Application of ZaSPL7 gene in regulation and control of hairy body and parthenocarpy of plant

Abstract

The invention discloses application of a ZaSPL7 gene in regulation and control of hairy bodies and parthenocarpy of plants, and belongs to the technical field of gene engineering. The gene ZaSPL7 for regulating and controlling organ development of the green prickleyash is separated from cDNA of the green prickleyash and is overexpressed in a plant, the number and density of epidermal hair of the transgenic plant are obviously increased, meanwhile, the number of fruit seeds is obviously reduced, even part of plants have seedless fruit phenotypes, and the unisexual setting rate is increased. The results show that the green prickleyash SPL7 gene may coordinate epidermal hair and fruit development by integrating development stage signals, and has important application value in regulation and control of epidermal appendages (such as thorns and hair) formation and apomixis, and the mechanism provides a new idea for simultaneous improvement of plant stress resistance (by reducing the thorns) and fruit commodity (by seedless). Important theoretical basis and technical support are provided for breeding of the green prickleyash, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and specifically relates to ZaSPL7 The application of genes in regulating plant trichomes and parthenocarpy. Background Art

[0002] Sichuan peppercorn( Zanthoxylum spp. ) is an important economic crop with medicinal and edible properties in my country, among which Zanthoxylum bungeanum ( Z. armatum Zanthoxylum bungeanum (Zanthoxylum bungeanum) holds a prominent position in the condiment and traditional Chinese medicine market due to its unique numbing flavor and aroma. However, the dense, hard thorns covering the green Zanthoxylum bungeanum plant make seedling cultivation, management, and harvesting expensive. In recent years, under the dual pressures of fluctuating market prices and rising labor costs, pepper farmers have been forced to cut back on their orchards to mitigate losses, posing a serious challenge to the sustainable development of the industry. Therefore, understanding the mechanism of thorn formation and cultivating thornless varieties have become key needs to overcome industry bottlenecks.

[0003] The thorns of green pepper belong to skin thorns, which are formed by the specialized cells of epidermis or cortex and have no direct connection with the vascular system. At present, the research on plant skin thorns is still in its infancy, and only the LOG 、 WOX3 、 NAC93 、 MYB86 Epidermal hairs are the protruding structures of plant epidermal cells, some of which are specialized into defensive prickles. They are also epidermal appendages. Studies have shown that epidermal hair development is affected by SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) gene family regulation, for example, in Arabidopsis, SPL9 miR156 inhibits epidermal hair formation by activating the negative regulators TCL1 and TRY, while miR156 inhibits epidermal hair formation by targeting SPL Genes regulate their expression levels, thereby affecting the temporal and spatial distribution of epidermal hairs. Similar mechanisms have also been verified in the development of cotton fibers (specialized epidermal hairs), indicating that SPL However, existing studies have mostly focused on SPL3 、 SPL9 etc. members, SPL7 Their functions in the formation of epidermal appendages such as epidermal hairs or prickles have not yet been clearly elucidated, especially in woody economic crops, where there is a lack of systematic research. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide ZaSPL7 The application of genes in regulating plant trichomes and parthenocarpy can solve the technical problems of low efficiency and long cycle of traditional breeding methods.

[0005] To achieve the above object, the first aspect of the present invention is to provide a gene ZaSPL7 Application of genes in regulating plant trichomes and parthenocarpy ZaSPL7 The nucleotide sequence of the gene is shown in SEQ ID NO: 1. ZaSPL7 The amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows: Further, the application is to overexpress ZaSPL7 Genes, which realize the regulation of plant trichomes and parthenocarpy.

[0007] Furthermore, overexpression ZaSPL7 After the gene was introduced, the number and density of plant epidermal hairs increased, the number of fruit seeds decreased, and the parthenocarpy rate increased.

[0008] Furthermore, overexpression ZaSPL7 After the gene was introduced, the number and density of hairs on the leaves, stems, flowers and fruits of plants increased, and the number of fruit seeds decreased or even disappeared.

[0009] Furthermore, the plant is a dicotyledonous plant.

[0010] Furthermore, the plant is a tomato.

[0011] The second aspect of the present invention is to provide a method comprising the above-mentioned gene ZaSPL7 plasmid.

[0012] The third aspect of the present invention is to provide a method comprising the above-mentioned gene ZaSPL7 overexpression vector.

[0013] The fourth aspect of the present invention is to provide a method comprising the above-mentioned gene ZaSPL7 of engineered bacteria.

