Application of tomato SlNF-YC9 gene and encoding protein thereof in regulating drought tolerance of tomato

Abstract

The invention relates to the technical field of biology, and particularly provides application of a tomato SlNF-YC9 gene with a nucleotide sequence as shown in SEQ ID NO: 1, a tomato SlNF-YC9 protein with an amino acid sequence as shown in SEQ ID NO: 2 or a biological material containing the tomato SlNF-YC9 gene with the nucleotide sequence as shown in SEQ ID NO: 1 in regulation and control of tomato drought tolerance or cultivation of drought-tolerant tomatoes. And a new gene resource is provided for improvement of drought-resistant varieties of tomatoes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a tomato SlNF-YC9 gene and a protein encoded therein in regulating drought resistance of tomatoes. Background Art

[0002] Tomato (Solanum lycopersicum L.), as an important economic vegetable crop, is widely planted around the world. Its fruit is not only delicious, but also rich in nutritional value, containing a variety of vitamins and trace mineral nutrients that are beneficial to the human body. Tomato fruits are rich in vitamin C, vitamin A, vitamin K, and minerals such as potassium and iron, which make them play an important role in promoting health and preventing diseases. Tomato cultivation has a long history and originated in the Andes Mountains in South America. After a long period of domestication and breeding, there are now thousands of different varieties. These varieties have their own characteristics in appearance, color, size and taste, meeting the needs of different regions and consumers. From cherry tomatoes to ox heart tomatoes, from red to yellow, green and even black, the diversity of tomatoes demonstrates the wisdom and creativity of human agricultural civilization.

[0003] In agricultural production, tomato cultivation has certain requirements for environmental conditions. Drought, in particular, as a common natural disaster, has a serious impact on the growth and yield of tomatoes. Therefore, studying and utilizing tomato drought-resistant genes and improving varieties through molecular biology and genetic engineering are of great significance for improving tomato drought resistance, stabilizing yields, and ensuring food safety.

[0004] In recent years, scientists have made significant progress in the study of tomato drought resistance genes. For example, the role of genes such as CPK27 and BBX18 in tomato drought resistance has been discovered. These research results provide theoretical basis and technical support for the breeding of new drought-resistant tomato varieties. Through technical means such as whole genome association analysis, researchers are constantly exploring and analyzing the genetic basis of tomato drought resistance, in order to reduce the impact of drought on tomato yields in future agricultural production and promote the sustainable development of the tomato industry.

[0005] Therefore, creating new drought-resistant tomato materials and cultivating new drought-resistant varieties are of great significance to ensuring stable and increased tomato production. Summary of the invention

[0006] In view of the problem that there is a lack of drought-resistant tomato varieties in the prior art, the present invention provides an application of the tomato SlNF-YC9 gene and its encoded protein in regulating the drought resistance of tomatoes. Studies have found that transgenic plants overexpressing SlNF-YC9 are more drought-resistant, and the drought resistance of tomato plants obtained by knocking out the SlNF-YC9 gene using gene editing technology is significantly reduced, providing genetic resources for the improvement of drought-resistant tomato varieties.

[0007] Nuclear Factor-Y (NF-Y) is also known as CCAAT binding factor (CBF) and heme-activator proteins (HAP). NF-Y includes three subclass members: NF-YA, NF-YB, and NF-YC. In animals, a single subunit is usually encoded by a single gene, but in plants, it exists as a gene family. For example, in corn, there are 14, 18, and 18 NF-YA, NF-YB, and NF-YC members, respectively (Zhang et al., 2016), and in Arabidopsis, there are 10, 13, and 13 subunits, respectively (Laloum et al., 2013). NF-Y transcription factors not only regulate plant growth and development, but also play an important role in drought response. For example, PdNF-YB21, which is specifically expressed in poplar roots, can enhance the drought tolerance of overexpressing poplar plants by increasing the content of abscisic acid in the roots (Zhou et al., 2020). Soybean GmNF-YC14 / GmNF-YB2 / GmNF-YA16 form a trimer, regulate and enhance the expression of ABA receptor GmPYR1, thereby enhancing ABA signal transduction, and ultimately improving the drought tolerance of transgenic soybeans (Yu et al., 2021). Under normal conditions and drought stress, overexpression of sweet orange CsNF-YA5 in tobacco significantly reduced H2O2 production and increased photosynthetic rate (Pereira et al., 2018). Although some progress has been made in the study of NF-Y transcription factors, there are few reports on the involvement of tomato NF-YC members in tomato drought tolerance.

