Application of the tomato SlPIF8b gene in regulating tomato fruit coloring and ripening

Abstract

This invention discloses the application of the tomato SlPIF8b gene in regulating tomato fruit coloring and ripening, relating to the fields of genetic engineering and molecular biology. The tomato SlPIF8b gene has the nucleotide sequence shown in SEQ ID NO: 2. This application utilizes gene editing technology to knock out the SlPIF8b gene in tomatoes, which promotes the accumulation of lycopene and carotenoids in the tomato fruit, accelerates fruit coloring, and simultaneously promotes ethylene accumulation and softening, thus accelerating fruit ripening. This provides new germplasm resources for breeding new tomato varieties that promote fruit coloring and ripening, and has good potential application value. It also provides important theoretical basis and technical support for studying the molecular mechanisms of tomato ripening and improving the quality and nutritional value of tomato fruits.

Description

Technical Field

[0001] This application relates to the fields of genetic engineering, molecular biology and physiology, and in particular to the application of the tomato SlPIF8b gene in regulating tomato fruit coloring and ripening. Background Technology

[0002] Tomato (Solanum lycopersicum L.) belongs to the Solanaceae family and is a widely cultivated plant worldwide, enjoying immense popularity. The ripening of tomato fruit involves a series of physiological processes, including changes in color, an increase in flavor compounds, and fruit softening. These changes make ripe tomatoes edible, making them an important source of nutrition in the human diet. As a climacteric fruit, the tomato has advantages such as a short ripening cycle, ease of genetic transformation, and a relatively small genome. Furthermore, with the advancement of gene editing technology, genomics, and molecular genetics, the tomato has become a model organism for studying the development and ripening of fleshy fruits.

[0003] Tomato ripening is regulated by transcription factors. While the physiological and biochemical changes during tomato ripening have been relatively well understood, the transcriptional regulation of fruit ripening remains a challenge and a hot topic. Screening and identifying new transcription factors related to fruit ripening is crucial for studying tomato fruit ripening. This will 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 greenhouse production, low temperatures and weak light often lead to poor tomato fruit coloring and decreased nutritional quality. In recent years, with the development of LED lights and the application of supplemental lighting technology, tomato fruit coloring and nutritional quality have improved. However, how light signals regulate tomato fruit coloring and nutrient accumulation remains unclear. Phytochrome interaction factors (PIFs) belong to the 15th subfamily of the bHLH family of transcription factors and are key transcription factors for transmitting light signals in plants. Phytochrome interaction factors (PIFs) mainly participate in processes such as seed germination, chlorophyll synthesis, photomorphogenesis, shade response, and stress response by directly binding to the G-box (CACGTG) or PBE-box (CACATG) on the promoters of target genes. While there is considerable research on PIF transcription factors in Arabidopsis thaliana, research on them in tomato is still in its early stages. Light signals play a crucial role in tomato fruit coloring and ripening, but the function of the phytochrome interaction factor SlPIF8b in tomato fruit development and its effect on fruit color at maturity remains unclear. Summary of the Invention

[0004] The purpose of this application is to provide an application of the tomato SlPIF8b gene in regulating tomato fruit coloring and ripening, so as to promote tomato fruit ripening and improve the appearance and quality of tomato fruit.

[0005] To achieve the above objectives, the technical solutions adopted in the embodiments of the present invention are as follows:

[0006] In a first aspect, embodiments of the present invention provide the application of the 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.

[0007] Secondly, embodiments of the present invention provide the application of the protein encoded by the tomato SlPIF8b gene in regulating tomato fruit coloring and ripening, wherein the amino acid sequence of the protein encoded by the SlPIF8b gene is shown in SEQ ID NO: 1.

[0008] Furthermore, knocking out the expression of the SlPIF8b gene in tomatoes can promote tomato fruit coloring and ripening.

[0009] Furthermore, the gene knockout technology is specifically as follows:

[0010] The sgRNA sequence for the CRISPR / Cas9 editing target was designed as shown in SEQ ID NO: 3. Primers were artificially synthesized based on the sgRNA sequence and constructed into the CRISP / Cas9 vector.

