Tomato thioesterase gene SlFATB2 and its application

Abstract

This invention discloses a tomato thioesterase gene. SlFATB2 and its application in regulating tomato growth, development, and disease resistance, the aforementioned SlFATB2 The gene has the nucleotide sequence shown in SEQ ID No. 1. Its CDS sequence was cloned using bioinformatics combined with molecular biology techniques, and a strong promoter was constructed. SlFATB2 Overexpression vectors were used to obtain transgenic tomato plants overexpressing the vectors. The CRISPR / Cas9 knockout system was used to obtain... SlFATB2 Gene knockout tomato transgenic lines. Experiments have confirmed that knockout... SlFATB2 This leads to pollen shrinkage, fruit and seed shrinkage, and susceptibility to gray mold; overexpression SlFATB2 It can enhance resistance to gray mold without affecting normal growth. This invention clarifies that... SlFATB2 The gene function can be precisely improved in tomato agronomic traits and disease resistance through gene editing or overexpression, and its application prospects are broad.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and breeding technology, specifically relating to the gene encoding thioesterase in the tomato fatty acid synthesis pathway. SlFATB2 Its application is particularly suitable for improving tomato fertility, fruit and seed traits, and resistance to gray mold through gene editing or overexpression technology, providing new targets and technical solutions for tomato molecular breeding. Background Technology

[0002] tomato( Solanum lycopersicum As a globally important economic crop and a classic model species, tomato has advantages such as a small genome and short growth cycle, and is widely used in basic plant science research. Various volatile derivatives derived from fatty acids not only affect the flavor and quality of tomato fruits, but also play a crucial role in interspecific signal transduction, fungal and insect resistance processes.

[0003] The de novo fatty acid synthesis pathway in plants is highly conserved. Starting with acetyl-CoA as the substrate, it is carboxylated by acetyl-CoA carboxylase to form malonyl-CoA. Then, malonyl-CoA:ACP malonyltransferase transfers it to the acyl carrier protein (ACP) to generate malonyl-ACP, providing a direct substrate for carbon chain elongation. Subsequently, under the action of fatty acid synthase complexes, through multiple condensation and reduction reactions, intermediate products such as 16:0-ACP, 18:0-ACP, and 18:1-ACP are gradually formed. Finally, acyl-ACP thioesterase catalyzes the hydrolysis of thioester bonds to release free fatty acids. This step is a crucial link in fatty acid synthesis, and its activity directly determines the fatty acid composition of plant cells. Based on substrate specificity, plant thioesterases can be divided into two categories: FATA and FATB. FATB primarily hydrolyzes saturated acyl-ACP, exhibiting the highest hydrolytic activity towards 16:0-ACP, which is a major source of saturated fatty acids such as palmitic acid. It also possesses some hydrolytic activity towards unsaturated 18:1-ACP. In recent years, FATB-encoding genes in various plants, including soybean and rapeseed, have been cloned and identified, leading to significant progress in functional studies. However, research on lipid metabolism networks in tomatoes lags far behind other crops. Currently, only a small number of lipid-related genes have been cloned and identified, and systematic enzymatic characterization and physiological function analysis of key thioesterase-encoding genes involved in the de novo fatty acid synthesis pathway in tomatoes have not yet been conducted. Although existing studies have confirmed the important roles of thioesterase genes in fatty acid synthesis, stress resistance, and growth and development regulation in other plants, the specific regulatory mechanisms of these genes in tomato regarding growth, development, and resistance remain unclear.

[0004] In-depth exploration of tomato thioesterase gene resources, clarifying their functions and application value, and filling the gap in basic research on tomato lipid metabolism are of great significance for improving the regulatory network of tomato fatty acid synthesis and promoting the advancement of tomato molecular breeding technology. Summary of the Invention

[0005] This invention addresses the gap in existing functional studies of tomato thioesterase genes. Based on the cloning and identification of a novel gene affecting tomato development and disease resistance, it provides a method for utilizing the tomato thioesterase gene. SlFATB2 Applications in regulating male reproduction and disease resistance in tomatoes.

[0006] The objective of this invention is achieved through the following technical solution: A tomato thioesterase gene SlFATB2 Its application in regulating tomato pollen development, fruit size, seed size, and resistance to gray mold, the aforementioned SlFATB2 The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2.

