Beta-ketoester acyl-CoA reductase KCR gene and application thereof in improving salt tolerance of plants

Abstract

The invention relates to the technical field of plant genetic engineering. The invention provides a beta-ketoester acyl-CoA reductase KCR gene, wherein the nucleotide sequence of the KCR gene is as shown in SEQ ID NO. 1. The invention also provides a recombinant vector. The invention also provides a recombinant bacterium. The invention also provides an application of the KCR gene, the recombinant vector or the recombinant bacterium in improving the salt tolerance of plants. The invention also provides application of the KCR gene, the recombinant vector or the recombinant bacterium in plant salt tolerance genetic improvement. The invention further provides a method for obtaining the salt-tolerant plant. After the KCR gene is over-expressed in the grape callus, the salt tolerance of the obtained transgenic grape callus is obviously improved. And after the gene is over-expressed in arabidopsis thaliana, the obtained transgenic arabidopsis thaliana epidermis wax is accumulated and the salt tolerance is obviously increased, so that a new gene resource can be provided for salt tolerance improvement and germplasm resource breeding of a grape cultivation variety.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to β-ketoacyl-CoA reductase KCR Genes and their application in improving salt tolerance in plants. Background Art

[0002] Grape( Vitis vinifera L. ) is one of the top ten fruits in the world. It has strong adaptability and high economic benefits. It is a widely cultivated fruit tree variety in my country. However, soil salinization is common in my country's main grape producing areas such as Ningxia, Xinjiang, the old Yellow River course, and the Jiaodong Peninsula. Soil salinization seriously affects the plant's physiological activities such as photosynthesis, oxidation balance, and transpiration, resulting in a decrease in grape yield and quality. Therefore, improving the adaptability of grapes to salinized soils and achieving salt-tolerant cultivation are important issues that need to be urgently addressed in grape production.

[0003] Under salt stress, multiple physiological and biochemical processes of plants change, which ultimately affect plant growth and development. Under salt stress, plants will cope with salt stress by maintaining ion homeostasis, accumulating soluble substances, and enhancing antioxidant defense systems. Salt-alkali stress affects the transport of ions and cannot provide energy to plants, thereby inhibiting plant photosynthesis. Osmotic regulating substances in plants, such as soluble sugars, soluble proteins, and proline, regulate intracellular osmotic pressure, improve water retention capacity, and maintain normal physiological metabolism of plants. Both biotic and abiotic stresses can trigger the production of reactive oxygen species (ROS). Under salt stress, ROS levels will increase dramatically, and excessive ROS will cause great damage to proteins, lipids, and nucleic acids, leading to cell damage and even death. In addition, salt-alkali stress can induce the expression of related resistance genes, which are mainly involved in osmotic regulation, ion homeostasis, oxidative activity, and hormone signal transduction.

[0004] Plant cuticle wax is a waxy substance covering the epidermis of terrestrial plants. It is an important barrier between plants and the external environment. It plays an important role in maintaining water balance, resisting pests and diseases, preventing mechanical damage, reducing ultraviolet radiation and maintaining fruit quality. Cuticle wax is mainly composed of membrane very long-chain fatty acids (VLCFAs), alcohols, aldehydes, esters and alkane organic matter. Its synthesis process involves the coordinated participation of multiple enzymes, mainly including long-chain acyl-CoA synthetase, β-ketoacyl-CoA synthase, β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydratase, acyl-CoA reductase and wax ester synthase. The corresponding coding gene is LACS1, KCS1, KCR1, CER1, CER2, CER4 and WSD1 wait.

[0005] In plants, the biosynthesis of VLCFAs is divided into two stages. The first stage is the de novo synthesis of fatty acids in plastids, which is completed by condensation, reduction, dehydration, and reduction under the catalysis of fatty acid synthase (FAS) to form free fatty acids. The second stage is the extension of the carbon chain. The carbon chain extension enzyme is a multi-enzyme complex composed of four different enzymes: β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and trans-2,3-enoyl-CoA reductase (ECR). These four enzymes are involved in the process of fatty acid carbon chain extension in turn, and finally generate very long chain fatty acids. VLCFAs play an important role in participating in the growth and development of plants and coping with various stresses. In jute, CcNAC1 interacts with KCS, ultimately promoting early flowering and enhancing drought tolerance. AKR2A can affect the biosynthesis of VLCFAs in Arabidopsis by interacting with KCS1, thereby participating in the regulation of cold tolerance. GbKCS14 The synthesis of VLCFAs responds to low temperature in the early stage of hair raising in sea island cotton. However, there is no research on the relationship between KCR and salt tolerance of grape plants. Summary of the invention

[0006] The present invention aims to provide a β-ketoacyl-CoA reductase KCR (NCBI Gene ID accession number: PP588439) gene and its application in improving plant salt tolerance. The present invention firstly KCR The gene was cloned and its expression characteristics were analyzed. Then its distribution in cells was observed by subcellular localization. A plant expression vector was constructed, grape callus and Arabidopsis were transformed, and the positive seedlings were identified. The salt tolerance was analyzed by salt stress treatment. The experiment showed that overexpression KCR Genes give transgenic grape callus higher salt tolerance.

