Plant glandular hair head gene GHF1, its expression product, expression vector and application

Abstract

The present invention discloses a plant glandular hair head gene GHF1 The present invention identifies a gene that can negatively regulate the development of plant glandular hair heads. GHF1 , constructed its overexpression vector and gene editing vector, and carried out tobacco transformation respectively to obtain the corresponding overexpression strains and gene knockout plants; the epidermal hair morphology identification and leaf chemical composition analysis showed that GHF1 The gene can negatively regulate the occurrence and development of the glandular hair head of plants, and has important application value in the regulation of glandular hair development and the synthesis of glandular hair-specific substances.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, specifically to the gene encoding the plant glandular hair head. GHF1 , its expression products, expression vectors and applications. Background Art

[0002] The structure and function of Arabidopsis thaliana's epidermal hairs are relatively simple. They are single-celled, glandless structures without secretory capabilities, and the molecular network underlying their morphology has been elucidated. Unlike Arabidopsis thaliana, most terrestrial plants, such as the Solanaceae, Leguminosae, Asteraceae, Cannabaceae, and Lamiaceae, possess a more complex and diverse epidermal hair structure. These multicellular hairs contain both glandular hairs (guard hairs) and glandular hairs with secretory functions, and their morphology is complex. While the morphology of glandular hairs varies across plant families, their basic structure is similar, consisting of a base, a stalk, and a glandular head, with only the glandular head having both synthetic and secretory functions.

[0003] As a physical barrier between plants and the environment, epidermal hairs reduce water and heat loss, buffer direct sunlight, and protect against pathogens and insects. Glandular hairs, as secretory organs, promote the excretion of heavy metals and reduce their accumulation within the plant. Furthermore, glandular hairs, known as "plant chemical factories," can specifically synthesize and release a large number of secondary metabolites. These metabolites not only play a vital role in plant adaptation to environmental stress but also have significant commercial value as pharmaceutical precursors, flavors and fragrances, food additives, and insecticides. For example, artemisinin, a sesquiterpene lactone specifically synthesized in the glandular hairs of Artemisia annua, is currently the most effective treatment for malaria. Menthol monoterpenes in mint glandular hairs have anti-inflammatory, analgesic, and bactericidal properties. Pyrethroids, specifically synthesized in pyrethrum glandular hairs, are commonly used broad-spectrum insecticides. Siberian diterpenes specifically synthesized in tobacco glandular hairs have antifungal and anti-aphid properties. Phenylpropanoids are the primary components of glandular hairs in the Lamiaceae family and are involved in defense responses and insect pollination. In addition, the flavonoids, fatty acid derivatives and acyl glycosides synthesized by the glandular head have the function of protecting against ultraviolet radiation and preventing microbial invasion.

[0004] Tobacco, a model plant for molecular biology and genetic engineering research, has both guard hairs and glandular hairs. Glandular hairs account for about 85% of the total epidermal hairs, and the secretion of glandular hairs accounts for about 60% of the chemical composition of the entire leaf surface, making it an ideal plant for studying the molecular mechanism of glandular hair formation. The epidermal hairs of common tobacco include three types: (1) long-stalked glandular hairs, which are composed of a 4-6 cell glandular stalk and a 1-6 cell glandular head. The glandular head is rich in chloroplasts and specifically synthesizes cypermethrin diterpenes, lysine diterpenes, and sucrose esters; (2) short-stalked glandular hairs, which are composed of a stalk cell and an 8-16 cell glandular head. The glandular head does not contain chloroplasts and specifically synthesizes leaf resistance proteins; (3) guard hairs, which have no glandular cells and no synthesis or secretion capabilities.

[0005] Currently, there are increasing reports on genes regulating glandular trichome development, but no reports on genes regulating the development of the glandular trichome head. Therefore, the molecular regulatory network of glandular trichome development is still unclear. Therefore, identifying genes regulating the development of plant glandular trichome heads and studying the molecular regulatory network of plant glandular trichome development are of great significance for studying the developmental regulation of glandular trichome tissue and the formation of glandular trichome-specific synthetic substances.

[0006] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of the present invention is to provide a gene that can regulate the occurrence of plant glandular hair heads GHF1 , and use its expression vector to transform plants, in order to solve the current technical problems of insufficient research on the genes that cause glandular hair head formation and unclear molecular regulatory networks of glandular hair formation, laying the foundation for the study of the molecular mechanism of glandular hair formation.

