabidopsis thaliana plants.

[0301] 6. CAT Activity Assay

[0302] (1) Test method

[0303] Catalase (CAT) is widely present in plant tissues and is one of the important protective enzymes. Its function is to remove H2O2 produced during metabolism to avoid the oxidative damage of H2O2 accumulation to cells. Therefore, its activity is related to the stress resistance of plants.

[0304] The determination method refers to the literature [Yufeng Yang, Shikai Guan, Hong Zhai, Shaozhen He, Qingchang Liu. Development and evaluation of a storage root-bearing sweet potato somatic hybrid between Ipomoea batatas (L.) Lam. and I. triloba L. Plant Cell, Tissue and Organ Culture, 2009, 99: 83-89], and the CAT activity of Arabidopsis plants was detected. The Arabidopsis plants were Arabidopsis plants without stress treatment for 2 weeks in the above potted plant identification and Arabidopsis plants treated with salt for 1 week in the above potted plant identification. The experiment was repeated three times, and the results were averaged.

[0305] (2) Test results

[0306] The experimental results are shown in Figure 6 The results showed that the CAT activity of transgenic Arabidopsis thaliana L1, L2, L3 and L4 plants was significantly higher than that of wild-type Arabidopsis thaliana plants.

[0307] 7. APX Activity Assay

[0308] (1) Test method

[0309] Ascorbate peroxidase (APX) is one of the important antioxidant enzymes in plant active oxygen metabolism, especially the key enzyme for removing H2O2 in chloroplasts, and is also the main enzyme for vitamin C metabolism. APX activity can be used as a physiological and biochemical indicator of plant stress resistance, and its activity is related to the stress resistance of plants.

[0310] The determination method is based on the literature [Hong Zhai, Feibing Wang, Zengzhi Si, Jinxi Huo, Lei Xing, Yanyan An, Shaozhen He, Qingchang Liu. A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato. Plant Biotechnology Journal, 2016, 14: 592-602], and the APX activity of Arabidopsis plants is detected. The Arabidopsis plants are Arabidopsis plants without stress treatment for 2 weeks in the above potted identification and Arabidopsis plants treated with salt for 1 week in the above potted identification. The experiment needs to be repeated three times, and the results are averaged.

[0311] (2) Test results

[0312] The experimental results are shown in Figure 6 Middle G (Normal is blank control, Salt stress is salt stress, Drought stress is drought stress). The results showed that the APX activity of transgenic Arabidopsis thaliana L1 plants, L2 plants, L3 plants and L4 plants was significantly higher than that of wild-type Arabidopsis thaliana plants.

[0313] 8. POD activity assay

[0314] (1) Test method

[0315] Peroxidase (POD) activity can be used as a physiological and biochemical indicator of plant stress resistance. The lower the POD activity, the greater the degree of damage to the plant caused by adversity.

[0316] The determination method refers to the literature [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana. Molecular Genetics and Genomics, 2016, 291: 1545-1559], and the POD activity of Arabidopsis plants is detected. The Arabidopsis plants are Arabidopsis plants without stress treatment for 2 weeks in the above potted identification and Arabidopsis plants treated with salt for 1 week in the above potted identification. The experiment needs to be repeated three times, and the results are averaged.

[0317] (2) Test results

[0318] The experimental results are shown in Figure 6 The results showed that the POD activity of transgenic Arabidopsis thaliana L1, L2, L3 and L4 plants was significantly higher than that of wild-type Arabidopsis thaliana plants.

[0319] The results of physiological and biochemical indexes showed that overexpression of the AeLCYE gene significantly improved the salt tolerance of transgenic Arabidopsis plants.

[0320] <110>Huaiyin Institute of Technology

[0321] <120>Plant carotenoid biofortification and stress resistance-related protein AeLCYE and its encoding gene and application

[0322] <160>2

[0323] <210> SEQ ID NO 1

[0324] <211>1605

[0325] <212>DNA

[0326] <21 3> Okra (Abelmoschus esculentus L.)

[0327] <400>1

[0328]

[0329]

[0330] <210> SEQ ID NO 2

[0331] <211>534

[0332] <212>PRT

[0333] <213>Okra (Abelmoschus esculentus L.)

[0334] <400>2

[0335]

Claims

1. Use of a protein consisting of the amino acid sequence shown in SEQ ID NO 2 in improving the carotenoid content and / or salt tolerance of plants, characterized in that: The carotenoids include lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene, and the plant is Arabidopsis thaliana.

2. Use of an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene as shown in SEQ ID NO 1 for improving the carotenoid content and / or salt tolerance of plants, characterized in that: The carotenoids include lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene, and the plant is Arabidopsis thaliana.

3. A method for obtaining a plant with a high carotenoid content and / or high salt tolerance, characterized in that: Overexpressing the AeLCYE gene in the plant, wherein the AeLCYE gene sequence is shown in SEQ ID NO 1; The construction method of the AeLCYE gene overexpression vector used is: the DNA fragment of the AeLCYE gene is connected to the pYPx245 plasmid containing a double 35S promoter, and the expression vector also contains gusA Reporter gene and intronic kanamycin resistance marker gene; The carotenoids include lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene; The plant is Arabidopsis thaliana.

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