CN116286947A

CN116286947A - Method for promoting tomato maturation and quality improvement

Info

Publication number: CN116286947A
Application number: CN202310139749.3A
Authority: CN
Inventors: 周杰; 周灿彧; 王开心; 喻景权
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-02-08
Filing date: 2023-02-08
Publication date: 2023-06-23

Abstract

The invention discloses a method for promoting tomato maturation and quality improvement, which is used for silencing or knocking out XERICO genes in tomatoes, wherein the XERICO genes are XERICO1 genes and/or XERICO3 genes. The method can promote the maturation of tomatoes, improve the quality, particularly promote the maturation of tomatoes to be advanced, increase the yield of fruit ethylene, improve the quality of tomato fruits and the like.

Description

Method for promoting tomato maturation and quality improvement

Technical Field

The invention relates to the technical field of biology, in particular to a method for promoting tomato maturation and quality improvement.

Background

Ubiquitin proteasome pathway (UPS) is a highly selective protein degradation pathway in eukaryotic organisms, which specifically includes: firstly, under the condition of ATP energy supply, ubiquitin is activated by ubiquitin activating enzyme E1, then the ubiquitin is transferred onto ubiquitin conjugated enzyme E2 and is connected with cysteine residue of active site of E2 through a thioester bond, and E2 can directly transfer ubiquitin onto lysine residue of target protein. E3 ubiquitin ligase is capable of coupling ubiquitin with cognate substrates, having an important role in the ubiquitin pathway. All E3 have the ability to link target proteins and specific E2. Protein-specific post-translational modifications often serve as markers for recognition by their corresponding ubiquitin ligase E3. E3 ubiquitin ligases can be divided into four types, HECT, RING, U-box and RBR, by structure and function.

Tomato (Solanum lycopersicum l.) is an annual or perennial herb in the Solanaceae (Solanaceae) genus, an aliased tomato, a foreign persimmon, a vegetable crop cultivated the most widely and consumed worldwide, and due to its relatively short growth cycle, the facility cultivation technology is mature, and is an important respiratory jump fruit, becoming one of the important modes of plant molecular research.

The early research results show that the E3 ubiquitin ligase can play different regulating and controlling roles under different plants and plant growth and development stages or stress. For example, inhibition of AtATL78 in arabidopsis increases tolerance to cold stress, decreases tolerance to drought stress (Kim, so Jin and Kim, woo Taek (2013), suppression of Arabidopsis RING E, ubiquitin ligase AtATL, increases tolerance to cold stress and decreases tolerance to drought stress, FEBS Letters,587, doi: 10.1016/j.febset.2013.06.038); the over-expression of CHYR1 can promote and regulate stomatal movement and improve drought resistance (Shuangcheng Ding, bin Zhang, feng Qin, arabidopsis RZFP34/CHYR1, a Ubiquitin E3 Ligase, regulates Stomatal Movement and Drought Tolerance via SnRK2.6-Mediated Phosphorylation, the Plant Cell, volume 27,Issue 11,November 2015,Pages 3228-3244, https:// doi. Org/10.1105/tpc.15.00321); overexpression of XERICO can improve drought tolerance in Arabidopsis (Ko, J.—H., yang, S.H. and Han, K.—H. (2006), upregulation of an Arabidopsis RING-H2 gene, XERICO, confers drought tolerance through increased abscisic acid biosynthesis. The Plant Journal,47:343-355.Https:// doi.org/10.1111/j.1365-313X.2006.02782. X); overexpression of OsCTR1 in rice can improve drought tolerance (LIM, S.D., LEE, C.and JANG, C.S. (2014), osCTR1 in trafficking inhibition of interactions.plant Cell Environ,37:1097-1113.Https:// doi.org/10.1111/pce.12219); inhibiting OsHTASInhibiting heat resistance of rice (Jianping Liu, cuicui Zhang, chuchu Wei, xin Liu, mugui Wang, feifei Yu, qi Xie, jumin Tu, the RING Finger Ubiquitin E3 Ligase OsHTAS Enhances Heat Tolerance by Promoting H) ₂ O ₂ -Induced Stomatal Closure in Rice,Plant Physiology,Volume 170,Issue 1,January 2016,Pages429–443,https://doi.org/10.1104/pp.15.00879)。

However, the functions of RING type E3 ubiquitin ligase genes SlXERICO1 and SlXERICO3 in the tomato leaf in the aspects of fruit ripening and quality improvement are not reported yet.

Disclosure of Invention

The invention provides a method for promoting tomato maturation and quality improvement, which can inhibit expression of tomato XERICO1 genes and/or XERICO3 genes by CRISPR/Cas9 technology, generate XERICO1, XERICO3 and XERICO1/3 (jointly inhibit XERICO1 genes and XERICO3 genes) mutant plants, promote tomato maturation, improve quality, specifically promote tomato maturation in advance, increase fruit ethylene yield, improve tomato fruit quality and the like.