[0008] The first objective of the present invention is to provide an application of a tomato SlNF-YC9 gene in regulating tomato drought resistance, wherein the nucleotide sequence of the tomato SlNF-YC9 gene is shown in SEQ ID NO: 1; and regulating the overexpression of the tomato SlNF-YC9 gene to improve tomato drought resistance.

[0009] The second object of the present invention is to provide an application of the tomato SlNF-YC9 gene in cultivating drought-resistant tomatoes, the nucleotide sequence of the tomato SlNF-YC9 gene is shown in SEQ ID NO: 1; to construct a transgenic tomato plant overexpressing the tomato SlNF-YC9 gene to improve the drought resistance of tomatoes.

[0010] Furthermore, the transgenic tomato plant is constructed by Agrobacterium-mediated method.

[0011] Furthermore, the Agrobacterium-mediated method includes constructing a recombinant vector for overexpressing the tomato SlNF-YC9 gene, transforming Agrobacterium using the recombinant vector, and the initial vector of the recombinant vector is pHellsgate2.

[0012] The third object of the present invention is to provide an application of tomato SlNF-YC9 protein in regulating tomato drought resistance. The amino acid sequence of tomato SlNF-YC9 protein is shown in SEQ ID NO:2.

[0013] The fourth object of the present invention is to provide the use of tomato SlNF-YC9 protein in cultivating drought-tolerant tomatoes, and the amino acid sequence of tomato SlNF-YC9 protein is shown in SEQ ID NO:2.

[0014] The fifth objective of the present invention is to provide a biological material containing a nucleotide sequence such as the tomato SlNF-YC9 gene of SEQ ID NO: 1 for use in regulating tomato drought tolerance.

[0015] The sixth object of the present invention is to provide a biological material containing a tomato SlNF-YC9 gene of a nucleotide sequence such as SEQ ID NO: 1 for use in cultivating drought-tolerant tomatoes.

[0016] Furthermore, the biological material is a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

[0017] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0018] The study found that transgenic plants overexpressing SlNF-YC9 were more drought-resistant, while the drought resistance of tomato plants obtained by knocking out the SlNF-YC9 gene using gene editing technology was significantly reduced. The present invention provides a tomato SlNF-YC9 gene with a nucleotide sequence as shown in SEQ ID NO: 1, a tomato SlNF-YC9 protein with an amino acid sequence as shown in SEQ ID NO: 2, or a biological material containing a tomato SlNF-YC9 gene with a nucleotide sequence as shown in SEQ ID NO: 1 for use in regulating tomato drought resistance or cultivating drought-resistant tomatoes, providing new gene resources for improving drought-resistant tomato varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the results of detecting the expression level of SlNF-YC9 in tomato leaves of the wild-type tomato variety M82 in Example 1 after drought treatment for different days and rehydration after 5 days of treatment.

[0020] Figure 2 This is a graph showing the expression level of the SlNF-YC9 gene in the wild-type plants and overexpression plants in Example 3.

[0021] Figure 3 This is a diagram showing the sequencing results of the T0 generation knockout plants obtained in Example 3; wherein, Figure 3 A represents the gene editing site map of the T0 generation knockout plants; Figure 3 B represents the genetic difference map between the wild-type plant, the knockout line KO-13 and the knockout line KO-23; Figure 3 C represents the amino acid difference map between the wild-type plant, the knockout line KO-13 and the knockout line KO-23.

[0022] Figure 4 The drought stress phenotype diagram of the overexpression strain, knockout strain and wild-type plant AC under normal and drought treatments in Example 4; among them, OE-5 and OE-10 are overexpression strains, and KO-13 and KO-23 are knockout strains.

