Application of SlSYP121 gene and protein coded by SlSYP121 gene in tomato leaf shape development

Abstract

The invention is suitable for the technical field of molecular biology, and provides an SlSYP121 gene and application of a protein coded by the SlSYP121 gene in tomato leaf shape development. The nucleotide sequence of the SlSYP121 gene is as shown in SEQ ID No.1 (Sequence Identifier Number 1). The amino acid sequence of the SYP121 protein coded by the SlSYP121 gene is as shown in SEQ ID No. 2. According to the invention, the SlSYP121 overexpression vector is successfully introduced into a plant body, and a positive overexpression plant is screened out. Compared with a wild type plant, the positive overexpression plant has a leaf growth and development form different from that of the wild type plant, the phenomena that the leaf notch degree is reduced, and the epicotyl length and the leaf mucoid glandular hair length are reduced occur, and the morphological change lays a foundation for subsequent overexpression plant stress resistance experiments.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to application of SlSYP121 gene and protein encoded thereby in tomato leaf shape development. Background Art

[0002] The leaves in plant organs are an important bridge connecting to the external environment. Through photosynthesis, respiration, transpiration and other pathways, the leaves provide the basis for the growth of the entire plant, and are closely related to the growth, nutrition, yield, quality, and resistance of the plant. Among them, the morphological structure of the leaves is one of the most obvious manifestations of plant growth and development. Due to genetic and environmental factors, leaf shapes have evolved into a wide variety of forms, which makes them play an important role in the plant's response to adverse stress.

[0003] Tomato (Solanum lycopersicum L.) is native to South America and was originally a wild berry in South America. The United States, Russia, Italy and China are the main producers. In China, tomatoes are cultivated in both the north and south, with rich varieties and good taste. It is one of the most commonly cultivated fruits and vegetables in the world. As an indeterminate growth crop, the diversity of tomato leaf morphology can make the plant have different spatial extension differences, thereby regulating the water loss and temperature changes on the leaf surface, and resisting adversities such as low temperature, drought, and high salt.

[0004] The formation of leaf morphological characteristics is a very complex physiological and biochemical process, which is regulated by the expression of many functional genes and transcriptional regulatory factors. Transcription factors (such as KNOX, WOX and CUC), hormone regulatory genes (such as PIN1, IPT7, CKX3, etc.) and miRNA (such as miR164, miR139, etc.) affect leaf morphology by generating complex regulatory networks at different time stages and specific tissue regions. Leaf morphology of tomatoes is an important agronomic trait related to plant stress resistance and yield potential. Discovering genes related to leaf growth and development and applying them in breeding research to screen target varieties is conducive to improving plant stress resistance, thereby improving resource utilization and agricultural yield. Therefore, understanding the related factors and genetic basis of leaf margin morphogenesis is of great significance to tomato breeding and the molecular regulatory mechanism of leaf margin morphological development.

[0005] Higher plant cells complete the exchange of substances and information inside and outside the cell through their unique endomembrane system and vesicle transport mechanism, and the membrane fusion in the plant cell vesicle transport process is mainly mediated by SNARE proteins. Since long-term evolution, SNARE proteins have played their respective functions in different positions. Although there are many reports about the plant SNARE protein family, they are mainly concentrated in biotic stress and abiotic stress. In terms of plant growth and development, especially in terms of leaf morphology development, there are relatively few functional studies on SNARE proteins. For this reason, the present invention proposes the application of SlSYP121 gene and its encoded protein in tomato leaf shape development. Summary of the invention

[0006] The object of the present invention is to provide an application of the SlSYP121 gene and the protein encoded by it in tomato leaf shape development, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The application of the SlSYP121 gene and the protein encoded by it in tomato leaf shape development is characterized in that the nucleotide sequence of the SlSYP121 gene is shown as SEQ ID No.1, and the amino acid sequence of the SYP121 protein encoded by the SlSYP121 gene is shown as SEQ ID No.2.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention successfully introduced the SlSYP121 overexpression vector into the plant body and screened out positive overexpression plants. Compared with wild-type plants, the leaf growth and development morphology of the positive overexpression plants is different from that of the wild-type plants, with reduced leaf notching, reduced epicotyl length and reduced leaf viscous glandular hair length. These morphological changes lay the foundation for subsequent experiments on stress resistance of overexpression plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Amplification of the tomato SlSYP121 fragment.

[0012] Figure 2 For Escherichia coli colonies.

[0013] Figure 3 The results are verified by PCR of E. coli colonies.

[0014] Figure 4 The results were verified by Agrobacterium colony PCR.

[0015] Figure 5 This is the genetic transformation of SlSYP121 in tomato.

[0016] Figure 6 PCR identification of T0 generation transgenic plants.

