Pectin methylesterase gene participating in low-methyl esterification pectin generation

Abstract

The invention discloses a pectin methylesterase gene participating in low-methyl-esterification pectin generation, the pectin methylesterase gene participates in low-methyl-esterification pectin generation during loquat storage, and the nucleotide sequence of the pectin methylesterase gene is shown as SEQ ID No.1. The invention also discloses a preparation method of the pectin methylesterase gene. The method can regulate and control the synthesis of the low-methyl-esterification pectin in the loquat fruits, so that the hardness of the loquat fruits is influenced.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a pectin methylesterase gene involved in the production of low-methyl esterified pectin. Background Art

[0002] Loquat (Eriobotrya japonica) is an important economic fruit tree. Its fruit is popular among consumers for its soft texture and rich nutrition. However, loquat fruit is very prone to chilling injury and lignification after harvest, which leads to the decline of fruit quality and affects its commercial value and storage and transportation. Especially under low temperature storage conditions, the cell wall of the fruit will change, causing pectin degradation and hardening of texture.

[0003] Pectin methylesterase (PME) is an important cell wall modifying enzyme that affects the texture and structure of plant cell walls by regulating the degree of methyl esterification of pectin. PME plays an important role in the ripening and development of plant fruits. Studies have shown that it is closely related to changes in fruit texture in many plants. However, there are few studies on PME-related genes in loquat fruit, especially the molecular mechanisms of these genes and their functions in fruit storage.

[0004] The functions of PME genes have been systematically studied in plants such as Arabidopsis, tomato and grapes, and these studies have shown that PME affects the softening, ripening and cold storage stability of fruits by regulating the degradation of pectin in the cell wall. However, research on PME genes in loquat fruit and their role in the low-temperature storage of fruit is still in its infancy, and no clear reports have been made yet.

[0005] At present, the mechanism of pectin degradation and cold damage lignification in loquat fruit is not fully understood, and no research has revealed which PME genes are involved in the lignification process of loquat fruit. The increase of low-methylated pectin can combine with calcium ions in loquat, resulting in an increase in the firmness of loquat fruit, thereby reducing the quality of loquat (Huang W, Shi Y, Yan H, et al. The calcium-mediated homogalacturonan pectin complexation in cell walls contributes the firmness increase in loquat fruit during postharvest storage[J]. Journal of Advanced Research, 2023, 49: 47-62.). Summary of the invention

[0006] The purpose of the present invention is to provide a pectin methylesterase gene involved in the production of low-methylated pectin, which can regulate the synthesis of low-methylated pectin in loquat fruit, thereby affecting the hardness of loquat fruit.

[0007] The technical solution adopted by the present invention to solve its technical problem is: A pectin methylesterase gene involved in the production of low-methylated pectin. The pectin methylesterase gene is involved in the production of low-methylated pectin during loquat storage. The nucleotide sequence of the pectin methylesterase gene is shown in SEQ ID No.1.

[0008] The amino acid sequence of the protein encoded by the pectin methylesterase gene is shown in SEQ ID No.2.

[0009] Application of a pectin methylesterase gene involved in the production of low-methylated pectin in regulating the synthesis of low-methylated pectin in loquat fruit.

[0010] Application of a pectin methylesterase gene involved in the production of low-methylated pectin in regulating the firmness of loquat fruit.

[0011] The beneficial effects of the present invention are as follows: the present invention discovers a pectin methylesterase gene Ej00031073 involved in the generation of low-methylated pectin during the storage of loquats, and immunohistochemical results verify that overexpression of the gene has a function of affecting the formation of low-methylated pectin, and more low-methylated pectin can be produced in loquat fruits; by inhibiting the expression of the gene at room temperature, the synthesis of low-methylated pectin can be significantly reduced, thereby reaching or being better than the effect of 0°C preservation on low-methylated pectin, and utilizing the characteristics of the gene to better resist the damage of loquat cold damage and lignification, and maintain the taste of the loquat. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the fluorescence image of immunohistochemistry verification of Ej00031073 overexpression gene treatment and control; where: merged represents the overall shooting situation, AF represents autofluorescence, JIM5 represents the specific fluorescence of low methylated pectin, and DIC represents bright field.

[0013] Figure 2 This is a comparison chart of gene expression levels detected by quantitative PCR between the Ej00031073 gene overexpression treatment and the control.

