CN118638817A - Application of tomato SlPIF8b gene in regulating tomato fruit coloration and ripening - Google Patents

Abstract

The invention discloses an application of a tomato SlPIF b gene in regulating and controlling tomato fruit coloring and ripening, and relates to the fields of genetic engineering and molecular biology, wherein the tomato SlPIF b gene has the nucleotide sequence shown in SEQ ID NO:2, and a nucleotide sequence shown in the following formula. The application is that the SlPIF b gene is knocked out from tomatoes by utilizing a gene editing technology, so that the accumulation of lycopene, carotenoid and the like in tomato fruits can be promoted, the coloring of the tomato fruits is accelerated, the ethylene accumulation and softening process is promoted, and the fruit ripening is promoted. Provides new germplasm resources for cultivating new varieties which promote the coloration and maturation of tomato fruits, has better potential application value, and simultaneously provides important theoretical basis and technical support for researching the molecular mechanism of tomato maturation and improving the quality and nutritive value of tomato fruits.

Description

Application of tomato SlPIF8b gene in regulating tomato fruit coloration and ripening

Technical Field

The present application relates to the fields of genetic engineering, molecular biology and physiological technology, and in particular to the application of a tomato SlPIF8b gene in regulating the coloring and ripening of tomato fruits.

Background Art

Tomato (Solanum lycopersicum.L) belongs to the Solanaceae crop. It is a widely planted plant around the world and is deeply loved by people. The ripening of tomato fruit is accompanied by a series of physiological processes, including changes in color, increase in flavor substances, and softening of the fruit. These changes make the ripe tomato fruit edible and an important nutritious fruit in the human diet. As a respiratory climacteric fruit, tomato has the advantages of short cycle, easy genetic transformation and small genome. In addition, with the maturity of gene editing technology, genome informatics and molecular genetics, tomato has become a model organism for studying the development and ripening of fleshy fruits.

The ripening of tomatoes is regulated by transcription factors. The physiological and biochemical changes in the ripening process of tomatoes have been studied relatively thoroughly, but the transcriptional regulation of fruit ripening is still a difficult and hot topic. Screening and identifying new transcription factors related to fruit ripening is an important part of studying tomato fruit ripening. This can not only improve the transcriptional regulatory network of tomato fruit ripening, but also provide a theoretical basis for obtaining tomato varieties with higher nutritional value and better storage characteristics through transgenic technology in the future. Low temperature and weak light in facility production often lead to poor coloring of tomato fruits and reduced nutritional quality. In recent years, with the development of LED lights, the application of facility lighting technology has improved the coloring and nutritional quality of tomato fruits, but how light signals regulate tomato fruit coloring and the accumulation of nutrients is still unclear. Phytochrome interacting factors PIFs (PHYTOCHROMEINTERACTING FACTORS) belong to the 15th subfamily of the transcription factor bHLH family and are key transcription factors for plants to transmit light signals. Phytochrome interacting factors (PIFs) mainly participate in seed germination, chlorophyll synthesis, photomorphogenesis, shade response, and stress response by directly binding to the G-box (CACGTG) or PBE-box (CACATG) on the promoter of the target gene. PIFs transcription factors have been widely studied in Arabidopsis, but research in tomatoes has just begun. Light signals play an important role in tomato fruit coloring and ripening, but the function of the phytochrome interacting factor SlPIF8b in tomato fruit development and its role in fruit ripening color remain unclear.

Summary of the invention

The purpose of the present application example is to provide an application of a tomato SlPIF8b gene in regulating tomato fruit coloring and ripening, so as to promote tomato fruit ripening and improve tomato fruit appearance quality.

To achieve the above purpose, the technical solution adopted by the embodiment of the present invention is as follows:

In a first aspect, an embodiment of the present invention provides an application of a tomato SlPIF8b gene in regulating tomato fruit coloring and ripening, wherein the nucleotide sequence of the SlPIF8b gene is shown in SEQ ID NO:2.

In a second aspect, an embodiment of the present invention provides an application of a protein encoded by a tomato SlPIF8b gene in regulating the coloring and ripening of tomato fruits. The amino acid sequence of the protein encoded by the SlPIF8b gene is shown in SEQ ID NO: 1.

