Application of SlTCP1 and SlTCP30 genes in tomato leaf shape improvement

Abstract

The invention provides application of SlTCP1 and SlTCP30 genes in tomato leaf shape improvement, and belongs to the technical field of molecular breeding. According to the method, through operation on a specific gene, precise, efficient and stable bidirectional regulation and control on the tomato compound leaf complexity can be realized. A brand-new core gene resource and a technical scheme are provided for cultivating new tomato varieties meeting different agricultural requirements, for example, high-photosynthetic-efficiency complex leaf-shaped varieties can be directionally cultivated to improve the yield, or simplified leaf-shaped varieties are bred to adapt to mechanical harvesting and high-density planting, so that the breeding efficiency and accuracy are remarkably improved, and the economic benefit is increased. And the method has clear commercial development value and application prospect.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to the application of the SlTCP1 and SlTCP30 genes in the improvement of tomato leaf shape. Background Technology

[0002] As the primary organ for photosynthesis in plants, the morphology and structure of leaves directly affect the light energy utilization efficiency, canopy ventilation and light penetration, and final yield of crops. In Solanaceae crops such as tomatoes, leaves are typically compound leaves, composed of multiple leaflets. The complexity of these leaves (such as the number and arrangement of leaflets) is one of the key traits determining plant type and adaptability. Therefore, genetic improvement of tomato compound leaf development to cultivate varieties with ideal leaf shapes is of great significance to modern agriculture.

[0003] Currently, the plant science community knows that a family of transcription factors called TCPs plays a crucial role in regulating leaf development, particularly organ morphogenesis and differentiation. In tomatoes, the functions of some TCP family members (such as LANCEOLATE) have been preliminarily revealed; for example, functionally enhanced mutations in LANCEOLATE lead to leaf simplification. Furthermore, some small RNAs (such as miR319) have been found to increase leaf complexity by inhibiting the expression of multiple TCP genes. However, existing techniques still have significant limitations: for example, regulation via small RNAs often affects multiple target genes simultaneously, lacking specificity and potentially introducing unintended effects; while some known TCP genes, when their expression is inhibited, can increase leaf complexity, artificially enhancing their expression does not effectively achieve leaf simplification—that is, there is a lack of effective, bidirectionally operable single-gene targets. This makes it difficult to precisely and efficiently design leaf shapes according to specific needs (such as high light efficiency or suitability for machine harvesting) in breeding practice. Therefore, discovering new key genes with more defined functions and stronger regulatory efficacy is urgently needed to overcome the current technical bottlenecks in leaf shape breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of SlTCP1 and SlTCP30 genes in tomato leaf shape improvement, which solves the technical bottleneck of insufficient precision in regulation and inability to achieve efficient bidirectional regulation in existing tomato leaf shape improvement technologies.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a breeding method to increase the complexity of tomato compound leaves, comprising knocking out the SlTCP1 gene and the SlTCP30 gene in the tomato genome, wherein the CDS sequence of the SlTCP1 gene is shown in SEQ ID NO.1 and the CDS sequence of the SlTCP30 gene is shown in SEQ ID NO.3.

[0007] Preferably, the gene editing system is a CRISPR / Cas9 system;

[0008] The SlTCP1 gene was knocked out using a guide RNA targeting the sequence shown in SEQ ID NO.17;

[0009] The SlTCP30 gene was knocked out using a guide RNA targeting the sequence shown in SEQ ID NO.18.

[0010] The present invention also provides a CRISPR-Cas9 gene editing composition for knocking out the SlTCP1 and SlTCP30 genes in tomato, comprising a first guide RNA and a second guide RNA, wherein the first guide RNA targets the sequence shown in SEQ ID NO.16 and the second guide RNA targets the sequence shown in SEQ ID NO.17.

[0011] The present invention also provides a recombinant expression vector comprising the above-described gene editing composition.

[0012] The present invention also provides a kit comprising the above-described gene editing composition or recombinant expression vector.

[0013] This invention also provides the application of the above-mentioned recombinant expression vector or kit in tomato breeding.

[0014] The present invention also provides a breeding method for reducing the complexity of tomato compound leaves, comprising introducing or overexpressing the SlTCP1 gene or the SlTCP30 gene in tomato plants, wherein the CDS sequence of the SlTCP1 gene is shown in SEQ ID NO.1 and the CDS sequence of the SlTCP30 gene is shown in SEQ ID NO.3.

[0015] Preferably, the overexpression is achieved by operatively linking the coding sequence shown in SEQ ID NO.1 or SEQ ID NO.3 to a plant constitutive promoter and transferring it into a tomato plant.