[0017] Figure 7 Partial PCR identification of T1 transgenic plants.

[0018] Figure 8 Expression level analysis of SlSYP121 transgenic line.

[0019] Fig. 9 This is a schematic diagram of tomato leaf notching, where L is leaf length, Leaflengthd is the degree of notching, and d>0.3mm is counted as one notch.

[0020] Fig.10 Comparison of leaf shapes between wild-type and overexpressing tomato plants during the "two leaves and one heart" period.

[0021] Fig.11 This is the statistical result of leaf shape index.

[0022] Fig.12 It is the statistical result of leaf notch degree / leaflet longitudinal length.

[0023] Fig.13 It is the statistical result of the number of leaf notches / longitudinal length of leaflets.

[0024] Fig.14 Epicotyl length.

[0025] Fig.15 Comparison of epicotyl length between wild-type and overexpression tomato plants.

[0026] Fig.16 Comparison of epicotyl lengths between wild-type and overexpressed tomato plants; A to C are the epicotyl lengths of wild-type plants, and D to F are the epicotyl lengths of three SlSYP121 overexpression lines, OE#1, OE#4, and OE#5.

[0027] Fig.17 Comparison of the length of the mucilaginous glandular hairs of wild-type and overexpressing tomato plants (scale bar: 500 μm); A to C are the lengths of the mucilaginous glandular hairs of wild-type plants, and D to F are the lengths of the mucilaginous glandular hairs of the three SlSYP121 overexpressing strains OE#1, OE#4, and OE#5, respectively. DETAILED DESCRIPTION

[0028] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0029] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0030] Example 1, Construction of SlSYP121 overexpression vector;

[0031] 1. Cloning of the full-length SlSYP121 gene: Total RNA was extracted from the leaves of wild-type tomato Micro Tom (MT), and reverse transcribed to obtain cDNA, using the obtained cDNA as a template to amplify the CDS region of SlSYP121. Primers for the SlSYP121 gene were designed using NCBI, and protective bases were added at both ends of the restriction site, and sent to Kumei Biotechnology for primer sequence synthesis.

[0032] The primers for gene cloning and amplification are as follows:

[0033] Table 1 Primers for gene cloning and amplification

[0034]

[0035] The amplification reaction system is as follows:

[0036] Table 2 Amplification reaction system

[0037]

[0038] The reaction procedure is as follows:

[0039] Table 3 Reaction procedure

[0040]

[0041] After obtaining the target fragment, gel recovery was performed to obtain Inserts, which were stored at -20°C for future use. Figure 1 The 912 bp CDS sequence fragment of the SlSYP121 gene obtained by gel recovery is shown.

[0042] 2. Enzyme digestion: The expression vector plasmid empty vector pBWA(V)HS is digested with restriction endonucleases BsaI / Eco31I and the product is recovered. The system is as follows:

[0043] Table 4 System

[0044]

[0045] Incubate at 37°C for 2 hours. Take 5 μL of the product after enzyme digestion and load it on 1.0% agarose gel. Perform electrophoresis to verify the effect of enzyme digestion. After verification, purify the remaining enzyme digestion product with a DNA purification kit to obtain Linearized vector.

[0046] 3. Connection: Connect the purified restriction vector and the target fragment Inserts through seamless cloning. The connection system is as follows:

[0047] Table 5 Connection system

[0048]

[0049] Mix gently, react at 50℃ for 15min, and place the centrifuge tube on ice for a few seconds. The recombinant product can be stored at -20℃ or used directly for transformation.

[0050] 4. Transform E. coli with the recombinant product;

[0051] (1) Take 50 μL of Trans-T1 competent cells and thaw them on ice.

[0052] (2) Add 2 μL of the recombinant product to the competent medium, flick to mix, and place on ice for 30 min.

[0053] (3) Heat shock in a 42°C water bath for 40 seconds, then quickly move to an ice bath and cool for 2 to 3 minutes (without shaking).

[0054] (4) Add 900 μL of antibiotic-free LB liquid culture medium to each centrifuge tube, mix well, and shake at 37°C for 1 h (180-200 rpm).

[0055] (5) Centrifuge at 6000 rpm for 1 min to collect the bacteria, discard part of the supernatant, and keep about 100 μL of the transformed competent cells in a Kan + LB plate. Spread evenly with a sterile glass rod, place upright to dry, and place upside down in a 37°C incubator for 12 to 16 hours (overnight). Observe the colonies.

[0056] 5. Colony PCR verification: Pick a single colony of E. coli in the clean bench for colony PCR ( Figure 2 and Figure 3 ), mark the serial number, and select the corresponding colony and add it to 10mL LB liquid culture medium (Kan + ). Place in a 37℃ shaker, 200rpm, for 12h. Take 6μL of PCR product and load it on agarose gel electrophoresis. The positive colonies detected are sent to Kumei Biotech for sequencing. Take the positive bacterial solution and mix it with 50% glycerol in a ratio of 1:1 and store it in a -80℃ refrigerator for later use.

