CN119061024A

CN119061024A - MdHb1 gene and application of coded protein thereof in regulating postharvest apple fruit softening process

Info

Publication number: CN119061024A
Application number: CN202411579001.6A
Authority: CN
Inventors: 胡大刚; 孙权; 赵玉文; 赵婷婷; 韩朋良; 楚天歌; 陈泽
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2024-11-07
Filing date: 2024-11-07
Publication date: 2024-12-03

Abstract

The present invention discloses an application of an MdHb1 gene and its encoded protein in regulating the softening process of post-harvest apple fruit, and belongs to the technical field of plant genetic engineering. The present invention screens a non-symbiotic hemoglobin gene MdHb1 through metabolome and transcriptome association analysis, and for the first time studies and finds that there is a significant negative correlation between the expression of the MdHb1 gene and the hardness of the fruit during the post-harvest storage of apples; in vitro incubation experiments with substrates are used to prove that MdHb1 can catalyze the degradation of protopectin into soluble pectin; and instantaneous injection experiments with apple fruit are used to prove that MdHb1 can hydrolyze protopectin into soluble pectin, thereby promoting the softening of post-harvest apple fruit. The research results of the present invention provide new ideas for cultivating new storage-resistant apple varieties in the improvement of apple germplasm resources, which helps to reduce the storage loss of apples and improve production efficiency.

Description

MdHb1 gene and application of coded protein thereof in regulating postharvest apple fruit softening process

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to an application of MdHb gene and coding protein thereof in regulating softening process of picked apple fruits.

Background

Fruit softening is an important indicator of the intrinsic quality of apple fruits and is also an important feature during fruit ripening and post-harvest storage. The picked apples are excessively softened, so that the storage period and shelf life of the apples are shortened, the fruits are accelerated to rot and deteriorate, and the commodity value and economic benefit of the apples are seriously damaged.

Hemoglobin (Hb) is ubiquitous in all organisms, and there are at least three different types of Hb in plants, which can be classified as symbiotic, non-symbiotic, and truncated Hb. MdHb1 is a non-symbiotic hemoglobin gene that can act as a scavenger of Nitric Oxide (NO) in the prior art, oxidizing NO to NO ^3- under cold stress conditions, thereby regulating the level of NO. The expression of non-symbiotic hemoglobin has a certain correlation with the disease-resistant process of plants, and can prevent verticillium wilt caused by potato late blight or verticillium dahliae infection. Softening of apple-based respiratory-modified fruits is mainly related to pectin degradation, however, the role of non-symbiotic hemoglobin in postharvest fruit softening has not been studied.

Along with the appearance of the off-season fruit market, how to cope with softening of the picked apples, and the off-season fruit market is a urgent need for development of apple industry, and is a problem to be solved by scientific researchers. Therefore, the research on the molecular mechanism of softening the picked apples has important significance for maintaining the quality of the picked apples, prolonging the storage period and the shelf life of the apples and promoting the extension and development of the apple industry chain.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide an application of MdHb gene and coded protein thereof in regulating the softening process of picked apple fruits.

In order to achieve the above purpose, the invention adopts the following technical scheme:

In a first aspect of the invention there is provided the use of the MdHb gene to modulate the postharvest fruit softening process, said MdHb gene being a DNA molecule as set out in i) or ii) below:

i) The nucleotide sequence is a DNA molecule shown in SEQ ID NO. 1;

ii) a DNA molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.

The fruit is apple fruit.

Preferably, the apple fruit softening process is promoted by injecting MdHb gene over-expression vector into picked apples to improve the expression of apple MdHb gene;

Or inhibiting the expression of the apple MdHb1 gene by injecting MdHb gene silencing expression vector into the picked apples, so as to delay the apple fruit softening process.

In a second aspect of the invention, the application of MdHb gene-encoded protein in regulating postharvest apple softening process is provided, and the amino acid sequence of apple MdHb gene-encoded protein is shown as SEQ ID NO. 2.

The MdHb gene coded protein regulates the softening process of the picked fruits by catalyzing the degradation of protopectin into soluble pectin.

Preferably, the apple fruit softening process is promoted by increasing the activity of the protein encoded by the apple MdHb1 gene;

or delay the softening process of apple fruits by inhibiting the activity of protein encoded by apple MdHb gene

In a third aspect of the invention, a method is provided for growing a new variety of apples that is shelf stable by silencing MdHb gene in a wild type apple variety or mutating MdHb gene in a wild type apple variety.