[0014] The fifth aspect of the present invention is to provide a preparation for regulating plant trichomes and parthenocarpy, the preparation comprising one or more of the following: a. The first aspect of the present invention disclosed ZaSPL7 Gene; b. the plasmid disclosed in the second aspect of the present invention; c. the overexpression vector disclosed in the third aspect of the present invention; and d. The engineered bacteria disclosed in the fourth aspect of the present invention.

[0015] The beneficial effects of the present invention are: 1. The present invention isolates genes that regulate the organ development of Zanthoxylum bungeanum from Zanthoxylum bungeanum cDNA ZaSPL7 , which is encoded SPL7 Transcription factors are specifically and highly expressed in the developing tissues of the prickles. ZaSPL7The gene overexpression vector was introduced into wild-type tomato plants for genetic transformation and it was found ZaSPL7 The number and density of epidermal hairs of transgenic plants with overexpressed genes increased significantly, specifically in leaves, stems, flowers and fruits. At the same time, the number of fruit seeds decreased significantly, and some plants even had seedless fruit phenotypes, and the parthenocarpy rate increased. This shows that green pepper SPL7 The gene may coordinate the development of epidermal hairs and fruits by integrating developmental stage signals, and has important application value in regulating the formation of epidermal appendages (such as thorns and hairs) and apomixis. This mechanism provides a new idea for simultaneously improving plant stress resistance (by reducing thorns) and fruit commerciality (by making it seedless), providing an important theoretical basis and technical support for green pepper breeding, and has broad application prospects.

[0016] 2. Compared with the reported SPL9 Different genes, the present invention found ZaSPL7 Overexpression of the gene did not inhibit epidermal hairs but promoted their proliferation, revealing SPL This discovery fills the gap between the functional diversity of family members and ZaSPL7 Genetic gaps in epidermal appendage regulation through targeted regulation ZaSPL7 The gene can accurately improve plant morphology and fruit characteristics, providing a direct molecular target for the breeding of thornless green pepper varieties and the development of parthenocarpy technology, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 for ZaSPL7 Phylogenetic tree of genes; Figure 2 Multiple sequence alignment of ZaSPL7 protein; Figure 3 For the overexpression vector K303- ZaSPL7 Constructed electropherogram; Figure 4 For WT and 4 ZaSPL7 Comparison of the overall phenotypes of transgenic plants; Figure 5 for ZaSPL7 relative expression levels of genes in transgenic plants; Figure 6 WT and ZaSPL7 Local comparison of epidermal hairs at different parts of transgenic plants; Figure (A) is a local comparison of epidermal hairs on leaves, Figure (B) is a local comparison of epidermal hairs on stem segments, Figure (C) is a local comparison of epidermal hairs on flower buds, Figure (D) is a local comparison of epidermal hairs on fruit peels, Figure (E) is a comparison of epidermal hairs on leaves under an electron microscope, and Figure (F) is a comparison of epidermal hairs on stem segments under an electron microscope. Figure 7 WT and ZaSPL7 Statistics of the number of hairs on the leaf epidermis of transgenic plants; Figure 8 WT and ZaSPL7 Statistics of the number of short hairs on the leaf epidermis of transgenic plants; Figure 9 WT and ZaSPL7 Statistics of the length of short hairs on the leaf epidermis of transgenic plants; Figure 10 WT and ZaSPL7 Statistics of the number of hairs on the stem epidermis of transgenic plants; Figure 11 WT and ZaSPL7 Statistics of the number of short hairs on the stem epidermis of transgenic plants; Figure 12 WT and ZaSPL7 Statistics of the length of short hairs on the stem epidermis of transgenic plants; Figure 13 for ZaSPL7 Transgenic plant epidermal hairs GL2 Gene expression analysis graph; Figure 14 for ZaSPL7 Transgenic plant epidermal hairs MYC1 Gene expression analysis graph; Figure 15 for ZaSPL7 Transgenic plant epidermal hairs MYC2 Gene expression analysis graph; Figure 16 for ZaSPL7 Phenotype of seedless fruit in transgenic plants; Figure 17 for ZaSPL7 Statistical results of the number of fruit seeds of transgenic plants. DETAILED DESCRIPTION

[0018] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0019] ZaSPL7 The nucleotide sequence of the gene is as follows:

