Salt stress positive regulation gene BcCRK35 and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a salt stress forward regulation gene BcCRK35 and application thereof. The nucleotide sequence of the BcCRK35 gene is as shown in SEQ ID NO. 1; the amino acid sequence is as shown in SEQ ID NO. 2; the gene can be used for regulating and controlling the salt tolerance of saccharomycetes and regulating and controlling the salt tolerance of non-heading Chinese cabbages. The gene is over-expressed in yeast, so that the tolerance of the yeast to salt stress is enhanced; the gene is knocked out or silenced, so that the salt tolerance of the non-heading Chinese cabbage is reduced. According to the invention, overexpression of constructed BcCRK35 yeast proves that the gene plays a positive regulation role in salt stress. Meanwhile, after the BcCRK35 gene is silenced, the tolerance of a silenced plant to salt stress is reduced. The invention lays a theoretical foundation for researching a plant salt stress response mechanism, also provides a gene resource for cultivating a new variety of salt-tolerant non-heading Chinese cabbage, and has a relatively good potential application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a salt stress positive regulatory gene BcCRK35 and its applications. Background Technology

[0002] Salt stress is one of the major abiotic stress factors restricting global crop production. With global climate change and the continued development of irrigated agriculture, soil salinization is becoming increasingly serious, with the global area of ​​saline soils reaching approximately 9.5 × 10⁻⁶. 6 km 2 The area of ​​saline soil in my country is approximately 7.5 × 10⁻⁶. 5 km 2 Salt stress severely restricts the sustainable development of agricultural production. Non-heading Chinese cabbage (Brassicarapa ssp. chinensis, also known as bok choy or Chinese cabbage), an important leafy vegetable crop, originated in China. It is highly adaptable, has a short growth cycle, and high nutritional value, making it a popular vegetable in southern my country and playing a vital role in the "vegetable basket" project. It is increasingly becoming a globally recognized vegetable. However, the yield and quality of non-heading Chinese cabbage are easily affected by salt stress, manifesting as growth inhibition, ion toxicity, and oxidative damage.

[0003] Salt stress affects non-heading Chinese cabbage throughout its entire growth cycle. Studies have shown that with increasing salt stress, the germination rate of seeds, root length, dry and fresh weight of roots, cotyledons, and hypocotyls, as well as respiration rate, all significantly decrease in non-heading Chinese cabbage. Under salt stress, the magnesium content in the leaves of non-heading Chinese cabbage... 2+ Ca 2+ and K + The content decreased, while Na + Increased Cl- content leads to ion imbalance. Furthermore, salt stress affects photosynthetic properties, reducing photosynthetic rate and pigment content, disrupting the stability of photosynthetic system II, and affecting pigment fluorescence properties. Oxidative damage induced by salt stress manifests as disorder of the antioxidant enzyme system, with abnormal activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT).

[0004] Although some salt tolerance-related genes in non-heading Chinese cabbage have been identified, such as the S44 gene (which helps enhance plant salt tolerance), the BrTLP gene family (especially BrTLP14, which responds most strongly to salt stress), and the BrNAC transcription factor gene (related to cold resistance and may be involved in salt tolerance response), the genetic mechanism of salt tolerance in non-heading Chinese cabbage is extremely complex, controlled by multiple genes and resulting from the synergistic effects of multiple genes. Currently, little is known about the molecular mechanisms of specific responses to salt stress in different cell types of non-heading Chinese cabbage. For example, studies have shown that root hairs are the most sensitive cell population to high salt stress. Salt stress inhibits root hair development, leading to decreased expression of the mature root hair-specific gene BcIRT2 and reduced iron uptake by the roots. Single-cell level studies have found that high salt and osmotic stress inhibit the transformation of differentiating root hairs into mature root hairs in a similar way, preventing root hairs from performing environmental responses and ion uptake functions—a non-adaptive response.