A method for promoting tomato maturation and quality improvement comprises silencing or knocking out XERICO gene in tomato, wherein the XERICO gene is XERICO1 gene and/or XERICO3 gene.

The nucleotide sequence of the XERICO1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the XERICO3 gene is shown as SEQ ID No. 2.

Preferably, the method may specifically include the steps of:

(1) Constructing a gene silencing or knocking-out vector which is a plant expression vector with a sequence for silencing or knocking-out the XERICO1 gene with a base sequence shown as SEQ ID No.1 and/or the XERICO3 gene with a base sequence shown as SEQ ID No. 2;

(2) Introducing the gene silencing or knocking-out vector in the step (1) into tomato cells, and silencing or knocking-out the XERICO1 gene with the base sequence shown in SEQ ID No.1 and/or the XERICO3 gene with the base sequence shown in SEQ ID No. 2.

In a preferred embodiment, the method for promoting tomato maturation and quality improvement comprises introducing a CRISPR/Cas9 vector of tomato XERICO1 gene and/or XERICO3 gene into target tomato to inhibit expression, thereby obtaining mutant plants of XERICO1 gene and/or XERICO3 gene.

The inhibition of expression of the XERICO1, XERICO3 and XERICO1/3 genes in the tomato of interest may be any method that reduces the expression of the XERICO1, XERICO3 and XERICO1/3 genes in the tomato of interest. In a specific embodiment of the invention, the inhibition of expression of the XERICO1, XERICO3 and XERICO1/3 genes in tomato of interest is achieved by constructing CRISPR/Cas9 vector of the genes and further by means of Agrobacterium infection.

Further preferably, the method specifically comprises the steps of:

1) Constructing agrobacterium tumefaciens (Agrobacterium tumefaciens) engineering bacteria containing tomato XERICO1 genes and/or XERICO3 genes CRISPR/Cas9 vectors;

2) And (3) transforming the agrobacterium tumefaciens engineering bacteria into a target tomato explant to prepare a tomato XERICO1 gene and/or a mutant plant of XERICO3 gene inhibition expression.

Preferably, in step 1), the agrobacterium tumefaciens engineering bacterium is agrobacterium GV3101 strain.

Preferably, in the step 2), the explant is cotyledon in which the seed is germinated for 6 to 8 days.

In the specific method, preferably, the tomato XERICO1 gene and/or the mutant plant of XERICO3 gene inhibition expression is subjected to normal growth management to obtain transgenic F2 generation with stable inheritance and seeds after the transgenic F2 generation.

The concrete manifestations of the method for promoting tomato maturation comprise that the tomato fruit maturation speed is increased, the tomato fruit maturation time is shortened, and the like, and the concrete manifestations of promoting tomato quality improvement comprise that the content of fructose and glucose in the tomato fruit is increased, the content of citric acid and malic acid is reduced, and the like.

In one experimental protocol, the specific steps are as follows:

1. constructing agrobacterium tumefaciens engineering bacteria containing tomato XERICO1, XERICO3 and XERICO1/3 gene CRISPR/Cas9 vectors;

2. preparing XERICO1, XERICO3 and XERICO1/3 gene mutant plants by carrying out mediated transformation on the target tomato explant by the agrobacterium tumefaciens engineering bacteria;

3. performing normal growth management on the XERICO1, XERICO3 and XERICO1/3 gene mutant plants to obtain transgenic F2 generation with stable inheritance and seeds after the generation;

4. experiments are carried out by using transgenic F2 generation and later seeds, and the condition of the mature stage of the tomato plant fruit is observed.

The lower the expression level of XERICO1 and XERICO3 genes in tomatoes is, the faster the ripening of tomato fruits is, and the better the quality is.

The XERICO1, XERICO3 and XERICO1/3 genes can be introduced into tomato of interest by recombinant expression vectors containing the genes, and the recombinant expression vectors can be pFGC1008, pFGC5941, pCAMBIA1300, pBI121 and the like or other derivative plant expression vectors, and when the plant expression vectors are used for constructing the recombinant vectors, constitutive, tissue-specific or inducible promoters can be used.

The tomato XERICO1 and XERICO3 genes are specifically as follows:

mature sequences of tomato XERICO1 and XERICO3 are obtained from a Solanaceae Genomics Network (https:// solgenomics. Net /) database, and the base sequences are shown in SEQ ID NO: 1. 2.

The invention provides application of tomato XERICO1 genes and XERICO3 genes in regulating tomato fruit ripening and quality, wherein the nucleotide sequence of the XERICO1 genes is shown as SEQ ID No.1, and the nucleotide sequence of the XERICO3 genes is shown as SEQ ID No. 2.

Compared with the prior art, the invention has the beneficial effects that:

the invention suppresses expression of tomato XERICO1 gene and XERICO3 gene by CRISPR/Cas9 technology, generates XERICO1, XERICO3 and XERICO1/3 mutant plants, can promote the maturation of tomato fruits, improves the quality of the fruits, and is particularly characterized by promoting the mature to be advanced, increasing the yield of fruit ethylene and improving the quality of tomato fruits.