[0007] To solve the above problems, the present invention provides the following technical solutions: In a first aspect, the present invention provides a β-ketoacyl-CoA reductase KCR Gene (hereinafter referred to as " KCR Gene" or " KCR”), the nucleotide sequence of the β-ketoacyl-CoA reductase KCR gene is shown in SEQ ID NO.1. The gene contains an open reading frame of 963 bp, 320 amino acids, and the KCR The gene is induced by salt and plays a role in plant tolerance to salt stress. The molecular formula of the KCR gene is C 1622 H 2584 N 426 O 436 S 13 , the predicted molecular weight is 3.54 kDa, the isoelectric point is 8.59, and the instability coefficient is 26.37.

[0008] β-ketoacyl-CoA reductase as described above KCR Gene, the β-ketoacyl-CoA reductase KCR The protein sequence encoded by the gene is shown in SEQ ID NO.2.

[0009] The KCR amplification primers, the forward primer is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4.

[0010] In a second aspect, the present invention provides a recombinant vector carrying the β-ketoacyl-CoA reductase KCR gene as described above.

[0011] In a third aspect, the present invention provides a recombinant bacterium, comprising the recombinant vector described above.

[0012] In a fourth aspect, the present invention provides a β-ketoacyl-CoA reductase as described above KCR Application of the gene, the recombinant vector as described above or the recombinant bacteria as described above in improving the salt tolerance of plants.

[0013] In the present invention, salt tolerance refers to the tolerance of plant materials to 100-250 mM NaCl salt treatment.

[0014] The application as described above, wherein the application is at least one of the following (1) to (4): (1) KCR Transient overexpression of the gene in plants significantly increased the accumulation of phenols, wax esters, and alkanes in leaves; (2) KCR Transient gene silencing in plants significantly reduced the accumulation of phenols, wax esters, and alkanes in leaves; (3) KCR The gene was overexpressed in grape callus. Under salt stress, the overexpressed grape callus increased the activities of SOD, POD, and CAT compared with wild-type grape callus; and reduced the content of MDA, H2O2, and superoxide anion accumulation. (4) KCR The gene was overexpressed in grape callus and significantly increased the expression of salt-responsive genes under salt stress. CAX, HATP, HKT2, HPP, SOS1, SORK The amount of expression.

[0015] In the above application, the plant material used for transient transformation is the leaves of 'Zuoyouhong' grape, and the grape callus is induced from 'Seedless White' grape. In a fifth aspect, the present invention provides a β-ketoacyl-CoA reductase as described above KCR The application of the gene, the recombinant vector or the recombinant bacteria in genetic improvement of plant salt tolerance. Preferably, the experimental material used for transient transformation of the plant is 'Zuoyouhong' grape, and the experimental material used for the study of salt tolerance of grape callus is 'Seedless White' grape callus and Arabidopsis thaliana.

[0016] The application as described above, wherein the application is at least one of the following (a) to (b): (a) KCR The gene is overexpressed in callus, and the callus is induced into seedlings; (b) KCR Genes are overexpressed in plants, and offspring are bred and screened.

[0017] In a sixth aspect, the present invention provides a method for improving plant salt tolerance, comprising: KCR The nucleotide sequence of the gene SEQ ID NO.1 was constructed into an expression vector to form a recombinant expression vector, and then the recombinant expression vector was transformed into a strain to obtain a strain carrying KCR The recombinant strain of the gene is then used to infect plants, causing the plants to carry the KCR genes, and ultimately through KCR The expression of the gene regulates the accumulation of epidermal wax and the biosynthesis of VLCFAs, thereby regulating the salt tolerance of the plant. Optionally, the plant part infected by the recombinant strain is a grape leaf, a grape callus or an Arabidopsis plant. Optionally, the plant is a 'Zuoyouhong' grape.

[0018] Compared with the existing technology, the effects and advantages of the present invention are: 1. The present invention will KCR After the gene was overexpressed in grape callus, the salt tolerance of the transgenic grape callus was significantly increased. After the gene was overexpressed in Arabidopsis, the transgenic Arabidopsis obtained had significantly increased epidermal wax accumulation and salt tolerance, which can provide new gene resources for the salt tolerance improvement of grape cultivars and the selection of germplasm resources.