[0016] The present invention also provides the application of a plant expression vector containing the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.3 in reducing the complexity of tomato compound leaves.

[0017] The present invention also provides a method for high-throughput screening of genes regulating the development of tomato compound leaves, characterized by comprising the following steps: treating tomato cells with the above-mentioned gene editing composition, screening to obtain mutants with loss of function of SlTCP1 and SlTCP30 genes, and identifying changes in the complexity of compound leaves of the mutants.

[0018] The beneficial effects of this invention are:

[0019] This invention reveals the core regulatory functions of the SlTCP1 and SlTCP30 genes in the development of tomato compound leaves and provides specific technical solutions for precise genetic manipulation of them, achieving significant beneficial effects at multiple levels.

[0020] At the technical level, the most significant contribution of this invention lies in achieving reliable, powerful, and stable bidirectional precise regulation of the complexity of tomato compound leaves, successfully overcoming the bottlenecks of existing technologies. Specifically, by knocking out the SlTCP1 and SlTCP30 genes, plants with significantly more complex compound leaf structures (including the number of leaflets and higher-level compound leaf orders) can be obtained; while by overexpressing either gene, leaf simplification can be efficiently driven, resulting in a simpler leaf shape. This achievement fundamentally solves the dilemma of ineffective overexpression, unidirectional regulation, or insufficient efficacy of similar genes (such as SlTCP24 / 29). The regulatory effect of this invention is not only clear and phenotypically stable and heritable, but its double mutant produces a stronger simplification effect than known mutants of the same type, while overexpression of a single gene can achieve a significant simplification effect. Compared to the "coarse regulation" mode of simultaneously inhibiting multiple genes through miR319, this invention operates on a highly specific key single target, effectively avoiding non-target effects and significantly improving the precision and biosafety of regulation.

[0021] At the breeding application level, this invention provides customized molecular breeding solutions to meet the diversified needs of modern agriculture. Using the mutant materials of this invention, varieties with more complex compound leaves and higher photosynthetic efficiency can be selectively bred to improve population photosynthetic efficiency and yield. Using overexpression materials, varieties with simplified leaf shapes can be bred to adapt to high-density planting and mechanized harvesting, reducing production costs. This invention transforms tomato leaf shape improvement from the traditional model relying on phenotypic selection to precise and efficient design based on defined genotypes. By providing clear molecular targets and operational tools, it is expected to significantly shorten the breeding cycle and improve breeding efficiency. Simultaneously, the genetic materials created by this invention (including various mutants and overexpression lines) are themselves unprecedented novel germplasm resources that can serve as core parents, rapidly deriving new tomato varieties adapted to different ecological environments and market demands through hybridization breeding.

[0022] At the commercial development level, varieties with different leaf shapes bred based on SlTCP1 / SlTCP30 regulation technology can precisely meet differentiated market demands (e.g., complex leaf shapes are suitable for fresh food production, while simplified leaf shapes are suitable for the processing industry), and have broad market prospects and expected economic benefits. The specific regulation technology system established around these two key genes has formed a complete solution with clear technological barriers, laying a solid foundation for building an intellectual property protection system and subsequent technology development.

[0023] At the level of advancing the discipline, the findings of this invention deepen our understanding of the regulatory network of the TCP transcription factor family, revealing differences in functional hierarchy and efficacy among members. It indicates that SlTCP1 / 30 plays a central role in this network, providing new theoretical clues for elucidating the fine regulatory mechanisms of compound leaf development. Furthermore, the genetic material created in this invention, exhibiting a continuously adjustable leaf shape lineage, provides an ideal research model system for studying fundamental biological questions such as plant organ size and morphogenesis.

[0024] In summary, this invention not only overcomes the key bottlenecks of insufficient regulatory tools and single and incomplete schemes in existing technologies, but more importantly, it provides powerful core gene resources and tools for the precise design breeding of plant architecture in tomatoes and other crops, realizing a leap from "random selection" to "directed design". It has important value and far-reaching impact in both scientific exploration and industrial application. Attached Figure Description

[0025] Figure 1 This is the spectrum of the 1300-3*flag overexpression vector used in this invention;

[0026] Figure 2 This is a schematic diagram illustrating the construction of the SlTCP1 and SlTCP30 co-knockout vector in the embodiment;

[0027] Figure 3 The image shows the Sanger sequencing results of the sltcp1 / sltcp30 double mutant.