Furthermore, the coloring and ripening of tomato fruits were promoted by knocking out the expression of the SlPIF8b gene in tomatoes.

Further, the gene knockout technology is as follows:

The sgRNA sequence of the CRISPR/Cas9 editing target was designed as shown in SEQ ID NO: 3, and primers were artificially synthesized according to the sgRNA sequence and constructed into the CRISP/Cas9 vector;

The target site fragment of the SlPIF8b gene is amplified using target primers, and the gene fragment to be transferred is inserted into a linearized transformation vector, connected, and then transferred into Escherichia coli. After extracting the plasmid, the SlPIF8b gene knockout vector is obtained.

Furthermore, the primer sequences of the SlPIF8b gene target fragment are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

Furthermore, the transfer vector is a pCBSG012-slu61-DSG-bsai plasmid, and the pCBSG012-slu61-DSG-bsai plasmid is cut with BasI for linearization.

In a third aspect, an embodiment of the present invention provides a gene that promotes tomato fruit coloring and ripening. The tomato fruit coloring and ripening accelerating gene is obtained by knocking out the SlPIF8b gene of claim 1. The sequence of the SlPIF8b gene is shown in SEQ ID NO: 2.

In a fourth aspect, an embodiment of the present invention provides a method for promoting coloring and ripening of tomato fruits, the method comprising:

Step A1: Transform the SlPIF8b gene knockout vector into Agrobacterium;

Step A2: Infect tomato plants with Agrobacterium transformed with the SlPIF8b gene knockout vector.

In a fifth aspect, an embodiment of the present invention provides a method for inhibiting coloring and ripening of tomato fruits, the method comprising:

Step B1: extracting tomato total RNA, reversely transcribing to obtain cDNA, using cDNA as a template, SlPIF8b-OE-F and SlPIF8b-OE-R as primers, amplifying SlPIF8b gene, constructing the amplified product into a plant gene overexpression vector with a 35S promoter, and obtaining a recombinant expression vector, and transferring the SlPIF8b gene overexpression vector into Agrobacterium, wherein the nucleotide sequences of the primers SlPIF8b-OE-F and SlPIF8b-OE-R are shown in SEQ ID NO.6 and SEQ ID NO.7;

Step B2: Infecting tomato plants with Agrobacterium transformed with the SlPIF8b gene overexpression vector.

The technical solution provided by the embodiments of the present application may have the following beneficial effects:

Compared with the prior art, the present invention provides a knockout vector capable of knocking out the SlPIF8b gene, and the knockout vector can be used to knock out the SlPIF8b gene, thereby promoting the coloring and ripening of tomato fruits.

The present invention utilizes the SlPIF8b gene knockout vector to knock out the SlPIF8b gene to obtain plants that promote tomato fruit coloring and maturity, which is more economical and effective than traditional breeding methods, is a good method for creating improved tomato quality and maturity, and greatly shortens the breeding period; the SlPIF8b gene knockout strain promotes fruit coloring and maturity, and improves nutritional quality and appearance quality.

The present application constructs tomato SlPIF8b gene knockout and overexpression plants by genetic means, and regulates the expression level of the gene SlPIF8b to study its regulatory mechanism for tomato fruit coloring and ripening. The results show that tomato SlPIF8b gene knockout plants increase the accumulation of carotenoids and lycopene in tomato fruits, induce ethylene accumulation, and reduce fruit hardness, thereby promoting tomato fruit coloring and ripening. Therefore, the tomato SlPIF8b gene negatively regulates the accumulation of carotenoids and lycopene in tomato fruits, while inhibiting the synthesis of ethylene, thereby inhibiting the coloring and ripening of tomato fruits. Using gene editing and other technologies to mutate this gene is of great significance for improving the accumulation of tomato nutrients and the ripening of fruits. At the same time, the analysis of the function of this gene has an important theoretical basis and technical support for regulating the color and ripening of tomato fruits using supplementary lighting and other technologies.

The SlPIF8b protein and its encoding gene provided by the present invention provide gene resources for breeding new tomato varieties with higher nutritional value and better storage characteristics, have good potential application value, and can regulate the nutritional and appearance quality of tomatoes and the time to market of tomato fruits through gene editing and transgenic technology, and at the same time lay a theoretical foundation and technical support for the molecular mechanism of regulating the nutritional quality and maturity of tomatoes by using light signals.

