CN112322644B

CN112322644B - Application of tomato SlSPY gene in controlling tomato fruit ripening process

Info

Publication number: CN112322644B
Application number: CN202011353823.4A
Authority: CN
Inventors: 周艳虹; 徐进; 姜小春; 蔡溧聪; 喻景权
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2022-06-10
Anticipated expiration: 2040-11-26
Also published as: CN112322644A

Abstract

The application discloses an application of a tomato SlSPY gene in controlling a tomato fruit ripening process, wherein the expression level of the tomato SlSPY gene is increased by a gene overexpression technology; the nucleotide sequence of the tomato SlSPY gene is as follows: SEQ ID NO: 1. The tomato SlSPY overexpression can improve the sensitivity of tomato fruits in a green ripe stage to ethylene, accelerate the color change of the tomato fruits and promote the fruit softening and the accumulation of carotenoids such as lycopene. In the SPY gene knockout plant, the color change and softening processes of tomato fruits are inhibited, and the accumulation of carotenoids such as lycopene is less than that of wild fruits in the same period.

Description

Application of tomato SlSPY gene in controlling tomato fruit ripening process

Technical Field

The application relates to the fields of genetic engineering, molecular biology, physiology and the like, in particular to application of a SlSPY gene in promoting tomato fruit ripening.

Background

Tomatoes (Solanum lycopersicum L.) belong to the solanaceae family, annual or perennial herbs of the genus lycopersicon, and are vegetables widely cultivated in the world. The tomato fruit ripens through a series of complex physiological and biochemical processes, wherein the color, the flavor, the aroma, the texture and the nutrition are all changed violently, so that the tomato fruit is rich in nutrition and becomes an important nutritional fruit in human diet. Tomato fruits are climacteric fruits, whose ripening process is usually accompanied by a sharp increase in ethylene content, and thus ethylene plays an important role in the ripening process of tomato fruits. However, the role of the various components of the ethylene signaling pathway in the ripening process of tomato fruits needs to be further investigated.

Meanwhile, tomatoes have small genomes and short growth periods, and have far genetic relationship with model plants such as arabidopsis, rice, corn and the like, so that the tomatoes become model plants for researching development and maturity of fleshy fruits. Moreover, thanks to the completed fine sequence analysis of the whole genome of cultivated tomatoes, the research of tomato functional genomics and molecular genetics is deepened. SlSPY, as an O-GlcNAc transferase in tomato, has a very high similarity to animal OGT sequences. The phenomenon of O-GlcNAc glycosylation has been the focus of glycobiology since its discovery in 1984 by professor John Hopkins university, USA. The O-GlcNAc modified proteins are involved in a variety of physiological processes and many diseases in the human body are associated with the level of O-GlcNAc modification, such as obesity, cancer, diabetes, etc. SPY-like is highly conserved in the genome of many higher plants as an OGT specific to higher photosynthetic plants. However, the studies of O-GlcNAc modification in plants are rarely reported. Previous studies have shown that SPY can act as a negative regulator of GA signaling. In Arabidopsis, the phenotype of spy mutants is very similar to that obtained by over-administration of gibberellins. However, the role of SlSPY in the ripening of tomato fruits has not been reported. The role of SlSPY as an O-GlcNAc transferase in the ripening of tomato fruits has yet to be investigated.

Disclosure of Invention

The application aims to provide the application of the SlSPY gene in promoting the fruit ripening of tomatoes so as to solve the problem that the marketing time of tomatoes can be adjusted.

According to the embodiment of the application, the application of the tomato SlSPY gene in controlling the ripening process of tomato fruits is provided, and the expression level of the tomato SlSPY gene is increased through a gene overexpression technology; the nucleotide sequence of the tomato SlSPY gene is as follows: SEQ ID NO: 1.

The tomato SlSPY gene in the example is any one of the following nucleotide sequences 1) to 4):

1) SEQ ID NO: 1;

2) SEQ ID NO:1 by substituting, deleting and/or adding one or more nucleotides and has the function of not changing the prokaryotic nucleotide sequence;

3) under stringent conditions with SEQ ID NO:1 under stringent conditions of hybridization at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS, and washing the membrane with the solution;

4) a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence of 1), 2) or 3) and encodes the same functional protein.

The protein obtained by encoding the tomato SlSPY gene has one of the following amino acid sequences:

1) SEQ ID No: 2;

2) SEQ ID No:2 by substituting, deleting and/or adding one or more amino acids, and has equivalent activity, and the protein derived from the protein 1).