Drawings

FIG. 1 is a schematic representation of CRISPR/Cas9 vector construction for large fragment deletion. Wherein, FIG. 1A is a diagram showing the position structure of the gRNAs of the vector; FIGS. 1B, 1C and 1D show the positions and sequences of two gRNAs on tomato XERICO1, XERICO3 and XERICO1/3 genomes, with the gRNA specific recognition sequences underlined.

FIG. 2 is a diagram of mutant plant identification. FIG. 2A shows two mutant lines obtained from SlXERICO1, slxerico1#3 and slxerico1#5, respectively; FIG. 2B shows two mutant lines obtained from SlXERICO3, slxerico3#2 and slxerico3#4, respectively; FIG. 2C shows two mutant lines obtained for SlXERICO1 and SlXERICO3, slXerico1/3#1 and slXerico1/3#2, respectively.

FIG. 3 shows fruit ripening of wild type (wild-type) and tomato SlXERICO1, slXERICO3 and SlXERICO1/3 mutants. The Slxerico1-1 corresponding mutant strain, slxerico1#3, slxerico1-2 corresponding mutant strain, slxerico1#5 in FIG. 3; the corresponding mutant strain slxerico3#2 of the slxerico3-1 and the corresponding mutant strain slxerico3#4 of the slxerico3-2; slxerico1/3 corresponds to the mutant line Slxerico1/3#1. Wherein, FIG. 3A is the fruit ripening time of SlXERICO1, slXERICO3 and SlXERICO1/3 mutants, dpa is the days after flowering, the data shown in the graph are the average of four replicates, and the standard error is shown by the vertical line; fig. 3B shows fruit phenotypes of SlXERICO1, slXERICO3 and SlXERICO1/3 mutants, scale = 1cm.

FIG. 4 shows the fruit ethylene yield of wild type (wild-type) and tomato XERICO1, XERICO3 and XERICO1/3 mutant plants. Wherein dpa is the number of days after flowering. The data shown in the figures are the average of four replicates, with standard error shown by the vertical bars. The stars indicate that the different strains differed from the wild type by a significant level of 5% using the Turkey test.

FIG. 5 is the fruit hardness of wild type (wild-type) and tomato XERICO1, XERICO3 and XERICO1/3 mutant plants. Wherein dpa is the number of days after flowering. The data shown in the figures are the average of four replicates, with standard error shown by the vertical bars. The stars indicate that the different strains differed from the wild type by a significant level of 5% using the Turkey test.

FIG. 6 shows pigment content of wild type (wild-type) and tomato XERICO1, XERICO3 and XERICO1/3 mutant plants. Wherein, FIG. 6A shows carotenoid content, and FIG. 6B shows lycopene content. dpa is the day after flowering, the data shown in the figure are the average of three replicates, standard error is shown by vertical bars. The stars indicate that the different strains differed from the wild type by a significant level of 5% using the Turkey test.

FIG. 7 shows sugar acid content of wild type (wild-type) and tomato XERICO1, XERICO3 and XERICO1/3 mutant plants. Wherein, fig. 7A is the soluble sugar content, fig. 7B is the organic acid content, and fig. 7C is the sugar acid ratio. dpa is the day after flowering, the data shown in the figure are the average of three replicates, standard error is shown by vertical bars. The stars indicate that the different strains differed from the wild type by a significant level of 5% using the Turkey test.

Detailed Description

The invention will be further elucidated with reference to the drawings and to specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The methods of operation, under which specific conditions are not noted in the examples below, are generally in accordance with conventional conditions, or in accordance with the conditions recommended by the manufacturer.

The experimental materials, reagents and the like used in the following examples are all commercially available unless otherwise specified. The experimental material was selected from the tomato cultivar Alisa Craig (Solanum lycopersicum l.cv).

Example 1

Cloning and vector construction of tomato SlXERICO1/3 genes.

