CN121182891B - An SlZFP11 gene and its application in regulating crop storage tolerance - Google Patents

Abstract

The invention discloses SlZFP gene and application thereof in regulating crop storability, belonging to the technical field of genetic engineering. According to the invention, through CRISPR/cas9 gene editing technology, the endogenous gene SlZFP of the tomato is knocked out in the tomato, and the content of glucose, fructose, sucrose and other sugar substances and cellulose in tomato fruits obtained by the knocked-out strain is increased, so that the sweet taste of the tomato fruits is greatly enhanced, the quality of the fruits is improved, the water loss rate is reduced, the storability is obviously increased, the storage period of the tomato is prolonged, and gene resources and strategies are provided for innovation of tomato germplasm resources.

Description

SlZFP11 gene and application thereof in regulating crop storability

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to SlZFP gene and application thereof in regulating and controlling crop storability.

Background

Tomato (Solanum lycopersicum) is used as a typical respiratory transition fruit, and ethylene is increased sharply in the post-ripening process, so that rapid softening and post-harvest decay are easily caused, and great loss is caused. Although the softening can be delayed by simply and strongly inhibiting ethylene or key maturation factors, the problems of difficult storage resistance and cooperative promotion of flavor are difficult to achieve due to the contradiction that the accumulation of color and flavor substances is inhibited and storage resistance and bad taste are easy to occur.

In recent years, genetic engineering and molecular breeding means provide a new way for improving the comprehensive quality of tomatoes. Zinc finger proteins (Zinc Finger Proteins, ZFPs) are used as an important transcription regulatory factor and widely participate in the regulation of growth and development, stress response and fruit ripening in plants. ZFPs regulate downstream gene expression through their specific DNA binding domains, especially playing a key role in abiotic stress and hormonal signaling networks. Studies have shown that certain ZFP members affect multiple biological processes such as cell wall modification, sugar metabolism, and antioxidant substance synthesis, showing potential in the field of multi-trait synergistic regulation. However, the specific function of the SlZFP gene in tomato has not been clarified, and its regulatory mechanism in fruit ripening, quality formation and post-harvest storability has yet to be resolved.

Therefore, starting from key transcription factors, the function of SlZFP in the aspect of the collaborative improvement of the quality and the storability of the tomato fruits is mined, which is not only helpful for deepening the understanding of a fruit ripening regulation network, but also provides a new thought for breaking the negative correlation between the storability and the flavor, and provides effective gene resources and technical approaches for realizing the quality upgrading and the postharvest loss of the fruits and vegetables.

Disclosure of Invention

The invention aims to solve the technical problems of insufficient quality and short shelf life of tomatoes by providing SlZFP gene and application thereof in regulating and controlling crop storability.

In order to achieve the aim, the technical scheme adopted by the invention is that the SlZFP gene is applied to regulating and controlling the storability of tomatoes, the nucleotide sequence of the SlZFP gene is shown as SEQ ID NO. 1, and the SlZFP gene can also regulate and control the dry matter and sugar content of tomatoes.

Based on the technical scheme, the invention can also be improved as follows:

further, the amino acid sequence of the protein encoded by SlZFP gene is shown as SEQ ID NO. 2.

Further, by inhibiting or knocking out SlZFP gene, the dry matter and sugar content of tomato is increased, while the storability of tomato is improved.

The invention has the beneficial effects that the endogenous gene SlZFP of the tomato is knocked out in the tomato by using the CRISPR/cas9 gene editing technology, and the glucose, fructose, sucrose and other sugar substances and cellulose contents in tomato fruits obtained by a strain after the knocking out are increased, so that the sweet taste of the tomato fruits is greatly enhanced, the quality of the fruits is improved, the water loss rate is reduced, the storability is obviously increased, the preservation period of the tomato is prolonged, and the gene resources and strategies are provided for innovation of tomato germplasm resources, so that the demands of consumers can be better met.

