A kiwifruit drought-resistant gene AeNAC17 and its application

Abstract

The present invention relates to the field of genetic engineering technology, and provides a kiwifruit drought-resistant gene AeNAC17 and its application. The kiwifruit drought-resistant gene AeNAC17, and the nucleic acid molecule of the kiwifruit drought-resistant gene AeNAC17 is any one of the following nucleic acid molecules S1 to S2: S1, a nucleic acid molecule whose coding region is the nucleic acid molecule shown in SEQ ID NO: 1; S2, a nucleic acid molecule whose nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO: 1. Through the above technical solution, the problem of poor drought resistance of kiwifruit in the related art is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and specifically, to a kiwifruit drought-resistant gene AeNAC17 and its application. Background Art

[0002] Drought seriously affects the growth and development process of plants, as well as the yield and quality of crops. Drought has become one of the most serious factors restricting agricultural production. Improving the adaptability of crops to adversity through genetic engineering methods is a key issue that needs to be solved in the process of cultivating new crop varieties. In recent years, with the continuous in-depth understanding of plant metabolism, physiology, biochemistry and ecological adaptation under adversity stress such as drought, the process of how plants respond to adversity stress has been gradually revealed. Especially with the continuous development of molecular biology techniques and theories, people can explore the resistance mechanism of plants to adversity stress such as drought at the molecular level of gene composition, expression regulation and signal transduction, providing new means for improving the tolerance of crops to adversity stress by using genetic engineering means. Due to the complexity of plant stress resistance traits, it is difficult to effectively and directly improve plant stress resistance by using traditional breeding methods. With the development of molecular biology, genetic engineering means have opened up a new way for plant stress resistance breeding, but the screening of highly effective stress resistance genes has become the primary factor restricting the application of plant stress resistance genetic engineering.

[0003] Kiwifruit ( Actinidia chinensis Planch ) is an important cash crop in the world. Because it is rich in vitamin C and beneficial to the body, it is deeply loved by consumers. As the origin of kiwifruit, China has a long cultivation history. At present, it has become the country with the largest planting area in the world and is also an important way to increase farmers' income. Cultivating kiwifruit varieties with excellent quality and drought resistance is an important measure to ensure the safe development of the national kiwifruit industry. The excavation of kiwifruit drought-resistant genes is the basis of kiwifruit drought-resistant molecular breeding. Summary of the Invention

[0004] The present invention provides a kiwifruit drought-resistant gene AeNAC17 and its application, which solves the problem of poor drought resistance of kiwifruit in related technologies.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention provides a kiwifruit drought-resistant gene AeNAC17, and the nucleic acid molecule of the kiwifruit drought-resistant gene AeNAC17 is any one of the following nucleic acid molecules S1 to S2:

[0007] S1: a nucleic acid molecule whose coding region is the nucleic acid molecule shown in SEQ ID NO: 1;

[0008] S2: a nucleic acid molecule whose nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO: 1.

[0009] As a further technical solution, the amino acid sequence is as shown in SEQ ID NO: 2.

[0010] The present invention also provides a recombinant plasmid, which contains the kiwifruit drought-resistant gene AeNAC17.

[0011] As a further technical solution, it is any one of the following recombinant plasmids A1 to A2:

[0012] A1. A recombinant plasmid obtained by inserting the nucleic acid molecule shown in SEQ ID NO: 1 into the recognition site of the restriction endonuclease BamHI of the pCAMBIA1302 vector;

[0013] A2. A recombinant plasmid obtained by inserting the nucleic acid molecule shown in SEQ ID NO: 1 between the recognition sites of the restriction endonucleases BamHI and XbaI of the pCAMBIA1302 vector.

[0014] The present invention also provides the application of the kiwifruit drought-resistant gene AeNAC17 or the recombinant plasmid or the expression cassette in at least one of the following B1 to B2:

[0015] B1. Regulating plant drought resistance;

[0016] B2. Cultivating transgenic plants with altered drought resistance.

[0017] As a further technical solution, the regulation of plant drought resistance is to increase plant drought resistance; the improvement of drought resistance is manifested in the overexpression of plant leaf discs.

[0018] As a further technical solution, the cultivation of transgenic plants with altered drought resistance is to cultivate transgenic plants with increased drought resistance.

[0019] As a further technical solution, the plant is any one of the following P1 to P4:

[0020] P1. Dicotyledonous plants;

[0021] P2. Plants of Actinidiaceae;

[0022] P3. Plants of Actinidia;

[0023] P4. Kiwifruit.

[0024] The present invention also provides a method for obtaining transgenic plants, which includes the following steps: introducing the recombinant plasmid into a recipient plant to heterologously express the kiwifruit drought-resistant gene AeNAC17 in the recipient plant, thereby obtaining transgenic plants.