[0023] Figure 5 The figure is a test result of drought tolerance of overexpression strains, knockout strains and wild-type plants AC under normal and drought treatments in Example 4; wherein, Figure 5 A represents the conductivity measurement result diagram; Figure 5 B represents the result of MDA content determination; Figure 5 C represents the result of SOD enzyme activity determination; Figure 5 D represents the result of POD enzyme activity assay; among them, OE-5 and OE-10 are overexpression strains, and KO-13 and KO-23 are knockout strains. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] Example 1

[0027] Screening of drought-responsive gene SlNF-YC9

[0028] The wild-type tomato variety M82 that grew normally to the age of one month was used for drought treatment at 25℃, 16h light / 20℃, 8h dark. Tomato leaves were sampled after 1d, 2d, 3d, 4d, and 5d of drought and rehydration for transcriptome sequencing analysis.

[0029] Results: Figure 1As shown in the figure, analysis of the transcriptome results showed that the FPKM value of SlNF-YC9 expression was 3.45 times that of CK at 1 day of drought treatment and 5.11 times that of CK at 2 days. It then maintained a high expression until the expression level dropped to the level before drought treatment after rehydration.

[0030] Example 2

[0031] Construction of SlNF-YC9 overexpression and knockout vectors

[0032] Construction of SlNF-YC9 overexpression vector: Specific primers OE-SlNF-YC9-F (sequence: TTGGAGAGGACACGCTCGAGATGGATCAACATGGAAATGGACAGC) and OE-SlNF-YC9-R (sequence: CATTAAAGCAGGACTCTAGATTAAGAATCTGAGGGTGGTTGC) were designed according to the sequence. The SlNF-YC9 fragment was amplified using a high-fidelity DNA polymerase (Nanjing Novozyme Super-Fidelity DNA Polymerase, Catalog No.: P501-d1), and then the pHellsgate2 vector was double-digested with XbaI and XhoI to linearize it. The SlNF-YC9 fragment was ligated to the linearized vector using a homologous recombination enzyme (Nanjing Novozyme ClonExpress Ultra OneStep Cloning Kit, Catalog No.: C115-01) to obtain an overexpression vector driven by a 35S promoter. The recombinant plasmid was sent to Xi'an Qingke Company for sequencing, and the results of the detection primer sequences (F-GACGCACAATCCCACTATCC; R-GCTTCTGGATTCCTGTAATCTAAGC) were found to be completely consistent with the sequence published in the tomato genome. The nucleotide sequence of SlNF-YC9 is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2.

[0033] The SlNF-YC9 gene sequence, SEQ ID NO.1 is as follows:

[0034] ATGGATCAACATGGAAATGGACAGCCTCCAGGTATTGGAGTCGTTACTAGCTCAGCTCCAATATATGGTGCTCCATACCAAGCTAACCAAATGGCAGGGCCCTCTCCTCCTGCAGTTTCAGCTGGTGCAATTCAATCTCCTCAAGCAGCTGGTCTTGCTGCTTCGTCAGCTCAGATGGCGCAACATCAGCTCGCTTATCAGCACATTCATCAGCAGCAGCAACAACAGTTGCAGCAACAACTCCAGACTTTCTGGGCAAATCAATATCAAGAAATCGAGCATGTTACTGATTTCAAGAATCATAGCTTGCCATTGGCAAGGATCAAGAAAATCATGAAAGCGGATGAAGATGTTAGGATGATATCTGCTGAAGCACCAGTCGTATTTGCTCGTGCCTGTGAGATGTTCATACTTGAATTGACACTGCGTGCATGGAACCACACTGAGGAGAACAAAAGGAGGACGCTGCAGAAAAATGATATCGCTGCAGCCATAACAAGGACTGACATATTTGATTTCTTAGTTGACATTGTCCCAAGAGAGGACTTGAAAGATGAGGTGCTTGCAACAATTCCTAGAGGAACGCTTCCTGTTGGAGGCCCAACTGAGGGTCTGCCATTCTATTATGGCATGCCACCACAATCTGCTCAACCGATTGGAGCTCCAGGGATGTACATGGGAAAGCCTGTCGATCAAGCTCTGTATGCCCAGCAGCCCCGCCCATATATGGCACAGCCAATTTGGCCCCAGCAGCAGCAACCACCCTCAGATTCTTAA。