[0019] 2. The β-ketoacyl-CoA reductase KCR of the present invention participates in the response of grape to salt stress by regulating the synthesis of cuticular waxes, explores the key genes of VLCFAs and cuticular waxes in response to grape salt stress, analyzes their biological functions and mechanisms of action, utilizes relevant gene resources to improve the salt tolerance of grape cultivars and select germplasm resources, and develops regulatory technologies to enhance the salt resistance of fruit trees, which is of great significance for improving the quality and efficiency of salt-tolerant cultivation of grapes and other fruit trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a technical flow chart of the present invention; Figure 2 For the present invention KCR Gene expression characteristics analysis diagram: A is the expression characteristics in different tissues and organs of grapes (roots, buds, tendrils, stems, leaves, and fruits); B is the expression characteristics at different treatment times; Figure 3 This is a schematic diagram of KCR subcellular localization of the present invention; Figure 4 For the present invention KCR Schematic diagram of the effects of transient overexpression and transient silencing on the epidermal wax morphology and content of grape leaves under salt stress (WT is wild-type grape leaves; OEKCR is transient overexpression KCR grape leaves; pTRV2-KCR is transient silencing KCR grape leaves); where: A is the wild-type, transient overexpression under non-salt stress and salt stress KCR and momentary silence KCR Wax morphology of grape leaf epidermis; B is wild type and transient overexpression under non-salt stress and salt stress KCR and momentary silence KCR Contents of VLCFAs components in grape leaves; C is wild type and transient overexpression under non-salt stress and salt stress KCR and momentary silence KCR Wax content of grape leaf epidermis; Figure 5 For the present invention KCR Schematic diagram of salt treatment phenotype and identification of overexpressed grape callus; A is before and after salt treatment KCR Phenotypes of overexpressing grape callus and wild-type grape callus; B is KCR Identification of overexpressed grape callus; C is a schematic diagram of the content of each component of VLCFAs; Figure 6 For the present invention KCRSchematic diagram of the determination of physiological indicators of overexpressed grape callus treated with salt; wherein: A is the malondialdehyde content of grape callus before and after low temperature treatment; B is the superoxide anion content of grape callus before and after salt treatment; C is the hydrogen peroxide content of grape callus before and after salt treatment; D is the SOD activity of grape callus before and after salt treatment; E is the POD activity of grape callus before and after salt treatment; F is the CAT activity of grape callus before and after salt treatment; Figure 7 AF is a salt-responsive gene in grape callus before and after salt treatment ( CAX, HATP, HKT2, HPP, SOS1, SORK ) expression level; Figure 8 For the present invention KCR Heterologous overexpression Arabidopsis plants; wherein: A is overexpression Arabidopsis selected by kanamycin; B is KCR Heterologous overexpression Arabidopsis expression detection; C is KCR Identification of heterologous overexpression Arabidopsis lines; Fig. 9 For the present invention KCR Schematic diagram of salt treatment phenotype and survival rate of heterologous overexpression Arabidopsis plants; A is the phenotype and germination rate of heterologous overexpression Arabidopsis seeds before and after salt treatment; B is the phenotype and taproot length of heterologous overexpression Arabidopsis seedlings and wild-type Arabidopsis seedlings before and after salt treatment; C is the phenotype of heterologous overexpression Arabidopsis seedlings and wild-type Arabidopsis seedlings before and after salt treatment; Fig.10 For the present invention KCR The morphological structure and content of epidermal wax in heterologous overexpressed Arabidopsis seedlings; A is the epidermal wax morphological structure before and after salt treatment of Arabidopsis; B is the content of each component of VLCFAs before and after salt treatment of Arabidopsis; C is the epidermal wax content of Arabidopsis before and after salt treatment; Fig.11 For the present invention KCR Schematic diagram of the measurement of physiological indicators of heterologous overexpressed Arabidopsis seedlings under salt treatment; where: A is the relative water content of Arabidopsis before and after salt treatment; B is the water loss rate of Arabidopsis leaves before and after salt treatment; Fig.12 A is the relative permeability of cell membrane of Arabidopsis before and after salt treatment; B is the malondialdehyde content of Arabidopsis before and after salt treatment; C is the superoxide anion content of Arabidopsis before and after salt treatment; D is the SOD activity of Arabidopsis before and after salt treatment; E is the POD activity of Arabidopsis thaliana before and after salt treatment; F is the CAT activity of Arabidopsis before and after salt treatment. DETAILED DESCRIPTION

[0021] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Figure 1 The present invention is a technical flow chart, the grape of the present invention KCR The gene was cloned and its expression characteristics were analyzed. Its distribution in cells was then observed through subcellular localization. A plant expression vector was constructed, grape callus and Arabidopsis were transformed, and their positive seedlings were identified. Their salt tolerance was analyzed through salt stress treatment.

[0025] Example 1 KCR cloning Grape KCR The cloning of the full-length cDNA of the gene was carried out using the cDNA of wine grape Zuoyouhong as a template and amplified using a high-fidelity enzyme. The amplification primer sequences were SEQ ID NO. 3 (forward primer) and SEQ ID NO. 4 (reverse primer) as shown below: Forward primer: 5'-ATGGAACTTTGTTTCATGGAAAAGCTCC -3', Reverse primer: 5'-CTCCTGCTTCCTAGACCCTTTGAGTT -3', The amplified product was purified and recovered using a gel recovery kit. The purified product was connected to the pTOPO-Blunt vector (purchased from Beijing PolyMei Biotechnology Co., Ltd.) using DNA cloning technology. The ligated product was then transformed into DH5α competent cells (purchased from Sangon Biotechnology Co., Ltd.), plated, shaken, and then positively identified. After obtaining the positive clone, it was sent to the biological company for sequencing. According to the sequencing results, KCR The full-length gene sequence. The sequencing results showed that the KCR sequence size was 963 bp, encoding 320 amino acids, and the molecular formula of KCR was C 1622 H 2584 N426 O 436 S 13 , the predicted molecular weight is 3.54 kDa, the isoelectric point is 8.59, and the instability coefficient is 26.37.