[0057] The colony PCR system is as follows:

[0058] Table 6 Colony PCR system

[0059]

[0060] The reaction system is as follows:

[0061] Table 7 Reaction system

[0062]

[0063] The plasmid was extracted from the correctly sequenced E. coli to obtain the successfully connected plasmid pBWA(V)HS-SYP121, which was stored at -20°C.

[0064] 6. Transformation of Agrobacterium;

[0065] (1) Thaw Agrobacterium GV3101 at room temperature or in the palm of your hand for a while, and place it on ice for a while.

[0066] (2) Prepare in advance an ice box, liquid nitrogen, a water bath at 37°C, ultra-clean, sterile centrifuge tubes, and LB liquid culture medium (without resistance).

[0067] (3) In a clean bench, take 50 μL of Agrobacterium GV3101 competent cells, add 1–2 μL of successfully connected plasmid, and flick gently to mix.

[0068] (4) Place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0069] (5) Ultra-clean: Add 700 μL of antibiotic-free LB liquid culture medium, pipette to mix, and shake in a shaker at 28°C for 2 to 3 h.

[0070] (6) Centrifuge at 10,000 rpm for 1 min to collect bacteria and clean: take about 100 μL of the supernatant and apply it on Kan + 、Rif + Seal the plate, place it upright to dry, and then place it upside down in a 28°C incubator for 2 days. Observe the colonies.

[0071] (7) Pick a single Agrobacterium colony in the clean bench for colony PCR verification ( Figure 4 ), screen the positive clones, and place them in a shaking incubator at 28°C for future use.

[0072] Example 2, Agrobacterium-mediated genetic transformation of tomato;

[0073] 1. Seed disinfection: Soak the seeds in clean water for 2-3 hours in advance. In a sterile environment, wash with 75% alcohol for 30 seconds, shake with NaClO for 13 minutes, and rinse with sterile water 5-6 times.

[0074] 2. Sowing: sterilize the sterilized tomato seeds and sow them in 1 / 2 culture medium. Incubate in dark at 24℃ for 3-4 days. When the tomato seeds turn white, move the culture medium to 24℃, 16h light / 8h dark. 7-8 days after sowing, the two cotyledons of the seedlings are fully expanded and can be used for the experiment.

[0075] 3. Preparation and pre-culture of explants: After the cotyledons of the germinated tomato seedlings are fully expanded, the petioles and tips of the cotyledons are removed with a scalpel, and the middle part is cut into 2 to 3 sections and inoculated into the pre-culture medium, and pre-cultured at 24°C for 2 to 3 days.

[0076] 4. Agrobacterium infection and co-cultivation: Pick Agrobacterium and place it in the infection solution. Culture it with shaking at 28℃ until OD600≈0.8. Dilute the Agrobacterium resuspension until OD600=0.3-0.4, infect the leaves for 10-15min, inoculate the dried explants in the co-culture medium, and culture them in the dark at 24℃ for 2d.

[0077] 5. Screening and differentiation rooting: Inoculate the recovered callus into the screening medium and culture it at 24℃, 16h / 8h light / dark for 15-30 days; inoculate the selected callus into the differentiation medium and culture it at 24℃, 16h / 8h light / dark for 30-40 days. When the seedlings to be differentiated grow to about 2-3cm, cut them off from the callus and inoculate them into 1 / 2MS rooting medium and culture them at 24℃, 16h / 8h light / dark for 10-15 days.

[0078] 6. Transplantation: After 1 to 2 weeks of rooting culture, open the cover of the rooted tissue culture seedlings and harden them for 1 day, then transplant them to vermiculite for growth.

[0079] Example 3, identification and phenotypic observation of SlSYP121 transgenic plants;

[0080] 1. Obtaining transgenic plants: In order to further study the function of the SISYP121 gene, the Agrobacterium tumefaciens that had been transferred into pBWA(V)HS-SISYP121 was used to genetically transform wild-type tomatoes using the Agrobacterium-mediated leaf disc method. After the root system of the plant was fully developed, the transgenic strains obtained were hardened and cultured to obtain T0-generation transgenic tomato plants overexpressing SISYP121 ( Figure 5 ). The positive seedlings of T0 generation tissue culture seedlings were identified, and the positive plants of T0 generation were transferred from vermiculite to the substrate, and the seeds were harvested to obtain T1 generation. After further screening of positive seedlings, T2 generation seeds were sown, and T2 generation seeds were used as research objects for experiments. Wild-type MT seeds and T2 generation seeds of the same period were germinated in a dark environment for 3 days. After the seeds turned white, they were sown in plug trays and cultured at 24℃ and 16 / 8h. When the plants grew to "two leaves and one heart", the leaf growth and development phenotype was observed.