[0011] The target fragment of the SlPIF8b gene was amplified using target primers. The gene fragment to be transformed was inserted into a linearized transformation vector, ligated, and then transformed into E. coli. After plasmid extraction, the SlPIF8b gene knockout vector was obtained.

[0012] Furthermore, the primer sequences for the SlPIF8b gene target fragment are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0013] Furthermore, the transfer vector is the pCBSG012-slu61-DSG-bsai plasmid, and the pCBSG012-slu61-DSG-bsai plasmid is linearized by BasI digestion.

[0014] Thirdly, embodiments of the present invention provide a gene that promotes tomato fruit coloring and ripening. The tomato fruit coloring and ripening acceleration gene is obtained by knocking out the SlPIF8b gene as described in claim 1. The sequence of the SlPIF8b gene is shown in SEQ ID NO: 2.

[0015] Fourthly, embodiments of the present invention provide a method for promoting the coloring and ripening of tomato fruits, the method comprising:

[0016] Step A1: Transform the SlPIF8b gene knockout vector into Agrobacterium;

[0017] Step A2: Infect tomato plants with Agrobacterium tumefaciens that has been transferred into the SlPIF8b gene knockout vector.

[0018] Fifthly, embodiments of the present invention provide a method for inhibiting the coloring and ripening of tomato fruits, the method comprising:

[0019] Step B1: Extract total RNA from tomato, reverse transcribe to obtain cDNA, use cDNA as template, and SlPIF8b-OE-F and SlPIF8b-OE-R as primers to amplify the SlPIF8b gene. Construct the amplification product into a plant gene overexpression vector with a 35S promoter to obtain the recombinant expression vector. Transform the SlPIF8b gene overexpression vector into Agrobacterium tumefaciens. 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.

[0020] Step B2: Infect tomato plants with Agrobacterium tumefaciens that has been transformed into the SlPIF8b gene overexpression vector.

[0021] The technical solutions provided by the embodiments of this application may include the following beneficial effects:

[0022] Compared with the prior art, the present invention provides a knockout vector that can knock out the SlPIF8b gene. The SlPIF8b gene can be knocked out using the knockout vector to promote tomato fruit coloring and ripening.

[0023] This invention utilizes the SlPIF8b gene knockout vector to knock out the SlPIF8b gene, obtaining plants that promote tomato fruit coloring and ripening. This method is more economical and effective than traditional breeding methods, and is a good way to improve tomato quality and ripening, greatly shortening the breeding cycle. The SlPIF8b gene knockout lines promote fruit coloring and ripening, improving nutritional and appearance quality.

[0024] This application constructs tomato SlPIF8b gene knockout and overexpression plants using genetic methods, and studies the regulatory mechanism of the SlPIF8b gene on tomato fruit coloring and ripening by regulating its expression level. The results show that SlPIF8b gene knockout plants increase the accumulation of carotenoids and lycopene in tomato fruits, induce ethylene accumulation, and reduce fruit firmness, 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 ethylene synthesis, thus inhibiting tomato fruit coloring and ripening. Mutating this gene using gene editing and other technologies is of great significance for improving the accumulation of nutrients and ripening of tomatoes. Furthermore, elucidating the function of this gene provides important theoretical basis and technical support for using techniques such as supplemental lighting to regulate tomato fruit color and ripening.

[0025] The SlPIF8b protein and its encoding gene provided by this invention provide gene resources for cultivating new tomato varieties with higher nutritional value and better storage characteristics. It has good potential application value. It can regulate the nutritional and appearance quality of tomatoes and regulate the market time of tomato fruits through gene editing and transgenic technology. At the same time, it lays a theoretical foundation and technical support for the molecular mechanism of regulating the nutritional quality and ripening of tomatoes using light signals.

[0026] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0027] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0028] Figure 1 This is a diagram showing the sequencing results of the target site in the SlPIF8b gene knockout tomato plant in Example 3 of the present invention.

[0029] Figure 2 This is a graph showing the relative expression level of the SlPIF8b gene in the SlPIF8b gene overexpressing tomato lines in Example 3 of the present invention.

[0030] Figure 3 This shows the overexpression of SlPIF8b, SlPIF8b gene mutation, and color change of wild-type fruit in Example 4 of the present invention.