The invention has the beneficial effects that:

According to the invention, a non-symbiotic hemoglobin gene MdHb is screened through metabolome and transcriptome association analysis, and a first study shows that a remarkable negative correlation exists between the expression of MdHb gene and the fruit hardness in the storage process after apples are picked, a substrate in-vitro incubation experiment is utilized to prove that MdHb1 can catalyze protopectin to degrade into soluble pectin, and an apple fruit instant injection experiment is utilized to prove that MdHb1 can hydrolyze protopectin into soluble pectin to promote apple fruit softening. The research result of the invention provides a new idea for cultivating a new variety of apples which are durable in storage in the improvement of apple germplasm resources, is beneficial to reducing the storage loss of apples and improving the production benefit.

Drawings

FIG. 1 shows cloning of MdHb gene.

FIG. 2 shows MdHb gene bioinformatics analysis, wherein FIG. 2A shows analysis of three-dimensional structures of MdHb protein and AtHb protein, and FIG. 2B shows analysis of amino acid sequence alignment of MdHb1 and homologous genes thereof.

FIG. 3 is a graph showing the determination of softening related index during post-harvest storage of apple fruits. Wherein, A in figure 3 is the apple fruit stored after picking, B in figure 3 is the fruit hardness, C in figure 3 is the fruit protopectin content, and D in figure 3 is the fruit soluble pectin content.

FIG. 4 shows the correlation analysis between MdHb and fruit softening during postharvest storage of apples, wherein A in FIG. 4 shows MdHb gene expression during postharvest storage of apples, and B in FIG. 4 shows the correlation analysis between MdHb and fruit softening.

FIG. 5 is a subcellular localization analysis of MdHb protein.

FIG. 6 is an in vitro incubation experiment of MdHb to catalyze the conversion of protopectin to soluble pectin. Wherein, A in FIG. 6 is the enzyme activity curve of PGEX-4T empty vector control, and B in FIG. 6 is the enzyme activity curve of MdHb.

FIG. 7 shows a transient injection experiment of MdHb fruits for regulating softening of fruits, wherein A in FIG. 7 shows the transient injection of apple fruits, B in FIG. 7 shows the relative expression of MdHb, C in FIG. 7 shows fruit hardness detection, D in FIG. 7 shows protopectin content detection, and E in FIG. 7 shows soluble pectin content detection.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As mentioned above, fruit softening is an important feature in the process of fruit ripening and post-harvest storage, and the post-harvest fruits of apples are excessively softened, so that the storage period and shelf life of the fruits are shortened, and the fruits are accelerated to rot and deteriorate, thereby seriously damaging the commodity value and economic benefit of the fruits.

The softening of fruits of the respiratory type, mainly apples, is mainly related to pectin degradation. Along with degradation of protopectin in the cell wall, the content of soluble pectin increases, the intercellular adhesion decreases, and the fruit is softened finally. MdHb1 is a non-symbiotic hemoglobin gene, and the role of non-symbiotic hemoglobin in postharvest fruit softening has not been studied in the prior art.

Based on the above, the application provides an application of MdHb gene and its coding protein in regulating the softening process of picked apple fruits, and the application uses cDNA obtained by reverse transcription of total RNA extracted from apple pulp as a template to carry out PCR amplification. A new fruit softening related hemoglobin gene in apples is cloned, and is found to have the highest similarity with Arabidopsis AtHb1 through comparison and analysis with a related gene Blast in Arabidopsis, so that the gene is named MdHb1, and the nucleotide sequence of the gene is shown as SEQ ID NO.1, and is specifically as follows:

SEQ ID NO.1:

ATGGAAGGCAAAGTTTTCACAGAAGAACAGGAAACACTGGTGGTGAAGTCATGGGGTGTGATGAAGAAGAACGCTGCTGAATTGGGCCTTAAGTTCTTCTTAAAGATCTTTGAAATTGCACCATCAGCTCAGAAGCTGTTCTCTTTCTTGAGGGACTCTGACATTCCTCTTGAGAAGAACCCAAAGCTCAAGCCTCACGCCATGTCTGTATTTGTTATGACTTGTGAATCTGCAGTTCAACTCAGGAAAGCAGGCAAGGTTACAGTGAGAGAGTCAACCTTGAAAAGATTAGGTGGTGTCCACTTCAAGTCTGGAGTGGTAGATGAACATTATGAGGTGACCAAGTTCGCATTGTTGGAAACCATAAAGGAGGCCTTACCGGAAATGTGGTCACCGGAGATGAAGAATGCTTGGGGAGAAGCTTATGATCAGTTGGTTGCTGCTATAAAATCAGAAATGAAGCCTCCCCTTAATTAG.