[0008] According to the first aspect of the present disclosure, a plant glandular hair head gene is provided. GHF1 , whose nucleotide sequence is shown in SEQ ID NO.1, GHF1 The CDS of the gene is 729 bp in length, encoding 242 amino acids and a stop codon, and has a typical TIFY conserved domain.

[0009] According to the second aspect of the present disclosure, there is provided a plant glandular hair head gene GHF1 The encoded protein has an amino acid sequence as shown in SEQ ID NO.2.

[0010] According to the third aspect of the present disclosure, a method is provided which contains the plant glandular hair head gene GHF1 or an expression vector containing a fragment thereof.

[0011] According to the fourth aspect of the present disclosure, the plant glandular hair head gene GHF1 , the encoding protein or the expression vector is used to regulate the occurrence of plant glandular hair heads and / or the content of glandular hair secretions.

[0012] In some embodiments of the present disclosure, the application includes the following steps:

[0013] (1) Cloning the plant glandular hair head gene GHF1 , and after inserting the strong promoter 35S upstream, an overexpression vector was obtained;

[0014] (2) The overexpression vector is transformed into a recipient plant and cultivated to obtain a transgenic plant in which the heads of the long-stalked glandular hairs have disappeared.

[0015] In some embodiments of the present disclosure, the application includes the following steps:

[0016] (1) Design based on conserved sequences sgRNA Primers, construct the plant glandular hair head generation gene GHF1 Gene editing vectors;

[0017] (2) The gene editing vector is transformed into a recipient plant and cultivated to obtain a transgenic plant with enlarged heads of long-stalked glandular hairs.

[0018] In some embodiments of the present disclosure, the recipient plant comprises a plant of the Solanaceae family.

[0019] According to the fifth aspect of the present disclosure, the plant glandular hair head gene GHF1 , the encoded protein or the expression vector is used to regulate the type of plant epidermal hair.

[0020] According to the sixth aspect of the present disclosure, the plant glandular hair head gene GHF1 Or the expression vector is used in the construction or breeding of transgenic plants.

[0021] One or more technical solutions provided in the embodiments of this application have at least any of the following technical effects or advantages:

[0022] Screening identified GHF1 The gene encodes 242 amino acids and a stop codon, has a typical TIFY conserved domain, and is expressed at the highest level in the epidermal hairs of leaves and stems. Based on the identification and detection of the phenotypic and physiological characteristics of plants overexpressing and knocking out the gene, the results showed that compared with the control plants, GHF1 The long-stalked glandular hairs of the overexpressing strains were almost gone, and the main types of epidermal hairs were short-stalked glandular hairs and guard hairs. GHF1 The heads of the long-stalked glandular hairs of the knockout plants became larger, and the main types of epidermal hairs were short-stalked glandular hairs and long-stalked glandular hairs; and GHF1 The secretion content of glandular hairs in knockout plants was significantly increased. GHF1 The overexpression strains showed significantly reduced secretion levels of glandular trichomes. This gene negatively regulates the development of long-stalked glandular trichome heads, thus having important implications for the regulation of glandular trichome tissue development and the formation of trichome-specific synthetic substances. This provides a foundation for further research into the molecular mechanisms of glandular trichome development in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the prediction result of the conserved domain of tobacco GHF1 protein in one embodiment of the present application.

[0024] Figure 2 In one embodiment of this application, tobacco GHF1 Analysis of gene tissue expression characteristics.

[0025] Figure 3 In one embodiment of this application, tobacco GHF1 Detection diagram of gene overexpression strains; A: DNA-PCR gel electrophoresis diagram; B: GHF1 Expression analysis of overexpression lines.

[0026] Figure 4 In one embodiment of this application, tobacco GHF1 Analysis diagram of gene mutant sequencing; K326: control strain; K326-M: GHF1 Gene mutant plants.

[0027] Figure 5 These are pictures of epidermal hair phenotype observation in one embodiment of the present application; wherein, A: epidermal hair morphology observation results; B: epidermal hair density statistical analysis results; C: long-stalked glandular hair head diameter statistical analysis results.