[0026] The sequenced CDS sequence is SEQ ID NO.1: ATGGAACTTTGTTTCATGGAAAAGCTCCAAACTCAGCCACTCTGGGTTATTGTGCTCTTTGCTGTGGGATGTTTATCGATTTTGAAATCTTTCCTAGCTATCCTCAATGGGGTCTACGTTTGTTTTCTCAGACCGGGCAAGAATCTCAAGAAATACGGGTCGTGGGCACTCGTGACTGCTCCCACGGACGGTATCGGAAAGGGTTTTGCCTTTGAATTGGCTCGAAAAGGGCTTAATCTGGTATTGGTGGGTCGGAATCCGGATAAGCTGAAGGATGTGTCCGATGCGATTCAATCCAAATATGGGAAGACCCAGATCAAGGCTGTTGTGGTTGATTTTGCTGGTGATATTTCCGAGGGTGTTCTGAAGATTCGAGAAGCTATTGAAGGACTGGATGTTGGGGTTTTGATCAATAATGTGGGGGTTTCATATCCATATGCCAGGTTTTTTCATGAAGTGGATGATGAGCTGTTGAAGAATTTGATTAAAGTGAATGTCGAGGGCACCACGAAGGTCACACAGGCTGTTCTTCCAGGGATGCTCAAGAGGAAGAAGGGTGCAATTGTCAATATTGGTTCAGGTGCTGCTATTGTGATTCCTTCAGATCCTCTTTATGCTGTTTATGCGGCCACAAAAGCGTATATTGATCAATTCTCAAGGTGCCTCTATGTTGAATATAAGAATAGCGGGATTGATGTGCAGTGTCAGGTTCCACTGTATGTGGCCACTAAGATGGCATCAATCAGGAGATCTTCCTTCTTAGTTCCCTCATCAGATGGCTATGCCCGTGCAGCCATTCGCTGGATAGGGTATGAACCACGCTGCACTCCTTATTGGCCCCATGTCCTTATCTGGGGTCTGGCATGCCAATTACCAGAGTGTGCTATTGATGCATGGCGCCTGGGATTCAACCTTAAGATTAGAAAGAGGGGACAACTCAAAGGGTCTAGGAAGCAGGAGTAG。

[0027] The amino acid sequence is: SEQ ID NO.2: MELCFMEKLQTQPLWVIVLFAVGCLSVLKSFLAILNGVYVCFLRPGKNLKKYGSWALVTAPTDGIGKGFAFELARKGLNLVLVGRNPDKLKDVSDAIQSKYGKTQIKAVVVDFAGDISEGVLKIREAILGLDVGVLINNVGVSYPYARFFHEVDDELLKN LIKVNVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYAVYAATKAYIDQFSRCLYVEYKNSGIDVQCQVPLYVATKMASIRRSSFLVPSSDGYARAAMRWIGYEPRCTPYWPHVLIWGLACQLPECAIDVWRLGFNLKIRKRGQLKGSRKQE.

[0028] Example 2 Salt treatment KCR Analysis of expression characteristics of 1. Experimental methods: One-month-old grape Zuoyouhong and Shuangyou resistant tissue culture seedlings, and Chardonnay and Cabernet Sauvignon sensitive tissue culture seedlings cultivated in a culture room with a light intensity of 200mmol / (m2.s), a light / dark cycle of 16 / 8h, and a temperature of 25±1℃ were treated with 150mM NaCl and their roots were soaked. The sampling time points were 0h, 3h, 6h, 9h, 12h, and 24h. After taking leaves at each time point, they were quickly frozen in liquid nitrogen and then placed in a refrigerator at 80℃ for subsequent gene expression pattern analysis.

[0029] One-year-old branches of "Zuoyouhong" were selected from different tissue organs (roots, stems, buds, tendrils, leaves and fruits) of grapes, 0.1 g of each was frozen in liquid nitrogen, and then placed in a refrigerator at 80°C for subsequent analysis of tissue expression characteristics.

[0030] Total RNA of grapes was extracted by CTAB method, and the first strand of cDNA was synthesized by M MLV reverse transcription kit (purchased from Shanghai Shangbao Biotechnology Co., Ltd.) as a template. KCR The salt treatment and tissue expression characteristics of the gene were analyzed, and the Real time PCR program was: 95℃60s, 95℃10s, 58℃20s, 72℃15s, 40 cycles. Each sample was repeated 3 times, and the reaction was performed using TaKaRa PCR fluorescence quantitative analyzer.

[0031] Actin in grape was used as the internal reference gene (forward primer: 5' ATAGAAGCAGCAAGGGA 3'; reverse primer: 5' TGAGGCTCTTACTAATG 3'). -△△CT The algorithm calculates gene expression.

[0032] KCR real-time quantitative primers (forward primer: 5' TGTGCTCTTTGCTGTGGGAT 3'; reverse primer: 5' GCCCACGACCCGTATTTCTT 3').

[0033] 2. Experimental results and analysis: from Figure 2 It can be seen that KCR Analysis of tissue expression characteristics revealed that Figure 2 ADisplay KCR Significant expression in leaves and fruits. Figure 2 B shows that the expression level in resistant varieties was significantly higher than that in sensitive varieties.