[0005] In recent years, researchers have explored various strategies to improve crop salt tolerance, including multi-omics approaches, microbial-mediated strategies, and genetic engineering. However, the effect of introducing individual functional genes through molecular breeding on improving plant stress resistance is very limited, and even with the combined introduction of multiple genes, the coordination between these genes remains a challenge. Some protein factors that play a regulatory role in signal transduction networks, especially kinase proteins, can initiate signal transduction networks through the expression of a single gene, activating the transcription and expression of numerous downstream functional genes and the activation of functional proteins, thereby achieving a comprehensive improvement in crop stress resistance. However, the genetic mechanism of salt tolerance in non-heading Chinese cabbage is complex, and the multi-gene synergistic regulatory network has not yet been systematically elucidated. Therefore, it is urgent to systematically identify key genes for salt tolerance in non-heading Chinese cabbage, analyze their molecular regulatory networks, and develop effective molecular breeding techniques to provide gene resources and theoretical basis for cultivating new salt-tolerant non-heading Chinese cabbage varieties. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a salt stress positive regulatory gene BcCRK35 and its application.

[0007] The objective of this invention is achieved through the following technical solution: a salt stress positive regulatory gene, wherein the gene is BcCRK35, and the nucleotide sequence is shown in SEQ ID NO.1.

[0008] As a preferred technical solution, the amino acid sequence of the BcCRK35 gene is shown in SEQ ID NO.2.

[0009] As a preferred technical solution, overexpression of the BcCRK35 gene enhances the tolerance of yeast to salt stress.

[0010] The aforementioned BcCRK35 gene is used to regulate the salt tolerance of non-heading Chinese cabbage.

[0011] As a preferred technical solution, knocking out or silencing the BcCRK35 gene can reduce the salt tolerance of non-heading Chinese cabbage.

[0012] Primer pairs for amplifying the BcCRK35 gene, the primer pairs including a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO.3; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0013] A biomaterial capable of regulating the expression level of the BcCRK35 gene includes a biomaterial overexpressing the BcCRK35 gene and a biomaterial knocking out or silencing the BcCRK35 gene; the nucleotide sequence of the BcCRK35 gene is shown in SEQ ID NO.1.

[0014] As a preferred technical solution, the biological material overexpressing the BcCRK35 gene is one or more of a recombinant expression vector containing the BcCRK35 gene or a transgenic cell line containing the BcCRK35 gene, and the basic backbone of the recombinant expression vector is the pYES2-NTB vector.

[0015] As a preferred technical solution, the biological material for knocking out or silencing the BcCRK35 gene is one or more of a recombinant vector for knocking out or silencing the BcCRK35 gene or a transgenic cell line for knocking out or silencing the BcCRK35 gene, and the basic backbone of the recombinant expression vector is the pK7GWIWG2 vector (I).

[0016] The application of the aforementioned primer pairs or biological materials in regulating the salt tolerance of non-heading Chinese cabbage.

[0017] This invention has the following advantages: It provides a salt stress positive regulatory gene, BcCRK35, and its application. The BcCRK35 gene is a salt stress positive regulatory gene that can be used to regulate the salt tolerance of yeast and the salt tolerance of non-heading Chinese cabbage. This invention confirms the positive regulatory role of the BcCRK35 gene in salt stress through overexpression of constructed BcCRK35 yeast. Simultaneously, silencing the BcCRK35 gene leads to a decrease in the salt stress tolerance of the silenced plants. The BcCRK35 gene obtained by this invention lays a theoretical foundation for studying the plant salt stress response mechanism and also provides a gene resource for breeding new salt-tolerant non-heading Chinese cabbage varieties, possessing good potential application value. Attached Figure Description

[0018] Figure 1 This is an analysis diagram of the cis-acting elements of the BcCRK35 gene.

[0019] Figure 2This is a graph showing the expression analysis of the BcCRK35 gene under salt stress.

[0020] Figure 3 Phenotypic diagram of yeast strains overexpressing the BcCRK35 gene under salt stress.

[0021] Figure 4 The figure shows the expression detection results of the BcCRK35 gene in plants with silenced BcCRK35 gene under salt stress.

[0022] Figure 5 The growth phenotypes of the negative control and the non-heading Chinese cabbage with BcCRK35 gene silence under salt stress are shown.

[0023] Figure 6 The figures show the fresh weight and root volume of individual plants of Chinese cabbage that is a negative control under salt stress and a non-heading Chinese cabbage with BcCRK35 gene silence.

[0024] Figure 7 The graph shows the relative conductivity and malondialdehyde content of the negative control and BcCRK35 gene-silenced non-heading Chinese cabbage under salt stress.

[0025] Figure 8 Na+ was used as a negative control under salt stress and as a measure of Na+ in non-heading Chinese cabbage with BcCRK35 gene silencing. + and K + Content chart. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following: Unless otherwise specified, the experimental materials and reagents used in the present invention are all consumables and reagents that are conventionally available from commercial sources.