The coded amino acid sequence is shown as SEQ ID NO.2, and is specifically as follows:

SEQ ID NO.2:

MEGKVFTEEQETLVVKSWGVMKKNAAELGLKFFLKIFEIAPSAQKLFSFLRDSDIPLEKNPKLKPHAMSVFVMTCESAVQLRKAGKVTVRESTLKRLGGVHFKSGVVDEHYEVTKFALLETIKEALPEMWSPEMKNAWGEAYDQLVAAIKSEMKPPLN.

expression analysis found that MdHb gene expression was positively correlated with fruit softening caused by degradation of protopectin to soluble pectin during post-harvest storage of apple fruits. Subcellular localization analysis found MdHb that was localized to the cell wall. The in vitro incubation experimental analysis of the substrate shows that MdHb protein can specifically catalyze the conversion of protopectin into soluble pectin. The experimental result of the instant injection of the apple fruits shows that the excessive expression MdHb of 1 promotes the conversion of the protopectin into the soluble pectin, accelerates the softening of the picked apple fruits, and the silencing MdHb1 remarkably delays the conversion of the protopectin into the soluble pectin and maintains the hardness of the fruits.

In summary, the present invention has found a novel fruit softening related hemoglobin MdHb, which catalyzes the degradation of protopectin to soluble pectin, resulting in softening of apple fruits.

The following describes the embodiments of the present application in further detail with reference to examples. The following detailed description is illustrative and is not intended to provide further explanation of the application, but is not intended to limit the scope of the application.

EXAMPLE 1 cloning of the MdHb1 Gene

S1, extracting total RNA of apple pulp by using a polysaccharide polyphenol RNA extraction kit (RNApre Pure Plant Plus Kit, TIANGEN, DP 441)

50Mg of apple pulp frozen at-80 ℃ is taken, ground into powder in liquid nitrogen, transferred into a 1.5ml centrifuge tube, added with 500 mu L of lysis solution SL (containing 25 mu L of beta-mercaptoethanol) carried by the kit, deproteinized, DNA removed and rinsed, and finally dissolved into 30 mu L of ddH ₂ O.

RNA integrity was checked using agarose at 1% concentration and RNA concentration and purity were determined using Nanodrop 2000.

S2 PCR amplification MdHb full Length 1

Mu.l of total RNA was taken and reverse transcribed according to the instructions of TRANSSCRIPT ONE-Step gDNA Removal AND CDNA SYNTHESIS SuperMix (Trans), and the reaction was diluted 5-fold as template for PCR.

The total volume of the PCR reaction system is 50 mu L, which comprises 4 mu L of cDNA template, 4 mu L of dNTP, 2.5 mu L of cloning MdHb gene full-length coding region sequence primer, 10 mu L of 5 XHF Buffer, 28.5 mu L of ddH ₂ O and 0.5 mu L of Phusion high-fidelity enzyme. Amplification was performed according to the reaction procedure shown in table 1.

Wherein, the sequence primer of the full-length coding region of the clone MdHb gene is shown as SEQ ID NO. 3-SEQ ID NO. 4:

SEQ ID NO.3(F): ATGGAAGGCAAAGTTTTCACAGAA;

SEQ ID NO.4(R): ATTAAGGGGAGGCTTCATTTCTGA。

TABLE 1 reaction procedure

The PCR products were subjected to agarose gel electrophoresis detection (FIG. 1), the PCR products with correct detection were purified and recovered, the recovered fragments were connected to pLB vector (TIANGEN) cloning vector, transformed into DH 5. Alpha. And cultured in LB medium (the formula of LB medium: 1L medium contains 10g tryptone, 5g yeast extract, 10g sodium chloride and 12g agar), and positive clones were sequenced by Beijing qing Biotechnology Co., ltd. BLAST analysis was performed on the sequencing results.

S3 sequence alignment analysis

Sequence analysis is carried out on MdHb1 and homologous protein sequences in other species, and the MdHb and homologous genes thereof are found to contain a conserved globin-like structural domain. Furthermore, by comparing MdHb1 with the three-dimensional structure of the AtHb1 protein in arabidopsis, the two proteins were found to be highly similar in three-dimensional structure, indicating that Hb proteins maintain functional similarity during independent evolution (fig. 2).