[0034] The above results show that KCR The gene is induced by salt stress and may play a role in salt stress tolerance in grape.

[0035] Example 3 KCR subcellular localization analysis 1. Experimental methods: The CDS fragment of KCR was amplified using the plasmid pTOPO-Blunt-KCR as a template and fused to the vector pSuper1300GFP (purchased from Shanghai Beino Biotechnology Co., Ltd.). The recombinant plasmid of pSuper1300 KCR GFP was then transformed into Agrobacterium GV3101 (purchased from Sangon Biotechnology Co., Ltd.) and then identified by colony PCR. Positive bacteria were selected for subsequent experiments. The constructed pSuper1300 KCR GFP and the positive control pSuper1300 GFP were injected into tobacco leaves, respectively, and the subcellular localization of KCR was observed using a laser confocal microscope after 3 days of expression.

[0036] 2. Experimental results and analysis: from Figure 3 It can be seen that KCR is distributed on the endoplasmic reticulum.

[0037] Example 4 Transient overexpression KCR Morphology and content of wax in grape leaf epidermis Cut off the leaves of the grape tissue culture seedlings, rinse them twice with sterile water and then use them for transient transformation infection. The operation is as follows: (1) p19 (purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.), pSuper1300 GFP and the target gene recombinant vector Agrobacterium GV3101 were streaked onto LB solid medium (containing 50 mg / L Rif and 50 mg / L Kan) and incubated at 28°C for 2-3 days to obtain monoclonal colonies; (2) Pick one monoclonal colony from each strain and place it in 10 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan. Incubate at 28°C, 220 rpm, and shake for 12-24 hours to fully activate the bacteria. (3) The activated Agrobacterium culture liquid was inoculated into the same LB liquid medium at a volume ratio of 1:30, and cultured at 28°C, 220r / min for 10-12h. The cells were collected by centrifugation at 4000r / min, and MES buffer (10mmol / LMES, 10mmol / LMgCl2, 150μmol / LAS, pH=5.6-5.7) was added to suspend the cells, and the OD600 was adjusted to 1.0; (4) Mix p19 and pSuper1300 GFP, and p19 and target gene bacterial solution in a volume ratio of 1:1, mix well and incubate in a dark incubator at 28°C for 2-3 hours to prepare the infection solution; (5) Use a syringe needle to lightly poke a few small holes on the grape leaves, completely immerse them in the prepared Agrobacterium infection solution, vacuum evacuate for 10 minutes, quickly release the air to allow the Agrobacterium to penetrate the grape leaves, and repeat 3 times. Then let it stand for 15 minutes, take out the infected leaves and wipe the bacterial solution, and spread them flat on a large dish soaked with filter paper with sterile water. Cultivate at room temperature, shade it for 1 day, and then culture it under normal light for 2-3 days, and then carry out subsequent experiments.

[0038] Example 5 Instantaneous silencing of KCR grape leaf epidermal wax morphology and content 1. Vector construction The CDS fragment of KCR was amplified using the pTOPO-Blunt-KCR plasmid as a template and connected to the pTRV2 vector (purchased from Changsha Abiwei Biotechnology Co., Ltd.). The constructed vector was transferred into the GV3101 competent cell after being sequenced correctly.

[0039] 2. VIGS infection Cut off the leaves of the grape tissue culture seedlings, rinse them twice with sterile water and then use them for transient transformation infection. The operation is as follows: (1) pTRV1 (purchased from Beijing Coolbo Technology Co., Ltd.), pTRV2 (purchased from Beijing Coolbo Technology Co., Ltd.) and the target gene recombinant vector Agrobacterium GV3101 were streaked onto LB (containing 50 mg / L Rif and 50 mg / L Kan) solid medium, and monoclonal colonies were obtained after inverted culture at 28°C for 2-3 days; (2) Pick one monoclonal colony from each strain and place it in 10 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan. Incubate at 28°C, 220 rpm, and shake for 12-24 hours to fully activate the bacteria. (3) The activated Agrobacterium culture liquid was inoculated into the same LB liquid medium at a volume ratio of 1:30, and cultured at 28°C, 220 r / min for 10-12 h. The culture was then centrifuged at 4000 r / min to collect the cells. MES buffer (10 mmol / L MES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6-5.7) was added to suspend the cells, and the OD600 was adjusted to 1.0. (4) Mix pTRV1 and pTRV2, and pTRV1 and target gene bacterial solution in a volume ratio of 1:1, mix well and culture in a dark incubator at 28°C for 2-3 hours to prepare the infection solution; (5) Use a syringe needle to lightly poke a few small holes on the grape leaves, completely immerse them in the prepared Agrobacterium infection solution, vacuum evacuate for 10 minutes, quickly release the air to allow the Agrobacterium to penetrate the grape leaves, and repeat 3 times. Then let it stand for 15 minutes, take out the infected leaves and wipe the bacterial solution, and spread them flat on a large dish soaked with filter paper with sterile water. Cultivate at room temperature, shade it for 1 day, and then culture it under normal light for 2-3 days, and then carry out subsequent experiments.