Drawings

FIG. 1 is a map of a constructed FastCas98 expression vector;

FIG. 2 shows the results of electrophoretic detection of SlZFP gene CRISPR/cas9 knock-out plants;

FIG. 3 is a schematic diagram of gene editing of SlZFP gene knockout line;

FIG. 4 shows the water loss rate detection results of wild type tomato and SlZFP11 knockout line tomato fruits;

FIG. 5 shows the comparison of the storage experiments of wild type tomato and SlZFP11 knockout line tomato fruits;

FIG. 6 shows the results of dry matter content detection of fruits of wild type tomato and SlZFP11 knockout line tomato in red ripe stage;

FIG. 7 shows the results of sucrose content detection of fruits of wild type tomato and SlZFP11 knockout line tomato in red ripe stage;

FIG. 8 shows the results of the detection of fructose content in fruits of wild type tomato and SlZFP11 knockout line tomato in red ripe stage;

FIG. 9 shows the results of the glucose level detection of fruits of wild type tomato and SlZFP11 knockout line tomato in red ripe stage;

FIG. 10 shows the results of detecting genes SlLIN5 related to sugar metabolism in tomato fruits of wild type tomato and SlZFP gene knockout strain;

FIG. 11 shows the results of detecting genes SlLIN related to sugar metabolism in tomato fruits of wild type tomato and SlZFP11 knockout line;

FIG. 12 shows the results of detecting the genes SlSUS related to sugar metabolism in tomato fruits of wild type tomato and SlZFP11 knockout line.

Detailed Description

The following description of the specific embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, and the examples are not intended to be limiting, and the reagents or apparatus used are not intended to be limiting, and are conventional products available for commercial purchase. It should be understood that the invention is not limited to the specific embodiments, but is capable of numerous modifications within the spirit and scope of the invention as hereinafter defined and defined by the appended claims as will be apparent to those skilled in the art all falling within the true spirit and scope of the invention as hereinafter claimed.

The nucleotide sequence of SlZFP gene is as follows:

ATGGAGAATGATTTTCCTAATTCTTCCAACTCCCAAAAATACCAAATAACATGGGGTCCTGATGGAGGCAAAATAAGCCAAGTCAAGTGTTATAGATGTAGCTTTTGTAAAAGAGGTTTCTCTAATGCACAAGCTCTAGGTGGACACATGAATATCCATAGAAAAGATAGAGCCAAGCTTAGAGAAATCTCGATCGAGACTTCAGATCATATCAAGAAGTTTGTCAGTCCTTCTTCTCCCGATCATATTCAGGCACTATCCAGTACCGATGAATTAATATTGCAACACGATATATCTAGCGATGACATGAGTAACAACCCCTCGAAAAGGCCATGTGTTACACTTGAAGAACAACATCATAATCATAATCATCATCATCATATTTCGAAAGAAAAAGATGAAAATCATGAATTGATTATTGGAGGTGATGTTTTACAATTACCTTTGTTTGTGGACTCTCCATCAAAAGAAGAAATTAATAAGGGCATGCAATTGAGTGTTGATGATTCAAAATTGGACCTTGAGCTTCGATTAGGACCAGAACCATAG（SEQ ID NO：1）.

the amino acid sequence of the protein encoded by SlZFP gene is as follows:

MENDFPNSSNSQKYQITWGPDGGKISQVKCYRCSFCKRGFSNAQALGGHMNIHRKDRAKLREISIETSDHIKKFVSPSSPDHIQALSSTDELILQHDISSDDMSNNPSKRPCVTLEEQHHNHNHHHHISKEKDENHELIIGGDVLQLPLFVDSPSKEEINKGMQLSVDDSKLDLELRLGPEP（SEQ ID NO：2）.

The primer sequences used in the following examples are shown in Table 1.

TABLE 1 primer sequence listing

EXAMPLE 1 construction of the CRISPR/cas9 Gene knockout System of SlZFP Gene

1. Design of target sequence of SlZFP gene

Target site design was performed using the http:// cas9.Cbi. Pku. Edu. Cn/index. Jsp website. And (3) evaluating the sequence, position, GC content, potential off-target position and other relevant information of all candidate targets in the target design result, wherein the GC content is selected to be 45% -70%, and the position is located in the first 2/3 region (after ATG but not on the last exon) of the CDS of the gene, and is not matched with at least three bases of any other positions in the genome.