Example 2 construction of MdHb1 Gene overexpression vector MdHb-IL 60 and silencing vector MdHb-TRV

According to a conventional vector construction method, a MdHb gene fragment is amplified by using a primer shown in SEQ ID NO. 5-SEQ ID NO.6 and cloned into a TRV2 vector for silencing MdHb gene, and a silencing vector MdHb-TRV is obtained, wherein the TRV1 vector is used as an auxiliary plasmid. The primer shown in SEQ ID NO. 3-SEQ ID NO.4 is used for amplifying MdHb full-length coding sequence of the gene and cloning the coding sequence into an IL60 vector for over-expression of the gene to obtain an over-expression vector MdHb-IL 60, and the IL60-1 vector is used as an auxiliary plasmid.

SEQ ID NO.5(F): ATTAAGGGGAGGCTTCATTTCT;

SEQ ID NO.6(R): AAAAGATTAGGTGGTGTCCACTT。

Example 3 expression of MdHb A and positioning of MdHb A protein during post-harvest storage of apples

In order to clarify the relationship between MdHb and postharvest apple fruit softening, the present example measured the change in post-harvest apple fruit softening related index (a in fig. 3). As shown in fig. 3, fruit hardness gradually decreased with prolonged storage time (B in fig. 3), protopectin content gradually decreased (C in fig. 3), and soluble pectin content significantly increased (D in fig. 3), indicating that apple fruits gradually softened during post-harvest storage.

In addition, the expression of MdHb gene in whole apple postharvest storage process was examined in this embodiment. Real-time fluorescent quantitative PCR (RT-qPCR) of MdHb gene was performed using the primers shown in SEQ ID No. 7-SEQ ID No.8, and the results showed that MdHb gene expression gradually increased as the fruit was gradually softened during storage (FIG. 4A). Through correlation analysis, a high correlation exists between MdHb gene expression and fruit softening, which indicates that MdHb gene participates in postharvest apple fruit softening process (B in FIG. 4).

SEQ ID NO.7(F): GTCTGGAGTGGTAGATGAACAT;

SEQ ID NO.8(R): CCAACTGATCATAAGCTTCTC。

The present example further examined the localization of MdHb protein. 35Spro: mdHb1-GFP overexpression vector is constructed based on MdHb1 gene full length, 35Spro: mdHb1-GFP is expressed in tobacco (Nicotiana benthamiana) leaves by using an agrobacterium transient infection method, and imaging observation is carried out after three days of normal culture. Prior to imaging, tobacco epidermal cells were stained with Propidium Iodide (PI) dye for 5min, used as a cell wall indicator, and then observed using a high resolution laser confocal microscope (LSM 880, zeiss, meta, jena, germany) to obtain fluorescence images. As shown in FIG. 5, the GFP green fluorescence emitted by MdHb-GFP fusion proteins overlapped the cell wall stained red with PI dye, indicating that MdHb1 was localized to the cell wall.

Example 4 in vitro substrate incubation experiments and apple fruit transient injection experiments

To verify the function of MdHb gene to regulate fruit softening, the present invention provides a MdHb function verification experiment in example 4. Specifically, the method comprises (1) in vitro substrate incubation experiments, verifying that MdHb protein has the function of catalyzing the conversion of protopectin into soluble pectin, and (2) apple fruit instant injection experiments, verifying that MdHb catalyzes the degradation of protopectin into soluble pectin, and finally promoting the softening of apple fruits.

(1) In vitro substrate incubation experiments

The MdHb 'gene containing BamH I and 3' containing Xho I cleavage site was ligated into E.coli expression vector PGEX-4T vector. The recombinant plasmid was transformed into BL21 (DE 3) competent cells and induced to express with 50. Mu.M IPTG (Isopropyl thiogalactoside, isopopyl. Beta. -D-Thiogalactoside) at 16℃for 20 hours. Bacterial cells were lysed by sonication in 140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂HPO₄ and 1.8 mM KH ₂PO₄ (1 XPBS, pH 7.4). The MdHb1 soluble protein was purified using glutathione sepharose beads (Thermo Scientific, san Jose, CA, USA). Standard MdHb in vitro enzyme assays were performed in 100. Mu.L of a reaction mixture containing 10. Mu.g of pectin substrate, 1 XPBS buffer (pH 7.4) and purified MdHb enzyme.