[0007] Furthermore, the application includes two methods: one is to use CRISPR-Cas9 gene editing technology to specifically knock out the tomato genome. SlFATB2 The first method involved using the gene to produce tomato plants with shriveled pollen, smaller fruits and seeds, and susceptibility to gray mold; the second method involved overexpressing the gene via a strong promoter-mediated overexpression vector transformation technique. SlFATB2 Genes were used to obtain tomato plants that are resistant to gray mold and have growth and development consistent with the wild type.

[0008] As can be seen from the above technical solution, compared with the prior art, the present invention has the following advantages and effects: The present invention utilizes gene editing technology and genetic transformation to specifically knock out the thioesterase gene in tomatoes. SlFATB2 mutant materials of the thioesterase gene were obtained. fatb2 ; obtained by overexpression vectors with added strong promoters FATB2-OE Overexpression of genetic material. A series of experiments demonstrated that, compared to wild-type control tomato plants, fatb2 Pollen fertility and size, fruit weight, and seed size were all significantly reduced. FTAB2 No significant differences were found in pollen fertility and size, fruit weight, and seed size in the overexpressed materials, indicating that the thioesterase gene plays an important regulatory role in the male reproductive development of tomato and further affects fruit and seed development. Inoculation of detached leaves with *Botrytis cinerea* and statistical analysis of lesion area showed that, compared with wild-type MT, fatb2 The mutant showed a significantly increased susceptibility to the disease.

[0009] The thioesterase gene sequence provided by this invention can not only be used to study the role of genes in plant growth and male reproductive development, but is also related to the resistance of tomatoes to gray mold, providing a research basis and scientific evidence for improving crop resistance to gray mold through biofortification. Attached Figure Description

[0010] Figure 1 for SlFATB2 Image showing gene mutant editing patterns and quantitative results; Figure 2 Tomato thioesterase gene SlFATB2 Analysis and statistical graphs of pollen viability and morphological phenotypic correlation of mutant and overexpression materials; Figure 3 Tomato thioesterase gene SlFATB2 FDA-PI staining fertility phenotypes and statistical graphs of mutants and overexpression materials; Figure 4 Tomato thioesterase gene SlFATB2 Fruit and seed development phenotypic results and statistical graphs of mutants and overexpression materials; Figure 5 Tomato thioesterase gene SlFATB2 Phenotypic and statistical results of resistance to gray mold in mutants and overexpression materials. Detailed Implementation

[0011] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise defined, experimental methods not specifically described in the following embodiments are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press), or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products. Primer synthesis and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0012] All technical and scientific terms used herein have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0013] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1 SlFATB2 Gene sequence acquisition and cloning Literature was consulted, and based on the fatty acid sequence information in Arabidopsis thaliana, tomato thioesterases were searched using the tomato SL4.0 database on the Phyozome website (https: / / phytozome-next.jgi.doe.gov / ). SlFATB2The gene's CDS and protein sequence information, the relevant nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0015] Example 2 SlFATB2 Acquisition of gene mutants and overexpression genetic materials Following standard molecular biology operating procedures, sequence information was aligned, and CRISPR target and CDS primers were designed. The target gene CDS sequence from tomato MT leaf cDNA was cloned using high-fidelity polymerase. The CRISPR target primers and CDS sequence were constructed into the pEXT06 / G plant expression vector, first transformed into *E. coli* DH5α, and sequenced to verify successful construction. Subsequently, the successfully constructed plasmid was transformed into *Agrobacterium* GV3101 to obtain an *Agrobacterium* strain for tissue culture infection. Stable tomato gene mutants and overexpressed genetic materials were obtained using the *Agrobacterium* transformation method for phenotypic analysis.

[0016] The thioesterase gene was confirmed to have been obtained through gene sequencing. SlFATB2 Gene editing materials fatb2 as well as FATB2-OE Overexpression material. Gene expression results were analyzed by qRT-PCR. fatb2 Gene expression levels in the overexpression material were lower than in the wild type, while expression levels in different overexpression lines were significantly higher than in the wild type. Figure 1 See also Figure 1 In the figure, A represents the thioesterase gene in the obtained tomato genetic material. SlFATB2 Gene sequencing alignment results; B is SlFATB2 exist fatb2 Changes in mRNA expression levels in the material; C is SlFATB2 exist FATB2-OE Changes in mRNA expression levels in the material.