[0028] Figure 4 The image shows the DNA identification results of the 1300-SlTCP1-3*flag and 1300-SlTCP30-3*flag overexpression lines;

[0029] Figure 5 Figure showing the qRT-PCR identification results of the 1300-SlTCP1-3*flag and 1300-SlTCP30-3*flag overexpression lines;

[0030] Figure 6 Figure 1 shows the morphological phenotypes of leaves in plants with SlTCP1 / SlTCP30 co-knockout and overexpression. Detailed Implementation

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The main sequences involved in the embodiments are:

[0033] CDS and protein sequences of SlTCP1 (Solyc02g077250)

[0034]

[0035] SlTCP1 protein sequence:

[0036] MNHQFQVSDEEDGSEEEEEEEVFQENDIGNLQSHYQNQQMPQSLCKKPEKWANFTVSEQELNKGTRRMKPKRAKTDVIEGHGGRIIRATGRKDRHSKVSTAKGPKDRRVRLSPNNTAIQFYDVQDRLGYDRPSKAIDWLIKEAKAAIDALGEFPNNFHSTKLNPQK MQYSFDQEQSPEFSQENRGVPNSECGVQDNQQEVNYDIPNLFSSSDGFKIPFLSDLQSHPHGHFLNFQSLQDDTVLSSGNHHQGRFFTTTSVNHFPSVLSQNQVFSHREPLQSSFFPLMSDPLSTQLETLSYGFSNDGFSGIISSASRIQGEEEQATFFTAS, as in SEQ Shown as ID NO.2.

[0037] 3. CDS and protein sequences of SlTCP30 (Solyc10g008780)

[0038] SlTCP30 CDS sequence:

[0039]

[0040] SlTCP30 protein sequence:

[0041] MKHHLEVSNENEDKSSSSSQQDDDDDENKDEFFQEILQSDYQHMQQWPNYFTPPSSTLVPKKQNNTQCPSHKEETKKVSLKRKPKRAKQDVMEVHGSRIVRATGRKDRHSKVSTATGPKDRRVRLSPNTAIQFYDVQDRLGYDRPSKAIDWLIKEAQRAINALEKAPFQVFSNKINSTNKALNVRNVENEVGQFALEQSWSADKKTELEELARRSSKLNATVQYSTDQFPEGCYKEQSSKVCQANATQIVNSSANGSKIQSFCQIQTHPQAHLHSQTKRQSPNDAKIQSFCEYQTHPQSHFHSQTKKQSPNVAKTLSFCEMQTPSQGHFHSQTKNQPTNDAKIQSFCQLQTHPQSHFQSETKNQSPNVAKIQSFGELQTTPQSHFHSQTQKQSTNDAKIQSFCELQTHPQGHFHSETKNQSPNVAKVQSFCELQTHPQSHFQSETKKQSPNVAKIQSFCELQTPPLSHFQSETKNQSTQLGNFFSFQSCHQNEPISFSGDHHHGFSFSPQNFPTVLGQNQMFNYQREPLQSTSNFPQTISYSNNIGFANDGLLGLSSAPITQQQQQQDEQGSISTTLLHYQD, as shown in SEQ ID NO.4.

[0042] Examples

[0043] 1. Construction of sltcp1 / tcp30 double knockout mutants

[0044] 1.1 Using the vector pUC-AtU6-29 as a template, PCR amplification was performed using primers sgTCP1-F: 5'-acggtctccTGATTGAGTGAACAAGAATTGAACAAGTTTTAGAGCTAGAAATAG-3' (as shown in SEQ ID NO.5) and sgTCP30-R: 5'-acggtctccaaacAGTCACAAAGAGGAAACCAACAATCTCTTAGTCGACTC-3' (as shown in SEQ ID NO.6) to obtain the sgTCP1-proAtU6-29-sgTCP30 module, followed by purification of the PCR product;

[0045] 1.2 The PCR product and vector pDS016-AtU6 obtained above were digested with BsaI enzyme; after purification, the digested product was ligated with T4 DNA ligase and transformed into Escherichia coli DH5α. The gene knockout vector pDS016-T1 / T30 was constructed by Sanger sequencing verification.

[0046] 1.3 The above-mentioned vector pDS016-T1 / T30 was transformed into Agrobacterium strain GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number AC1001; the transformation process was in accordance with the instruction manual) using the freeze-thaw method. Multiple pDS016-T1 / T30 transgenic lines were obtained through Agrobacterium-mediated leaf disc genetic transformation (the tomato variety used was Ailsa Craig), which involves Agrobacterium infecting tomato cotyledons, inducing callus differentiation, and redifferentiating into shoots.