CRISPR/Cas9 vector construction

The full length of the tomato SlXERICO1/3 gene is 459bp and 456bp respectively, and each consists of a single exon. The sgRNA of the SlXERICO1/3 gene is screened respectively by using a CRISPR-P (http:// CRISPR. Hzau. Edu. Cn/CRISPR2 /) website and selecting a U6 promoter. Wherein, the SlXERICO1/3 gene CRISPR/Cas9 vector respectively selects two sgRNAs at the exons of the gene, each sgRNA has a forward primer and a reverse primer, and the forward primer of the first sgRNA selected by each vector is a universal forward primer, and the sequence of the PCR primer is shown in table 1. FIG. 1 is a schematic representation of CRISPR/Cas9 vector construction for large fragment deletion. Wherein, FIG. 1A is a diagram showing the position structure of the gRNAs of the vector; FIGS. 1B, 1C and 1D show the results of two gRNAs inTomato XERICO1, XERICO3 and XERICO1/3 genome, and the gRNA specific recognition sequences are underlined. The specific construction process is as follows: and taking the tRNA carrier as a template, respectively carrying out PCR of two targets by using KOD high-fidelity enzyme, and purifying a PCR product, wherein the fragment size is about 200 bp. Multi-fragment recombination was accomplished using Golden Gate assembly kit (BsaI-HFv 2) (NEB, E1601). The recombinant system comprises Golden Gate Assembly Mix, PHEE401 vector, target PCR purified product and T4 DNA ligase buffer, and ddH is used ₂ O was filled to 20. Mu.L and the PCR program set up was referred to the kit instructions. The recombinant product is subjected to thermal transfer of trans5 alpha escherichia coli competent cells (TransGen, CD 201), and is subjected to shaking table 200rpm activation for 1h at 37 ℃ as described in the specification, supernatant is removed by centrifugation, 100-200 mu L of liquid is remained, precipitation is carried out by resuspension, and the obtained product is uniformly coated on a solid LB culture medium containing 50mg/L kanamycin, and is cultured overnight at 37 ℃. After single spots grow out, the spots are picked up and shaken slightly, bacterial liquid PCR is carried out by using general M13-F and M13-R primers, positive clones are identified and sequenced, and the positive plasmids are named pHEE401-SlXERICO1, pHEE401-SlXERICO3 and pHEE401-SlXERICO1/3 respectively.

TABLE 1 PCR primer sequences to construct CRISPR/Cas9 vectors

2. Tomato total RNA extraction

The tomato total RNA is extracted by using a plant total RNA extraction kit (Tiangen, DP 419), and the steps are as follows:

(1) Grinding 0.1g of blade in liquid nitrogen, adding 1mL of lysate RZ, and mixing by vortex;

(2) Standing at room temperature for 5min to completely separate nucleic acid protein complex;

(3) Centrifuging at 12000rpm for 5min at 4deg.C, removing supernatant, and transferring into a new RNase-free centrifuge tube;

(4) Adding 200 mu L of chloroform, covering a tube cover, vigorously shaking for 15s, and standing at room temperature for 3min;

(5) Centrifugation at 12000rpm for 10min at 4℃the samples were divided into three layers: a yellow organic phase, an intermediate layer and a colourless aqueous phase, RNA being predominantly in the upper aqueous phase, the volume of the aqueous phase being approximately 50% of the volume of the lysate RZ reagent used. Transferring the water phase into a new pipe for the next operation;

(6) Slowly add 0.5 times volume of absolute ethanol, mix well (precipitation may occur at this time). Transferring the obtained solution and the precipitate into an adsorption column CR3, centrifuging at 12000rpm for 30s at 4 ℃, and discarding the waste liquid in a collecting pipe;

(7) Adding 500 mu L deproteinized solution RD into an adsorption column CR3, centrifuging at 4 ℃ and 12000rpm for 30s, and discarding the waste liquid;

(8) Adding 600 mu L of rinsing liquid RW into an adsorption column CR3, standing at room temperature for 2min, centrifuging at 4 ℃ and 12000rpm for 30s, and discarding the waste liquid;

(9) Repeating the operation step (8);

(10) Placing the adsorption column into a 2mL collecting pipe, centrifuging at 12000rpm for 2min at 4 ℃ to remove residual waste liquid;

(11) Drying the adsorption column in an ultra clean bench for 5min, transferring into a new RNase-Free centrifuge tube, adding 50 μl of RNase-Free ddH ₂ O, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min at 4 ℃;

(12) Determination of OD with ultraviolet Spectrophotometer ₂₆₀ /OD ₂₈₀ Detecting the content and purity of the RNA sample, wherein the concentration is 781 ng/. Mu.L and the OD ₂₆₀ /OD ₂₈₀ ＝2.10。

3. Gene cloning and construction of agrobacterium tumefaciens engineering bacteria

Reverse transcription kit (Vazyme, R223) was used to remove genomic DNA from total RNA and reverse into cDNA. The obtained CRISPR/Cas9 vector pHEE401-SlXERICO1, pHEE401-SlXERICO3 and pHEE401-SlXERICO1/3 are respectively transferred into agrobacterium tumefaciens GV3101 to respectively obtain agrobacterium tumefaciens engineering bacteria A containing the tomato SlXERICO1 gene CRISPR/Cas9 vector, and agrobacterium tumefaciens engineering bacteria B containing the tomato SlXERICO3 gene CRISPR/Cas9 vector and agrobacterium tumefaciens engineering bacteria C containing the tomato SlXERICO1/3 gene CRISPR/Cas9 vector.

Example 2

Constructing tomato SlXERICO1/3 gene mutant plants.

The leaf disc method is utilized, tomato cotyledons are infected through agrobacterium mediation, target vectors CRISPR/Cas9, namely pHEE401-SlXERICO1, pHEE401-SlXERICO3 and pHEE401-SlXERICO1/3 are respectively transformed into the tomato cotyledons, hygromycin is utilized for screening, and PCR amplification generation sequencing is utilized for screening mutant plants.

The method comprises the following specific steps:

1) Configuration of culture Medium

Sowing culture medium: 2.15g/L MS powder+100 mg/L inositol+10 g/L sucrose+8 g/L agar. The pH was 5.8.