[0031] Figure 4 This is a schematic diagram showing the results of SlPIF8b overexpression, SlPIF8b gene mutation, and wild-type fruit color difference index in Example 4 of the present invention.

[0032] Figure 5This is a schematic diagram showing the results of SlPIF8b overexpression, SlPIF8b gene mutation, and changes in carotenoids in wild-type fruits in Example 4 of the present invention.

[0033] Figure 6 This is a schematic diagram showing the changes in lycopene content in wild-type fruits after overexpression of SlPIF8b, SlPIF8b gene mutation, and other factors in Example 4 of the present invention.

[0034] Figure 7 This is a comparison diagram of the changes in fruit firmness in wild-type fruit with overexpression of SlPIF8b, SlPIF8b gene mutation, and wild-type fruit in Example 4 of the present invention.

[0035] Figure 8 The results show the changes in ethylene release from wild-type fruits due to overexpression of SlPIF8b, SlPIF8b gene mutation, and other factors in Example 4 of this invention. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0037] Unless otherwise specified, all materials, reagents, etc., used in the following examples are commercially available. The implementation of this invention will utilize conventional botanical techniques, tissue culture, molecular biology, biological physiology and biochemistry, DNA recombination, and bioinformatics techniques that are readily apparent to those skilled in the art. These techniques are fully explained in the literature.

[0038] Example 1: Construction of SlPIF8b gene overexpression vector

[0039] To understand the molecular mechanism of tomato fruit ripening, the SlPIF8b gene was cloned from the tomato genome. Based on the coding region sequence analysis, specific primers SlPIF8b-OE-F and SlPIF8b-OE-R were designed, and restriction enzyme sites (Asc I and SalI) were added to the primers, as shown in SEQ ID NO. 6 and 7, respectively. The SlPIF8b fragment was amplified by PrimerSTAR high-fidelity PCR. The PCR-amplified fragment and the vector were then digested with enzymes, and the SlPIF8b fragment was ligated into pFGC1008-HA to obtain a plant overexpression vector. The recombinant plasmid was sent to Qingke Company for sequencing confirmation. The nucleotide sequence of the obtained SlPIF8b gene is shown in SEQ ID No. 2; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID No. 1. The results show that the cloned sequence is consistent with the sequence published in Solgenomics (Solyc10g018510).

[0040] SEQ ID NO.6 is as follows:

[0041] ttggcgcgcc atggattatg aagtagcaga

[0042] SEQ ID NO.7 is as follows:

[0043] acgcgtcgac atttttaaag ccaagatttg

[0044] SEQ ID No. 2 is as follows:

[0045] atggattatg aagtagcaga gctaaaatgg gaaaagggag aggtagtgat gcatgggtta

[0046] ggtcctccag gcgtgccttg ttattataag cctttatcga ctccttctcc aacaaaatac

[0047] acgtgggacg ataagccaca tgctgctgca ggtggcacac ttgaatccat agtgaaccaa

[0048] gctacgactc ataatattga catcggtgac gagggtggtg atgatgatga tttagtgagt

[0049] tggtttgatg attgtcttcc tgaaacgtcc atggatattg tggccgtagt tccaacaagt

[0050] tgtactaact ataatcaaca agtgccccg tccacacgtg ttgcatcatg cagtggtgat

[0051] gcagagatgg cacgtgtggg aatgggatct agctttgagg aaatatcgga agactttgag

[0052] aatcaagagg ctaagaactt gatcggctca atggtatacg agggcaaaaa taacactgtg

[0053] agcccgggag agacaagttt gggtgaggaa agagtactta caacaacatc tacctttaat

[0054] cataataaaa ggaagacact gaataatcat gatagcagag gtcaggagtc gagagataat

[0055] gaggatgagg atgagaaaaa aagatccaaa atttcttcat tttcaacaaa aaggtgcaga

[0056] gttgctgcta ctcacaacca gtctgaacga aaaagaagag acaagataaa ccaaaggttg

[0057] aagacattgc agaagttagt tccaacatcg agtaagactg atacggcatc aatgttagat

[0058] gaggtgatag aatatttgaa gcaactacga gctcaagtta aagccatgag catgatgatt

[0059] catgttaaca tgcagccacc ccctatgatg ttaccaaata tggcattcca acaacaa

[0060] caacaatttc aaatgtcaat gatggggatg gctagaccca tcgatgtcaa tgcccttagc

[0061] agccccaaca taacaacaat cccatcgatt ctccatacca ccgcaccctc taatttcaat

[0062] aaccctccta ttgcctcccc tggagctgat cctttagctt ccttggtcgc agtacgccaa

[0063] ttatcacagc ctatgacgat ggatgcttat agcaggatgg cagcattgta ccaacaatat

[0064] ctacagtcaa atgcaaatct tggctttaaa aattga

[0065] SEQ ID No.1 is as follows:

[0066] Ala Thr Gly GlyAla Thr ThrAla Thr GlyAlaAla Gly ThrAla Gly

[0067] Cys Ala GlyAla Gly Cys ThrAlaAlaAlaAla Thr Gly Gly GlyAla

[0068] AlaAlaAla Gly Gly GlyAla GlyAla Gly Gly ThrAla Gly Thr Gly

[0069] Ala Thr Gly Cys Ala Thr Gly Gly Gly Thr ThrAla Gly Gly Thr Cys

[0070] Cys Thr Cys Cys Ala Gly Gly Cys Gly Thr Gly Cys Cys Thr Thr Gly

[0071] Thr ThrAla Thr ThrAla ThrAlaAla Gly Cys Cys ThrThr ThrAla<000016​Thr Cys GlyAla Cys Thr Cys Cys Thr Thr Cys Thr Cys Cys AlaAla

[0073] Cys AlaAlaAlaAla ThrAla Cys Ala Cys Gly Thr Gly Gly GlyAla

[0074] Cys GlyAla ThrAlaAla Gly Cys Cys Ala Cys Ala Thr Gly Cys Thr

[0075] Gly Cys Thr Gly Cys Ala Gly Gly Thr Gly Gly Cys Ala Cys Ala Cys

[0076] Thr Thr GlyAlaAla Thr Cys Cys Ala ThrAla Gly Thr GlyAlaAla

[0077] Cys Cys AlaAla Gly Cys ThrAla Cys GlyAla Cys Thr Cys Ala Thr

[0078] AlaAla ThrAla Thr Thr GlyAla Cys Ala Thr Cys Gly Gly Thr Gly

[0079] Ala Cys GlyAla Gly Gly Gly Thr Gly Gly Thr GlyAla Thr GlyAla

[0080] Thr GlyAla Thr GlyAla ThrThr ThrAla Gly Thr GlyAla Gly Thr

[0081] Thr Gly Gly ThrThr Thr GlyAla Thr GlyAla Thr Thr Gly Thr Cys

[0082] Thr Thr Cys Cys Thr GlyAlaAlaAla Cys Gly Thr Cys Cys Ala Thr

[0083] Gly GlyAla ThrAla Thr Thr Gly Thr Gly Gly Cys Cys Gly ThrAla

[0084] Gly Thr Thr Cys Cys AlaAla Cys AlaAla Gly Thr Thr Gly ThrAla

[0085] Cys ThrAlaAla Cys ThrAla ThrAlaAla Thr Cys AlaAla Cys Ala

[0086] Ala Gly Thr Gly Cys Cys Cys Cys Cys Gly Thr Cys Cys Ala Cys Ala

[0087] Cys Gly Thr Gly Thr Thr Gly Cys Ala Thr Cys Ala Thr Gly Cys Ala

[0088] Gly Thr Gly Gly Thr GlyAla Thr Gly Cys Ala GlyAla GlyAla Thr

[0089] Gly Gly Cys Ala Cys Gly Thr Gly Thr Gly Gly GlyAlaAla Thr Gly

[0090] Gly GlyAla Thr Cys ThrAla Gly Cys Thr Thr Thr GlyAla Gly Gly

[0091] AlaAlaAla ThrAla Thr Cys Gly GlyAlaAla GlyAla Cys ThrThr

[0092] Thr GlyAla GlyAlaAla Thr Cys AlaAla GlyAla Gly Gly Cys Thr

[0093] AlaAla GlyAlaAla Cys ThrThr GlyAla Thr Cys Gly Gly Cys Thr

[0094] Cys AlaAla Thr Gly Gly ThrAla ThrAla Cys GlyAla Gly Gly Gly

[0095] Cys AlaAlaAlaAlaAla ThrAlaAla Cys Ala Cys Thr Gly Thr Gly

[0096] Ala Gly Cys Cys Cys Gly Gly GlyAla GlyAla GlyAla Cys AlaAla

[0097] Gly Thr Thr Thr Gly Gly Gly Thr GlyAla Gly GlyAlaAlaAla Gly

[0098] Ala Gly ThrAla Cys Thr ThrAla Cys AlaAla Cys AlaAla Cys Ala

[0099] Thr Cys ThrAla Cys Cys Thr ThrThrAlaAla Gly Cys Ala ThrAla

[0100] Ala ThrAlaAlaAlaAla Gly GlyAlaAla GlyAla Cys Ala Cys Thr

[0101] GlyAlaAla ThrAlaAla Thr Cys Ala Thr GlyAla ThrAla Gly Cys

[0102] Ala GlyAla Gly Gly Thr Cys Ala Gly GlyAla Gly Thr Cys GlyAla

[0103] GlyAla GlyAla ThrAlaAla Thr GlyAla Gly GlyAla Thr GlyAla

[0104] Gly GlyAla Thr GlyAla GlyAlaAlaAlaAlaAlaAlaAla GlyAla

[0105] Thr Cys Cys AlaAlaAlaAla Thr Thr Thr Cys Thr Thr Cys Ala Thr

[0106] Thr Thr Thr Cys AlaAla Cys AlaAlaAlaAlaAla Gly Gly Thr Gly

[0107] Cys Ala GlyAla Gly Thr Thr Gly Cys Thr Gly Cys ThrAla Cys Thr

