Slgh1 gene, mRNA, protein and biological material for regulating and controlling tomato fruit shape and fruit weight characters and application of Slgh1 gene, mRNA, protein and biological material

Abstract

The invention discloses a Slgh1 gene, mRNA, protein and a biological material for regulating and controlling tomato fruit shape and fruit weight characters and application of the Slgh1 gene and the biological material, and relates to the technical field of molecular breeding. The nucleotide sequence of the gene Slgh1 provided by the invention is as shown in SEQ ID NO: 1, the full-length sequence of mRNA is as shown in SEQ ID NO: 3, and the amino acid sequence of protein is as shown in SEQ ID NO: 5. When the gene Slgh1 is expressed in tomatoes, tomato fruits can be lengthened, and the fruit weight is increased. The gene Slgh1 provided by the invention is obtained by performing gene knockout on the basis of a tomato endogenous gene SlGH1. After the normal SlGH1 gene is mutated, the gene expression level is obviously reduced; compared with the wild type, the fruit shape is changed from round fruit to long fruit, and the fruit weight is obviously increased. The invention provides a new thought and genetic material for quantitative improvement of tomato quality and yield-increasing breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, specifically to a Slgh1 gene, mRNA, protein, and biomaterials that regulate tomato fruit shape and weight traits, and their applications. Background Technology

[0002] tomato( Solanum lycopersicum ;2n=24) belongs to the Solanaceae family ( Solanaceae Tomato genus ( Lycopersicon Tomatoes are one of the world's most important vegetable crops. The fruit, a berry, is the most important part of the tomato's economic traits and can be eaten raw, cooked, or processed.

[0003] Tomato fruit shape is an important agronomical and economic trait, a key determinant of tomato appearance quality, and a crucial goal for genetic improvement. Fruit shape diversity is the result of directional selection for fruit size during domestication, and also reflects adaptation to the processing needs of modern agriculture and people's pursuit of high-quality fruit appearance. Long, slender tomatoes are suitable for canning and are easier to transport on industrial conveyor belts; large, flat tomatoes are the preferred choice for making hamburgers and sandwiches; while pear-shaped, extra-long, and bell-shaped tomatoes satisfy people's pursuit of high-quality appearance.

[0004] Fruit weight is a crucial factor influencing fruit yield. Identifying genes related to fruit weight is beneficial for breeding high-yielding, high-quality tomato varieties, meeting growing market demand and generating significant economic benefits. However, currently, only SUN, OVATE, LC, FAS, and fs8.1 have undergone functional analysis of fruit morphogenesis, and the complex mechanisms of tomato fruit morphogenesis remain unclear. Summary of the Invention

[0005] The purpose of this invention is to provide a Slgh1 gene, mRNA, protein, and biological material for regulating tomato fruit shape and weight traits, and its application. Compared with the wild gene, the gene can cause fruit lengthening and increased fruit weight, and can be applied to tomato breeding to provide new genetic material for breeding.

[0006] The objective of this invention can be achieved through the following technical solutions: A Slgh1 gene that regulates the fruit shape and weight traits of tomatoes, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The mRNA transcribed from the Slgh1 gene has the nucleotide sequence shown in SEQ ID NO.3.

[0008] The protein encoded by the Slgh1 gene has the amino acid sequence shown in SEQ ID NO.5.

[0009] Biological materials expressing the Slgh1 gene include any one of the following: a recombinant expression vector containing the Slgh1 gene, a recombinant microorganism containing the Slgh1 gene, an expression cassette containing the Slgh1 gene, or a host cell containing the Slgh1 gene; the host cell includes bacterial or fungal cells.

[0010] Application of the Slgh1 gene, Slgh1 gene transcribed mRNA, Slgh1 gene encoded protein, or biological materials in regulating tomato fruit shape and weight: expressing the Slgh1 gene, Slgh1 gene transcribed mRNA, Slgh1 gene encoded protein, or transferring biological materials into tomato plants to increase tomato fruit length and fruit weight.