Nursing medium: 4.44g/L MS powder+30 g/L sucrose+100 mg/I inositol+1.3 g/L thiamine hydrochloride+0.2 mg/L2, 4-D+200mg/L KH ₂ PO ₄ +0.1mg/L KT+7.5g/L agar. The pH was 5.8.

2Z selection regeneration Medium: 4.44g/L MS powder+30 g/L sucrose+100 mg/L inositol+2 mg/L ZR+300mg/L timentin+6mg/L hygromycin. The pH was 5.8.

0.2Z selection regeneration Medium: 4.44g/L MS powder+30 g/L sucrose+100 mg/L inositol+0.2 mg/L ZR+300mg/L timentin+6mg/L hygromycin. The pH was 5.8.

Rooting medium: 4.44g/L MS powder+30 g/L sucrose+100 mg/L inositol+300 mg/L timentin+6mg/L hygromycin. The pH was 5.8.

Liquid MS 0.2 medium: 4.44g/L MS powder+20 g/L sucrose+100 mg/L inositol+0.2 mg/L thiamine hydrochloride. The pH was 5.8. Agrobacterium for suspension infection.

YEB medium: 5g beef extract, 5g peptone, 1g yeast extract, 5g sucrose, 0.5g MgSO ₄ ·7H ₂ O, constant volume to 1L with distilled water, adjusting pH to 7.0, and autoclaving at 121deg.C for 20 min. Each liter of the YEB solid culture medium is added with 15g of agar powder, and other components are the same as the liquid culture medium.

2) Culture of aseptic seedlings

Tomato seeds were soaked in tap water (or with shaker 28 ℃ 200 r/min) for 6-8 h, then sterilized with 75% alcohol for 30s, then sterilized in 10% NaClO for 15min (with shaker 28 ℃ 200 r/min), rinsed 3 times with sterilized distilled water and transferred to sterilization vessel, inoculated in 1/2 MS medium. Culturing at 25deg.C in dark, transferring into light culture room, and culturing at 25deg.C under 16h light/8 h dark with 1800lx light intensity.

3) Preparation of explants and cultivation of Agrobacterium

After the seeds germinate for about a week, when the cotyledons are stretched and the true leaves are not grown, the cotyledons of the aseptic seedlings are cut into two sections by a new scalpel, the cotyledons are provided with a small petiole, and the cotyledons are spread in a nursing culture medium for preculture for 24 hours (the cotyledons are protected from light and are obtained overnight, and the overgrowth is easily caused by overlength of nursing culture time). Single colonies of Agrobacterium were picked on LB plates containing antibiotics and inoculated into 30mL of LB containing antibiotics (150 mL Erlenmeyer flask), and cultured overnight at 28℃at 200r/min to mid-log (OD 600. Apprxeq.1.0, about 16-24 h). Shaking bacteria, cutting cotyledon (inoculating for 12-20 h).

4) Conversion regeneration

Taking out the engineering bacteria A-C from refrigerator at-80 deg.C, activating on YEB plate containing corresponding antibiotic, picking single colony of Agrobacterium, inoculating in 2mL YEB containing antibiotic, shaking at 28 deg.C and 200rpm for overnight culture, expanding 30mL at 1:100, shaking at 28 deg.C and culturing at 200rpm for over night to OD ₆₀₀ =0.8 to 1.0. Centrifuging the cultured agrobacterium for 10min at 4000r/min at 4 ℃; the supernatant was discarded, and 15mL of suspension medium MS 0.2 was added to suspend the cells for further use. The precultured cotyledon explant is transferred to a sterilized culture dish poured with 15mL of MS 0.2, the suspended bacterial liquid is poured in, the cotyledon explant is infected for 2-3 min in a dark place, and the culture dish is gently rocked. The explant is gently fished up by forceps, the explant is transferred to a sterilized filter paper, residual bacterial liquid is sucked up, the residual bacterial liquid is transferred to the sterilized filter paper, the back surface of the explant is flatly paved back to the original nursing culture medium, and the explant is co-cultured in darkness at 22 ℃ for 48 hours.

The co-cultured explant is transferred to a 2Z culture medium from the right side upwards, is cultured at 25 ℃ under 16h illumination/8 h dark photoperiod, the fresh 2Z culture medium is replaced every two weeks, the browned explant is excised after differentiation and bud emergence, and the differentiated bud is transferred to a 0.2Z culture medium for selective culture. Fresh medium was changed every three weeks.

5) Rooting culture and transplanting

When the regenerated buds grow to about 1cm, cutting off the buds (without cutting off the buds so as not to hurt rooting parts), and putting the buds into a rooting culture medium for rooting. Hardening seedlings of transformed seedlings which have good rooting and grow to about 5cm after 2 weeks, and transplanting the seedlings into a nutrition pot with turf, vermiculite=3:1 matrix to obtain tomato SlXERICO1/3 gene mutant plants.