[0035] The SlNF-YC9 protein sequence, SEQ ID NO.2, is as follows:

[0036] MDQHGNGQPPGIGVVTSSAPIYGAPYQANQMAGPSPPAVSAGAIQSPQAAGLAASSAQMAQHQLAYQHIHQQQQQQLQQQLQTFWANQYQEIEHVTDFKNHSLPLARIKKIMKADEDVRMISAEAPVVF ARACEMFILELTLRAWNHTEENKRRTLQKNDIAAAITRTDIFDFLVDIVPREDLKDEVLATIPRGTLPVGGPTEGLPFYYGMPPQSAQPIGAPGMYMGKPVDQALYAQQPRPYMAQPIWPQQQQPPSDS.

[0037] Construction of CRISPR / Cas9 vector containing SlNF-YC9 specific sgRNA: According to the genome sequence of SlNF-YC9, the sgRNA target sequence was designed using the CRISPR target design website (http: / / crispr.hzau.edu.cn / CRISPR / ). The SlNF-YC9 specific knockout target sequence is CATGGAAATGGACAGCCTCC. After annealing, the target sequence is connected with the AtU6-sgRNA-AtUBQ-Cas9 vector linearized by BbsI restriction enzyme digestion, and the plasmid is extracted after transformation of E. coli. The constructed Cas9 recombinant plasmid and pCAMBIA2301 vector are both double-digested with EcoRI and HindIII, and the plasmid is extracted after transformation of E. coli after ligation. The final vector is sent to Xi'an Qingke Company for sequencing.

[0038] Example 3

[0039] Obtaining tomato SlNF-YC9 gene overexpression and knockout plants

[0040] The correctly sequenced overexpression vector and knockout vector were transformed into Agrobacterium GV3101. The tomato cotyledons were infected with 30 mL of Agrobacterium GV3101 culture liquid transformed with SlNF-YC9 overexpression vector and knockout vector at OD 0.6, and then callus induction was performed using tomato screening medium, adventitious bud differentiation was performed using subculture medium, and rooting culture was performed using rooting medium, and finally rooted tissue culture seedlings were obtained.

[0041] Tomato screening medium: Weigh 4.43g MS powder (Phytotech, M519), dissolve in 800mL ultrapure water, add 30g sucrose, and dilute to 1000mL. Adjust pH to 5.8, add 7.5g agar, autoclave at 121℃ for 21min, and add 0.1mg / L IAA + 2mg / L zeatin (ZR) + 100mg / L kana + 360mg / L TMT after cooling.

[0042] Callus induction culture conditions: 16h light / 8h dark, 25 degrees, culture for 14 days.

[0043] Tomato subculture medium: Weigh 4.43g MS powder (Phytotech, M519), dissolve in 800mL ultrapure water, add 30g sucrose, and dilute to 1000mL. Adjust pH to 5.8, add 7.5g agar, autoclave at 121℃ for 21min, and add antibiotics 0.2mg / L zeatin (ZR) + 100mg / L kana + 360mg / L TMT after cooling.

[0044] Adventitious bud differentiation culture conditions: 16h light / 8h dark, 25 degrees, culture for 14 days.

[0045] Rooting medium: Weigh 4.43g MS powder (Phytotech, M519), dissolve in 800mL ultrapure water, add 30g sucrose, 2mg / L IBA, and dilute to 1000mL. Adjust pH to 5.8, add 7.5g agar, autoclave at 121℃ for 21min, and add antibiotics 360mg / L TMT + 50mg / L kana after cooling.

[0046] Rooting culture conditions: 16h light / 8h dark, 25 degrees, culture for 21 days.

[0047] Primers were used to detect positive overexpression plants, and real-time fluorescence quantitative PCR was used to detect the expression level of overexpression plants. Figure 2 As shown, the expression levels of SlNF-YC9 in the two overexpression lines were approximately 90.5 and 38.3 times that of the wild-type plants, respectively, indicating that the overexpression lines were successfully obtained. The target site sequences of the candidate knockout plants were amplified by PCR using Taq enzyme and sent to Xi'an Qingke Company for sequencing to detect whether editing occurred. After sequencing, it was found that the KO-13 plant had a 1 base deletion and the KO-23 plant had a 1 base insertion, both of which had frameshift mutations and caused premature termination of translation ( Figure 3 ). The above results indicate that knockout plants were successfully obtained.