[0017] Example 3 Tomato Thioesterase Gene SlFATB2 Phenotypic analysis of pollen fertility and size in mutants and overexpression materials Alexandrite staining method: Prepare Alexandrite stain solution (anhydrous ethanol, malachite green, glycerol, acid fuchsin, orange yellow G, glacial acetic acid, phenol), stain for 30 minutes, and then observe the pollen color under a microscope to analyze and count pollen fertility.

[0018] Scanning electron microscopy observation: The morphology and wrinkling of the exine of mature tomato pollen were observed using scanning electron microscopy. The dried pollen material was smeared on a metal stage and then observed and photographed using a cryo-scanning electron microscope after being sprayed with gold.

[0019] FDA-PI fluorescence staining method; pollen fertility was observed and recorded 20 min after staining using a fluorescence microscope.

[0020] The results of pollen Alexandrite staining and scanning electron microscopy showed that, compared with the wild type, fatb2 Pollen fertility and size were significantly reduced, accompanied by significant shrinkage of pollen grains; FTAB2 No significant differences were found in pollen fertility, morphology, and size among the overexpressed materials. (See also...) Figure 2 In the figure, A and F are the phenotypic images of pollen Alexandrite staining and scanning electron microscopy of the control group MT tomato material; B and G are the tomato thioesterase gene. SlFATB2 Phenotypic images of pollen from mutant materials after Alexandrine staining and scanning electron microscopy; C and H are phenotypic images of pollen from materials overexpressing the tomato thioesterase gene SlFATB2 after Alexandrine staining and scanning electron microscopy; D is the phenotypic image of the tomato thioesterase gene in the Alexandrine staining experiment. SlFATB2 Results of fertility changes in pollen from mutants and overexpression materials; E shows the changes in pollen diameter during the Alexander staining experiment; I shows the changes in the proportion of abnormal pollen morphology recorded by scanning electron microscopy; L shows the changes in pollen long axis length recorded by scanning electron microscopy.

[0021] Further FDA-PI staining results showed that, compared to the wild type, fatb2 The fertility of the pollen was significantly reduced, while FATB2 There was no significant difference in overexpressed materials. (See also...) Figure 3 In the figure, A is the phenotype of pollen FDA-PI staining in the control group MT tomato material; B is the tomato thioesterase gene. SlFATB2 Phenotypic diagram of pollen from mutant materials stained with FDA-PI; C represents the tomato thioesterase gene. SlFATB2 Phenotypic diagram of pollen FDA-PI staining of overexpression material; D represents the tomato thioesterase gene in the FDA-PI staining experiment. SlFATB2 Results of changes in the activity of pollen from mutants and overexpression materials.

[0022] Example 4 Tomato Thioesterase Gene SlFATB2 Phenotypic analysis of fruit weight and seed size in mutants and overexpression materials During the tomato ripening period, mature fruits were collected for growth and development phenotypic analysis. The results showed that compared with the wild type... fatb2 Both fruit weight and seed size were significantly reduced; while ​ There was no significant difference in overexpressed materials. (See also...) ​ In the diagram, A represents the tomato thioesterase gene. ​ Phenotypic diagrams of fruit size in mutants and overexpressed materials; B represents the tomato thioesterase gene. ​ Phenotypic diagram of seed size of mutants and overexpression materials; C represents the tomato thioesterase gene. ​ Results of changes in fruit weight in mutants and overexpressed materials; D represents the tomato thioesterase gene. ​Results of changes in seed area of ​​mutants and overexpression materials.

[0023] Example 5 Tomato Thioesterase Gene ​ Phenotypic analysis of gray mold resistance in mutants and overexpression materials Botrytis cinerea was cultured under light for about 2 weeks, and the spores were washed off and the final concentration was set at 10⁶ spores / ml. 10 µl of the spore suspension was inoculated onto 4-week-old tomato leaves of uniform size, and cultured under light in a closed environment. After 48 h of inoculation, photos were taken and the area of ​​the lesions was counted.

[0024] The results showed that compared with wild-type MT, ​ Susceptibility to disease is significantly increased, while ​ Overexpression materials exhibited a significant disease-resistant phenotype. (See also...) ​ In the figure, A is a phenotypic diagram of the leaf lesion area after gray mold infection in the control group MT tomato material; B is the tomato thioesterase gene. ​ Phenotypic diagram of leaf lesion area after infection with the mutant gray mold; C represents the tomato thioesterase gene. ​ Phenotypic diagram of leaf lesion area after gray mold infection in overexpressed materials; D represents the tomato thioesterase gene. ​ Results of changes in leaf lesion area after infection with gray mold by mutants and overexpression materials.