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

BRIEF DESCRIPTION OF THE 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 present application.

FIG. 1 is a diagram showing the sequencing results of the target sites of the SlPIF8b gene knockout tomato plants in Example 3 of the present invention.

FIG. 2 is a graph showing the relative expression level of the SlPIF8b gene in the SlPIF8b gene overexpressing tomato strains in Example 3 of the present invention.

FIG. 3 shows the color change of fruits of overexpressed SlPIF8b, SlPIF8b gene mutation and wild type in Example 4 of the present invention.

4 is a schematic diagram of the color difference index results of overexpressed SlPIF8b, SlPIF8b gene mutation and wild-type fruits in Example 4 of the present invention.

5 is a schematic diagram of the changes in carotenoids in fruits of overexpressed SlPIF8b, SlPIF8b gene mutation and wild type in Example 4 of the present invention.

6 is a schematic diagram showing the changes in lycopene content in fruits overexpressing SlPIF8b, SlPIF8b gene mutation and wild type in Example 4 of the present invention.

7 is a comparison of the changes in fruit firmness of overexpressed SlPIF8b, SlPIF8b gene mutants and wild type fruits in Example 4 of the present invention.

FIG. 8 shows the results of the changes in ethylene release from fruits overexpressing SlPIF8b, SlPIF8b gene mutations, and wild-type fruits in Example 4 of the present invention.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial sources, and the present invention will be implemented using conventional botanical techniques, tissue culture, molecular biology, biological physiology and biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the literature.

Example 1: Construction of SlPIF8b gene overexpression vector

In order to understand the molecular mechanism of tomato fruit ripening, the SlPIF8b gene was cloned from the tomato genome. According to the analysis of the coding region sequence, specific primers SlPIF8b-OE-F and SlPIF8b-OE-R were designed, and restriction enzyme sites (Asc I and SalI) were added to the primers respectively. The sequences are shown in SEQ ID NO.6 and 7. The SlPIF8b fragment was amplified by PrimerSTAR high-fidelity enzyme PCR, and then the PCR amplified fragment and the vector were digested, and the SlPIF8b fragment was connected to pFGC1008-HA to obtain a plant overexpression vector. The above recombinant plasmid was sent to Qingke Company for sequencing confirmation, and the nucleotide sequence of the obtained gene SlPIF8b is shown in SEQ ID No.2; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.1. The results showed that the cloned sequence was consistent with the sequence published in Solgenomics (Solyc10g018510).

SEQ ID NO.6 is as follows:

ttggcgcgcc atggattatg aagtagcaga

SEQ ID NO.7 is as follows:

acgcgtcgac atttttaaag ccaagatttg

SEQ ID No. 2 is as follows:

atggattatg aagtagcaga gctaaaatgg gaaaagggag aggtagtgat gcatgggtta

ggtcctccag gcgtgccttg ttattataag cctttatcga ctccttctcc aacaaaatac

acgtgggacg ataagccaca tgctgctgca ggtggcacac ttgaatccat agtgaaccaa

gctacgactc ataatattga catcggtgac gagggtggtg atgatgatga tttagtgagt

tggtttgatg attgtcttcc tgaaacgtcc atggatattg tggccgtagt tccaacaagt

tgtactaact ataatcaaca agtgcccccg tccacacgtg ttgcatcatg cagtggtgat

gcagagatgg cacgtgtggg aatgggatct agctttgagg aaatatcgga agactttgag

aatcaagagg ctaagaactt gatcggctca atggtatacg agggcaaaaa taacactgtg

agcccgggag agacaagttt gggtgaggaa agagtactta caacaacatc tacctttaag

cataataaaa ggaagacact gaataatcat gatagcagag gtcaggagtc gagagataat

gaggatgagg atgagaaaaa aagatccaaa atttcttcat tttcaacaaa aaggtgcaga

gttgctgcta ctcacaacca gtctgaacga aaaagaagag acaagataaa ccaaaggttg

aagacattgc agaagttagt tccaacatcg agtaagactg atacggcatc aatgttagat

gaggtgatag aatatttgaa gcaactacga gctcaagtta aagccatgag catgatgatt

catgttaaca tgcagccacc ccctatgatg ttaccaaata tggcattcca acaacaacaa

caacaatttc aaatgtcaat gatggggatg gctagaccca tcgatgtcaa tgcccttagc

agccccaaca taacaacaat cccatcgatt ctccatacca ccgcaccctc taatttcaat

aaccctccta ttgcctcccc tggagctgat cctttagctt ccttggtcgc agtacgccaa

ttatcacagc ctatgacgat ggatgcttat agcaggatgg cagcattgta ccaacaatat

ctacagtcaa atgcaaatct tggctttaaa aattga

SEQ ID No. 1 is as follows:

Ala Thr Gly GlyAla Thr ThrAla Thr GlyAlaAla Gly ThrAla Gly

Cys Ala GlyAla Gly Cys ThrAlaAlaAlaAla Thr Gly Gly GlyAla

AlaAlaAla Gly Gly GlyAla GlyAla Gly Gly ThrAla Gly Thr Gly

Ala Thr Gly Cys Ala Thr Gly Gly Gly Thr ThrAla Gly Gly Thr Cys

Cys Thr Cys Cys Ala Gly Gly Cys Gly Thr Gly Cys Cys Thr Thr Gly

Thr ThrAla Thr ThrAla ThrAlaAla Gly Cys Cys ThrThr ThrAla

Thr Cys GlyAla Cys Thr Cys Cys Thr Thr Cys Thr Cys Cys AlaAla

Cys AlaAlaAlaAla ThrAla Cys Ala Cys Gly Thr Gly Gly GlyAla

Cys GlyAla ThrAlaAla Gly Cys Cys Ala Cys Ala Thr Gly Cys Thr

Gly Cys Thr Gly Cys Ala Gly Gly Thr Gly Gly Cys Ala Cys Ala Cys

Thr Thr GlyAlaAla Thr Cys Cys Ala ThrAla Gly Thr GlyAlaAla

Cys Cys AlaAla Gly Cys ThrAla Cys GlyAla Cys Thr Cys Ala Thr

AlaAla ThrAla Thr Thr GlyAla Cys Ala Thr Cys Gly Gly Thr Gly

Ala Cys GlyAla Gly Gly Gly Thr Gly Gly Thr GlyAla Thr GlyAla

Thr GlyAla Thr GlyAla ThrThr ThrAla Gly Thr GlyAla Gly Thr

Thr Gly Gly ThrThr Thr GlyAla Thr GlyAla Thr Thr Gly Thr Cys

Thr Thr Cys Cys Thr GlyAlaAlaAla Cys Gly Thr Cys Cys Ala Thr

Gly GlyAla ThrAla Thr Thr Gly Thr Gly Gly Cys Cys Gly ThrAla

Gly Thr Thr Cys Cys AlaAla Cys AlaAla Gly Thr Thr Gly ThrAla

Cys ThrAlaAla Cys ThrAla ThrAlaAla Thr Cys AlaAla Cys Ala

Ala Gly Thr Gly Cys Cys Cys Cys Cys Gly Thr Cys Cys Ala Cys Ala

Cys Gly Thr Gly Thr Thr Gly Cys Ala Thr Cys Ala Thr Gly Cys Ala

Gly Thr Gly Gly Thr GlyAla Thr Gly Cys Ala GlyAla GlyAla Thr

Gly Gly Cys Ala Cys Gly Thr Gly Thr Gly Gly GlyAlaAla Thr Gly

Gly GlyAla Thr Cys ThrAla Gly Cys Thr Thr Thr GlyAla Gly Gly

AlaAlaAla ThrAla Thr Cys Gly GlyAlaAla GlyAla Cys ThrThr

Thr GlyAla GlyAlaAla Thr Cys AlaAla GlyAla Gly Gly Cys Thr

AlaAla GlyAlaAla Cys ThrThr GlyAla Thr Cys Gly Gly Cys Thr

Cys AlaAla Thr Gly Gly ThrAla ThrAla Cys GlyAla Gly Gly Gly

Cys AlaAlaAlaAlaAla ThrAlaAla Cys Ala Cys Thr Gly Thr Gly

Ala Gly Cys Cys Cys Gly Gly GlyAla GlyAla GlyAla Cys AlaAla

Gly Thr Thr Thr Gly Gly Gly Thr GlyAla Gly GlyAlaAlaAla Gly

Ala Gly ThrAla Cys Thr ThrAla Cys AlaAla Cys AlaAla Cys Ala

Thr Cys ThrAla Cys Cys Thr ThrThrAlaAla Gly Cys Ala ThrAla

Ala ThrAlaAlaAlaAla Gly GlyAlaAla GlyAla Cys Ala Cys Thr

GlyAlaAla ThrAlaAla Thr Cys Ala Thr GlyAla ThrAla Gly Cys

Ala GlyAla Gly Gly Thr Cys Ala Gly GlyAla Gly Thr Cys GlyAla

GlyAla GlyAla ThrAlaAla Thr GlyAla Gly GlyAla Thr GlyAla

Gly GlyAla Thr GlyAla GlyAlaAlaAlaAlaAlaAlaAla GlyAla

Thr Cys Cys AlaAlaAlaAla Thr Thr Thr Cys Thr Thr Cys Ala Thr

Thr Thr Thr Cys AlaAla Cys AlaAlaAlaAlaAla Gly Gly Thr Gly

Cys Ala GlyAla Gly Thr Thr Gly Cys Thr Gly Cys ThrAla Cys Thr

Cys Ala Cys AlaAla Cys Cys Ala Gly Thr Cys Thr GlyAlaAla Cys

GlyAlaAlaAlaAlaAla GlyAlaAla GlyAla GlyAla Cys AlaAla

GlyAla ThrAlaAlaAla Cys Cys AlaAlaAla Gly Gly ThrThr Gly

AlaAla GlyAla Cys Ala ThrThr Gly Cys Ala GlyAlaAla Gly Thr

ThrAla Gly Thr Thr Cys Cys AlaAla Cys Ala Thr Cys GlyAla Gly

ThrAlaAla GlyAla Cys Thr GlyAla ThrAla Cys Gly Gly Cys Ala

Thr Cys AlaAla Thr Gly Thr ThrAla GlyAla Thr GlyAla Gly Gly

Thr GlyAla ThrAla GlyAlaAla ThrAla Thr Thr Thr GlyAlaAla

Gly Cys AlaAla Cys ThrAla Cys GlyAla Gly Cys Thr Cys AlaAla

Gly Thr ThrAlaAlaAla Gly Cys Cys Ala Thr GlyAla Gly Cys Ala

Thr GlyAla Thr GlyAla ThrThr Cys Ala Thr Gly Thr ThrAlaAla

Cys Ala Thr Gly Cys Ala Gly Cys Cys Ala Cys Cys Cys Cys Cys Thr

Ala Thr GlyAla Thr Gly ThrThrAla Cys Cys AlaAlaAla ThrAla

Thr Gly Gly Cys Ala ThrThr Cys Cys AlaAla Cys AlaAla Cys Ala

Ala Cys AlaAla Cys AlaAla Cys AlaAla Thr Thr Thr Cys AlaAla

Ala Thr Gly Thr Cys AlaAla Thr GlyAla Thr Gly Gly Gly GlyAla

Thr Gly Gly Cys ThrAla GlyAla Cys Cys Cys Ala Thr Cys GlyAla

Thr Gly Thr Cys AlaAla Thr Gly Cys Cys Cys Thr ThrAla Gly Cys

Ala Gly Cys Cys Cys Cys AlaAla Cys Ala ThrAlaAla Cys AlaAla

Cys AlaAla Thr Cys Cys Cys Ala Thr Cys GlyAla Thr Thr Cys Thr

Cys Cys Ala ThrAla Cys Cys Ala Cys Cys Gly Cys Ala Cys Cys Cys

Thr Cys ThrAlaAla Thr Thr Thr Cys AlaAla Thr AlaAla Cys Cys

Cys Thr Cys Cys ThrAla Thr Thr Gly Cys Cys Thr Cys Cys Cys Cys

Thr Gly GlyAla Gly Cys Thr GlyAla Thr Cys Cys Thr ThrThr Ala

Gly Cys Thr Thr Cys Cys Thr Thr Gly Gly Thr Cys Gly Cys Ala Gly

ThrAla Cys Gly Cys Cys AlaAla Thr ThrAla Thr Cys Ala Cys Ala