[0014] Figure 3 This is a comparison of the formation of low-methyl pectin in loquat fruit after different treatments; where: merged represents the overall shooting situation, AF represents autofluorescence, JIM5 represents the specific fluorescence of low-methyl pectin, and DIC represents bright field. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described in detail below through specific embodiments.

[0016] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0017] Example 1: Obtaining and identifying the full-length loquat Ej00031073 gene 1. Loquat tissue material: Loquat fruits were harvested (in April and May each year, from an orchard in Lanxi, Zhejiang Province, China), frozen with liquid nitrogen and placed in a -80°C refrigerator. Three biological replicates were set for each sample.

[0018] 2. RNA extraction and cDNA synthesis: The loquat fruit samples were ground into powder in liquid nitrogen, and the total RNA was extracted by the CTAB method. After the electrophoresis test was qualified, the DNA was removed according to the instructions of TURBO DNAase Kit (Ambion), and 1.0gRNA was taken and reverse transcribed into cDNA according to the requirements of iScript cDNA Synthesis Kit (Bio-Rad).

[0019] 3. Obtaining the Ej00031073 gene: The loquat samples were sent to Beijing Novogene Technology Co., Ltd. for RNA sequencing to obtain an RNA-seq database. In the obtained RNA-seq database, genes related to PME were searched. Based on the expression trends of these genes, a possible Ej00031073 gene was screened from seven candidate genes through comparison, and the full-length sequence was determined by BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and cloning primers were designed. The upstream primer sequence was cgctctagaactagtggatccATGGCTTCTAAGTTCTTTCATCTTGTG (SEQID No.3), and the downstream primer sequence was gtcgacggtatcgataagcttTCAAACCAAACCACCAGTGTAAGG (SEQ ID No.4). Then, PCR amplification was performed using the Novogene high-fidelity enzyme kit. The PCR reaction system was 50μL: 25μL 2 × Phanta-MasterMix, 2 μL of upstream and downstream primers (10 μM), 4 μL of cDNA, 13 μL of H2O. The reaction procedure was: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 58°C for 30 s; extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min, and storage at 4°C. The target gene Ej00031073 was obtained, and the sequence homology of the Ej00031073 gene was compared on NCBI. The homology with the PME gene in Arabidopsis was 72%, and no related gene was reported.

[0020] The sequence of Ej00031073 is:

[0021] The amino acid sequence of the protein encoded by Ej00031073 is as follows: MASKFFHLVKVSSFLIIFHFLSSRSLADVPLNTPLPPETICKSTPHPSYCTSVLPHNNESVYDFGRFSVQRALSESHKLLDLYEKYLQKGSSLTNPAIQALEDCKQLALLNIDFLLSSLETVNKTSDVLPILDADDVQTLLSAILTNHQTCSDGIASLPSSAGSVLGDLSVPLSNNTKLYSTSLALFTKGWVPKDKNGVPKQPKRQFKFGKGRLNLKMSSNARAIYDSAINHRGRRLLQVGDEEVLVKDIVVVSQDGSGNFTTINQAIAVAPNNSVASGGYFMIYITAGVYEEYVSIISQKKYLLFIGDGINQTIITGHNSVGDGSTTFNSATLAVVAQGFMAVNITVRNTAGPSKGQAVALRSGADFSVFYSCSFEGYQDTLYTHSLRQFYRECDIYGTVDFIFGNAAVVLQNCNIYPRQPNQGQSNPITAQGRTDPNQNTGTSIHNCTITPTPDLASSNYTVKTYLGRPWKEYSRTVYMQTFMGSLIDPAGWLAWSGDFALSTLYYAEYNNTGPGSNTTNRVTWPGYHVINATVAANFTVSNFLLGDNWLPDTGVPYTGGLV (SEQ ID No.2).

