Cucumber csghd protein and application of the coding gene thereof in regulating vitamin c synthesis

Abstract

The application discloses a cucumber CsGDH protein and application of a coding gene thereof in regulation and control of vitamin C synthesis, and an amino acid sequence of the CsGDH protein is shown as SEQ ID NO. 2. The application discloses a gene sequence of cucumber L-galactose dehydrogenase CsGDH and a coding protein thereof for the first time, and verifies a key function of the CsGDH in vitamin C synthesis, thereby providing a new gene resource for research on regulation and control of vitamin C metabolism.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and genetic engineering technology, specifically relating to an L-galactose dehydrogenase (CsGDH) derived from cucumber and its encoding gene, as well as the application of this protein and gene in regulating plant vitamin C synthesis and cultivating plant varieties with high vitamin C content. Background Technology

[0002] Vitamin C (Vc), also known as ascorbic acid, is an important antioxidant and essential nutrient in plants, playing a vital role in human health. Since the human body cannot synthesize vitamin C, it must be obtained from food, especially fresh fruits and vegetables. Cucumbers are a widely cultivated and consumed vegetable globally, primarily eaten raw, making them an ideal source of vitamin C. Therefore, increasing the vitamin C content of cucumber fruits is of great significance for improving their nutritional quality.

[0003] However, research on the molecular regulatory mechanisms of vitamin C synthesis in cucumber fruits remains relatively weak. In particular, there are no clear reports or effective technical solutions regarding the specific biological function of the L-galactose dehydrogenase (GDH) gene in cucumbers, its expression regulation, or how to use this gene for genetic modification to increase the vitamin C content of cucumbers. Therefore, in-depth exploration and identification of key genes regulating vitamin C synthesis in cucumbers, and the development of their application methods, have significant theoretical and practical value for the molecular breeding of high-vitamin C cucumbers. Summary of the Invention

[0004] The technical problem this invention aims to solve is the lack of effective gene resources and technical methods for increasing vitamin C content in cucumbers in the existing technology. This objective is achieved through the following technical means:

[0005] This invention provides an L-galactose dehydrogenase (CsGDH) protein derived from cucumber, the amino acid sequence of which is shown in SEQ ID NO.2. The CsGDH protein of this invention can efficiently catalyze the synthesis of vitamin C, and the expression level of its encoding gene is significantly positively correlated with the vitamin C content in cucumber fruit.

[0006] This invention first provides a gene encoding cucumber L-galactose dehydrogenase, named CsGDH, whose nucleotide sequence is shown in SEQ ID NO.1. The CsGDH protein encoded by this gene has a conserved domain of L-galactose dehydrogenase.

[0007] Another aspect of the present invention provides a recombinant vector containing the gene for cucumber L-galactose dehydrogenase.

[0008] Another aspect of the present invention provides a recombinant engineered bacterium containing the gene for cucumber L-galactose dehydrogenase.

[0009] This invention also relates to the application of the above-mentioned cucumber L-galactose dehydrogenase gene in regulating the vitamin C content of plants, wherein the plant is cucumber.

[0010] This invention also relates to a method for increasing the vitamin C content of cucumbers, the method comprising: constructing a plant overexpression vector of the cucumber L-galactose dehydrogenase gene, and transforming cucumbers by Agrobacterium-mediated transformation to obtain transgenic cucumber lines with stable overexpression of the cucumber L-galactose dehydrogenase gene.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] This invention provides new gene resources: For the first time, the gene sequence of cucumber L-galactose dehydrogenase CsGDH and its encoded protein are disclosed, and its key function in vitamin C synthesis is verified, providing new gene resources for research on vitamin C metabolic regulation.

[0013] The application effects are clear and stable: This invention, through the construction of stably inherited overexpression and gene-edited cucumber lines, directly demonstrates that the expression level of the CsGDH gene can stably and positively regulate the vitamin C content in cucumber fruits. Overexpression significantly increases vitamin C content, while gene editing leading to loss of function reduces the content. This conclusion, based on stably inherited transgenic materials, provides a reliable basis for subsequent genetic improvement.