[0011] The application of the Slgh1 gene, its transcribed mRNA, its encoded protein, or biological materials in tomato breeding aims to increase the length and weight of tomato fruits by transferring the Slgh1 gene, its transcribed mRNA, its encoded protein, or biological materials into tomato plants.

[0012] The Slgh1 gene is used to regulate tomato fruit shape and weight traits.

[0013] As a further aspect of the present invention, the specific method of application is to express the Slgh1 gene in tomato plants through Agrobacterium-mediated genetic transformation.

[0014] As a further aspect of the present invention: the preferred variety of tomato plant is the wild-type round-fruited tomato, Yellow Cherry 22 (wild-type tomato Y22).

[0015] The beneficial effects of this invention are: The gene Slgh1, which regulates the shape and weight of tomato fruit, provided by this invention, was obtained by gene knockout mutation based on the gene SlGH1. After the gene SlGH1 is mutated into the gene Slgh1, the tomato fruit shape becomes longer (the fruit shape index increases, and the round fruit becomes a long fruit) and the fruit weight increases compared to the wild type. This invention provides new ideas and genetic materials for breeding. Attached Figure Description

[0016] The invention will now be further described with reference to the accompanying drawings.

[0017] Figure 1 Comparison of the shape of fruits from the Slgh1 gene mutant gh1 mutant and wild-type tomato Y22 at different developmental stages, with a scale bar of 1 cm; Figure 2 Statistical graph of fruit shape index of Slgh1 gene mutant material gh1 mutant and wild-type tomato Y22 at different developmental stages; Figure 3 A statistical graph showing the fruit weight of the Slgh1 gene mutant gh1 and wild-type tomato Y22 at different developmental stages; Figure 4 The expression profiles of the Slgh1 and SlGH1 genes in various tissues of tomato are shown; (a) shows the expression of the SlGH1 gene in the wild-type Y22 material, and (b) shows the expression of the Slgh1 gene in the mutant material gh1. MG represents tomato fruit at the green ripening stage and RR represents tomato fruit at the ripening stage. Figure 5 The diagram shows the comparison of the primary sequence and tertiary spatial structure of the Slgh1 protein in the Slgh1 gene mutant material with that of the wild-type tomato SlGH1 protein; where (a) is the comparison of the primary structure of the protein, (b) is the tertiary structure model of the SlGH1 protein, (c) is the tertiary structure model of the Slgh1 protein, and (d) is the comparison of the tertiary structure of the SlGH1 protein and the Slgh1 protein. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The nucleotide sequence of the Slgh1 gene is shown in SEQ ID NO.1. It is used to regulate the shape and weight of tomato fruits. The Slgh1 gene is obtained by gene mutation based on the wild-type SlGH1 gene. Tomato materials resulting from the Slgh1 gene mutation are represented by lowercase gh1. The nucleotide sequence of the wild-type SlGH1 gene is shown in SEQ ID NO.2. The wild-type amino acid sequence is shown in SEQ ID NO.6.

[0020] The T1 site “TGCACTTAATTACCGGCCG” and the T2 site “ACTTATGGTGGGAAGGCAG” in SEQ ID NO.2 are gene editing targets.

[0021] Gene editing targets were designed using the CRISPR-P 2.0 website (http: / / cbi.hzau.edu.cn / CRISPR2 / ), and experimental procedures were performed in accordance with the literature “CRISPR-P 2.0: an improved CRISPR-Cas9tool for genome editing in plants”.

[0022] Mutant materials were obtained using CRISPR / Cas9 technology and tomato genetic transformation technology. Transgenic materials were obtained by following the genetic transformation steps in the reference "Identified trans-splicing of YELLOW-FRUITED TOMATO 2 encoding the PHYTOENESYNTHASE 1 protein alters fruit color by map-based cloning, functional complementation and RACE".