Gly Cys Cys ThrAla Thr Gly Ala Cys GlyAla Thr Gly GlyAla Thr

Gly Cys Thr ThrAla Thr Ala Gly Cys Ala Gly GlyAla Thr Gly Gly

Cys Ala Gly Cys Ala Thr Thr Gly ThrAla Cys Cys AlaAla Cys Ala

Ala ThrAla Thr Cys ThrAla Cys Ala Gly Thr Cys AlaAla Ala Thr

Gly Cys Ala AlaAla Thr Cys Thr Thr Gly Gly Cys Thr Thr ThrAla

AlaAla AlaAla Thr Thr Gly Ala Pro

It should be noted that the plant overexpression vector is suitable for dicotyledonous plants, such as tomatoes, eggplants, peppers and the like.

Example 2: Construction of SlPIF8b gene editing (knockout) vector

According to the gene sequence of SlPIF8b, the CRISPR target and primers were designed using the CRISPR-P website. The target fragment sequence of the SlPIF8b gene is shown in SEQ ID NO.3. The primer sequences of the target are shown in SEQ ID NO.4 and SEQ ID NO.5. The synthesized target primer sequence was annealed and connected to SlU61, which was then connected to the linearized cloning vector pCBSG012-slu61-DSG-bsai. The above recombinant plasmid was sent to Qingke Company for sequencing confirmation.

SEQ ID NO.3 is as follows:

aagccacatg ctgctgcagg

SEQ ID NO.4 is as follows:

atgcatgggt taggtcctcc

SEQ ID NO.5 is as follows:

catctctgca tcaccactgc

Example 3: Construction and detection of tomato SlPIF8b transgenic plants

The constructed plant vector and CRISPR gene editing vector were transferred into Agrobacterium GV3101 by Agrobacterium-mediated genetic transformation, and tomato cotyledons were infected. Tissue culture seedlings were obtained by callus induction, resistance induction differentiation and rooting culture. T2 generation mutant seeds and overexpression seeds were tested for kanamycin resistance and chloramphenicol resistance, and 3/4 strains with resistance and the remaining 1/4 without resistance were selected, indicating that the overexpression vector with the target gene was inserted in a single copy in the strain. These plants were removed and then harvested individually.

The overexpression positive transgenic plants were verified by qRT-PCR technology, and the results showed that the expression level of SlPIF8b was upregulated 200 times compared with the wild type (Figure 2). The positive SlPIF8b mutant transgenic plants were verified by PCR and sequencing technology, and it was found that pif8b#28 added a base at the target site and mutated near the original adjacent motif (PAM), causing SlPIF8b to stop translating (Figure 1).

Example 4: Statistics of tomato fruit color change time in SlPIF8b gene-edited and overexpressed plants

After the tomato materials bloomed, the blooming flowers were marked with dates, and the color change of different transgenic materials was recorded 31-51 days after flowering (Figure 3). The color change of tomato fruits with SlPIF8b gene mutation (slpif8b#28) was significantly earlier than that of wild-type fruits, while the color change of tomato fruits with SlPIF8b gene overexpression (SlPIF8b-OE#69) was delayed compared with wild-type fruits (WT), indicating that the SlPIF8b gene inhibits the color change time of tomato fruits.

Example 5: Determination of the color difference index of tomato fruit in SlPIF8b gene-edited and overexpressed plants

The color changes of fruits of different materials were detected 51 days after flowering. L*, a* and b* were detected using a Konica Minolta CR-400 colorimeter in CIE mode. The L* value represents brightness, the a* value represents green-red, and the b* value represents blue-yellow. The color was calculated as the ratio of a*/b*. We randomly selected 6 fruits from each material and tested them at 4 positions near the equator of the fruit. The results showed that compared with the wild-type fruit (WT), the color difference index of tomato fruits with SlPIF8b gene overexpression (SlPIF8b-OE#69) was lower with the increase of days after flowering, the color was obviously greener, and matured later; while the color difference index of tomato fruits of the SlPIF8b gene mutant (slpif8b#28) was higher, the fruit changed color faster, and the color was redder (Figure 4), which indicated that the SlPIF8b gene negatively regulated the color of tomato fruits.