[0014] Broad Application Prospects: The application method described in this invention improves the vitamin C nutritional quality of cucumbers and other fruits and vegetables through molecular breeding. Compared with traditional hybridization breeding, it is more targeted and has a shorter breeding cycle. This technology has significant application value in meeting consumers' demand for high-quality fruits and vegetables and in addressing the problem of insufficient vitamin C intake in the human body. Attached Figure Description

[0015] Figure 1 Analysis results of CsGDH stable overexpression transgenic cucumber lines. Figure A shows the relative expression level of the CsGDH gene in the overexpression lines, and Figure B shows the vitamin C content of the fruits of the corresponding lines. The results show that compared with the wild type (WT), the overexpression line (CsGDH-OE) has significantly increased CsGDH expression and vitamin C content.

[0016] Figure 2Analysis results of CsGDH gene-edited homozygous mutant cucumber lines. Figure A shows the CsGDH gene editing status in the mutant lines, and Figure B shows the vitamin C content of the corresponding line's fruit. The results show that the vitamin C content of the gene-edited mutant line (CsGDH-CR) is significantly reduced compared with the wild type (WT). Detailed Implementation

[0017] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0018] Example 1: Cloning and Sequence Analysis of the CsGDH Gene

[0019] Materials: The fruit of cucumber (Cucumis sativus L.) cultivar '9930' was used as the material.

[0020] Total RNA extraction and cDNA synthesis: Total RNA was extracted from cucumber fruits using an RNA extraction kit and then reverse transcribed to synthesize first-strand cDNA.

[0021] Gene cloning: Using cDNA as a template, PCR amplification was performed using specific primers (forward primer: 5'-ATGGCGGTTTCTTATCCCAAG-3'; reverse primer: 5'-TCAGCTGTTTTGGATTCCACTTG-3'). The PCR products were recovered by agarose gel electrophoresis, ligated into the pMD19-T vector, transformed into *E. coli* DH5α, and plated on LB agar containing ampicillin. Positive clones were screened by PCR amplification using the aforementioned gene cloning-specific primers and sequenced for verification. The correctly sequenced CsGDH gene nucleotide sequence is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2.

[0022] SEQ ID NO.1

[0023] ATGGCGGTTTCTTATCCCAAGCTCCCCTTGCGGGAACTGGGAAACACTGGCCTCAAAACCAGCAGTGTCGGCTTTGGTGCTTCCCCTCTAGGCAGCGTCTTCAGCCCCGTTTCTGAGGAGGATGCCGTCGCTGCCGTTCGTGAAGCCTTTTGTCTTGGCATCAACTTCTTCGACACCTCCCCGTACTATGGAAGGAACTTATCAGAAAAGATGCTTGGTAAGGGACTGAAAGCTCTAGGAGTTCCAAGGAGTGAGTATATAGTGTCAACAAAGTGTGGGAGATATGGTGAGGGTTTTGATTTCAGTGCTGAGAGGGTGACAAGGAGCATTGACGAGAGTTTGGCTAGGCTACAACTAGATTACGTTGATATACTGCATTGCCATGATATTGAATTTGGATCTCTTGATCAGATTGTGAACGAGACGATTCCTGCACTTCAAAAGCTAAAAGAAGCTGGGAAGACTCGTTTCATTGGTATCACAGGATTACCGTTGGAAATATTTACGTATGTGCTTGATCGAGTACCACCCGGCACCGTTGATGTGATTCTTTCATATTGTCACTACAGTATTAATGATTCAACATTGTTAGATTTACTACCTTACTTGAAGAGTAAGGGAGTTGGAATAATCAGTGCTTCTCCTCTAGCAATGGGACTTCTAACTGATCAAGGTCCCCCAGAATGGCACCCAGCTTCACCGGAATTAAAGTCTGCATGCCAAGCTGCGGCTGCTCATTGTAGAAAGAAAGGAAAAAATATTTCAAAGTTAGCCATCCAATACAGTCTTGTGAACAAGGATATTTCAACGGTGCTGGTTGGCATGAACTCTGTCGGACAGGTGGAGGAAAACGTAGCTGCTGCTGAAGAACTTGCCACATTTGGGAGGGATGAGGAAACTCTGTCGGAGGTTGAAGCTATCCTTATCCCTGTCAAGAATCAGACATGGCCAAGTGGAATCCAAAACAGCTGA