Example 3

Molecular detection of transgenic plants tomato SlXERICO1/3 mutant plants were detected at the DNA level by PCR.

The DNA of tomato transgenic plant is extracted rapidly by CTAB method in small quantity, and the steps are as follows:

1. 50-100 mg tomato leaves are taken in a 1.5mL centrifuge tube, steel balls are added, quick-freezing is carried out in liquid nitrogen, and after the sample is ground into powder, 500 mu L of CTAB buffer is added;

2.55 ℃ water bath for 15min, and the mixing is reversed for several times during the period;

3. adding 500. Mu.L chloroform to isoamyl alcohol (24:1), mixing well by vortex, centrifuging, 12000rpm,5min;

4. transferring the supernatant to a new centrifuge tube, adding 1/10 volume of sodium acetate (3M) and 2 times of ice absolute ethyl alcohol, mixing uniformly by vortex, and precipitating for 1h at-20 ℃;

5. centrifuging at 12000rpm for 3min to obtain white precipitate under the bottom, which is DNA, and discarding supernatant;

6. adding 1mL of precooled 70% ethanol, shaking up and down, washing, centrifuging at 12000rpm for 1min, and discarding the supernatant;

7. repeating washing once, removing residual liquid as much as possible, blow-drying on a super clean bench, adding 50 μl ddH ₂ O is dissolved and stored at-20 ℃.

And respectively designing specific primers near the sequence positions of sgRNAs of the tomato SlXERICO1/3 genes to detect the change condition of the target gene sequence. The primers are as follows, and the lengths of the SlXERICO1/3 detection fragments are 731bp and 453bp respectively. And (5) comparing and screening the homozygous mutant of the SlXERICO1/3 according to the sequencing result of the PCR products. The PCR results were carried out and are shown in FIGS. 2A, 2B and 2C. Two lines of slxerico1#3 and slxerico1#5 of a slxerico1 mutant plant are shown in fig. 2A, wherein slxerico1#3 has 1 base inserted at exon 1 and slxerico1#5 has 16 bases deleted at exon 1, resulting in premature termination of the SlXERICO1 protein at 93 and 52 amino acids, respectively; two lines of slxerico3#2 and slxerico3#4 of the slxerico3 mutant plant are shown in fig. 2B, wherein slxerico3#2 lacks 1 base at exon 1 and slxerico3#4 lacks 2 bases at exon 1, leading to premature termination of the SlXERICO3 protein at 106 and 86 amino acids, respectively; FIG. 2C shows two lines of slXERICO1/3 mutant plants, slXerico1/3#1 and slXerico1/3#2, wherein slXerico1/3#1 lacks 1 base at exon 1 of slXERICO1 and 1 base at exon 1 of slXERICO3, such that the slXERICO1 and slXERICO3 proteins terminate prematurely at amino acids 98 and 106, respectively; slXERICO1/3#2 inserts 1 base at exon 1 of SlXERICO1 and 2 bases at exon 1 of SlXERICO3, resulting in premature termination of the SlXERICO1 and SlXERICO3 proteins at amino acids 59 and 86, respectively.

The specific primer sequences were as follows:

CRISPR-SlXERICO1-F：5′-TACTTGAAGTCCTATGTTTCTTCTCTA-3′(SEQ ID No.13)；

CRISPR-SlXERICO1-R：5′-CATTGGACATGTATCGTCCTCTTC-3′(SEQ ID No.14)；

CRISPR-SlXERICO3-F：5′-ATGGGACTCTCACCATATACGACTC-3′(SEQ ID No.15)；

CRISPR-SlXERICO3-R：5′-CATTGGACAAGTATCTTCCTCACCT-3′(SEQ ID No.16)。

example 4

Observing the mature stages of the obtained tomato XERICO1, XERICO3 and XERICO1/3 gene mutant plants, wherein the experimental materials are respectively as follows: wild type WT, mutant plants XERICO1-1, XERICO1-2, XERICO3-1, XERICO3-2, XERICO1/3. Wherein, the transgenic plants adopt F2 generation seeds with stable inheritance for subsequent experimental observation.

And performing phenotypic observation and physiological index measurement on the plants in the fruit period. The seedling stage of the plants is conventionally managed in a growth chamber, and when the wild WT grows to six leaves and one core, the plants of each plant system are transplanted to an artificial greenhouse in a unified manner. The culture conditions are as follows: 200 mu mol m ^-2 s ^-1 Light intensity (PPFD), 12h photoperiod, 25/20 ℃ (day/night), and Hoagland nutrient solution was irrigated every 2-3 d.

(1) Fruit ripening statistics

Counting the days from the sowing of the plants to the development of the first inflorescences and the days from the breaking of the colors of the fruits, and counting the days from the flowering of different strains to the breaking of the colors of the fruits. Taking pictures to record the fruit phenotype of different strains in the invention by taking the wild fully-expanded fruits in the green ripening period as the start. The results are shown in FIG. 3.

Note that: the data shown in fig. 3A are averages of four replicates. Standard errors are shown by vertical bars using Turkey test. .