Example 6: Determination of carotenoid content in tomato fruits of SlPIF8b gene-edited and overexpressed plants

Cut the peel tissue section from the tomato fruit near the equator, weigh the sample, weigh 1g, and then add liquid nitrogen to crush it into powder in a mortar and pestle. Add 10ml of hexane:acetone (6:4, v/v) to the sample to extract total carotenoids. Then centrifuge the sample at 4000g for 5min, and transfer the supernatant to a new tube after centrifugation. Immediately measure the absorbance of the supernatant at 450nm using a spectrophotometer, and the total carotenoid content is quantified according to the formula: carotenoid content (mg mL ^-1 ) = (4×OD450×10mL)g ^-1 . The above operation should be set up with more than 5 biological replicates. The results showed that compared with the wild-type fruit (WT), the total carotenoid content in the fruit overexpressing the SlPIF8b gene (SlPIF8b-OE#69) was significantly lower than that in the wild-type fruit (WT), while the total carotenoid accumulation in the tomato fruit of the SlPIF8b gene mutant (slpif8b#28) was higher than that in the wild-type fruit (Figure 5), indicating that the SlPIF8b gene negatively regulates the accumulation of carotenoids in the fruit.

Example 7: Determination of lycopene content in tomato fruits of SlPIF8b gene editing and overexpression plants

Cut the peel tissue from the equator of the fruit to obtain a 5mm wide sample. After grinding it thoroughly in liquid nitrogen, weigh 0.4-0.6g of the sample and place it in a 50mL centrifuge tube. Add 20mL of acetone (containing 0.05% 2,6-di-tert-butyl-p-cresol, BTH), 95% ethanol, and n-hexane (1:1:2, V/V) to the centrifuge tube. After placing the centrifuge tube in an ice box, put the ice box in a shaker and shake it at 180rpm for 15min. After the shaking is completed, add 3mL of ice deionized water to each centrifuge tube, mix it well, put the centrifuge tube back into the ice box, and shake it at 180rpm for 5min. After standing at room temperature for 5min for liquid phase separation, measure the absorbance of OD503 of the supernatant. The calculation formula is as follows: lycopene (mg/kg) = (OD503×31.2)/mg. The results showed that compared with the fruits of wild-type plants (WT), the lycopene content in the fruits of SlPIF8b gene overexpression (SlPIF8b-OE#69) was significantly reduced, while the lycopene content in the tomato fruits of the SlPIF8b gene mutant (slpif8b#28) was significantly higher than that in the wild-type fruits (Figure 6), indicating that the SlPIF8b gene negatively regulates the accumulation of lycopene in the fruit.

Example 8: Determination of tomato fruit firmness in SlPIF8b gene editing and overexpression plants

The fruit firmness of different materials was tested 51 days after flowering. A fruit texture analyzer (BROOKFIELD CT3; Middleboro, USA) equipped with a 2 mm diameter probe was inserted into the fruit about 7 mm deep. The firmness was recorded twice at the equator of each fruit, and two measurements were taken at 90° to each other. The results showed that the firmness of the fruit with SlPIF8b gene overexpression (SlPIF8b-OE#69) was significantly increased compared with the wild-type fruit (WT). The firmness of the fruit of the SlPIF8b gene mutant (slpif8b#28) was significantly less than that of the wild-type fruit (Figure 7), indicating that the SlPIF8b gene inhibits the ripening of tomato fruit.

Example 9: Determination of ethylene release in tomato fruits of SlPIF8b gene-edited and overexpressed plants

The ethylene content of fruits was determined by Shimadzu GC2010 gas chromatograph, activated alumina column (DB-5MS column, 30m×0.25mm×0.25μm), and flame ionization detector. Tomato fruits of different materials were placed in a 250mL beaker for 4h 51 days after flowering, and then 1ml of gas was extracted with a syringe and injected into the GC. Column temperature: 60℃; FID detector temperature: 130℃; carrier gas flow rate: 30mL·min ^-1 ; H2 flow rate: 1mL·min ^-1 ; air flow rate: 400mL·min ^-1 , split ratio: 35; pressure: 113.5kPa; injection time: 3min. The results showed that compared with the wild-type fruit (WT), the ethylene content in the fruit of the SlPIF8b gene mutant (slpif8b#28) was significantly increased, while the ethylene content in the fruit of the SlPIF8b gene overexpression (SlPIF8b-OE#69) was significantly decreased (Figure 8), indicating that the SlPIF8b gene inhibits the accumulation of ethylene content in tomato fruit.