[0108] Cys Ala Cys AlaAla Cys Cys Ala Gly Thr Cys Thr GlyAlaAla Cys

[0109] GlyAlaAlaAlaAlaAla GlyAlaAla GlyAla GlyAla Cys AlaAla

[0110] GlyAla ThrAlaAlaAla Cys Cys AlaAlaAla Gly Gly ThrThr Gly

[0111] AlaAla GlyAla Cys Ala ThrThr Gly Cys Ala GlyAlaAla Gly Thr

[0112] ThrAla Gly Thr Thr Cys Cys AlaAla Cys Ala Thr Cys GlyAla Gly

[0113] ThrAlaAla GlyAla Cys Thr GlyAla ThrAla Cys Gly Gly Cys Ala

[0114] Thr Cys AlaAla Thr Gly Thr ThrAla GlyAla Thr GlyAla Gly Gly

[0115] Thr GlyAla ThrAla GlyAlaAla ThrAla Thr Thr Thr GlyAlaAla

[0116] Gly Cys AlaAla Cys ThrAla Cys GlyAla Gly Cys Thr Cys AlaAla

[0117] Gly Thr ThrAlaAlaAla Gly Cys Cys Ala Thr GlyAla Gly Cys Ala

[0118] Thr GlyAla Thr GlyAla ThrThr Cys Ala Thr Gly Thr ThrAlaAla

[0119] Cys Ala Thr Gly Cys Ala Gly Cys Cys Ala Cys Cys Cys Cys Cys Thr

[0120] Ala Thr GlyAla Thr Gly ThrThrAla Cys Cys AlaAlaAla ThrAla

[0121] Thr Gly Gly Cys Ala ThrThr Cys Cys AlaAla Cys AlaAla Cys Ala

[0122] Ala Cys AlaAla Cys AlaAla Cys AlaAla Thr Thr Thr Cys AlaAla

[0123] Ala Thr Gly Thr Cys AlaAla Thr GlyAla Thr Gly Gly Gly GlyAla

[0124] Thr Gly Gly Cys ThrAla GlyAla Cys Cys Cys Ala Thr Cys GlyAla

[0125] Thr Gly Thr Cys AlaAla Thr Gly Cys Cys Cys Thr ThrAla Gly Cys

[0126] Ala Gly Cys Cys Cys Cys AlaAla Cys Ala ThrAlaAla Cys AlaAla

[0127] Cys AlaAla Thr Cys Cys Cys Ala Thr Cys GlyAla Thr Thr Cys Thr

[0128] Cys Cys Ala ThrAla Cys Cys Ala Cys Cys Gly Cys Ala Cys Cys Cys

[0129] Thr Cys ThrAlaAla Thr Thr Thr Cys AlaAla Thr AlaAla Cys Cys

[0130] Cys Thr Cys Cys ThrAla Thr Thr Gly Cys Cys Thr Cys Cys Cys Cys

[0131] Thr Gly GlyAla Gly Cys Thr GlyAla Thr Cys Cys Thr ThrThr Ala

[0132] Gly Cys Thr Thr Cys Cys Thr Thr Gly Gly Thr Cys Gly Cys Ala Gly

[0133] ThrAla Cys Gly Cys Cys AlaAla Thr ThrAla Thr Cys Ala Cys Ala

[0134] Gly Cys Cys ThrAla Thr Gly Ala Cys GlyAla Thr Gly GlyAla Thr

[0135] Gly Cys Thr ThrAla Thr Ala Gly Cys Ala Gly GlyAla Thr Gly Gly

[0136] Cys Ala Gly Cys Ala Thr Thr Gly ThrAla Cys Cys AlaAla Cys Ala

[0137] Ala ThrAla Thr Cys ThrAla Cys Ala Gly Thr Cys AlaAla Ala Thr

[0138] Gly Cys Ala AlaAla Thr Cys Thr Thr Gly Gly Cys Thr Thr ThrAla

[0139] AlaAla AlaAla Thr Thr Gly Ala Pro

[0140] It should be noted that the plant overexpression vector is suitable for dicotyledonous plants, such as tomatoes, eggplants, and peppers.

[0141] Example 2: Construction of the SlPIF8b gene editing (knockout) vector

[0142] Based on the SlPIF8b gene sequence, CRISPR targets 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 for the target are shown in SEQ ID NO.4 and SEQ ID NO.5. After annealing, the synthesized target primer sequences were ligated to SlU61, and then ligated to the linearized cloning vector pCBSG012-slu61-DSG-bsai. The recombinant plasmid was sent to Qingke Technology Co., Ltd. for sequencing confirmation.

[0143] SEQ ID NO.3 is as follows:

[0144] aagccacatg ctgctgcagg

[0145] SEQ ID NO.4 is as follows:

[0146] atgcatgggt taggtcctcc

[0147] SEQ ID NO.5 is as follows:

[0148] catctctgca tcaccactgc

[0149] Example 3: Construction and detection of SlPIF8b transgenic tomato plants

[0150] The constructed plant vector and CRISPR gene editing vector were transformed into Agrobacterium GV3101 using Agrobacterium-mediated genetic transformation. Tomato cotyledons were then infected, and tissue culture seedlings were obtained through callus induction, resistance-induced differentiation, and rooting culture. T2 generation mutant seeds and overexpression seeds were tested for kanamycin and chloramphenicol resistance, respectively. Lines with 3 / 4 resistance and the remaining 1 / 4 non-resistance were selected, indicating that the overexpression vector carrying the target gene was inserted as a single copy in these lines. These plants were then removed and individually harvested for seed.

[0151] The positive transgenic plants overexpressing SlPIF8b were verified using qRT-PCR technology. The results showed that the expression level of SlPIF8b was upregulated by 200-fold compared with the wild type. Figure 2 Using PCR and sequencing techniques to verify positive SlPIF8b mutant transgenic plants, it was found that the addition of a base at the target site of pif8b#28 resulted in a mutation near the original adjacent motif (PAM), causing SlPIF8b translation to cease. Figure 1 ).

[0152] Example 4: Statistics on the color change time of tomato fruits in plants with SlPIF8b gene editing and overexpression

[0153] After the tomato materials flowered, the open flowers were marked with their 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). This indicates that the SlPIF8b gene inhibits the color change time of tomato fruits.

[0154] Example 5: Determination of color difference index of tomato fruits from SlPIF8b gene-edited and overexpressed plants

[0155] The color changes of fruits from different materials 51 days after flowering were detected. L*, a*, and b* were measured using a Konica Minolta CR-400 colorimeter in CIE mode. L* values ​​represent lightness, a* values ​​represent green-red, and b* values ​​represent blue-yellow. Color was calculated as the ratio of a* / b*. Six fruits were randomly selected from each material, and measurements were taken at four locations near the equator. Results showed that compared to wild-type fruits (WT), tomatoes overexpressing the SlPIF8b gene (SlPIF8b-OE#69) had lower color difference indices, were significantly greener, and matured later with increasing days after flowering; while tomatoes with the SlPIF8b gene mutant (slpif8b#28) had higher color difference indices, faster color change, and were redder. Figure 4 This indicates that the SlPIF8b gene negatively regulates the color of tomato fruits.