[0020] ZaSPL7 The amino acid sequence of the protein encoded by the gene is as follows: MENGGYMFVREQSGNNNNGNSNSNNLAWENTWALNNHHSRFDLGSSSSFLYRNTTAETTNATLTEGNAAHALLFPHPTHRSLYAGVGSNLHPDPHLMCLKLGKRHYFEDSTAAPMGQRHVDGAGGYSMVKKGKPYYTYSGCGDGVGLSSSSSSAAAPVLVTPTATVPRCQVDGCHVALINSKDYHRRHKVCEMHSKAPKVMVLG LEQRFCQQCSRFHVVSEFDDTKRSCRRRLAGHNERRRKSSHDSLSRNSSQVNKLVSGRLRSPATGRALSLLSSSSSSSKAGSWVSSSDLSSRCSAALRELIAENRAAILARHLILDGRDWNNNANHEDHPQTGSDSMTSNHHYLYQQHQIMLTEQQQQQQQNNWERYQETNTHVTLDLMQAPSEEEECSELWTSLDDDHVV (SEQ ID NO: 2).

[0021] The primer sequences used in the following experiments are shown in Table 1.

[0022] Table 1 Primer sequence list

[0023] Example 1 Construction of overexpression vector 1. Using amplification primers ZaSPL7 -F (SEQ ID NO: 3) and ZaSPL7 -R (SEQ ID NO: 4) was amplified from the cDNA of Zanthoxylum bungeanum leaves. ZaSPL7 The full-length sequence of the gene was sequenced and its ORF was found to be 1212 bp (SEQ ID NO: 1) containing 403 amino acids (SEQ ID NO: 2).

[0024] The phylogenetic tree was then constructed and the results showed that ZaSPL7 With other species ZaSPL7 High homology ( Figure 1 ).Will ZaSPL7 Comparing the domain sequence with other homologous genes, we can find that ZaSPL7 The domains show a certain degree of consistency with the domain sequences of other homologous genes. ZaSPL7Within the domain, multiple sequences showed high consistency, indicating that this is a functionally important region ( Figure 2 ).

[0025] 2. Use Takara In Fusion kit to ZaSPL7 The gene was connected to the intermediate vector 8GWN and then sequenced. The correctly sequenced 8GWN- ZaSPL7 The plasmid was recombined with the plant overexpression vector CaMV35S-K303 to successfully construct the overexpression vector K303- ZaSPL7 The specific steps are: (1) Using the TAKARA In-Fusion® HD Cloning Kit, the amplification primers ZaSPL7 -F and ZaSPL7 The fragment amplified by -R was connected with the intermediate vector 8GWN vector after double digestion with NotⅠ and Sbfi, and then transformed into DH5α. The strain was spread on a plate containing 100μg / mL spectinomycin for screening, and the monoclonal strain was selected for colony PCR. The strain that amplified the target fragment was sent for sequencing. If the sequencing was correct, it could be identified as containing 8GWN- ZaSPL7 Plasmid-positive strains; (2) Shake the positive strain to extract the plasmid, use this plasmid as a template and use amplification primers ZaSPL7 -F and ZaSPL7 -R amplification was performed, and the plasmid with the correct amplified band was homologously recombined with the plant expression vector CaMV35S-K303 using the Gateway LR Clonase II kit. The product was transformed into DH5α and screened on a plate containing 100 μg / mL kanamycin. Monoclonal strains were selected and amplified using primers K303-F1 (SEQ ID NO. 5) and ZaSPL7 -R (SEQ ID NO. 4) amplification, and then colony PCR; when the amplified target band fragment size is consistent with the expected and is sequenced and verified, it can be identified as a positive strain, that is, the overexpression vector K303- ZaSPL7 ( Figure 3 ); 3. ZaSPL7 The gene overexpression vector was transformed into a microbial transformant (Agrobacterium tumefaciens GV3101) to prepare an engineered bacterium; specifically, the positive strain obtained (overexpression vector K303- ZaSPL7 ) shake the bacteria, extract the plasmid, which is the K303- SPL7The plasmid was transformed into Agrobacterium tumefaciens GV3101 and screened on a plate containing 100 μg / mL kanamycin and 50 μg / mL rifampicin. Monoclonal strains were selected for colony PCR verification. The strains that passed the verification were the ones containing the overexpression vector K303- ZaSPL7 of engineered bacteria.

[0026] Example 2 Obtaining transgenic plants 1. Insert the overexpression vector K303- ZaSPL7 Agrobacterium was used to transform tomato explants using the leaf disc method. The specific steps are as follows: (1) Pre-culture: Take a sterile cultured wild-type tomato plant and cut the young leaves into 0.5 cm 2 Explants were obtained by pre-culturing in MB medium containing 1.5 mg / L 2,4-dichlorophenylacetic acid (2,4-D) and 1 mg / L kinetin (KT) in the dark for 1-2 days.