Phenotype recordings were performed on the fruits photographed from green to red, as shown in FIG. 3, and the fruits of XERICO1, XERICO3 and XERICO1/3 mutant lines were found to mature faster than the wild type.

(2) Determination of fruit ethylene Release amount

The ethylene release rate of the tomato fruits in different maturity stages is measured, at least 2 fruits in different periods are weighed and placed in a 550mL sealed fresh-keeping box, and the tomato fruits are placed for 1h at normal temperature and in a dark place. More than 1mL of headspace gas in the cartridge was withdrawn from the rubber tube above each fresh-keeping cartridge with a syringe with 1mL of scale, and each cartridge was withdrawn 4 times as 4 biological replicates. And sequentially inserting needles on the prepared wood plugs according to corresponding labels, and injecting the samples into a gas chromatograph (Philips, UNICAM pro.GC) one by one after the detection samples are absorbed in each period. The analytical column used was a 1500X 4mm alumina glass column. The temperatures of the sample injector, the detector and the column passing are 130 ℃, 130 ℃ and 200 ℃ respectively. In the measurement, 1mL of gas was quantitatively injected for detection. According to the standard ethylene standard gas (10 mu L/L), the peak position is referenced, the corresponding peak time and peak area are obtained by using analysis software equipped with a gas chromatograph, the ethylene concentration in each sample is calculated, and the ethylene release value per kilogram of sample per hour is calculated. The results are shown in FIG. 4. The data shown in the figures are the average of four replicates, with standard error shown by the vertical bars.

As can be seen from FIG. 4, at 41 and 45 days after flowering, the ethylene yields of XERICO1, XERICO3 and XERICO1/3 mutant lines were higher than the wild type, indicating that the XERICO1, XERICO3 genes affected the release of fruit ethylene to some extent.

(3) Quality index measurement

(1) Fruit hardness measurement

The ethylene release rate of the tomato fruits at different maturity stages is measured, at least 3 fruits at different periods are cut off to be about 1cm thick peel at the top position of the fruit and the end position of the fruit stem, a GY-1 fruit sclerometer is used for measuring the hardness of pulp of the fruit, the probe of the sclerometer is perpendicular to the fruit surface and is pressed under a stable force, the probe penetrates into a diaphragm for 2mm, and the reading shown by the sclerometer is measured at the moment, namely the pulp hardness of the fruit and the front and the back are measured.

FIG. 5 is the fruit hardness of wild type (wild-type) and tomato XERICO1, XERICO3 and XERICO1/3 mutant plants.

Note that: dpa is the number of days post-flowering. The data shown in the figures are the average of four replicates, with standard error shown by the vertical bars. The stars indicate that the different strains differed from the wild type by a significant level of 5% using the Turkey test.

As can be seen from FIG. 5, the hardness of XERICO1, XERICO3 and XERICO1/3 mutant lines decreased more rapidly than that of the wild type at 41 and 43 days after flowering, indicating that the XERICO1, XERICO3 genes affected the hardness and quality of the fruits to some extent.

(2) Extraction and content detection of fruit carotenoid

0.5g of tomato pulp is weighed, fully ground by liquid nitrogen and transferred into a 2mL centrifuge tube. 350. Mu.L of methanol and 700. Mu. L, ddH2O 350. Mu.L of chloroform were added in this order, then mixed by vortexing, centrifuged at 10000g for 10min at 4℃with a centrifuge, and the chloroform phase was collected. The residual tube was then charged with 700. Mu.L of chloroform, and the extraction was repeated several times until colorless. The collected chloroform phases were combined and the collection tube was blow-dried with nitrogen. Then 350. Mu.L of a 6% KOH in methanol (w/v) was added to dissolve the precipitate, and the solution was treated at 60℃in the absence of light and derivatised for 30min. Chloroform 700. Mu.L and water 350. Mu.L were added, mixed by vortexing, centrifuged at 10000g for 5min at 4℃with a centrifuge, and the chloroform phase was collected. Adding 700 μl of water into chloroform phase, extracting, repeating for several times, and standing until the water layer is neutral. The chloroform phase collected was dried with nitrogen, and finally dissolved in 100. Mu.L of chromatographic grade ethyl acetate, and centrifuged at 14000g for 20min at 4℃to ensure complete sedimentation of the precipitate, and 150. Mu.L of the supernatant was used for High Performance Liquid Chromatography (HPLC). The above procedure should be set to more than 5 biological repeats.

The procedure for the injection of carotenoids is described below. Using an Alliance2695 system (Waters Corporation, USA), containing 2695 separation modules and 2996PDA detector, a 5 μmC30 reverse phase column (250 mm. Times.4.6 mm) and a 20 mm. Times.4.5 mm C30 pre-column (Waters Corp.) were provided. The temperature of the column is set to 25 ℃, the flow rate is regulated to 1mL/min, the sample injection volume is set to 20 mu L, and the wavelength range during detection is 220-600 nm. The mobile phase was gradient eluted with solvents a (methanol), B (80% methanol) and C (MTBE), with 0-6 min set to 95% a+5% B, 7-11 min set to 80% a+5% b+15% C, 12-32 min mobile phase set to 30% a+5% b+65% C, 48-50 min set to 95% a+5% B, then not changed to end, the whole procedure was about 60min. And then, carrying out data analysis by using Waters Empower software, and carrying out systematic analysis and identification on each carotenoid component according to the retention time and the absorption spectrum curve of the standard sample. The results are shown in FIG. 6.