[0025] The working principle and beneficial effects of the present invention are as follows:

[0026] In the present invention, the drought-resistant gene AeNAC17 of kiwifruit was discovered, and AeNAC17 was transiently overexpressed in kiwifruit leaf discs to obtain kiwifruit overexpressing the AeNAC17 gene. The kiwifruit overexpressing the AeNAC17 gene was subjected to drought treatment: the wet weight of the leaf discs in the experimental group was significantly higher than that in the negative control group, and the relative wet weight of the leaf discs was basically the same as that in the positive control group. Overexpressing the AeNAC17 gene significantly retained the water in the kiwifruit leaves. Therefore, the drought-resistant gene AeNAC17 of kiwifruit in the present invention has important application value, improves the drought resistance of kiwifruit, and overexpressing AeNAC17 significantly retains the water in the kiwifruit leaves, and also has important application value in the research of improving plant drought resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0028] Figure 1 For Embodiment 1 of the present invention, the evolutionary relationship of homologous genes of AeNAC17 in different species;

[0029] Figure 2 For Embodiment 1 of the present invention, the agarose gel electrophoresis result of the full-length cDNA of the AeNAC17 gene in the PCR amplification product;

[0030] Figure 3 For Embodiment 1 of the present invention, the drought treatment of kiwifruit leaf discs with transient overexpression;

[0031] Figure 4 For Embodiment 1 of the present invention, detecting the relative wet weight of leaf discs;

[0032] Figure 5 For Embodiment 1 of the present invention, detecting the relative expression levels of AeNAC17 and AeDREB2A genes in overexpressed leaf discs by real-time fluorescence quantitative PCR. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present invention.

[0034] In the following quantitative experiments of the embodiments, three repeated experiments are set, and the results are averaged.

[0035] The pCAMBIA1302 vector is described in the following literature: Rizwan HM, Yang Q, Yousef AF, et al. Establishment of a Novel and Efficient Agrobacterium-Mediated in Planta Transformation System for Passion Fruit (Passiflora edulis). Plants (Basel). 2021;10(11):2459. Published 2021 Nov 15. doi:10.3390 / plants10112459;

[0036] Obtaining the positive control AeDREB2A sequence: By referring to the literature (Reis RR, da Cunha BA, Martins PK, et al. Induced over-expression of AtDREB2A CA improves drought tolerance in sugarcane. Plant Sci. 2014;221-222:59-68. doi:10.1016 / j.plantsci.2014.02.003), it is known that the DREB2A gene is an important marker gene in Arabidopsis thaliana, and its drought resistance mechanism is clear. The homologous gene AeDREB2A (gene ID: Actinidia00973) sequence of this gene in kiwifruit was obtained by performing protein homology alignment in the kiwifruit database (https: / / kiwifruitgenome.org / ); the amino acid sequence of AeDREB2A is shown in SEQ ID NO:3.

[0037] The pCAMBIA1302 vector and the drought-sensitive kiwifruit variety 'Hongyang' involved in the following examples are germplasms cultivated in the greenhouse, and all other reagents and consumables are commercially available;

[0038] Exnase 5×CE II Buffer was purchased from Nanjing Novozymes Biotech Co., Ltd., product number: C112;

[0039] Green Taq Mix was purchased from Nanjing Novozymes Biotech Co., Ltd., product number P131-01.

[0040] Example 1

[0041] Obtaining the sequence of AeNAC17:

[0042] 1. Obtaining AeNAC17

[0043] ①Obtaining cDNA template

[0044] Select healthy and young leaves of Actinidia arguta, quickly place them in liquid nitrogen for quick freezing and grinding, extract total RNA of kiwifruit using Huayueyang Plant RNA Extraction Kit (product number: 0416 - 50), then synthesize cDNA using Thermo Fisher RevertAid™ FirstStrand cDNA Synthesis Kit (product number: K1662), and after diluting the cDNA 50 - fold, set it aside for use.

[0045] ②PCR amplification and identification of amplification products

[0046] First, design primers according to the CDS (coding sequence) region of the AeNAC17 gene in kiwifruit, as shown in Table 1 below.

[0047] Table 1 Primer sequences for recombinant vector construction

[0048]

[0049] Subsequently, use kiwifruit cDNA as a template for PCR amplification, perform agarose gel electrophoresis detection, check that the band size is correct, recover the gel and send it for sequencing, and finally obtain the sequence of AcNAC17. The nucleotide sequence of gene AcNAC17 is shown as SEQ ID NO:1, and the amino acid sequence of AcNAC17 is shown as SEQ ID NO:2. As Figure 1 shows the evolutionary relationship of homologous genes of the AeNAC17 gene in different species; as Figure 2 shows the agarose gel electrophoresis result of the full - length cDNA of the AeNAC17 gene of the PCR amplification product.