[0023] Example 1: Slgh1 gene sequence Gene editing target design was performed using the CRISPR-P 2.0 website (http: / / cbi.hzau.edu.cn / CRISPR2 / ) following the steps outlined in the literature "CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants". The sequence of the wild-type gene SIGH1 is shown in SEQ ID NO.2, where the target nucleotide sequence for gene editing at the T1 site is: TGCACTTAATTACCGGCCG, and the target nucleotide sequence for the T2 site is: ACTTATGGTGGGAAGGCAG.

[0024] Mutant materials were obtained using CRISPR / Cas9 technology and tomato genetic transformation technology. Transgenic materials were obtained by referring to the genetic transformation steps in the article "Identified trans-splicing of YELLOW-FRUITED TOMATO 2 encoding the PHYTOENE SYNTHASE 1 protein alters fruit color by map-based cloning, functional complementation and RACE".

[0025] After obtaining the genetically edited material, DNA was extracted from the sample and used as a template. Primers were designed targeting the SlGH1 gene, and PCR amplification was performed using PrimeSTAR Max DNA polymerase (TAKARA, Dalian, China). The PCR product was ligated into a blunt-ended pLB vector (TIANGEN, Shanghai, China) and sequenced using Sanger sequencing to finally obtain the mutant gene sequence. The nucleotide sequence is shown in SEQ ID NO. 1.

[0026] Example 2: A comparison of the tomato mutant material gh1 obtained through genetic transformation with the wild-type material Y22 showed that the shape of the mutant fruit changed from round to long at different developmental stages. Figure 1 The fruit shape index increased significantly. At maturity (42 DPA, 42 days after flowering), the average fruit shape index changed from 0.86 (0.8 < fruit shape index < 1, round fruit) to 1.14 (fruit shape index > 1, long fruit), and the average weight of a single fruit increased from 11.97g to 17.31g (an increase of about 45%).

[0027] ① Process of obtaining mutant materials Using Agrobacterium tumefaciens-mediated genetic transformation of wild-type tomato Y22, a mutant material gh1 with the SlGH1 gene edited in tomato was created. Tomato genetic transformation: the genetic transformation steps in the article "Identified trans-splicing of YELLOW-FRUITEDTOMATO 2 encoding the PHYTOENE SYNTHASE 1 protein alters fruit color by map-based cloning, functional complementation and RACE" were followed to obtain transgenic material.

[0028] ②Measure the fruit shape index and single fruit weight at different stages of fruit development. Fruit length and width of wild-type Y22 and gh1 mutant materials at different developmental stages were measured using vernier calipers, and fruit shape index was calculated. The results are as follows: Figure 2 As shown; the weight of fruits at different developmental stages was measured using an analytical balance, and the results are as follows. Figure 3 As shown in the figure, the asterisk * indicates a significant difference between the data (P<0.05).