[0027] Example 1: Cloning and Bioinformatics Analysis of the BcCRK35 Gene

[0028] Using the Unigene gene sequence obtained from the full-length transcriptome data of non-heading Chinese cabbage as a reference sequence, the full-length gene was obtained through whole-genome synthesis. Then, BLAST alignment was performed in the NCBI database and the Arabidopsis thaliana genome website, and combined with transcriptome annotation results, it was named the BcCRK35 gene. The BcCRK35 open reading frame (ORF) is 777 bp, encoding 258 amino acid residues, and the protein's molecular formula is C0. 1249 H 1962 N 326 O 386 S 17The total number of atoms is 3940, the relative molecular weight is 28.27 kDa, the theoretical isoelectric point is 5.05, the aliphatic coefficient is 78.22, the average hydrophobicity is -0.043, and the instability coefficient is 37.37. Analysis of the cis-regulatory elements of the BcCRK35 gene using the online tool PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) revealed that it contains multiple salt stress-related response elements, including DRE, MYB, MYC, and the W box. Specifically... Figure 1 As shown. The CDS sequence of the BcCRK35 gene is as follows:

[0029] ATGGTACGTTATTCCAACGTTTCCTTCTCCGGATCACTCCTCATGGAACCATCACAAGCTCTGTACAACACTGGAGATATCGGAGATACTGATGCTAATATGACGGTGTTCGATAGAGTCTATGATGAGTTAATGATCCGTACAATTACTGCAGCTTCAAACGGATCATCATCCTTTGAGCAAAAGTATTTCGCAGCTGAGGTCGCTTCGTTGACCAACTTAGAAACTATGTACGCAATGATGCAATGTACGCCTGATGTTTCTTCAGGGGACTGTGAGTTTTGTCTCGAGAAAAGCGTCGGCGAGTATTCTTCTTGTTGTCGCGGTAAACAAGGTGGTGCGGTTATAAGACCGAGTTGCTTTTTCCGGTGGGATTTGTATCCTTATGCTGGAGCTTTTGATAATGTTACATTGCCACCGGCTCGACCGCAGGCTTCATCTCCGCCACCGCCTTCTTTGAGTCCTCCCGTAAGCGATACGGCAAATACAACCGGCAAAGATCGAAAGATTATTTCAACAGGTATTATTGTGGCAATTGTTGTCCCTGCCGTCATTATGTTGGTACTACTAGTTGTAGGATTCATGGTTTGCAGGAAAAGGAAGTTATACCAAACAAATGAAGTTCGAGCTGGTGATGAGATCACAACAACACGCTCACTGCAATTTAGTTTTAAAATGATTAAAGATGCAACTGATACGTTTGCAGACAGTAATCTGATTGGCCGAGGTGGATTTGGTGAAGTTTACAAGGAACTCTTTCAACTGGAACTGAAGTAG SEQ ID NO.1 Amino acid sequence:

[0030] MVRYSNVSFSGSLLMEPSQALYNTGDIGDTDANMTVFDRVYDELMIRTITAASNGSSSFEQKYFAAEVASLTNLETMYAMMQCTPDVSSGDCEFCLEKSVGEYSSCCRGKQGGAVIRPSCFFRWDLYPY AGAFDNVTLPPARPQASSPPPPSLSPPVSDTANTTGKDRKIISTGIIVAIVVPAVIMLVLLVVGFMVCRKRKLYQTNEVRAGDEITTTRSLQFSFKMIKDATDTFADSNLIGRGGFGEVYKELFQLELK SEQ ID NO.2