2. Construction of SlZFP gene CRISPR/cas9 vector

(1) CRISPR/cas9 vector primer design for constructing SlZFP gene

ZFP11-Cas9 -F:CCAAAAATACCAAATAACATGGG(gRNA1)GTTTCAGAGCTATGCTGGAAACA(SEQ ID NO:5);

ZFP11-Cas 9-R ATAACATGGGGTCCTGATGGAGG (gRNA 2 reverse complement sequence) CAATCACTACTTCGACTCTAGCTGT (SEQ ID NO: 6).

(2) Amplification of the target fragment the amplification system is shown in Table 2.

TABLE 2 PCR amplification System

The amplification procedure was 98℃for 3min, (98℃for 10s,58℃for 20s,72℃for 30 s) 30 cycles, 72℃for 5min, and 4℃for preservation.

(3) FastCas9 vector cleavage was ligated to the fragment, and the ligation system is shown in Table 3.

TABLE 3 FastCas9 vector cleavage and fragment ligation System

The connection procedure was 37℃2min, 16℃5min.6 cycles. 1. Mu.L of transformed E.coli was taken for competence.

(4) Colony PCR verification

4 Single colonies of each gene are picked for PCR verification, positive colonies are amplified, sequencing is carried out by a sequencing company, and plasmids are extracted from bacterial liquid with correct sequencing. The correct recombinant plasmid is transformed into agrobacterium tumefaciens GV3101, and the agrobacterium tumefaciens GV3101 is screened on a flat plate culture medium containing 50 mug/mL of kanamycin and 100 mug/mL of rifampicin, and monoclonal is selected for colony PCR verification, and the strain after verification is the engineering bacterium of the recombinant expression vector.

The map of FastCas98 expression vector constructed is shown in FIG. 1.

EXAMPLE 2 obtaining SlZFP Gene knockout line plants

1. Transformation of tomato explants by leaf disc method using agrobacterium containing recombinant plasmid

(1) Seed disinfection

Placing wild tomato seeds into a sterile vessel, pouring 70% ethanol water solution to soak for 30s to disinfect the surfaces of the seeds, pouring the ethanol water solution, washing the seeds twice by using sterile water, pouring sodium hypochlorite solution with 5% of available chlorine content to soak for 15min, shaking continuously to ensure that each seed is fully disinfected, washing the seeds by sterile water for 3-4 times, pouring water in the seeds, transferring the seeds onto a seed culture medium, and placing the seeds into an illumination incubator for illumination culture at 25 ℃ for 14h, dark culture at 20 ℃ for 10h, illumination intensity of 250 mu mol.m ^-2•s^-1, relative humidity of 80%, and culture period of 8-10 days.

(2) Pre-culture of explants

After the seeds germinate for about 9-10 days, i.e. the best period for obtaining explants when the true leaves of the tomato seedlings are about to grow out, the seedlings are placed on sterile filter paper, the cotyledons and hypocotyls are cut with sterilized surgical blades, and then transferred to KCMS preculture medium for dark culture for one day.

(3) Agrobacterium infection explant

100. Mu.L of stored Agrobacterium tumefaciens GV3101 was added to 20mL of LB medium (containing 20. Mu.L, 50. Mu.g/mL kanamycin and 40. Mu.L, 100. Mu.g/mL rifampicin) and incubated overnight at 28℃at 230rpm until absorbance OD ₆₀₀ was about 0.8-1.0. 1mL of the bacterial liquid was aspirated, the supernatant was discarded after centrifugation at 5000rpm, the pellet was washed once with KCMS liquid medium, and the bacterial liquid was diluted to OD ₆₀₀ = 0.1 with KCMS liquid medium. A drop of agrobacterium dilution is dripped at the wound of the explant by using a pipetting gun, the bacterial liquid is not required to be sucked out, and the plate is sealed and then placed on a KCMS culture medium for dark culture for 2 days.

(4) Differentiation and rooting of explants

After 2 days of co-cultivation of the explants with Agrobacterium, the explants were transferred from KCMS medium to a preliminary screening medium (2Z) supplemented with 20. Mu.M trans-zeatin nucleosides, and the preliminary screening medium was changed every 15 days. When shoots to be differentiated were grown from the explants, the explants were transferred to medium (1Z) supplemented with 10. Mu.M trans-zeatin nucleosides and the medium was changed every 15 days. After culturing twice on 1Z medium, the differentiated callus grows buds with independent main stems, and is cut and inserted into rooting medium ENR for transplanting after rooting.