[0081] 2. Identification of transgenic positive seedlings: DNA was extracted from each generation of transgenic plants and PCR was performed, with pBWA(V)HS-SlSYP121 as the positive control and wild-type MT as the negative control. The plants with the same band as the positive control were positive plants. PCR amplification was performed, the PCR amplification primers were shown in Table 8, the PCR reaction system was the same as Table 7, and the pre-denaturation time was set to 3 min.

[0082] Table 8 PCR amplification primers

[0083]

[0084] In order to verify the success of transgenic plants, after obtaining T0 generation plants, the whole genome DNA of the plants was extracted and used as a template to perform PCR reaction using the universal upstream primer SlSYP121-Fd of the vector and the downstream primer SlSYP121-R of the gene. The PCR products were detected by agarose gel electrophoresis. In the electrophoresis results, M represents Marker, and lanes 1 to 14 ( Figure 6 ) and 1 to 20 ( Figure 7 ) are different overexpression plants, lanes 15 and 21 are positive controls, and lanes 16 and 22 are negative controls. According to the PCR identification results, it was found that the electrophoresis results of PCR for the 14 overexpression lines all showed bands, so they were judged to be positive, while the electrophoresis results of PCR for the wild-type lines did not show bands, so they were judged to be negative, indicating that all 14 overexpression lines were successfully transformed. In addition, we also performed PCR identification on the T1 generation plants, and the positive rate was about 85%, and some of them, such as Figure 7 shown.

[0085] 3. Expression and phenotype observation of wild-type MT and overexpressed SlSYP121 plants: In order to detect the expression of SlSYP121 in transgenic tomato materials and wild-type MT materials, real-time fluorescence quantitative PCR was used to detect gene expression. The extracted RNA samples were reverse transcribed without contamination. Fluorescence quantitative PCR was used for detection. Figure 8 It can be seen that the expression of SlSYP121 in the overexpressed plants (OE#1~OE#7) was significantly higher than that in the wild-type plant MT, and each line was overexpressed at least 4 times. Among them, the overexpression efficiency of SISYP121 gene in OE#1 and OE#7 plants of SISYP121 transgenic tomatoes was the highest, reaching about 40 to 60 times.

[0086] Analysis of tomato plant development is a process of staged changes. We selected the T2 generation transgenic material "two leaves and one heart" period and counted the leaf shape index, notch degree, and notch number of the first pair of true leaves. The standard notch depth was set to be no less than 2mm as one notch. In order to further analyze and represent the change in notch degree, the present invention uses the ratio of notch degree (d) to leaflet longitudinal length (L) to represent the leaf notch degree ( Fig. 9 ), the ratio of the number of notches to the longitudinal length of the leaflets represents the number of notches. It was found that the leaf shape index of the leaves of the overexpressing plants did not show significant differences compared with the background material MT ( Fig.11 ), but the number of leaf notches decreased and the degree of leaf notches weakened ( Fig.10 ). The ratio of notch degree to leaflet longitudinal length was 0.05-0.2 for overexpression plant materials and 0.3 for wild-type MT, with significant difference ( Fig.12 ); the ratio of notch number to leaflet longitudinal length, the value of overexpression plant material is 0.2-0.4, and the wild type MT is about 0.45, the difference is quite significant ( Fig.13 ).

[0087] The internode length of plants is an important agronomic trait that affects plant height, planting density, uprightness, resistance, light energy utilization, fruit setting, yield and tolerance to abiotic stress. Glandular hairs are also specialized structures of plant epidermal cells that have the functions of regulating leaf temperature and blocking pathogen attacks. Therefore, we also observed and measured the epicotyls of tomatoes and the sticky glandular hairs on leaves. We found that after the SlSYP121 gene was overexpressed in plants, the epicotyl length of tomatoes at the seedling stage was reduced compared with the wild-type MT ( Figures 14 to 16 ), the length of the epidermal glandular hairs of the leaves is also relatively reduced ( Fig.17 ), indicating that SlSYP121 may be involved in the development of tomato internode length and leaf glandular hairs, laying a foundation for further exploring the tolerance of SlSYP121 to abiotic stresses such as drought resistance.

[0088] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Application of SlSYP121 gene and the protein encoded by it in tomato leaf shape development, characterized in that: The nucleotide sequence of the SlSYP121 gene is shown in SEQ ID No.1, and the amino acid sequence of the SYP121 protein encoded by the SlSYP121 gene is shown in SEQ ID No.2.

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