It is understood that one skilled in the art can substitute, delete and/or add one or several amino acids to obtain a mutant sequence of the protein based on the amino acid sequence disclosed in the present invention without affecting its activity. It is understood that, considering the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

Further, the gene overexpression technology is specifically as follows:

extracting total RNA of the tomato, carrying out reverse transcription to obtain cDNA, amplifying an SlSPY gene by taking the cDNA as a template and F and R as primers, and constructing an amplification product on a plant overexpression vector; the nucleotide sequences of the primers F and R are shown as SEQ ID NO:3 and 4;

and (3) introducing the plant overexpression vector into a host cell, infecting a target plant by using the plant overexpression vector, and screening a positive transgenic plant to obtain a transgenic plant.

Further, the host cell is an escherichia coli cell or an agrobacterium cell, preferably the agrobacterium is EHA 105.

The technical scheme provided by the embodiment of the application can have the following beneficial effects:

according to the embodiments, the tomato SlSPY overexpression plant or the gene knockout plant is constructed by a gene means, the expression level of the gene SlSPY is regulated to study the regulation mechanism of the gene SlSPY on tomato fruit ripening, and the results show that the SlSPY overexpression can improve the sensitivity of tomato fruits on ethylene, promote tomato fruit color change, carotenoid accumulation and fruit hardness reduction, and further promote low fruit ripening. Thus, the tomato SlSPY gene promotes fruit ripening in tomato fruits by increasing their sensitivity to ethylene. The SlSPY gene provided by the invention provides gene resources for new early-maturing or late-maturing tomato varieties, has good potential application value, can adjust the marketing time of tomato fruits through a transgenic technology, and reduces the accumulation and rot of mature fruits.

The invention constructs a transgenic plant for overexpression and gene knockout of the tomato SlSPY gene for the first time and performs functional research. By marking the flowering time, the SlSPY gene is found to be capable of promoting the color change and the maturity of tomato fruits in the tomato fruits.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.

FIG. 1 shows the Western Blot detection result of the plant protein of the tomato line over-expressed by the SlSPY gene in example 3 of the invention.

FIG. 2 shows the sequencing result of the sgRNA sequence of the SlSPY gene knockout tomato line in example 3 of the present invention.

FIG. 3 shows the fruit color changes of the over-expressed SlSPY and SlSPY gene mutation and wild type tomato plants at 30 days, 35 days, 40 days, 45 days and 50 days after the flowers in example 4 of the present invention.

FIG. 4 shows the overexpression SlSPY and SlSPY gene mutation and the change of the content of carotenoids such as lycopene in the fruits 7 days after color breaking and color breaking of wild type tomato plants in example 5 of the invention.

Fig. 5 shows the fruit hardness changes of the over-expressed SlSPY, SlSPY gene mutation and wild type tomato plants at 30, 35, 40, 45 and 50 days after flowering in example 6 of the present invention.

FIG. 6 shows the fruit color change of the over-expressed SlSPY and SlSPY gene mutation and wild tomato plant after treating ethylene inhibitor (1-MCP), air or ethylene in example 7.

FIG. 7 shows the changes of carotenoid content such as lycopene in fruits after 1-MCP, air or ethylene treatment of the wild type tomato plants and the mutation of genes of the over-expressed SlSPY and SlSPY in example 7 of the present invention.

FIG. 8 shows the fruit hardness changes of the overexpression SlSPY and SlSPY gene mutation and wild type tomato plants after 1-MCP, air or ethylene treatment in example 7 of the invention.

Detailed Description

Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

Example 1: construction of SlSPY Gene overexpression vector

In order to regulate the time of marketing of tomato fruits, the SlSPY gene is cloned from tomato genome. Specific primers SlSPY-F and SlSPY-R are designed according to the sequence analysis of a coding region, and restriction enzyme sites (Asc I and Kpn I) are added to the primers respectively, and the sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4. Amplifying an SlSPY fragment by PrimerSTAR high-fidelity enzyme PCR, then carrying out enzyme digestion on the PCR amplified fragment and the vector, and connecting the SlSPY fragment to pFGC1008-HA to obtain an overexpression vector pFGC 1008-SlSPY-HA. The recombinant plasmid is sent to Shanghai company for sequencing confirmation, and the nucleotide sequence of the obtained gene SlSPY is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 2. The results showed that the cloned sequence was identical to the sequence published in Solgenomics (Solyc09g 010180).