(5) Identification of transgenic positive plants

The transgenic T0 generation seedlings are checked to see whether the seedlings are transgenic positive plants by using CRISPR/Cas9 vector detection primers Cas9-F (SEQ ID NO: 3)/Cas-R (SEQ ID NO: 4), and the detection results are shown in figure 2.

As shown in FIG. 2, the genomic DNA of T0 generation seedlings was used as a template for PCR amplification, and the fragment of 500bp size detected by electrophoresis was determined as a transgenic positive plant.

Then taking genomic DNA of T0 generation seedlings as a template, designing a detection primer ZFP11-Cas9 check-F (SEQ ID NO: 7)/ZFP 11-Cas9 check-R (SEQ ID NO: 8) containing target sites for PCR amplification, recovering a target strip after agarose gel electrophoresis, constructing a purified PCR product on a pEASY-Blunt-Zero cloning vector for monoclonal sequencing, carrying out 20 monoclonal sequencing on each corresponding transgenic positive plant, and comparing a sequencing result with a Wild Type (WT) sequence to obtain the editing situation of the T0 generation plants. As shown in FIG. 3, the SlZFP gene knockout lines (SlZFP-KO-L1 and SlZFP-KO-L2) were selected to have mutations in which five bases of gRNA2 were deleted and one base of gRNA1 was inserted, respectively.

Selecting T0 generation seeds which are sequenced to be heterozygous, screening by kanamycin, sowing continuously, analyzing the editing situation of T1 generation plants (the editing detection method is the same as that of T0 generation plants), selecting 2-3 homozygous lines with different editing forms, collecting the seeds, screening by kanamycin, sowing until the T2 generation seeds are used for subsequent research.

Example 3 Effect of the SlZFP Gene knockout on the storability of tomato fruits

1. Water loss rate determination of SlZFP gene knockout plants

WT and SlZFP11 knockout lines (SlZFP-KO-L1 and SlZFP-KO-L2) in the red ripe stage (7 days after color breaking) were picked, the tomato fruits were washed with distilled water and sterilized, and the fruits were stored under conditions of 60% humidity, 16 hours (25 ℃) light) +8 hours (18 ℃) dark for 10 days. The storage water loss rate was calculated by measuring the fresh weight of each fruit daily, with the following formula:

;

wherein a is the storage water loss rate,% >, b ₀ is the first day fresh weight of the fruit, g, and b _t is the fresh weight of the fruit after t days of storage, g.

The water loss rate measurement results are shown in FIG. 4. As shown in fig. 4, the water loss rate of SlZFP a 11 knockout tomato fruit was significantly reduced.

2. SlZFP11 Gene knockout plant storage experiments

WT and SlZFP11 knockout lines (SlZFP-KO-L1 and SlZFP-KO-L2) in the red ripe stage (7 days after color breaking) were picked, the tomato fruits were washed with distilled water and sterilized, and the fruits were stored under conditions of 60% humidity, 16 hours (25 ℃) light) +8 hours (18 ℃) dark for 10 days. The degree of collapse of the fruit was assessed on day 10. The storage results of tomato fruits are shown in FIG. 5.

As shown in FIG. 5, the SlZFP gene knockout tomato fruit has weaker epidermis collapse degree than the WT group, and has obviously improved storability.

In conclusion, the SlZFP gene in the tomato is knocked out by utilizing the gene editing technology, so that the cellulose content of the tomato fruit can be obviously improved, the water loss rate of the tomato fruit is reduced, and the storability of the tomato fruit is further enhanced, thereby improving the fruit quality. Provides good reference and reference for innovation of tomato germplasm resources, and aims to better meet the demands of consumers.

Example 4 Effect on tomato fruit Dry matter and sugar content after the SlZFP Gene was knocked out

1. Determination of the dry matter content of SlZFP Gene knockout fruit

Picking fruits of Wild Type (WT) and SlZFP knockout plants in the red ripe stage, setting 3 biological repetitions, washing the fruit surfaces with distilled water, wiping the tomato fruits dry, putting the tomato fruits into gauze, extruding the tomato fruits into a clean beaker, extruding the juice into a clean beaker, namely, a crude extract of soluble solids, and measuring the crude extract by using a handheld refractometer, wherein the dry matter results are shown in figure 6.