[0040] Example 6 Instantaneous transformation of KCR grape leaf epidermal wax morphology and content Grape leaves transiently overexpressing pSuper1300-KCR-GFP and grape leaves transiently silencing pTRV2-KCR were obtained by Agrobacterium infection and treated with 150 mM NaCl salt. The differences in VLCFAs and leaf epidermal wax content were analyzed by scanning electron microscopy and gas chromatography. Figure 4 shown.

[0041] from Figure 4 It can be seen that the results showed that under normal growth conditions, there was no significant difference in the morphology and content of epidermal wax in the leaves of transiently transformed grapes and empty vectors. However, after salt stress treatment, compared with the wild type, the VLCFAs content in the leaves of transiently overexpressed KCR grapes increased significantly, and the contents of VLCFAs components, especially C22 and C24, increased significantly. KCRThe content of VLCFAs in grape leaves was significantly reduced, and the content of VLCFAs components, especially C22 and C24, was significantly reduced ( Figure 4 B) After salt stress treatment, transient overexpression of KCR The wax crystals in the epidermis of grape leaves increased significantly, and the wax content in the epidermis increased significantly ( Figure 4 A, arrows indicate epidermal wax crystals), among which phenols, wax esters and alkanes increased significantly, while transient silence KCR The wax crystals in the epidermis of grape leaves were significantly reduced, and the epidermal wax content was reduced, especially the phenols, wax esters and alkanes were relatively reduced ( Figure 4 C).

[0042] The above results indicate that KCR regulates the synthesis of VLCFAs and cuticular waxes and participates in the process of plant resistance to salt stress.

[0043] Example 7 Genetic transformation of grape callus and identification of overexpression callus 1. Genetic transformation of grape callus (1) Preparation of plant materials The flower buds of grape variety 'seedless white' were used as experimental materials. They were cut into small pieces and placed in sterilized empty bottles. They were disinfected with 75% alcohol for 30 seconds, soaked and disinfected with 15-18% NaClO for 15 minutes, and washed with sterile water for 3-5 times. After disinfection, they were placed in the induction medium and cultured in the dark in a 25℃ light incubator. The induced grape embryonic callus was inoculated on the subculture medium and cultured in the dark in a 25℃ light incubator. It was subsequently used to obtain transgenic callus, observe low temperature phenotypes, and detect stress resistance physiological indicators. During this period, Agrobacterium bacterial liquid was prepared.

[0044] (2) Preparation of Agrobacterium infection solution In the clean bench, use a sterilized inoculation loop to pick up pSuper1300-KCR-GFP Agrobacterium stored at -80℃, streak on a culture medium containing 50mg / L Kan and Rif antibiotics, and place the culture medium in a 28℃ incubator for dark culture for 2 days. Take a sterilized 100mL conical flask and pour 50mL of liquid culture medium containing Kan and Rif antibiotics, scrape the grown Agrobacterium and dissolve it in the liquid culture medium, and shake at 28℃ 200r / min for 12h. Collect the bacteria and add liquid culture medium containing 20mg / LAS (Acetosyringone, acetosyringone) to adjust the concentration to OD600 value of 0.6-0.8.

[0045] (3) Infection and co-cultivation Place the well-growing grape embryonic callus in a culture dish containing filter paper, and use a pipette to drop the prepared Agrobacterium solution onto the grape callus. After standing for 5 minutes, take the filter paper to absorb the residual Agrobacterium solution to complete the infection. After removing the residual solution, place the grape callus evenly on the co-culture medium containing AS. Then, culture it in the dark at 25℃ for 3 days.

[0046] (4) Screening, culture and regeneration The grape embryonic callus cultured in the dark for 3 days was taken out and transferred to the screening medium containing 50 mg / L Hyp.

[0047] The formula of the culture medium used in the experiment is shown in Table 1. The prepared culture medium should be sterilized at 115℃ for 20 minutes, and after adding the filtered and sterilized antibiotics in the clean bench, it should be divided into culture dishes or conical bottles that have also been sterilized at 115℃ for 20 minutes, and sealed for later use. Figure 5 shown.

[0048] Table 1 Culture medium formula

[0049] 2. Identification of positive seedlings from Figure 5 It can be seen that the real-time fluorescence quantitative analysis of overexpression callus KCR The results showed that compared with the wild type, the overexpression callus KCR The relative expression level of Figure 5 B) The obtained positive transgenic callus was used for subsequent salt tolerance analysis.

[0050] Example 8 Analysis of salt tolerance of overexpressed grape callus Before salt treatment, there was no obvious phenotypic difference between wild-type and overexpression grape callus, but after 7 days of treatment with 0, 50, 100, and 150 mM NaCl, the phenotypes were observed and the differences between wild-type and overexpression grape callus were detected by gas chromatography. KCR Overexpression of VLCFAs in callus.

[0051] from Figure 5 As can be seen, the results showed that after treatment with 100 m M NaCl, most of the wild-type grape callus turned brown and black, and its growth was significantly inhibited. However, the overexpression grape callus was still golden in color and was less damaged ( Figure 5 A) Under salt stress, all components of VLCFAs increased, and KCR The increase in overexpression callus was significantly higher than that of wild type, especially C24 and C26, suggesting that KCR Salt stress promoted the accumulation of VLCFAs ( Figure 5C).