The reaction mixture was incubated at 50 ℃ for 15 minutes. 500. Mu.L of concentrated sulfuric acid was added to stop the reaction. The sample was then placed at 90 ℃ for 10 minutes. After cooling to room temperature, 100. Mu.L of 1.5 g/L carbazole was added. After thorough mixing, the reaction was carried out at room temperature for 30 minutes. The sample was diluted to 1 mL with distilled water and the absorbance at 530 nm was measured. As shown in FIG. 6, the results indicate that along with the extension of the in vitro incubation reaction time, mdHb protein gradually catalyzes the degradation of protopectin to soluble pectin.

(2) Apple fruit instant injection experiment

The MdHb gene overexpression vector MdHb-IL 60 and silencing vector MdHb-TRV constructed in example 2 are respectively transferred into an agrobacterium GV3101 strain by a heat shock transformation method, and are subjected to suspension culture overnight until OD ₆₀₀ =0.8-1.0, and are centrifuged at 5000 rpm for 5 min. The suspension thalli are sucked and beaten by 1mL sterile water, centrifuged 5 and min are carried out by 5000 and rpm, and the agrobacterium thalli are collected. Respectively suspending thalli by using proper sterile water, regulating the OD ₆₀₀ value to 0.6 by using the sterile water, adding 10mM MgCl ₂, 10mM MES and 100 mu M acetosyringone, and preparing MdHb-IL 60 target gene invasion solution or MdHb-TRV target gene invasion solution.

The infestation was gently injected into the prepared apple (red star apple) fruit using a sterile syringe of 1 mL. Wherein, IL60+TRV is a control group, 200 mu L of IL60 empty-load bacterial liquid+200 mu L of TRV (TRV 1 herein) empty-load bacterial liquid is injected into each apple of the control group, 200 mu L of TRV (TRV 1 herein) empty-load bacterial liquid+200 mu LMdHb1-IL60 target gene invasion liquid is injected into each apple of the MdHb1-IL60+TRV group, and 200 mu L of IL60 empty-load bacterial liquid+200 mu LMdHb1-TRV target gene invasion liquid is injected into each apple of the MdHb1-TRV+IL60 group. The injected red star apples were placed in an artificial culture room at 19 ℃ and in darkness for 10 days, and the pulp samples near the injection holes were sampled for subsequent detection (a in fig. 7).

As shown in fig. 7, RT-qPCR assays showed that MdHb gene expression was significantly higher in the MdHb-il60+trv fruit injected than the control (il60+trv) (B in fig. 7), mdHb gene expression was significantly lower in the MdHb1-trv+il60 fruit injected than the control (il60+trv) (B in fig. 7), hardness and protopectin content of the MdHb1-il60+trv fruit injected significantly lower than the control (il60+trv) accompanied by changes in MdHb gene expression, soluble pectin was significantly higher than the control (il60+trv) (C in fig. 7 and D in fig. 7), hardness and protopectin content of the MdHb-trv+il60 fruit injected significantly higher than the control (il60+trv), and soluble pectin was significantly lower than the control (il60+trv) (E in fig. 7). These results indicate that MdHb1 ultimately leads to fruit softening by promoting degradation of protopectin to soluble pectin in apple fruit bodies.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification made within the spirit and principles of the present application

Equivalent substitutions, modifications and the like are intended to be included in the scope of the present application.

Claims

1. Application of the MdHb1 gene in regulating the softening process of postharvest fruits, characterized in that the MdHb1 gene is a DNA molecule as shown in the following i) or ii):

i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. The use according to claim 1, characterized in that the fruit is apple fruit.

3. The use according to claim 1, characterized in that the expression of the MdHb1 gene is increased by injecting the MdHb1 gene overexpression vector into the harvested apples, thereby promoting the softening process of the apple fruit.

4. The use according to claim 1, characterized in that the softening process of apple fruit is delayed by injecting the MdHb1 gene silencing expression vector into the harvested apples to inhibit the expression of the apple MdHb1 gene.

5. Use of a protein encoded by the MdHb1 gene in regulating the softening process of post-harvest apples, wherein the amino acid sequence of the protein encoded by the apple MdHb1 gene is shown in SEQ ID NO.2.

6. The use according to claim 5, characterized in that the protein encoded by the MdHb1 gene regulates the softening process of post-harvest fruits by catalyzing the degradation of protopectin into soluble pectin.

7. The use according to claim 5, characterized in that the softening process of apple fruit is promoted by increasing the activity of the protein encoded by the apple MdHb1 gene.

8. The use according to claim 5, characterized in that the softening process of apple fruit is delayed by inhibiting the activity of the protein encoded by the apple MdHb1 gene.