From the test results of FIG. 6, it was found that the dry matter content in tomato fruits was significantly increased after knocking-out SlZFP gene.

2. Sugar content determination of SlZFP gene knockout plant fruit

And collecting fruit samples of wild and gene knockout plants in the red ripe stage for sugar content determination. 0.1g of fruit sample is weighed, the soluble sugar of tomato fruit is extracted by taking chromatographic pure (Ar) methanol as an extracting agent, and 3 biological repeats are arranged.

The specific process is that methoxyamine hydrochloride and N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) are used for derivatizing the sample. The measurement was performed using Shimadzu GC 2010 pro. The chromatographic parameters are SPL temperature of 250 ℃, split ratio of 10:1, chromatographic column flow of 1mL/min, heating program of 130 ℃ initial temperature, heating rate of 5 ℃ to 185 ℃, heating rate of 0.5 ℃ to 190 ℃ and heating rate of 8 ℃ to 300 ℃ finally, and keeping for 10min. The detector temperature was 320℃and the hydrogen flow was 40mL/min, the dry air flow was 400mL/min and the nitrogen flow was 40mL/min. The results of the glucose, sucrose and fructose content measurements are shown in fig. 7-9, wherein the abscissa indicates the fruit ripening time point, i.e. "Br" indicates the break period, i.e. the time when the fruit starts to change from green to pale yellow or orange, which is the starting point of the ripening of the tomato fruit, and "br+3" indicates the 3 rd day after breaking, i.e. the ripening period of the fruit from the beginning to the 3 rd day after breaking, and "br+7" indicates the 7 th day after breaking, which is the post-ripening period.

From the test results of fig. 7 to 9, it can be seen that the glucose, sucrose and fructose contents in tomato fruits are significantly increased after the SlZFP gene is knocked out.

3. Detection of gene expression related to influence of sugar metabolism in SlZFP gene knockout line tomato fruits

(1) Taking SlZFP gene knockout strain (SlZFP-KO-L1 and SlZFP-KO-L2) and Wild Type (WT) tomato fruits at the same development stage, wherein more than 3 biological repeats are respectively carried out;

(2) Frozen tissue was ground with liquid nitrogen, total sample RNA was extracted using the plant total RNA extraction kit (DP 432) from Tiangen RNAprep Pure, and its purity and integrity were checked. The purity (OD 260/280 is approximately equal to 1.8-2.0) and the integrity (clear electrophoresis band) of the RNA are detected by a micro ultraviolet spectrophotometer, which shows that the RNA purity is good. 1. Mu.g of RNA was used to synthesize cDNA by reverse transcription using a kit of Renzan HISCRIPT III ALL-in-one RT SuperMix Perfect for qPCR (R333-C1), and diluted for use.

(3) QRT-PCR detection was performed to calculate the relative expression level. Statistical analysis (t-test), P <0.05 was significant. The results of the detection of the expression of the gene related to the influence of the cell wall metabolism in the SlZFP gene knockout line tomato fruit are shown in fig. 10-12.

As shown in fig. 10-12, the expression levels of genes SlLIN, slLIN, and SlSUS3 associated with sugar metabolism were significantly increased in the SlZFP-KO-L1 and SlZFP-KO-L2 plant fruits, indicating that the SlZFP11 gene affects sugar content in tomato fruits by regulating the expression of the sugar metabolism gene.

In conclusion, the SlZFP gene in the tomato is knocked out by utilizing the gene editing technology, so that the sugar content of the tomato fruit can be obviously improved, the sweet taste of the tomato fruit is greatly enhanced, and the fruit quality is improved. Provides good reference and reference for innovation of tomato germplasm resources, and aims to better meet the demands of consumers.

Claims (1)

1. An application of the SlZFP11 gene in regulating the storage tolerance of tomatoes, characterized in that by inhibiting or knocking out the SlZFP11 gene, the dry matter and sugar content of tomatoes are increased, while the storage tolerance of tomatoes is also improved; the nucleotide sequence of the SlZFP11 gene is shown in SEQ ID NO: 1.