[0052] The above results suggest KCR Salt stress promoted the accumulation of VLCFAs and participated in the salt stress response of grapes.

[0053] Under normal conditions, wild-type and KCR There was no significant difference in the physiological parameters of overexpression callus. Figure 6 It can be seen that after 100mM NaC1 treatment, the results showed that compared with the wild-type callus, KCR The contents of SOD, POD and CAT in overexpressed grape callus were significantly increased ( Figure 6 DF), MDA content, superoxide anion content, and hydrogen peroxide content decreased ( Figure 6 AC).

[0054] This indicates that overexpression of KCR can promote the accumulation of osmotic substances in transgenic materials, reduce ROS content, reduce cell membrane lipid peroxidation, and improve the resistance of grapes to salt stress.

[0055] from Figure 7 It can be seen that before and after salt treatment, there was no significant difference in the relative expression of salt-responsive genes in wild-type grape callus and overexpression grape callus, but after salt treatment, compared with wild-type grape callus, the relative expression of salt-responsive genes in overexpression grape callus ( CAX, HATP, HKT2, HPP, SOS1, SORK ) was significantly upregulated ( Figure 7 Middle AF, the first from the left in each group is the wild-type grape callus without salt treatment, and the second is the overexpression without salt treatment. KCR Grape callus tissue, the third one is wild-type grape callus tissue treated with salt, and the fourth one is overexpressed in salt treatment KCR Grape callus).

[0056] In conclusion, the salt tolerance phenotype observation and physiological index determination showed that overexpression KCR Genes give transgenic grape callus higher salt tolerance.

[0057] Example 9 Identification of positive seedlings of overexpressed Arabidopsis Using the "inflorescence infection method", KCR was introduced into Arabidopsis thaliana by Agrobacterium, and the first-generation seeds obtained were screened using a medium containing kanamycin ( Figure 8 A), Real-time fluorescence quantitative analysis of Arabidopsis plants overexpressing KCR The results showed that compared with the wild type, the overexpression of Arabidopsis KCR The relative expression level of Figure 8 B) and semi-quantitative analysis of KCR Heterologous overexpression Arabidopsis lines were identified ( Figure 8 C), the obtained positive transgenic Arabidopsis plants were used for subsequent salt tolerance analysis. KCR The homozygous Arabidopsis lines with different expression levels were used for subsequent experiments. OEKCR-3, OEKCR-7 and OEKCR-9 .

[0058] Example 10 Analysis of salt tolerance of overexpressed Arabidopsis In order to further verify the biological function of KCR in salt stress response, KCR Heterologous overexpression in Arabidopsis thaliana. KCR Heterologous overexpression of Arabidopsis seeds and wild type and 14 days after germination KCR Heterologous overexpression Arabidopsis seedlings were treated with 100 mM NaCl to observe phenotypes and detect related physiological indicators; wild-type and non-bolting Arabidopsis seedlings that were about 4 weeks old were selected. KCR Heterologous overexpression Arabidopsis seedlings were treated with 250 mM NaCl for 7 days, and the phenotypes were observed and related physiological indices were detected. Scanning electron microscopy and gas chromatography were used to analyze the differences in VLCFAs and leaf epidermal wax content. Fig. 9 shown.

[0059] from Fig. 9 As can be seen, the results showed that under normal conditions, there was no significant difference in the morphology between wild type and heterologous overexpression Arabidopsis. However, under salt stress, KCR Heterologous overexpression of Arabidopsis thaliana plants showed obvious salt stress tolerance phenotypes, with seed germination rate and taproot length significantly higher than those of wild type, while the leaf wilting rate of Arabidopsis thaliana seedlings was significantly lower than that of wild type ( Fig. 9 medium AC).

[0060] from Fig.10 It can be seen that after salt stress treatment, compared with the wild type, KCR Heterologous overexpression of Arabidopsis thaliana increased the accumulation of wax crystals in leaves ( Fig.10 A, arrows indicate wax crystals). The content of VLCFAs increased significantly, and the content of VLCFAs components, especially C24 and C26, increased significantly ( Fig.10 B), and KCR The accumulation of wax in the leaf epidermis of heterologous overexpression Arabidopsis thaliana increased significantly, and the contents of various wax components increased.

[0061] GC MS test results also showed that after salt treatment, KCR The accumulation of wax in the leaf epidermis of heterologous overexpressed Arabidopsis increased significantly, and the contents of various wax components increased, among which alkanes increased significantly ( Fig.10 C).

[0062] The above results indicate that KCR regulates the synthesis of VLCFAs and cuticular waxes and participates in the process of plant resistance to salt stress.