FIG. 6 shows pigment content of wild type (wild-type) and tomato XERICO1, XERICO3 and XERICO1/3 mutant plants. Wherein, FIG. 6A shows carotenoid content, and FIG. 6B shows lycopene content.

Note that: dpa is the day after flowering, the data shown in the figure are the average of three replicates, standard error is shown by vertical bars. The stars indicate that mutant plants were up to 5% significantly different from wild type using the Turkey test.

(3) Determination of soluble sugar and organic acid content of fruit

0.2g of pulp is ground into powder by liquid nitrogen, 1mL of chromatographic methanol is added, vortex mixing is carried out, after heating for 15min at the temperature of 950rpm rotary metal bath 70 ℃, 10000g of normal temperature centrifuge is centrifuged for 10min, the supernatant is sucked into a 1.5mL centrifuge tube, 100 mu L of supernatant is taken from the supernatant into a new 1.5mL centrifuge tube, 20 mu L of ribitol (0.2 mg/mL) is added as an internal standard, vacuum drying is carried out for a plurality of hours at normal temperature, 400 mu L of freshly prepared methoxyamine hydrochloride solution (20 mg/mL, pyridine solution) is added, heating for 1.5h at the temperature of 950rpm rotary metal bath 37 ℃, and 600 mu L of bis (trimethylsilyl) trifluoroacetamide (1% trimethylchlorosilane) is added, and heating for 30min at the temperature of 37 ℃ is continued at 950 rpm. After the extraction is completed, the components are usedThe analytical instrument was Shimadzu GC-MS-QP2010 plus and the column was VF-5MS (30 m. Times.0.25 mm. Times.0.25 μm). The carrier gas was helium (He), and the column flow was 1mL/min ^-1 . Weigh with METTLER one ten thousandth balance.

FIG. 7 shows sugar acid content of wild type (wild-type) and tomato XERICO1, XERICO3 and XERICO1/3 mutant plants. Wherein, fig. 7A is the soluble sugar content, fig. 7B is the organic acid content, and fig. 7C is the sugar acid ratio. Compared with the wild type, the XERICO1, XERICO3 and XERICO1/3 mutant strains have higher fructose and glucose content, no obvious difference of sucrose and lower citric acid and malic acid content.

As can be seen from fig. 6 and 7, 41 days and 45 days after flowering, the XERICO1, XERICO3 and XERICO1/3 mutant lines have higher carotenoid and lycopene content, higher soluble sugar content, lower organic acid content and larger sugar acid ratio compared with the wild type, indicating that the XERICO1, XERICO3 genes affect the color and flavor quality of fruits to some extent.

The research results of the invention show that the lower the expression quantity of XERICO1 and XERICO3 genes in tomatoes is, the faster the ripening of tomato fruits is, and the quality of the fruits is better.

Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the foregoing description of the invention, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A method for promoting tomato maturation and quality improvement, which is characterized in that XERICO genes in tomatoes are silenced or knocked out, wherein the XERICO genes are XERICO1 genes and/or XERICO3 genes.

2. The method according to claim 1, wherein the nucleotide sequence of the XERICO1 gene is shown in SEQ ID No.1, and the nucleotide sequence of the XERICO3 gene is shown in SEQ ID No. 2.

3. The method according to claim 1, characterized in that it comprises the steps of:

4. The method according to claim 1, wherein the CRISPR/Cas9 vector of tomato xeraco 1 gene and/or xeraco 3 gene is introduced into tomato of interest for repression of expression to obtain mutant plants of xeraco 1 gene and/or xeraco 3 gene.

5. The method according to claim 4, characterized in that it comprises the steps of:

6. The method according to claim 5, wherein in step 1), the agrobacterium tumefaciens engineering bacterium is agrobacterium GV3101 strain.

7. The method according to claim 5, wherein in step 2), the explant is cotyledon from 6 to 8 days of seed germination.

8. The method according to claim 5, wherein the tomato XERICO1 gene and/or mutant plants in which XERICO3 gene is inhibited are subjected to normal growth management to obtain stably inherited transgenic F2 generation and later seeds.

9. The method according to claim 1, wherein the method comprises accelerating the ripening of tomato fruits, reducing the ripening time of tomato fruits, and accelerating the quality of tomato comprises increasing the fructose and glucose content of tomato fruits and reducing the citric acid and malic acid content.

10. The application of tomato XERICO1 gene and XERICO3 gene in regulating tomato fruit maturation and quality is characterized in that the nucleotide sequence of the XERICO1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the XERICO3 gene is shown as SEQ ID No. 2.