[0029] Example 3: RNA extraction and obtaining full-length mRNA Total RNA was extracted from the fruits of the mutant material gh1 and the wild-type material Y22 using the RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN, Shanghai, China). SMARTer RACE 5' / 3' kit (Clotech, MountainView, China) was used to extract RNA. A 5' and 3' RACE library was constructed in CA, USA. Conserved region sequence primers (forward primer F: 5'-ATGGCATTATCTTCACTGGAGGTTG-3'; reverse primer R: 5'-GAAGGCAGGGAAGGGTTATGACG-3'), 5'-specific RACE primers (5'-SP1R: 5'-GATTACGCCAAGCTTACTTCGTCATAACCCTTCCCTGCCT-3'; 5'-SP2R: 5'-GATTACGCCAAGCTTGTGTTCACCAGCATCGTTCTTCTCC-3'), and 3'-specific RACE primers (3'-SP1F: 5'-GATTACGCCAAGCTTTGGCATTATCTTCACTGGAGGTTGGGC-3'; 3'-SP2F: 5'-GATTACGCCAAGCTTTTGGAGAAGAACGATGCTGGTGAACAC-3') were designed according to the kit instructions to obtain the mutant Slgh1. The full-length sequences of mRNA and wild-type SlGH1 mRNA are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0030] Example 4: Comparison of gene expression profiles and protein sequences: Total RNA was extracted from different tissues (roots, stems, leaves, sepals, petals, stamens, pistils, green-ripe fruit (MG), and mature fruit (RR)) of the mutant material gh1 and the wild-type material Y22 using the RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN, Shanghai, China). cDNA libraries were obtained using the PrimeScript RT Master Mix reverse transcription kit. Quantitative real-time primers qSlgh1-F (5'-GGGAGCGGCGATACCGTAAAGC-3') and qSlgh1-R (5'-GGGAATGACTCTAGCACCAGCCA-3') were designed for Slgh1 gene expression, and quantitative real-time primers qSlGH1-F (5'-ATGGGAAACTACCTTTCGATTTCAT-3') and qSlGH1-R (5'-ACATTAGTGGCGTTGCTGAGTCGG-3') were designed for wild-type SlGH1 gene expression. The expression levels of transcript mRNA were then detected. Please refer to [link to relevant documentation]. Figure 4Compared with wild-type SlGH1, the relative expression level of Slgh1 in the mutant was significantly reduced in the ovary and mature fruit.

[0031] Sequence alignment of the encoded protein revealed that, compared to the protein encoded by the wild-type gene SlGH1, the protein encoded by Slgh1 is missing 79 amino acids at the N-terminus (Met1-Glu79) and 317 amino acids near the C-terminus (Tyr317). Figure 5 (As shown in Figure a). Through three-dimensional structure comparison, the Slgh1 protein may have lost a key functional domain, leading to a functional alteration. Figure 5 (As shown in the middle bd diagram).

[0032] As can be seen from the above embodiments, the Slgh1 gene, which regulates tomato fruit shape and weight, provided by the present invention, was obtained by gene mutation based on the SlGH1 gene. After the SlGH1 gene mutated into the Slgh1 gene, the tomato fruit shape became longer than that of the wild type, and the fruit weight increased significantly compared to the wild type. The present invention provides new ideas and genetic materials for breeding.

[0033] The foregoing has provided a detailed description of one embodiment of the present invention, but this description is merely a preferred embodiment and should not be construed as limiting the scope of the invention. All equivalent variations and modifications made within the scope of the claims of this invention should still fall within the patent coverage of this invention.

Claims

1. A Slgh1 gene that regulates the fruit shape and weight traits of tomatoes, characterized in that, The nucleotide sequence of the Slgh1 gene is shown in SEQ ID NO.

1.

2. An mRNA transcribed from the Slgh1 gene as described in claim 1, characterized in that, The nucleotide sequence of the mRNA is shown in SEQ ID NO.

3.

3. A protein encoded by the Slgh1 gene as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

5.

4. A biological material expressing the Slgh1 gene as described in claim 1, characterized in that, The biological material includes any one of the following: a recombinant expression vector containing the Slgh1 gene of claim 1, a recombinant microorganism containing the Slgh1 gene of claim 1, an expression cassette containing the Slgh1 gene of claim 1, or a host cell containing the Slgh1 gene of claim 1; wherein the host cell includes bacterial or fungal cells.

5. The application of the Slgh1 gene of claim 1, the mRNA of claim 2, the protein of claim 3, or the biomaterial of claim 4 in regulating tomato fruit shape and fruit weight, characterized in that, Expressing the Slgh1 gene of claim 1, the mRNA of claim 2, the protein of claim 3, or transferring the biological material of claim 4 into tomato plants increases the length and weight of tomato fruits.

6. The Slgh1 gene as described in claim 1 is applied to regulate the fruit shape and weight traits of tomatoes.

7. The application according to claim 6, characterized in that, The specific method of the application is as follows: expressing the Slgh1 gene in tomato plants through Agrobacterium-mediated genetic transformation.

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