[0047] 1.4 A primer was designed on both sides of the SlTCP1 and SlTCP30 target sites. Using the DNA of the positive line as a template, the target sequence was amplified. The mutation status of the target gene at the target site was detected by Sanger sequencing. Finally, multiple double mutant lines with simultaneous loss of function of SlTCP1 and SlTCP30 were obtained, such as sltcp1 / tcp30-#1, sltcp1 / tcp30-#3, and sltcp1 / tcp30-#4.

[0048] 2. Creation of SlTCP1 and SlTCP30 overexpression lines

[0049] 2.1 Using primer combinations flagTCP1-F: 5'-atttggagaggacagggtaccATGAATCATCAGTTTCAAGT-3' (as shown in SEQ ID NO.7) + flagTCP1-R: 5'-cgaactagtgtcgactctaga AGAAGCAGTAAAAAATGTAGCCTGCTCC-3' (as shown in SEQ ID NO.8) and flagTCP30-F: 5'- atttggagaggacagggtaccATGAAACATCATCTTGAAGTATCAAA-3' (as shown in SEQ ID NO.9) + flagTCP30-R: 5'-cgaactagtgtcgactctagaATCTTGATAATGTAGAAGAGTAGTTG-3' (as shown in SEQ ID NO.10), and using cDNA from the tomato variety Ailsa Craig as a template, the CDS sequences of SlTCP1 and SlTCP30 were amplified by PCR.

[0050] 2.2 The vector plasmid 1300-3*flag (image shown) Figure 1 (As shown) The samples were double-digested with KpnI and XbaI and homologously recombined with the purified products of SlTCP1 and SlTCP30, respectively. The samples were then transformed into E. coli DH5α and verified by Sanger sequencing to obtain the 1300-TCP1-3*flag and 1300-TCP30-3*flag overexpression vectors, respectively.

[0051] 2.3 Using the same transformation steps as in 1.3 above, multiple overexpression lines of SlTCP1 and SlTCP30 were obtained respectively;

[0052] 2.4 Extract leaf DNA from overexpressing lines and amplify the hygromycin resistance gene fragment of the vector by PCR using primer combination HYG-F: 5'-ATCGGCGAGTACTTCTACACAG-3' (as shown in SEQ ID NO.11) + HYG-R: 5'-GATCGAAAAGTTCGACAGCGTC-3' (as shown in SEQ ID NO.12) to identify positive lines at the DNA level.

[0053] 2.5 RNA was extracted from leaves and real-time quantitative PCR was performed using primer combinations qTCP1-F: 5'-CTGCAAAAAGCCAGAAAAATGG-3' (as shown in SEQ ID NO.13) + qTCP1-R: 5'-TACTGTGTCTGTCTTTCCTTCC-3' (as shown in SEQ ID NO.14) and qTCP30-F: '-GTCCAAGTCACAAAGAGGAAAC-3' (as shown in SEQ ID NO.15) + qTCP30-R: '-TAGCCTTACTAGGACGGTCATA-3' (as shown in SEQ ID NO.16) to measure the expression levels of SlTCP1 or SlTCP30 in overexpression lines. Positive lines with higher expression levels were selected for subsequent studies.

[0054] A schematic diagram of co-knockout vector construction is shown below. Figure 2 As shown.

[0055] The target sequence sgTCP1 (5'-AGTGAACAAGAATTGAACAA-3') (as shown in SEQ ID NO.17) is expressed by the AtU6-26 promoter driving the expression of the target sequence sgTCP30 (5'-TTGGTTTCCTCTTTGTGACT-3') (as shown in SEQ ID NO.18) driving the expression of the target sequence SlTCP30.

[0056] The results of the mutant Sanger sequencing are as follows Figure 3 As shown.

[0057] Sanger sequencing results showed that in the sltcp1 / sltcp30#1 line, there was a homozygous deletion of 5 bases in the SlTCP1 gene at the target sequence position, and a homozygous deletion of 19 bases in the SlTCP30 gene at the target sequence position. In the sltcp1 / sltcp30#2 line, there was a homozygous insertion of 1 base in the SlTCP1 gene at the target sequence position, and a homozygous insertion of 1 base in the SlTCP30 gene at the target sequence position. These sequencing results collectively indicate that sltcp1 / sltcp30#1 and sltcp1 / sltcp30#2 are both homozygous mutant lines of SlTCP1 and SlTCP30, which can be used for subsequent phenotypic observation.

[0058] DNA identification results of 1300-SlTCP1*3flag and 1300-SlTCP30*3flag overexpression lines are as follows: Figure 4 As shown.

[0059] DNA was extracted from the leaves of the overexpressing lines and amplified by PCR using 1300-3flag vector-specific primers. The results showed that among the 10 obtained SlTCP1-3*flag lines, all except #7 were negative lines and all the others were positive lines; among the 10 obtained SlTCP30-3*flag lines, all except #4 and #8 were negative lines and all the others were positive lines, which can be used for further analysis.