[0022] Example 2: Construction of the overexpression vector of the loquat Ej00031073 gene The pGreenII002962-SK empty vector (commercially available) was digested with BamHI and HindIII to obtain the vector fragment. The target gene fragment and the vector fragment were homologously recombinated to obtain the recombinant product, which was transformed into DH5a Escherichia coli competent cells by heat shock method and spread on LB solid medium containing 50mg / L kanamycin (LB solid medium formula: tryptone 10g / L, yeast extract 5g / L, NaCl 5g / L, agar 15g / L). After selecting positive monoclonal bacteria for bacterial inspection, the plasmid was extracted to obtain the recombinant plasmid and sequenced for verification. The recombinant plasmid was transformed into Agrobacterium. After the recombinant plasmid was transformed into Agrobacterium GV3101 strain by electroporation, it was spread on LB solid medium containing 50ug / L kanamycin and 25ug / L gentamicin. After culturing at 28℃ for 2 days, the positive monoclonal was picked and stored in a -80℃ refrigerator with glycerol after bacterial inspection. Preparation of Agrobacterium infection solution: Agrobacterium carrying recombinant plasmid or empty plasmid was streaked onto LB solid medium containing 50ug / L kanamycin and 25ug / L gentamicin, activated and cultured at 28℃ for 2 days, then transferred to medium containing the same antibiotics and continued to culture at 28℃ for 1 day. Scrape the cells and resuspend them in the infiltration solution, and adjust the concentration to OD 600 =0.75, and the overexpression vector and control were obtained for use.

[0023] Verification of homologous expression of Ej00031073 in loquat fruit: The overexpression vector and control were injected directly into loquat fruit in the green fruit stage, with at least three replicates. After 7 days of free growth, samples were collected, paraffin sections were made, and immunohistochemical verification was performed. The results were as follows Figure 1 As shown, SK is the control group, and the overexpression group is the result after our candidate gene is overexpressed. It can be clearly seen from the fluorescence results that in the control group SK, since the Ej00031073 gene is not overexpressed, there is very little low-methylated pectin, and the specific fluorescence is very weak (JIM5 channel), but in the overexpression group, the fluorescence is very strong. By comparison, it can be concluded that the overexpressed gene has a significant promoting effect on the synthesis of low-methylated pectin. It is verified that the Ej00031073 gene of the present invention is involved in the synthesis of low-methylated pectin in loquat fruit.

[0024] Quantitative PCR was further performed on the control group and overexpressed loquat fruits to observe the expression of genes, such as Figure 2 As shown: SK is the control group and OE is the overexpression group (7 replicates). The results show that the Ej00031073 gene was successfully overexpressed in the fruit. Figure 1 The staining visualization results correspond to those of FIG. , which verifies that the Ej00031073 gene of the present invention is involved in the synthesis of low-methylated pectin in loquat fruit.

[0025] Embodiment 3: The overexpression vector and PME inhibitor (phosphatase PME-1 inhibitor (AMZ30), commercially available) were injected directly into loquat fruits in the green fruit stage, with at least three replicates. After injection, they were allowed to grow freely for 7 days, and then samples were collected, paraffin sections were made, and immunohistochemical verification was performed. Finally, the inhibition was observed by fluorescence. At the same time, mature loquats were placed at 0℃ for 7 days as a comparison, and the results were as follows Figure 3 As shown by Figure 3 It can be seen that in the group injected with the phosphatase PME-1 inhibitor (AMZ30) that inhibits the expression of the Ej00031073 gene, since the expression of the Ej00031073 gene is inhibited, there is very little low-methylated pectin, and the specific fluorescence is very weak (JIM5 channel), but in the overexpression group, the fluorescence is very strong. The inhibition group can significantly reduce the synthesis of low-methylated pectin; at the same time, the formation of low-methylated pectin is also less under 0 degrees Celsius storage conditions, but the formation of low-methylated pectin in the inhibition group is even less. It can be seen that by inhibiting the expression of the Ej00031073 gene at room temperature, the synthesis of low-methylated pectin can be significantly reduced, thereby achieving a better effect on low-methylated pectin preservation than 0°C, and improving the taste and quality of loquat.

[0026] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A pectin methylesterase gene involved in the production of low-methylated pectin, characterized in that: The pectin methylesterase gene is involved in the production of low-methylated pectin during loquat storage, and the nucleotide sequence of the pectin methylesterase gene is shown in SEQ ID No.

1.

2. A pectin methylesterase gene involved in the production of low-methylated pectin according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the pectin methylesterase gene is shown in SEQ ID No.

2.

3. Use of a pectin methylesterase gene involved in the production of low-methylated pectin as claimed in claim 1 in regulating the synthesis of low-methylated pectin in loquat fruit.

4. Use of a pectin methylesterase gene involved in the production of low-methylated pectin as claimed in claim 1 in regulating the hardness of loquat fruit.

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