Example 2: construction of SlSPY Gene mutation vector

The SlSPY gene target sequence is designed by using a CRISPR-P website, and the specific sequence is shown as SEQ ID NO. 5 and is TGTAGTTCATGGCAAGTAAC. The synthesized target sequence is connected to Bbs I site of AtU6-sgRNA-AtUBQ-Cas9 vector after annealing, and then the newly obtained AtU6-sgRNA-AtUBQ-Cas9 fragment is connected to Hind III/Kpn I site of pCAMBIA1301 vector, so as to construct tomato SlSPY gene CRISPR expression vector. The recombinant plasmid was sent to Shanghai for sequencing confirmation.

Example 3: construction and detection of tomato SlSPY transgenic material

An overexpression vector pFGC1008: SlSPY-HA and a gene editing vector pCAMBIA1301: AtU6-sgRNA (SlSPY) -AtUBQ-Cas 9. Agrobacterium GV3101 is transformed, tomato cotyledon infection is performed, tissue culture seedling is obtained through callus induction, resistance inducing differentiation and rooting culture, T1 mutant seed and over-expressed seed are tested separately for kanamycin resistance and chloromycetin resistance, 3/4 strain with resistance and the rest 1/4 strain without resistance are selected, and the over-expression vector with the target gene connected to the strain is inserted in single copy form. These plants were removed and single harvest was performed. Western Blot is used for verifying an SLSPY overexpression positive transgenic plant, and the result shows that a wild type HAs no protein band, while an overexpression strain HAs a SlSPY-HA band (figure 1), PCR and sequencing technologies are used for verifying a positive SlSPY mutation transgenic plant, and spy #1 lacks 11 bases, spy #2 lacks 14 bases, mutations are respectively carried out at the 5 th base of an original adjacent motif (PAM), and translation is stopped due to the early appearance of a stop codon (figure 2).

Example 4: tomato fruit color-turning time statistics of SlSPY gene transgenic material

The blooming flowers were dated after the tomato material blossoming and the color changes of the different transgenic materials were recorded 30, 35, 40, 45 and 50 days after the flower (fig. 3). The color change of the SlSPY gene mutant tomato fruits was significantly delayed compared to wild-type fruits, which indicates that the SlSPY gene mutation delayed the color change of tomato fruits.

Example 5: determination of tomato fruit pigment content of SlSPY gene transgenic material

The fruit carotenoid content was measured 30, 35, 40, 45 and 50 days after the different materials were bloomed. The peeled tomato flesh was placed in liquid nitrogen, freeze-dried, then thoroughly ground, and 0.1g was weighed into a 2ml centrifuge tube. Adding 700. mu.L chloroform, 350. mu.L ddH₂O, 350. mu.L of methanol, vortexed thoroughly and mixed, centrifuged at 10000g for 10min at 4 ℃ and the chloroform phase is collected. The remaining residue tube was continued to add 700. mu.L of chloroform until the chloroform phase was colorless. The chloroform phases were combined and blown dry with nitrogen. Then, 350. mu.L of methanol solution (w/v) containing 6% KOH was added to dissolve the precipitate, and derivatization was carried out at 60 ℃ for about 30min in the dark. Add 350. mu.L of ddH₂O and 700. mu.L of chloroform, vortexed and mixed well, centrifuged at 10000g for 5min at 4 ℃ and the chloroform phase collected in a10 mL centrifuge tube. Adding 700. mu.L ddH into chloroform phase₂And (4) re-extracting for multiple times. The collected chloroform phase was blown dry with nitrogen and finally dissolved in 100. mu.L of chromatographic grade ethyl acetate, centrifuged at 14000g for 20min at 4 ℃ and 150. mu.L of the supernatant was taken for High Performance Liquid Chromatography (HPLC) analysis. The above operation should be set to more than 5 biological replicates.

The results show (fig. 4) that the carotenoid accumulation amount of lycopene and the like in the tomato fruits overexpressing SlSPY was greater than that in the wild type fruits 7 days after color breaking. The accumulation amount of carotenoid such as lycopene in SlSPY gene mutation tomato fruits is less than that of wild fruits

Example 6: tomato fruit hardness determination of SlSPY gene transgenic material

Fruit firmness was measured 30, 35, 40, 45 and 50 days after flowering for the different materials. Measuring the hardness of tomato fruit by Texture analyzer TA.XT.plus Texture Analyser (Stable Micro systems, UK), with probe diameter of 8.04mm, measuring distance of 12nm, speed before measurement of 13mm/s, speed during measurement of 1.5mm/s, and speed after measurement of 1.5mm/s13 mm/s. During measurement, three points are randomly selected on the equatorial plane of tomato fruits, tomato peels with the diameter of 1.5mm are cut off, each fruit is measured for three times, and the average value of the maximum pressure is taken as the hardness of the tomato fruits, and the unit is gram per square millimeter (g/mm)²)。

The results show (fig. 5) that in over-expressed SlSPY tomato fruits, the decrease in tomato fruit firmness is faster than wild type tomato fruits with increasing days after flowering, while the decrease in fruit firmness of SlSPY gene mutant tomatoes is slower than wild type tomato fruits.