[0156] Example 6: Determination of carotenoid content in tomato fruits from SlPIF8b gene-edited and overexpressed plants

[0157] Slices of pericarp tissue were cut from the upper part of the tomato fruit near the equator. The sample was weighed (1g) and then crushed into powder using liquid nitrogen in a mortar and pestle. Total carotenoids were extracted by adding 10ml of hexane:acetone (6:4, v / v) to the sample. The sample was then centrifuged at 4000g for 5min, and the supernatant was transferred to a new tube. The absorbance of the supernatant at 450nm was immediately measured using a spectrophotometer. The total carotenoid content was quantified using the formula: Carotenoid content (mg / mL) -1 )=(4×OD450×10mL)g -1The above operations should be performed in at least five biological replicates. Results showed that, compared to wild-type fruit (WT), the total carotenoid content of fruit overexpressing the SlPIF8b gene (SlPIF8b-OE#69) was significantly lower than that of wild-type fruit (WT), while the total carotenoid accumulation of tomato fruit from the SlPIF8b gene mutant (slpif8b#28) was higher than that of wild-type fruit. Figure 5 This indicates that the SlPIF8b gene negatively regulates the accumulation of carotenoids in fruits.

[0158] Example 7: Determination of lycopene content in tomato fruits from SlPIF8b gene-edited and overexpressed plants

[0159] The pericarp tissue was cut near the equator of the fruit to obtain a 5 mm wide sample. After thorough grinding in liquid nitrogen, 0.4–0.6 g of the sample was weighed and placed in a 50 mL centrifuge tube. 20 mL of a mixture containing acetone (containing 0.05% 2,6-di-tert-butyl-p-cresol, BTH), 95% ethanol, and n-hexane (1:1:2, V / V) was added to the centrifuge tube. The centrifuge tube was placed in an ice box, which was then placed in a shaker and shaken at 180 rpm for 15 min. After shaking, 3 mL of ice-cold deionized water was added to each centrifuge tube, mixed well, and the tube was returned to the ice box and shaken at 180 rpm for 5 min. After standing at room temperature for 5 min, the liquid phase was separated, and the absorbance of the supernatant was measured. The calculation formula is as follows: Lycopene (mg / kg) = (OD503 × 31.2) / mg. The results showed that, compared with wild-type plants (WT), the lycopene content in fruits overexpressing the SlPIF8b gene (SlPIF8b-OE#69) was significantly lower, while the lycopene content in tomatoes from the SlPIF8b gene mutant (slpif8b#28) was significantly higher than that in wild-type fruits. Figure 6 This indicates that the SlPIF8b gene negatively regulates the accumulation of lycopene in fruits.

[0160] Example 8: Determination of fruit firmness in tomato plants edited and overexpressed with the SlPIF8b gene.

[0161] Fruit firmness was assessed 51 days post-flowering for different materials. A fruit texture analyzer (BROOKFIELD CT3; Middleboro, USA) equipped with a 2 mm diameter probe was inserted approximately 7 mm into the fruit. Firmness was recorded twice at the equator for each fruit, with measurements taken at 90° intervals between each other. Results showed that fruits overexpressing the SlPIF8b gene (SlPIF8b-OE#69) exhibited significantly higher firmness compared to wild-type fruits (WT). Conversely, fruits from the SlPIF8b mutant (slpif8b#28) showed significantly lower firmness than wild-type fruits. Figure 7This indicates that the SlPIF8b gene inhibits the ripening of tomato fruits.