[0028] Figure 6 This is an analysis diagram of leaf secretions in one embodiment of the present application; wherein, A: epidermal hair glycolipid staining results; B: GC / MS analysis results of leaf chemical components. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are described below with reference to the accompanying drawings and examples. However, the following examples are only used to illustrate the present invention in detail and are not intended to limit the scope of the present invention in any way.

[0030] Unless otherwise specified, the plasmid vectors involved in the following examples are all conventional commercial vectors; the reagents involved are all commercially available conventional reagents unless otherwise specified; the experimental methods involved are all conventional methods unless otherwise specified.

[0031] Example 1: Genes Predominantly Expressed in Glandular Hairs GHF1 Cloning and tissue expression analysis

[0032] This example is based on the common tobacco ( Nicotiana tobacum ) glandular hair cDNA library (Cui et al. BMC Plant Biology 2011, 11:76), a dominant gene of tobacco glandular hair was screened out and named GHF1 .

[0033] 1. Tobacco genes GHF1 clone

[0034] (1) Take 2 g of tobacco leaves and grind them thoroughly in liquid nitrogen. Follow the steps of the KK Ultrafast Plant Total RNA Extraction Kit (Beijing Zhuangmeng Biotechnology) to obtain RNA solution. Follow the steps of the reverse transcription kit (HiScript II Q RTSuperMix for qPCR) to reverse transcribe the RNA into cDNA.

[0035] (2) Gene cloning: Design amplification primers and perform PCR amplification using cDNA as a template.

[0036] PCR system (10 μL): 5 μL Mix; 0.25 μL each of F / R; 0.5 μL cDNA; 4 μL ddH2O.

[0037] PCR amplification program: 95°C, 5 min; 95°C, 50 s; 58°C, 50 s; 72°C, 1 min; 35 cycles; 72°C, 10 min.

[0038] The amplification primers are (SEQ ID NO.3~4):

[0039] F: 5'-ATGTCAAGTTTGCAACTTTCTTC-3';

[0040] R: 5'-CTATAACGTGAAGTTGAGATCA-3'.

[0041] (3) After PCR amplification, gel electrophoresis was performed, and the results showed that there was a clear band at approximately 700 bp.

[0042] (4) Cut out the electrophoresis band in the gel and recover the PCR fragment using a DNA recovery kit.

[0043] (5) The recovered PCR fragments were ligated with the pMD19-T vector (Takara).

[0044] (6) The ligation product was transformed into Escherichia coli DH™ 5α competent cells by heat shock method, positive clones were screened using ampicillin-resistant LB plates, and plasmids were extracted for DNA sequencing.

[0045] 2. Prediction of sequence conserved domains

[0046] The amino acid sequence of tobacco GHF1 obtained by sequencing analysis was submitted to NCBI (https: / / www.ncbi.nlm.nih.gov / ) for tblastn analysis to predict the conserved domain of GHF1. Figure 1 As shown, the cloned tobacco GHF1The gene has a conserved TIFY domain, suggesting that it belongs to the plant-specific TIFY family.

[0047] 3. Tissue Expression Characterization Analysis

[0048] The roots, stems and leaves of the five-leaf tobacco variety K326 were collected, and the epidermal hairs of the leaves and stems were collected by liquid nitrogen freeze-brush method. Total RNA was extracted from the samples and reverse transcribed to synthesize cDNA. L2 5 as an internal reference gene, and semi-quantitative and qRT-PCR methods were used to analyze tobacco GHF1 Tissue expression characteristics of genes.

[0049] The amplification primers are (SEQ ID NO.5~8):

[0050] GHF1 -F: 5'-GGAAGAACAATCAACCAAGACAATG-3';

[0051] GHF1 -R: 5'-GGTATGGCGCTCTGGCCGTGTCTC-3';

[0052] L2 5-F: 5'-CCCCTCACCACAGAGTCTGC-3';

[0053] L2 5-R: 5'-TTCTAACTCCTGTTGTTGTGGGAA-3'.

[0054] PCR system for semi-quantitative gene analysis (10 μL): 5 μL Mix; 0.25 μL each of F / R; 0.5 μL cDNA; 4 μL ddH2O.

[0055] The PCR reaction program for semi-quantitative analysis of genes was as follows: 95°C for 5 min; 28 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and 72°C for 10 min.