[0024] SEQ ID NO.2

[0025] MAVSYPKLPLRELGNTGLKTSSVGFGASPLGSVFSPVSEEDAVAAVREAFCLGINFFDTSPYYGRNLSEKMLGKGLKALGVPRSEYIVSTKCGRYGEGFDFSAERVTRSIDESLARLQLDYVDILHCHDIEFGSLDQIVNETIPALQKLKEAGKTRFIGIT GLPLEIFTYVLDRVPPGTVDVILSYCHYSINDSTLLDLLPYLKSKGVGIISASPLAMGLLTDQGPPEWHPASPELKSACQAAAAHCRKKGKNISKLAIQYSLVNKDISTVLVGMNSVGQVEENVAAAEELATFGRDEETLSEVEAILIPVKNQTWPSGIQNS

[0026] Example 2: Effect of stable genetic transformation of the CsGDH gene on vitamin C content

[0027] Overexpression vector construction and stable transformation: The full-length CDS sequence of CsGDH was ligated into the plant overexpression vector pCAMBIA1305.4 using specific primers 5'-acgggggactctagaggatccATGGCGGTTTCTTATCCCAAG-3' and 5'-gtcatccttgtaatccacgtgGCTGTTTTGGATTCCACTTGGC-3' via double digestion (BamHI and PmlI) to construct the recombinant plasmid; the constructed overexpression vector was then transformed into Agrobacterium GV3101. Using cucumber cotyledons as explants, the target gene was integrated into the cucumber genome via Agrobacterium-mediated transformation. After screening, differentiation, and rooting, T0 generation transgenic plants were obtained. CsGDH gene expression was detected by GFP fluorescence signal observation and qRT-PCR (using gene-specific primers 5'-CAAAGTTAGCCATCCAATACA-3' and 5'-TCCTCATCCCTCCCAAA-3') to screen for a transgenic line stably overexpressing CsGDH (CsGDH-OE). The line was then propagated to obtain the T1 generation for phenotypic analysis. The results showed that compared with the wild-type (WT) control, the expression level of the CsGDH gene in the fruit of the overexpressing line was significantly increased. Figure 1 A), and the vitamin C content measured by LC-MS also increased significantly ( Figure 1 B).

[0028] Gene editing vector construction and stable transformation: Two specific sgRNA targets were designed against the first exon of the CsGDH gene, with sequences 5'-TTATCCCAAGCTCCCCTTGC-3' and 5'-AAAACCAGCAGTGTCGGCTT-3'. These were then constructed into the CRISPR / Cas9 gene editing vector pBSE402 using primers 5'-tcgaagtagtgattgTATCCCAAGCTCCCCTTGCgttttagagctagaaatagc-3' and 5'-ttctagctctaaaacAAGCCGACACTGCTGGTTTaatctcttagtcgactcta-3'. In this study, cucumbers were transformed using the same Agrobacterium-mediated transformation method as overexpression to obtain T0 generation transgenic plants. DNA was extracted from the T0 generation plants, and the fragment containing the target site was amplified by PCR and sequenced. The PCR primers were 5'-ATCAAGAGTGAAGGGAT-3' and 5'-TAGCCTAGCCAAACTCT-3' to identify the gene-edited plants. Through self-pollination, a line (CsGDH-CR) without the gene-editing vector but with a homozygous mutation at the target site was screened from the progeny for subsequent analysis. The results showed that, compared with the wild-type (WT) control, no functional expression of CsGDH was detected in the fruits of the homozygous gene-editing mutant line (CsGDH-CR), and the vitamin C content measured by LC-MS was also significantly reduced. Figure 2 ).

[0029] In summary, the experimental results of Example 2 fully demonstrate that the CsGDH gene plays a key positive regulatory role in regulating vitamin C synthesis in cucumber fruit, and that the vitamin C content in the fruit can be effectively altered through stable overexpression or gene editing technology.

[0030] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A cucumber CsGDH protein, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A gene encoding cucumber CsGDH, the nucleotide sequence of which is shown in SEQ ID NO.

1.

3. A recombinant vector containing the cucumber CsGDH gene as described in claim 2.

4. A recombinant engineered bacterium containing the cucumber CsGDH gene as described in claim 2.

5. The application of the cucumber CsGDH gene of claim 2 in regulating the vitamin C content of plants, wherein the plant is cucumber.

6. A method for increasing the vitamin C content of cucumbers, the method comprising: The vector described in claim 3 was constructed and cucumbers were transformed using Agrobacterium-mediated transformation to obtain transgenic cucumber lines with stable overexpression of the cucumber L-galactose dehydrogenase gene.

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