[0031] Example 2: Expression of the BcCRK35 gene under salt stress conditions

[0032] The non-heading Chinese cabbage used was commercially available four-season dwarf black-leaf sweet Chinese cabbage. Seedlings were raised using a sponge block method. When the seedlings reached three leaves and one bud, healthy seedlings with uniform growth were selected and transplanted into a hydroponic incubator. After transplanting, the seedlings were allowed to recover for 3 days before being subjected to salt stress treatment. The salt stress concentration used was 120 mmol / L NO3. - The control concentration was 7.5 mmol / L NO3. - NO3 - Half of the salt was provided by Ca(NO3)2·4H2O and half by KNO3. Leaves from the same location were collected at 0h, 0.5h, 1h, 3h, 6h, 12h, 24h, and 48h of salt stress treatment. Total RNA was extracted and reverse transcribed into cDNA using TransScript in a one-step process. TB... The Premix Ex Taq™ II FAST qPCR kit (TaKaRa) was used to detect relative gene expression using qRT-PCR. The reaction mixture (20 μL) consisted of: 10 μL of 2×TB Green Premix Ex Taq II Fast qPCR kit, 0.4 μL each of forward and reverse primers, 2 μL of cDNA template, and 7.2 μL of ddH2O. The reaction program was: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 60℃ extension for 30 s, 40 cycles, using 2... -ΔΔCt The expression levels of differentially expressed genes were calculated using a method with three biological replicates and three technical replicates per experiment. The BcActin gene (Hu Rong. Identification and functional analysis of the BcCAMTA transcription factor family in non-heading Chinese cabbage [D]. Nanjing Agricultural University, 2015.) was used as an internal reference gene. The primers for BcCRK35 expression amplification were:

[0033] F:5'-CCAACGTTTCCTTCTCCGGA-3'SEQ ID NO.3,

[0034] R:5'-TAACAACAGGGACGGCAGTA-3'SEQ ID NO.4.

[0035] Genetic testing results as follows Figure 2 As shown, the results indicate that the relative expression level of the BcCRK35 gene increased with the extension of salt stress treatment time, with a rapid increase at 3 h and a peak at 24 h, indicating that BcCRK35 is induced by high temperature stress and may be a potential salt stress response gene in non-heading Chinese cabbage.

[0036] Example 3: Construction of heterologous overexpression vector for BcCRK35 gene and salt tolerance analysis

[0037] The full-length CDS sequence of the target gene BcCRK35 was constructed into the pYES2-NTB vector (purchased from Nanjing Genscript Biotech Co., Ltd.), with BamHI and EcoRI restriction sites, to obtain the overexpression vector pYES2-NTB-BcCRK35. pYES2-NTB-BcCRK35 and the negative control pYES2-NTB were respectively transformed into yeast INVSC1. Following the manufacturer's instructions, the process included plate culture, liquid shaking culture, bacterial resuspension, centrifugation, water bath incubation, centrifugation for cell harvesting, suspension of the bacterial cells, mixing, and incubation at constant temperature. Positive clones of pYES2-NTB-BcCRK35 and the negative control pYES2-NTB were selected, resuspended in 2 mL of sterile water, and diluted to SG-U+0M NO3. - SG-U+0.5M NO3 - SG-U+1.0M NO3 - SG-U+1.3M NO3 - SG-U+1.5MNO3 - SG-U+2.0M NO3 - Place the plates on a 30°C incubator and incubate for 7 days. Observe the plates and take photos after 7 days. The results are as follows: Figure 3 As shown, the results revealed that the negative control was at SG-U+0M NO3. - SG-U+0.5M NO3 - SG-U+1.0M NO3 - It can grow on plates, in SG-U+1.3M NO3. - SG-U+1.5M NO3 - SG-U+2.0NO3 -The yeast cells could not grow on the plate. Compared with the negative control, pYES2-NTB-BcCRK35 showed more obvious growth and denser yeast cells, indicating that pYES2-NTB-BcCRK35 is salt-tolerant and can help yeast survive salt stress. This proves that the BcCRK35 gene plays a positive regulatory role in salt stress.

[0038] Example 4: Construction and Heat Resistance Analysis of BcCRK35 Gene Silent Vector

[0039] By recombination, a 273 bp non-conserved region of BcCRK35 (CDS505-777 bp) and its complementary fragment were used to replace the ccdB gene in the pK7GWIWG2(I) plasmid (purchased from Shanghai Zeye Biotechnology Co., Ltd.), resulting in the BcCRK35 silencing expression vector pK7GWIWG2(I)-BcCRK35. This vector was transformed into Agrobacterium GV3101, and after culturing, centrifugation, resuspending, and staining, BcCRK35-silencing expression plants were obtained and positive results were identified. Subsequently, the BcCRK35-silencing positive plants and their negative controls were treated with 120 mmol / L NO3. - The plants were treated for 21 days (nutrient solution was changed every 7 days). Phenotypic changes before and after treatment were photographed and recorded. Leaves were collected for growth and physiological index measurements. The control concentration was 7.5 mmol / L NO3. - Positive plant identification was performed using gene expression assays, following the same method as in "Example 2, Expression of BcCRK35 gene under salt stress conditions". Growth indicators were measured as follows: fresh weight was determined using an electronic balance, and root volume was measured using the water displacement method. Physiological indicators were measured as follows: relative conductivity was measured using a conductivity meter, malondialdehyde (MDA) content was determined using the thiobarbituric acid colorimetric method, and Na... + K + The content was determined using inductively coupled plasma mass spectrometry. All measurements were performed in triplicate.