[0025] The sequence described in this invention includes: SEQ ID No. 1: CDS sequence of the tomato thioesterase gene SlFATB2 atggcactca tgatgattag agatttatgc agtttacatt acccgattaa tttgtcgaac 60 acagagcttc aggtgaagga atgccggatt ctgaaatttg gttgtaatat gaagaagaaa 120 ttgatggcgg tgaacgctag cgcaagcgga agcggaaatt cgaagagtat tgaaacgatt 180 aacgggaaga agattaatgg tgttcatgta gggaaaaggg gaaatgtagt aattgaaccg 240 ggatcgtctt cgtcgtcgtc atcggtgaat cattcatata tgttggggaa ttttgtggat 300 gagcggttg tgtatagaca gtctttgtg attaggtctt atgaaattgg gcctgataaaa 360 actgctacaa tggaaactat catgaatctc cttcaggaga cagctctaaa tcatgtagca 420 agctcagggg ttggtagtaa tggatttggg gctacacgag agatgagcct taggaaactc 480 atatgggtag tcactcgcat acagattaaa gtcgaacaat atagctcctg gggagatgtg 540 gtagagatag atacatgggt agatgcagca ggtaaaaatg gaatgaggag ggattggatc 600 attcgtgact ccaacactgg caacatcatc acaaaagcaa caagcacgtg ggtgataatg 660 aacagagaaa caagaaggct atccaaaatc ccggagcagg tcaaagcaga agttcgacct 720 ttctacatca acaaatttgc aatacctact gcacaaattg attctgaaaa gattgagaaa 780 ctcaacgatg aaaccgctca aattatctct tccggcttag ctccgcggtg gagtgacatg 840 gatgccaatc aacatgtcaa caatgtcaaa tatattggat ggattttgga gagtgttccc 900 attaatgttt tagaagacag ccacttaatg agcttgacat tagagtatcg acgtgaatgt 960 caattgtcaa atgtgctgca gtccatgaca acaatgagag aatagcaac atcggatggt 1020 gatgaaaaca gtggaatgga atgcacacat ctgattcgta tggaggccga tcaaggagag 1080 gtggttcgag ccaggtccat atggcagcct aaacaatga 1119 SEQ ID No. 2: Protein amino acid sequence of tomato thioesterase gene SlFATB2 MALMMIRDLCSLHYPINLSNTELQVKECRILKFGCNMKKKLMAVNASASGSGNSKSIETINGKKINGVHVGKRGNVVIEPGSSSSSSVNHSYMLGNFVDERFVYRQSFVIRSYEIGPDKTATMETIMNLLQETALNHVASSGVGSNGFGATREMSLRKLIWVVTRIQIKVEQYSSWGDVVEIDTW VDAAGKNGMRRDWIIRDSNTGNIITKATSTWVIMNRETRRLSKIPEQVKAEVRPFYINKFAIPTAQIDSEKIEKLNDETAQIISSGLAPRWSDMDANQHVNNVKYIGWILESVPINVLEDSHLMSLTLEYRRECQLSNVLQSMTTMREIATSDGDENSGMECTHLIRMEADQGEVVRARSIWQPKQ In summary, the invention illustrated herein can be suitably practiced without any elements or limitations not specifically disclosed herein. Therefore, terms such as "comprising / including" should be understood as open-ended and without limitation. Furthermore, the terms and expressions used herein are descriptive rather than limiting, and are not intended to exclude any equivalent features of the shown and described features or portions thereof; however, it should be recognized that various modifications are possible within the scope of the invention. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can employ modifications and variations embodying the invention as disclosed herein, and such modifications and variations are considered to be within the scope of the invention.

Claims

1. Tomato thioesterase gene SlFATB2 Its application in regulating tomato pollen development, fruit size, seed size, and resistance to gray mold, the aforementioned SlFATB2 The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The application includes two methods: one is to use CRISPR-Cas9 gene editing technology to specifically knock out the tomato genome. SlFATB2 The first method involved using the gene to produce tomato plants with shriveled pollen, smaller fruits and seeds, and susceptibility to gray mold; the second method involved overexpressing the gene via a strong promoter-mediated overexpression vector transformation technique. SlFATB2 Genes were used to obtain tomato plants that are resistant to gray mold and have growth and development consistent with the wild type.

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