[0050] 2. Establishment of transient system for anti - ulcer disease gene

[0051] ①Construction of recombinant expression vector based on pCAMBIA1302 vector

[0052] According to the function of the pCAMBIA1302 vector, inserting the target gene after the 35S promoter has the function of gene over - expression. According to the available restriction enzyme sites (Nco I and Spe I) of the pCAMBIA1302 vector, use primer pairs with homologous restriction enzyme sites to re - PCR amplify the target gene fragment, purify the fragment and measure its concentration. The primer sequences for recombinant vector construction are shown in Table 2; taking AeNAC17 as an example, the other two genes have the same components except for different primers, and the amplification system is shown in Table 3.

[0053] Table 2 Primer sequences for recombinant vector construction

[0054]

[0055] Note: The underlined part is the corresponding restriction enzyme site.

[0056] Table 3 Amplification system

[0057]

[0058] Meanwhile, the pCAMBIA1302 empty vector Escherichia coli was activated and cultured with shaking to extract the plasmid, and the concentration was detected. Then, double digestion was performed on it. The double digestion operation system is shown in Table 4:

[0059] Table 4 Double digestion operation system

[0060]

[0061] Double digestion operation process: Digest at 37 °C for 1 h in a water bath. After digestion, place it at 85 °C for 10 min to inactivate the enzyme. Detect by 1.0% agarose gel electrophoresis and purify and recover the target fragment.

[0062] The mixture was placed in a PCR instrument and ligated at 37 °C for 30 min, and then transformed into Escherichia coli competent cells DH5α (Beijing Tsingke Biotechnology Co., Ltd., product number: TSC-C14), and recombinant screening and sequencing verification were carried out. Among them, the ligation system is shown in Table 5:

[0063] Table 5 Ligation system

[0064]

[0065] For the plasmid verified to be correct by sequencing, 0.5 μL was aspirated and cultured overnight with shaking at 37 °C and 200 rpm in 5 mL of LB / Kan+ liquid medium. The plasmid in Escherichia coli was extracted using a plasmid recovery kit (source: Beijing Zhuangmeng International Biotechnology Co., Ltd., product number: ZPK101-3). The recombinant plasmid pCAMBIA1302-AeNAC17 was transformed into Agrobacterium tumefaciens GV3101 (source: Shanghai Weidi Biotechnology Co., Ltd., product number: AC1001), cultured for 48 h. After being verified to be correct by PCR, a single colony was picked and placed in LB liquid medium (containing kanamycin and rifampicin) and cultured with shaking at 28 °C and 220 rpm for detection until OD600 = 0.6 - 0.8; the bacterial liquid was detected (the detection system is shown in Table 6, and the primer sequences are shown in Table 7 below). The detection program for the recombinant plasmid detection fragment with a size of 780 bp: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 3 min, 32 cycles; extension at 72 °C for 5 min.

[0066] Table 6 Detection system

[0067]

[0068] Table 7 Primer sequences for detecting recombinant plasmids

[0069]

[0070] ② Transient transformation of kiwifruit

[0071] The method for transient transformation of kiwifruit includes the following steps:

[0072] (1) Disinfection: Take the leaves of 'Hongyang' kiwifruit with normal growth, the same size and no disease spots (the kiwifruit grows in the greenhouse of Northwest A&F University). Place the collected leaves in a foam box. First, soak them in sodium hypochlorite (chemical formula: NaClO) with a concentration of 0.06% in the dark for 5 min, then wash them twice with 75% (v / v) alcohol, soak for 1 min before each wash. Wash them twice with sterile water. Then dry the excess water on the leaf surface with sterile filter paper.

[0073] (2) Leaf disc punching: Place the above-treated leaves on sterile filter paper and punch leaf discs with a sterile puncher with a pore size of 1 cm. When punching leaf discs, pay attention to avoiding the main veins and lateral veins of the leaves.

[0074] (3) Agrobacterium infection: Collect the above-mentioned Agrobacterium carrying the gene after shaking culture. Set up three groups of experiments, namely the blank control (OE-EV), the positive control group (OE-AeDREB2A), and the experimental group (OE-AeNAC17). Wash them twice with 10 mM MgCl2, and then resuspend them with the Agrobacterium infection solution MMA (0.2 mM AS (acetosyringone), 10 mM MgCl2, 10 mM MES with pH 5.6 (adjusted with NaOH)). Then adjust the OD600 concentration to 0.6 respectively and incubate in the dark for 3 h. Place the punched leaf discs in 50 mL centrifuge tubes containing the bacterial suspension respectively, mix well to make the leaf discs fully contact with the bacterial suspension. Use the vacuum method (0.2 Mpa) for vacuum infiltration (30 min) to make the bacterial suspension fully immerse into the leaf discs. Then take out the leaf discs, dry the surface moisture, place them on water agar with a mass concentration of 0.8%, and co-culture them in an incubator at 28 °C for 48 h.