Example 7: detection of sensitivity of tomato fruits made of SlSPY gene transgenic material to ethylene

The fruits of over-expression SlSPY gene tomatoes, SlSPY gene mutation tomatoes and wild type tomatoes in the green mature period 30 days after flowering are respectively treated with ethylene concentration of 500 mu LL^-1Air and 1-MCP concentration of 10 mu L L^-1After 24 hours, the tomato was placed in a ventilated room, the color change of the fruits was recorded after 10 days, and the carotenoid content of the fruits of the SlSPY gene tomatoes, the SlSPY gene mutant tomatoes and the wild-type tomatoes among different treatments was determined according to the method described in example 5, and the hardness change of the fruits of the SlSPY gene tomatoes, the SlSPY gene mutant tomatoes and the wild-type tomatoes among different treatments was determined according to the method described in example 6.

The results show that compared with wild fruits, the over-expressed SlSPY gene tomatoes have faster color change after being treated with ethylene (fig. 6), and a large amount of lycopene is accumulated (fig. 7), and the fruit hardness is rapidly reduced (fig. 8), while the SlSPY gene mutant tomatoes have slow color change (fig. 6), and carotenoid such as lycopene is less accumulated (fig. 7), and the fruit hardness is slowly reduced (fig. 8).

The transgenic tomato is constructed by the gene disclosed by the invention, and the resistance of the transgenic tomato to low-temperature stress can be improved. In order to facilitate the identification and screening of transgenic tomato plants, the vectors used may be processed, for example by adding plant selectable markers or antibiotic markers with resistance. The 3HA tag protein was added to the overexpression vector pFGC1008 described above.

The tomato SlSPY overexpression can improve the sensitivity of tomato fruits to ethylene, promote the color change of the tomato fruits, accumulate carotenoids and reduce fruit hardness, and further promote the low fruit ripening. Thus, the tomato SlSPY gene promotes fruit ripening in tomato fruits by increasing their sensitivity to ethylene. The SlSPY gene provided by the invention provides gene resources for new early-maturing or late-maturing tomato varieties, has good potential application value, can adjust the marketing time of tomato fruits through a transgenic technology, and reduces the accumulation and rot of mature fruits.

Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.

It will be understood that the present application is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.

Sequence listing

<110> Zhejiang university

Application of tomato SlSPY gene in regulation and control of tomato fruit ripening

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aaactgggaa ccatggcttg ccgacctgcg cctgtacagg tgacttggat tggataccct 1800

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acaccctctc ctgaagctgg gccagtgtgt cctgcacctg ctttatctaa tggcttcgtt 1980

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gttgatcttt tgcccttgat ccttctaaat catgatcata tgcaagcata ttccctcatg 2220

gacattagct tggatacttt tccttatgct ggaacaacca ctacatgtga atcactatac 2280

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Thr Ala Asn Asn Lys Leu Gly Thr Met Ala Cys Arg Pro Ala Pro Val

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Ser Ala Ile Gln Leu Ala Ser Asp Val Thr Ser Leu Ser Asn Leu Arg

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Lys Phe Thr Arg Asn Ile Glu Ser Ile Tyr Arg Ser Met Trp Arg Arg

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Tyr Cys Asp Gly Asp Val Pro Ser Leu Arg Arg Met Glu Leu Leu Gln

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Gln Gln Gln Thr Gln Thr Glu Ser Val Val Pro Glu Glu Ser Ser Val

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Asn Pro Ser Glu Arg Thr Ile Thr Ser Ala Pro Thr Asp Gly Ser Ile

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Lys Glu Asn Gly Phe Thr Ala Val Pro Ala Leu Ala Leu Lys Ser Ser

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Thr Ser Glu Glu Asn Gly Val Gln Ser Asn His Asn Gly Asn His Gly

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Asn Leu Ser

930

Claims

1. TomatoSlSPYApplication of gene in controlling tomato fruit ripening process, and tomato fruit ripening process is realized by gene overexpression technologySlSPYIncreased expression levels of the gene; the tomato isSlSPYThe nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.