[0063] from Fig.11 It can be seen that the detection KCR Water content of heterologous overexpressed Arabidopsis. Under normal conditions, there was no significant difference in relative water content between wild-type and overexpressed Arabidopsis strains ( Fig.11 A, the first one from the left in each group is the relative water content of wild-type Arabidopsis without salt treatment, the second, third and fourth are the relative water content of heterologous overexpression without salt treatment OEKCR-3 , OEKCR-7 and OEKCR-9 The relative water content of Arabidopsis thaliana. The fifth is the relative water content of wild-type Arabidopsis thaliana treated with salt. The sixth, seventh and eighth are the relative water content of heterologous overexpression treated with salt. OEKCR-3 , OEKCR-7 and OEKCR-9 Relative water content of Arabidopsis thaliana), after salt stress, the water loss rate of leaves of the three overexpression lines was lower than that of wild-type Arabidopsis. At 5 h of salt stress, the water loss rate of leaves of wild-type Arabidopsis reached 50%, while the water loss rate of leaves of overexpression lines was between 30% and 40% ( Fig.11 B); the relative water content of the overexpression strain was significantly higher than that of the wild type ( Fig.11 A).

[0064] from Fig.12 It can be seen that salt stress increased the relative permeability of Arabidopsis leaves and promoted the MDA content in Arabidopsis leaves, but the relative permeability of the cell membrane and MDA in the overexpression strain were significantly lower than those in WT ( Fig.12 A and Fig.12 B, the first one from the left in each group is the relative permeability and MDA content of wild-type Arabidopsis cells without salt treatment, the second, third and fourth are heterologous overexpression without salt treatment OEKCR-3 , OEKCR-7 and OEKCR-9 The relative permeability and MDA content of Arabidopsis cell membrane. The fifth is the relative permeability and MDA content of wild-type Arabidopsis cell membrane treated with salt. The sixth, seventh and eighth are heterologous overexpression treated with salt. OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis cell membrane relative permeability and MDA content). The SOD, POD, CAT activities and superoxide anion content in the overexpression strain were significantly higher than those in the wild type ( Fig.12 CF, the first one from the left in each group is the superoxide anion content, SOD activity, POD activity and CAT activity of wild-type Arabidopsis without salt treatment, the second, third and fourth are heterologous overexpression without salt treatment OEKCR-3 , OEKCR-7 and OEKCR-9Arabidopsis superoxide anion content, SOD activity, POD activity and CAT activity. The fifth is the superoxide anion content, SOD activity, POD activity and CAT activity of wild-type Arabidopsis treated with salt. The sixth, seventh and eighth are heterologous overexpression treated with salt. OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis superoxide anion content, SOD activity, POD activity and CAT activity).

[0065] The above results show that under salt stress, KCR can reduce water loss to maintain plant water content, reduce the accumulation of reactive oxygen species by increasing the activity of antioxidant enzymes in Arabidopsis leaves, and alleviate membrane oxidative damage caused by salt stress.

[0066] It should be pointed out that the specific implementation methods are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-mentioned embodiments, and there are many variations. Those skilled in the art who can obtain the invention clearly disclosed in the present invention or without objection based on the written description of the document should be considered as the scope of protection of this patent.

Claims

1. A β-ketoacyl-CoA reductase KCR A gene characterized by The β-ketoacyl-CoA reductase KCR The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The β-ketoacyl-CoA reductase according to claim 1 KCR A gene characterized by The β-ketoacyl-CoA reductase KCR The protein sequence encoded by the gene is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: Carrying the β-ketoacyl-CoA reductase as claimed in claim 1 KCR Gene.

4. A recombinant bacterium, characterized in that: Comprising the recombinant vector according to claim 3.

5. The β-ketoacyl-CoA reductase according to any one of claims 1 to 2 KCR Use of the gene, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in improving the salt tolerance of plants.

6. The use according to claim 5, characterized in that: The application is at least one of the following (1) to (4): (1) KCR Transient overexpression of the gene in plants significantly increased the accumulation of phenols, wax esters, and alkanes in leaves; (2) KCR Transient gene silencing in plants significantly reduced the accumulation of phenols, wax esters, and alkanes in leaves; (3) KCR The gene was overexpressed in grape callus. Under salt stress, the overexpressed grape callus increased the activities of SOD, POD, and CAT compared with wild-type grape callus; and reduced the content of MDA, H2O2, and superoxide anion accumulation. (4) KCR The gene was overexpressed in grape callus and significantly increased the expression of salt-responsive genes under salt stress. CAX, HATP, HKT2, HPP, SOS1, SORK The expression amount.

7. The use according to any one of claims 5 to 6, characterized in that: The plant is grape, Arabidopsis or tobacco.

8. The β-ketoacyl-CoA reductase according to any one of claims 1 to 2 KCR Use of the gene, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in genetic improvement of plant salt tolerance.

9. The use according to claim 8, characterized in that: The application is at least one of the following (a) to (b): (a) KCR The gene is overexpressed in callus, and the callus is induced into seedlings; (b) KCR Genes are overexpressed in plants, and offspring are bred and screened.

10. A method for improving plant salt tolerance, characterized in that: The β-ketoacyl-CoA reductase of claim 1 KCR The nucleotide sequence of the gene SEQ ID NO.1 was constructed into an expression vector to form a recombinant expression vector, and then the recombinant expression vector was transformed into a strain to obtain a strain carrying KCR The recombinant strain of the gene is then used to infect plants, causing the plants to carry the KCR genes, and ultimately through KCR The expression of genes regulates the accumulation of cuticular waxes and the biosynthesis of VLCFAs, thereby regulating the salt tolerance of plants.

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