[0060] The qRT-PCR identification results of the 1300-SlTCP1*3flag and 1300-SlTCP30*3flag overexpression lines are as follows: Figure 5 As shown.

[0061] Leaves were extracted from plants that tested positive by the above PCR assay, and real-time quantitative PCR (qRT-PCR) was performed using wild-type plants as controls. The results showed that the expression levels of the target gene (SlTCP1 / SlTCP30) in all positive lines were significantly higher than those in the wild type. Among them, SlTCP1-OE#1 showed the highest fold-regulation, averaging 18.1-fold. In contrast, among the SlTCP30 overexpression positive lines, SlTCP30-OE#7 showed the highest fold-regulation, averaging 52.2-fold. These results indicate that the created overexpression lines significantly upregulated the expression levels of SlTCP1 / SlTCP30 and can be used for further research.

[0062] Phenotypic observation of leaf morphology by SlTCP1 / SlTCP30 co-knockout and overexpression, results are as follows: Figure 6 As shown.

[0063] By observing the leaf phenotypes of overexpression lines and double mutants, this invention found that the leaves of plants overexpressing SlTCP1 and SlTCP30 were significantly simplified, while the leaf compound leaf development of the sltcp1 / sltcp30 double mutant was more complex. Statistical results showed that the number of leaflets in the overexpression lines of SlTCP1 and SlTCP30 was significantly lower than that in the wild type, while the number of leaflets in the sltcp1 / sltcp30 double mutant was significantly increased.

[0064] As demonstrated by the above embodiments, this invention provides two novel genes, SlTCP1 and SlTCP30, which have core regulatory functions in the development of tomato compound leaves. Through specific molecular biology techniques, loss-of-function double mutants of SlTCP1 and SlTCP30 and their respective overexpression transgenic plants were successfully created. Experimental results show that manipulating these two genes can produce potent and stable opposite phenotypes: the double mutant plants develop a much more complex compound leaf structure than the wild type; while plants overexpressing either gene exhibit significantly simplified leaf characteristics. This fully demonstrates that SlTCP1 / SlTCP30 can serve as efficient targets for bidirectional and precise regulation of tomato compound leaf complexity, providing key gene resources and feasible technical pathways for molecular design breeding of tomato plant architecture.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A breeding method for increasing the complexity of tomato compound leaves, characterized in that, This includes knocking out the SlTCP1 and SlTCP30 genes in the tomato genome, with the CDS sequence of the SlTCP1 gene shown in SEQ ID NO.1 and the CDS sequence of the SlTCP30 gene shown in SEQ ID NO.

3.

2. The method according to claim 1, characterized in that, The gene editing system is a CRISPR / Cas9 system; The SlTCP1 gene was knocked out using a guide RNA targeting the sequence shown in SEQ ID NO.16; The SlTCP30 gene was knocked out using a guide RNA targeting the sequence shown in SEQ ID NO.

17.

3. A CRISPR-Cas9 gene editing composition for knocking out the SlTCP1 and SlTCP30 genes in tomato, characterized in that, It includes a first guide RNA and a second guide RNA, wherein the first guide RNA targets the sequence shown in SEQ ID NO.17 and the second guide RNA targets the sequence shown in SEQ ID NO.

18.

4. A recombinant expression vector comprising the gene editing composition of claim 3.

5. A kit comprising the gene editing composition of claim 3 or the recombinant expression vector of claim 4.

6. The application of the recombinant expression vector of claim 4 or the kit of claim 5 in tomato breeding.

7. A breeding method for reducing the complexity of tomato compound leaves, characterized in that, This includes introducing or overexpressing the SlTCP1 gene or the SlTCP30 gene in tomato plants, wherein the CDS sequence of the SlTCP1 gene is shown in SEQ ID NO.1 and the CDS sequence of the SlTCP30 gene is shown in SEQ ID NO.

3.

8. The method according to claim 7, characterized in that, The overexpression is achieved by operatively linking the coding sequence shown in SEQ ID NO.1 or SEQ ID NO.3 to a plant constitutive promoter and transferring it into tomato plants.

9. The application of a plant expression vector containing the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.3 in reducing the complexity of tomato compound leaves.

10. A method for high-throughput screening of genes regulating tomato compound leaf development, characterized in that, The procedure includes the following steps: treating tomato cells with the gene editing composition of claim 3, screening for mutants with loss of function of the SlTCP1 and SlTCP30 genes, and identifying changes in the complexity of the compound leaves of the mutants.

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