[0162] Example 9: Determination of ethylene release from tomato fruits of SlPIF8b gene-edited and overexpressing plants

[0163] The ethylene content of fruits was determined using a Shimadzu GC2010 gas chromatograph (Japan), an activated alumina column (DB-5MS column, 30m × 0.25mm × 0.25μm), and a flame ionization detector. Tomato fruits from different materials, 51 days after flowering, were placed in 250mL beakers for 4 hours. Then, 1mL of gas was extracted using a syringe and injected into the GC. Column temperature: 60℃; FID detector temperature: 130℃; carrier gas flow rate: 30mL / min. -1 H2 flow rate is 1 mL·min -1 Air flow rate 400 mL / min -1 The split ratio was 35; the pressure was 113.5 kPa; and the injection time was 3 min. The results showed that compared with wild-type fruit (WT), the ethylene content in fruit from the SlPIF8b gene mutant (slpif8b#28) was significantly increased, while the ethylene content in fruit from SlPIF8b gene overexpression (SlPIF8b-OE#69) was significantly decreased. Figure 8 This indicates that the SlPIF8b gene inhibits the accumulation of ethylene in tomato fruits.

[0164] This invention constructs tomato SlPIF8b gene knockout plants using gene editing technology and then constructs SlPIF8b overexpression plants using transgenic technology. The results show that the SlPIF8b gene mutation can promote the accumulation of carotenoids and lycopene in tomato fruits, thereby promoting fruit coloring. Furthermore, the SlPIF8b gene mutation can promote ethylene accumulation, reduce fruit firmness, and thus advance fruit ripening. Therefore, the SlPIF8b gene provided by this invention is a key gene regulating tomato fruit ripening and carotenoid accumulation. SlPIF8b gene mutants can be constructed using gene editing and other technologies to improve tomato fruit coloring and the accumulation of nutrients such as carotenoids, achieving molecular design breeding and possessing significant application value.

[0165] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, the present invention is not limited to the above embodiments and many variations or modifications are possible, which will be obvious to those skilled in the art. Therefore, all such modifications or modifications made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. Tomato SlPIF8b The application of genes in regulating tomato fruit coloring and ripening, the aforementioned SlPIF8b The nucleotide sequence of the gene is shown in SEQ ID NO:

2. This application involves knocking out the gene using gene knockout technology. SlPIF8b Gene expression in tomatoes promotes fruit coloring and ripening.

2. According to claim 1, the gene knockout technology is specifically as follows: The sgRNA sequence for the CRISPR / Cas9 editing target was designed as shown in SEQ ID NO:

3. Primers were artificially synthesized based on the sgRNA sequence and constructed into the CRISP / Cas9 vector. Amplification of the target primers SlPIF8b The gene target fragment is inserted into a linearized transformation vector, ligated, and then transformed into E. coli. After plasmid extraction, the SlPIF8b gene knockout vector is obtained.

3. The application according to claim 2, wherein... SlPIF8b The primer sequences for the gene target fragment are shown in SEQ ID NO:4 and SEQ ID NO:

5.

4. In the application according to claim 2, the transfer vector is pCBSG012-slu61-DSG-bsai plasmid, and the pCBSG012-slu61-DSG-bsai plasmid is linearized by digestion with BasI.

5. A method for promoting the coloring and ripening of tomato fruits, the method comprising: Step A1: SlPIF8b Gene knockout vector was transferred into Agrobacterium; The SlPIF8b The nucleotide sequence of the gene is shown in SEQ ID NO: 2; Step A2: Transfer to the above SlPIF8b Agrobacterium, a gene knockout vector, infected tomato plants.

6. A method for inhibiting the coloring and ripening of tomato fruits, the method comprising: Step B1: Extract total RNA from tomatoes, reverse transcribe to obtain cDNA, and use the cDNA as a template... SlPIF8b -OE-F and SlPIF8b -OE-R is the primer, used for amplification. SlPIF8b The gene was amplified and its product was constructed into a plant gene overexpression vector with a 35S promoter. The resulting recombinant expression vector was used to... SlPIF8b The gene overexpression vector was transferred into Agrobacterium, wherein the primers... SlPIF8b -OE-F and SlPIF8b The nucleotide sequence of -OE-R is shown in SEQ ID NO. 6 and SEQ ID NO. 7; SlPIF8b The nucleotide sequence of the gene is shown in SEQ ID NO: 2; Step B2: Transfer to the above SlPIF8b Agrobacterium, a gene overexpression vector, infected tomato plants.

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