[0027] (2) Agrobacterium inoculation: Inoculate the overexpression vector K303- ZaSPL7 Agrobacterium GV3101 was cultured overnight in liquid LB medium at 28°C. The cells were then harvested by centrifugation at 6000 rpm for 5 minutes and suspended in liquid MB medium containing 1.5 mg / L 2,4-D, 1 mg / L KT, and 100 mmol / L acetosyringone (AS) to prepare the infection solution. Explants were then immersed in the infection solution for 8 minutes with continuous shaking. The explants were removed and placed on sterile filter paper to absorb excess liquid. Finally, the explants were incubated in co-cultivation medium (MB + 1.5 mg / L 2,4-D + 1 mg / L KT + 100 mmol / L AS) in the dark for 2 days.

[0028] (3) Bud induction and kanamycin resistance selection: After 2 days of dark culture, the explants were transferred to a selection medium (MB + 2 mg / L ZR (zeatin riboside) + 100 μg / mL kanamycin + 400 μg / mL cephalexin). The buds were induced at 25°C, 12 h photoperiod and 2000 Lux for 40-60 days. The culture medium was changed every 3-4 weeks to obtain kanamycin-resistant shoots.

[0029] (4) Root induction: The kanamycin-resistant shoots were transferred to 1 / 2MB medium supplemented with 0.1 mg / L indole 3-acetic acid (IAA), 100 mg / L activated carbon (AC) and 15 mg / L kanamycin (Kan) for root induction; after rooting, they were transplanted into soil to obtain 4 ZaSPL7 Tomato plants with gene overexpression ZaSPL7 -OX1, ZaSPL7 -OX2, ZaSPL7 -OX3 and ZaSPL7-OX4. Figure 4 As shown in the figure, it can be seen that the leaf thickness of the transgenic plants increased significantly, the density of the epidermal hairs of various organs changed significantly, and the number of epidermal hairs on leaves, stems, flower buds and fruit peels increased significantly.

[0030] 2. Identification of transgenic positive plants The BGMG Fast Total RNA / DNA Extraction Kit (BG0060) from Baoguang Biotechnology Co., Ltd. was used to extract the RNA from the young leaves of the plants to be tested (WT1, WT2, ZaSPL7 -OX1, ZaSPL7 -OX2, ZaSPL7 -OX3 and ZaSPL7 -OX4) genome, DNA / RNA was extracted and purified, followed by PCR identification of transgenic positive strains. PCR reactions were performed using Biolight Biotechnology's 2x Super PCR Mix (BG005M). The PCR amplification system is shown in Table 2. WT1 and WT2 represent two wild-type experimental groups grown under the same growth conditions.

[0031] Table 2 PCR amplification system

[0032] The amplification conditions were as follows: (1) amplification at 98°C for 2 min; (2) amplification at 98°C for 10 s, amplification at 55-60°C for 20 s, and amplification at 72°C for 2 min; 25-35 cycles were performed; (3) amplification at 72°C for 5 min.

[0033] Finally, overexpression identification was performed and the target gene was analyzed using qRT-PCR ZaSPL7 Actin was used as the internal reference gene, and three technical replicates were performed for each gene. The primer sequences used were qPCR-SPL7-F (SEQ ID NO. 6) and qPCR-SPL7-R (SEQ ID NO. 7).

[0034] Using a nucleic acid solution, a reverse transcription kit was used to remove the genome and reverse transcribed cDNA. The cDNA was synthesized using a high-efficiency reverse transcription kit (BG0070), and the qRT-PCR reaction was performed using a quantitative reagent (BG0014). After obtaining qRT-PCR data using a Bio-rad 96-well real-time quantitative PCR instrument, the data was initially processed using Bio-Rad CFX Manager. The relative expression values ​​were obtained using the 2-ΔΔct method, and the significance was calculated using a t-test. Finally, GraphPadPrism 7 was used to generate the graph ( Figure 5 ).