The present invention constructs tomato SlPIF8b gene knockout plants by gene editing technology, and constructs tomato SlPIF8b overexpression plants by transgenic technology. The results show that the SlPIF8b gene mutation can promote the accumulation of carotenoids and lycopene in tomato fruits, thereby promoting the color change of tomato fruits. In addition, the SlPIF8b gene mutation can promote ethylene accumulation, reduce fruit hardness, and thus make the fruit mature in advance. Therefore, the SlPIF8b gene provided by the present invention is a key gene for regulating tomato fruit maturation and carotenoid accumulation. The SlPIF8b gene mutant can be constructed by using technologies such as gene editing, thereby improving the coloring of tomato fruits and the accumulation of nutrients such as carotenoids, realizing molecular design breeding, and having good application value.

Although the present invention has been described in detail above by means of general description, specific embodiments and tests, the present invention is not limited to the above embodiments and may be subject to many modifications or improvements, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims (9)

1. Application of tomato SlPIF8b gene in regulating tomato fruit coloring and ripening, the nucleotide sequence of the SlPIF8b gene is shown in SEQ ID NO: 2. 2. Application of the protein encoded by the tomato SlPIF8b gene in regulating the coloring and ripening of tomato fruits. The amino acid sequence of the protein encoded by the SlPIF8b gene is shown in SEQ ID NO: 1. 3. The use according to claim 1 or 2, wherein the coloring and ripening of tomato fruits are promoted by knocking out the expression of the SlPIF8b gene in tomatoes. 4. According to the use of claim 3, the gene knockout technology is specifically as follows: The sgRNA sequence of the CRISPR/Cas9 editing target was designed as shown in SEQ ID NO: 3, and primers were artificially synthesized according to the sgRNA sequence and constructed into the CRISP/Cas9 vector; The target site fragment of the SlPIF8b gene is amplified using target primers, and the gene fragment to be transferred is inserted into a linearized transformation vector, connected, and then transferred into Escherichia coli. After extracting the plasmid, the SlPIF8b gene knockout vector is obtained. 5. The use according to claim 4, wherein the primer sequences of the SlPIF8b gene target fragment are shown in SEQ ID NO: 4 and SEQ ID NO: 5. 6. The use according to claim 4, wherein the transfer vector is pCBSG012-slu61-DSG-bsai plasmid, and the pCBSG012-slu61-DSG-bsai plasmid is cut with BasI for linearization. 7. A gene that promotes tomato fruit coloring and ripening, wherein the tomato fruit coloring and ripening accelerating gene is obtained by knocking out the SlPIF8b gene of claim 1, and the sequence of the SlPIF8b gene is shown in SEQ ID NO: 2. 8. A method for promoting coloring and ripening of tomato fruits, the method comprising: Step A1: Transform the SlPIF8b gene knockout vector into Agrobacterium; Step A2: Infect tomato plants with Agrobacterium transformed with the SlPIF8b gene knockout vector. 9. A method for inhibiting the coloring and ripening of tomato fruits, the method comprising: Step B1: extracting tomato total RNA, reverse transcription to obtain cDNA, using cDNA as a template, SlPIF8b-OE-F and SlPIF8b-OE-R as primers, amplifying SlPIF8b gene, constructing the amplified product into a plant gene overexpression vector with a 35S promoter, and obtaining a recombinant expression vector, and transferring the SlPIF8b gene overexpression vector into Agrobacterium, wherein the nucleotide sequences of the primers SlPIF8b-OE-F and SlPIF8b-OE-R are shown in SEQ ID NO.6 and SEQ ID NO.7; Step B2: Infecting tomato plants with Agrobacterium transformed with the SlPIF8b gene overexpression vector.