[0075] ③ Simulated drought treatment and calculation of relative leaf weight

[0076] After blotting dry the surface moisture of the overexpressed kiwifruit leaf discs, place them in a dry petri dish, and then place them in a light and ventilated incubator at 25 °C for simulated drought treatment. Weigh the mass of the leaf discs at 0 h and 6 h of treatment respectively, and then dry the leaves and weigh the dry weight of the leaf discs. Among them, the ratio of the difference between the mass and dry weight of the leaf discs weighed at 0 h and 6 h to the dry weight is the relative moisture weight (%). Set up three groups of experiments: blank control (OE-EV), positive control group (OE-AeDREB2A), and experimental group (OE-AeNAC17), with three experimental replicates in each group.

[0077] Test results:

[0078] The drought treatment results of the leaf discs of the three groups of kiwifruit, namely the blank control (OE-EV), the positive control group (OE-AeDREB2A), and the experimental group (OE-AeNAC17), are as Figures 3 - 4 shown. It can be seen from the figure that compared with the leaf discs of the experimental group and the positive control group, the leaf discs of the blank control (OE-EV) after 6 hours of drying treatment are more wilted. Moreover, the wet weight of the leaf discs in the experimental group is significantly higher than that in the negative control group, and the relative moisture weight of the leaf discs is basically the same as that in the positive control group, indicating that overexpression of the AeNAC17 gene significantly retains the moisture in the kiwifruit leaves.

[0079] ④ Gene transient overexpression analysis

[0080] Extract the RNA of the leaf discs of 9 treatments, using OE-AeDREB2A as the positive control and OE-EV as the blank control. Take three biological replicates for each treatment. Reverse transcribe the RNA into cDNA with a final concentration of 2.0 μg / μL according to the method of transient transformation of kiwifruit. Dilute the reverse-transcribed cDNA 50 times for subsequent RT-qPCR determination. Design specific primers for each gene according to Primer3plus, and the primers are synthesized by Xi'an Tsingke Biotechnology Co., Ltd. The primer sequences are shown in Table 8; the reaction system for qPCR is shown in Table 9.

[0081] Table 8 Primer sequences

[0082]

[0083] Table 9 Reaction system for qPCR

[0084]

[0085] Note: ChamQ SYBR qPCR Master Mix (Q311) is purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0086] Loaded onto the LightCycler 96 (Roche, Switzerland, product number: LightCycler96) according to the reaction system in Table 9. The program was set as follows: pre-denaturation at 95°C for 30 seconds; cycling reaction for 40 cycles, with 95°C for 8 seconds and 60°C for 15 seconds; melting curve, 95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds. The final data was statistically analyzed using Graphpad prism 8.0 software.

[0087] Test results:

[0088] As Figure 5 shown by the relative expression levels of the AeNAC17 and AeDREB2A genes detected by real-time fluorescence quantitative PCR in overexpressing leaf discs, it can be seen that compared with the blank control (OE-EV) and the positive control group (OE-AeDREB2A), the relative expression levels in the experimental group (OE-AeNAC17) were higher in transgenic kiwifruit, indicating that plants containing the AeNAC17 gene have excellent drought resistance.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a kiwifruit drought-resistant gene AeNAC17, a recombinant plasmid containing the kiwifruit drought-resistant gene AeNAC17, or an expression cassette containing the kiwifruit drought-resistant gene AeNAC17 in at least one of the following B1 to B2: B1. Increase the drought resistance of kiwifruit; B2. Cultivate transgenic kiwifruit with increased drought resistance; The nucleic acid molecule of the kiwifruit drought-resistant gene AeNAC17 is any one of the following nucleic acid molecules shown in S1 to S2: S1. The coding region is the nucleic acid molecule shown in SEQ ID NO: 1; S2. The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO:

1.

2. Use of a protein encoded by the kiwifruit drought resistance gene AeNAC17 according to claim 1 in increasing the drought resistance of kiwifruit or cultivating transgenic kiwifruit with increased drought resistance, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that The recombinant plasmid containing the kiwifruit drought-resistant gene AeNAC17 is a recombinant plasmid obtained by inserting the nucleic acid molecule shown in SEQ ID NO: 1 into the pCAMBIA1302 vector.

4. The application according to claim 1, characterized in that, Increase the drought resistance of kiwifruit by overexpressing the kiwifruit drought-resistant gene AeNAC17.

5. The application according to claim 1, characterized in that The method for obtaining the transgenic kiwifruit includes the following steps: introducing the recombinant plasmid into the recipient kiwifruit, enabling the kiwifruit drought-resistant gene AeNAC17 to be heterologously expressed in the recipient kiwifruit, and obtaining the transgenic kiwifruit.

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