[0048] Primers for constructing SlNF-YC9 knockout vector:

[0049] F GATTGCATGGAAATGGACAGCCTCC.

[0050] RAAACGGAGGCTGTCATTTCCATGC.

[0051] SlNF-YC9 knockout vector detection primers:

[0052] F CGGCGAGTTCTGTTAGGTCCTCTA.

[0053] R CTCCGTGGATACCGACCTTCCGC.

[0054] Primers for editing detection in SlNF-YC9 knockout plants:

[0055] F TCTTCGTGTGATTGATATCCAC.

[0056] RTGGTGCTTCAGCAGATATCATC.

[0057] SlNF-YC9 fluorescence quantitative primer:

[0058] F CGTTACTAGCTCAGCTCCAATA.

[0059] R GATGAATGTGCTGATAAGCGAG.

[0060] Example 4

[0061] Detection of drought tolerance in tomato SlNF-YC9 gene overexpression and knockout lines

[0062] Wild-type AC plants, overexpression lines, and knockout lines were all subjected to 12 days of continuous drought treatment. At the beginning of the experiment, overexpression plants and knockout plants showed similar phenotypes to AC plants. However, after 4 days of drought, AC plants began to show leaf wilting, knockout plants showed stronger wilting, while overexpression plants remained basically healthy ( Figure 4 ). Although all plants became wilted after 12 days of drought treatment, it was obvious that the knockout plants and AC plants showed more severe drought damage (such as stem breakage and petiole shedding) than the overexpression plants. The above results indicate that overexpression of SlNF-YC9 enhances the drought resistance of tomato plants. The overexpression plants showed increased activities of antioxidant enzymes SOD and POD and reduced membrane damage under drought stress, as shown by reduced electrolyte leakage and reduced malondialdehyde content, while the knockout lines showed the opposite ( Figure 5 However, under control conditions, no significant differences in these physiological and biochemical parameters were detected among AC, overexpression plants, and knockout plants.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of tomato SlNF-YC9 gene in regulating tomato drought tolerance, characterized in that: The nucleotide sequence of the tomato SlNF-YC9 gene is shown in SEQ ID NO: 1; regulating the overexpression of the tomato SlNF-YC9 gene to improve the drought tolerance of tomato.

2. Application of tomato SlNF-YC9 gene in breeding drought-tolerant tomatoes, characterized in that: The nucleotide sequence of the tomato SlNF-YC9 gene is shown in SEQ ID NO: 1; a transgenic tomato plant overexpressing the tomato SlNF-YC9 gene is constructed to improve the drought tolerance of tomatoes.

3. The use according to claim 1 or 2, characterized in that: Construction of transgenic tomato plants by Agrobacterium-mediated transgenesis.

4. The use according to claim 3, characterized in that The Agrobacterium-mediated method comprises constructing a recombinant vector for overexpressing the tomato SlNF-YC9 gene, and transforming Agrobacterium using the recombinant vector, wherein the initial vector of the recombinant vector is pHellsgate2.

5. Application of tomato SlNF-YC9 protein in regulating tomato drought tolerance, characterized in that: The amino acid sequence of tomato SlNF-YC9 protein is shown in SEQ ID NO:

2.

6. Application of tomato SlNF-YC9 protein in cultivating drought-tolerant tomatoes, characterized in that: The amino acid sequence of tomato SlNF-YC9 protein is shown in SEQ ID NO:

2.

7. Application of biological materials containing the tomato SlNF-YC9 gene with a nucleotide sequence such as SEQ ID NO: 1 in regulating tomato drought tolerance.

8. Use of biological materials containing the tomato S1NF-YC9 gene of the nucleotide sequence such as SEQ ID NO: 1 in cultivating drought-tolerant tomatoes.

9. The use according to claim 7 or 8, characterized in that The biological material is a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

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