[0040] Positive identification test results such as Figure 4 As shown, under salt stress, the expression level of the BcCRK35 gene in the silenced plants was significantly lower than that in the control plants, indicating that the BcCRK35 gene silencing system was successfully constructed. Phenotypic observation results are as follows... Figure 5 As shown, under salt stress treatment, compared with the control plants, the older leaves of the BcCRK35 gene-silenced plants first showed symptoms of mottled chlorosis, accompanied by leaf curling and drooping. In severe cases, the entire plant's leaves wilted and turned yellow. The negative-negative plants only showed mild growth inhibition and localized yellowing of leaf margins. The results of growth and physiological index measurements are as follows: Figure 6 and Figure 7As shown, the results indicated that under salt stress, the fresh weight and root volume of BcCRK35 gene-silenced plants were significantly lower than those of negative-positive plants. Under normal conditions, there was no significant difference in relative conductivity and MDA content between negative-positive plants and BcCRK35-silenced plants, while the relative conductivity and MDA content of both negative-positive plants and BcCRK35-silenced plants were significantly increased after salt treatment, with a greater increase in the BcCRK35-silenced plants. The results also showed that the relative conductivity and MDA content of salt-treated plants were significantly increased. + and K + The content was found, and the results were as follows: Figure 8 As shown, under normal growth conditions, the negative-affected plants and the BcCRK35 silent plants Na + and K + The Na content was basically the same, but after salt stress treatment, compared with the control treatment, the Na content in negative-negative plants and BcCRK35 silent plants was significantly higher. + Content and Na + / K + The ratios all increased significantly, while K + The content did not change significantly, and the Na content in BcCRK35 silent plants was also low. + / K + The ratio increased even more significantly.

[0041] The above results indicate that silencing the BcCRK35 gene leads to a decrease in the plant's tolerance to salt stress.

[0042] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A gene that positively regulates salt stress, characterized in that, The gene is BcCRK35, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The salt stress positive regulatory gene according to claim 1, characterized in that, The amino acid sequence of the BcCRK35 gene is shown in SEQ ID NO.

2.

3. The application of the BcCRK35 gene as described in claim 1 in regulating the salt tolerance of yeast, wherein overexpression of the BcCRK35 gene enhances the tolerance of yeast to salt stress.

4. The application of the BcCRK35 gene as described in claim 1 in regulating salt tolerance in non-heading Chinese cabbage.

5. The application according to claim 4, characterized in that, Knocking out or silencing the BcCRK35 gene reduces the salt tolerance of non-heading Chinese cabbage.

6. Primer pairs for amplifying the BcCRK35 gene, characterized in that, The primer pair includes a forward primer and a reverse primer, the nucleotide sequence of which is shown in SEQ ID NO.3; the nucleotide sequence of which is shown in SEQ ID NO.

4.

7. A biomaterial capable of regulating the expression level of the BcCRK35 gene, characterized in that, The invention includes biological materials that overexpress the BcCRK35 gene and biological materials that knock out or silence the BcCRK35 gene; the nucleotide sequence of the BcCRK35 gene is shown in SEQ ID NO.

1.

8. The biomaterial according to claim 7, characterized in that, The biological material overexpressing the BcCRK35 gene is one or more of a recombinant expression vector containing the BcCRK35 gene or a transgenic cell line containing the BcCRK35 gene, and the basic framework of the recombinant expression vector is the pYES2-NTB vector.

9. The biomaterial according to claim 7, characterized in that, The biological material for knocking out or silencing the BcCRK35 gene is one or more of a recombinant vector for knocking out or silencing the BcCRK35 gene or a transgenic cell line for knocking out or silencing the BcCRK35 gene, and the basic backbone of the recombinant expression vector is the pK7GWIWG2 vector (I).

10. The application of the primer pair of claim 6 or the biomaterial of claim 7 in regulating the salt tolerance of non-heading Chinese cabbage.

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