[0035] Example 3 Overexpression ZaSPL7 Phenotypic identification of transgenic plants 1. Analysis of epidermal hair density and morphology Wild-type (WT) and transgenic tomato plants ( ZaSPL7 -OX2) were cut into small pieces of about 0.5 cm², fixed with 2.5% glutaraldehyde for 4 hours, and then rinsed with phosphate buffered saline (PBS) three times for 10 minutes each; then fixed with 1% osmium acid for 1 hour, and rinsed with PBS three times; finally, dehydrated with 30%, 50%, 70%, 90% and 100% ethanol in sequence for 10 minutes each. The samples were placed in a critical point dryer and dried, and then gold-sprayed and observed and photographed using a scanning electron microscope. The length, branching and morphological characteristics of the epidermal hairs were analyzed by scanning electron microscopy images to evaluate the overexpression. ZaSPL7 Genetic influences on epidermal hair development.

[0036] The results are as follows Figure 6 As shown, the leaf thickness of transgenic plants was significantly increased compared with wild-type plants ( Figure 6 A), and the density of epidermal hairs of each organ changed significantly, with the number of epidermal hairs on leaves, stems, flower buds and fruit peels increasing significantly ( Figure 6 B- Figure 6 D). At the same time, the epidermal hair morphology of the transgenic plants also showed certain changes, such as a significant increase in the length of the trichomes ( Figure 6 E and Figure 6 F), which indicates ZaSPL7 Overexpression of the gene had a significant impact on the development and differentiation of epidermal hairs.

[0037] Then, the number of epidermal hairs per unit area (4mm² / 1mm²) at different locations was counted to compare the differences in epidermal hair density between transgenic plants and wild-type plants. Figure 7-12 As shown, it was found that the number of epidermal hairs (long hairs) on the leaf veins of transgenic plants increased by 25-30 times compared with the wild type, the number of long hairs in the stem segments increased by 5-6 times compared with the wild type, and the number of short hairs decreased.

[0038] Further analysis revealed that ( Figure 13-15 ), ZaSPL7 Gene overexpression plants ( ZaSPL7 -OX1 and ZaSPL7 -OX2) epidermal hair development-related genes such as GL2 、 MYC1 and MYC2 The expression of ZaSPL7 Genes may regulate epidermal hair development by regulating these genes.

[0039] 2. Fruit seedless trait detection Wild-type (WT) and transgenic tomato plants ( ZaSPL7 -OX2) fruits were cut open along their cross-sections to observe the distribution of seeds within the fruit. The number of seeds in each fruit was counted and compared between the transgenic and wild-type plants. The seeds were then removed from the fruit to observe their developmental status, assessing whether they were completely aborted or partially developed, and analyzing the effect of the SPL7 gene on seed formation.

[0040] The results are as follows Figure 16 As shown in the figure, the transgenic plants showed seedless fruit. The anatomical observation results showed that the number of seeds in the transgenic plants was significantly reduced, and some fruits were completely seedless. Figure 17 ), and found that the number of fruit seeds in the transgenic plants was zero, which indicates that ZaSPL7 Overexpression of the gene led to seed abortion and significantly increased the parthenocarpy rate of fruits.

[0041] The above results further confirmed ZaSPL7 The key role of genes in regulating trichomes and seedless fruit traits. The present invention overexpresses ZaSPL7 The gene not only significantly increases the number of epidermal hairs on the plant, but also makes the fruit seedless, providing important theoretical basis and technical support for the cultivation of thornless green pepper varieties and the development of parthenocarpy technology.

Claims

1. A gene ZaSPL7 The application of the method in regulating plant trichomes and parthenocarpy is characterized in that: The gene ZaSPL7 The nucleotide sequence of the gene is shown in SEQ ID NO:

1. ZaSPL7 The amino acid sequence of the encoded protein is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The application is through overexpression ZaSPL7 Genes, which realize the regulation of plant trichomes and parthenocarpy.

3. The use according to claim 2, characterized in that Overexpression ZaSPL7 After the gene was introduced, the number and density of plant epidermal hairs increased, the number of fruit seeds decreased, and the parthenocarpy rate increased.

4. The use according to any one of claims 1 to 3, characterized in that The plant is a dicotyledonous plant.

5. A method comprising the gene according to claim 1 ZaSPL7 plasmid.

6. A method comprising the gene according to claim 1 ZaSPL7 overexpression vector.

7. A method comprising the gene according to claim 1 ZaSPL7 of engineered bacteria.

8. A preparation for regulating plant trichomes and parthenocarpy, characterized in that: The preparation contains one or more of the following: a. The gene according to claim 1 ZaSPL7 ; b. The plasmid according to claim 5; c. The overexpression vector according to claim 6; and d. The engineered bacteria according to claim 7.

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