[0056] qRT-PCR reaction system (10 μL): 2×LightCycler®480 SYBR GreenⅠMaster, 5 μL; 10 μM Forward primer, 0.2 μL; 10 μM Reverse primer, 0.2 μL; cDNA, 1 μL; Nuclease-free H2O, 3.6 μL.

[0057] qRT-PCR reaction program: 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, 40 cycles.

[0058] The results are as follows Figure 2 As shown: GHF1 The gene was expressed at the highest level in the epidermal hairs of leaves and stems, but not in roots. GHF1 The gene is predominantly expressed in epidermal hairs.

[0059] Example 2 GHF1 Obtaining gene overexpression lines

[0060] 1. Construction of overexpression vector

[0061] tobacco GHF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the expression vector used is pCAMBIA-NPT vector:

[0062] (1) Selection Spe I and Nru The two restriction enzyme sites are ligase sites and are designed as follows GHF1 PCR amplification was performed using cloning primers with restriction enzyme cutting sites (see SEQ ID NO.9-10):

[0063] F: 5'-AGGACTAGTATGTCAAGTTTGCAACTTTCTTC-3',

[0064] R: 5'- GAGATCGCGACTATAACGTGAAGTTGAGATCA-3';

[0065] The restriction enzyme cutting sites marked in bold are Spe I and Nru I.

[0066] The PCR reaction system was as follows: 2 μL genomic DNA (100 ng / μL); 1 μL Primer Star DNA polymerase; 2 μL primer 1 (10 μM); 2 μL primer 2 (10 μM); 10 μL 5× PCR reaction buffer; 4 μL dNTPs (2.5 mM); 29 μL water; the total volume was 50 μL.

[0067] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; 35 cycles of 95°C for 50 s, 58°C for 50 s, and 72°C for 1 min; and extension at 72°C for 10 min.

[0068] (2) Gel electrophoresis was used to detect the PCR product, and the results showed that there was a clear band at the position of approximately 750 bp.

[0069] (3) Cut the gel to recover the target fragment and recover the DNA fragment.

[0070] (4) Use Spe I and Nru I respectively digested the recovered DNA fragments and pCAMBIA-NPT vector.

[0071] Enzyme digestion reaction system: 10 μL DNA (100 ng / μL); 1 μL restriction endonuclease 1 (15 U / μL); 1 μL restriction endonuclease 2 (15 U / μL); 5 μL restriction endonuclease reaction 10× Buffer; total volume is 50 μL; enzyme digestion is carried out at 37°C for 5 h.

[0072] (5) Perform gel electrophoresis and cut the gel to recover the gene and vector fragments.

[0073] (6) Ligating the recovered gene and vector fragments;

[0074] Ligation reaction system: 1 μL vector fragment; 3 μL GHF1 ; 1 μL T4 ligase; 1 μL 10× T4 ligase buffer; 4 μL water; total volume 10 μL; ligation at 16°C overnight.

[0075] (7) Heat shock transformation of Escherichia coli was performed, and Kan resistance was used for screening. Positive clones were selected to extract the recombinant vector and named 35S:GHF1 , and store at -20℃ for future use.

[0076] 2. Recombinant vector 35S:GHF1 Transformation of Agrobacterium

[0077] (1) Add 5 μl of plasmid (about 500 ng) to GV3101 Agrobacterium tumefaciens competent cells melted in an ice bath. After standing on ice for 30 min, transfer to liquid nitrogen for quick freezing for 5 min, and then heat shock in a 37°C water bath for 5 min.

[0078] (2) After heat shock, add 500 μL of non-resistant YEB liquid culture medium and activate in a shaker at 28°C and 160 rpm for 2 h.

[0079] (3) The bacterial solution was spread on YEB solid culture medium containing Kan and Rif and cultured inverted at 28°C in the dark for 3 days. A single colony was picked for bacterial solution PCR detection to obtain a positive colony.

[0080] 3. Obtaining transgenic plants

[0081] The leaves of the common tobacco cultivar K326 were transformed using the Agrobacterium-mediated method. Transgenic plants were screened on MS resistance medium containing 30 mg / L Kan. After obtaining T3 pure lines, leaf DNA was extracted and detected by PCR. 35S:GHF1The positive transformed lines were initially determined by qRT-PCR. GHF1 Gene expression level.

[0082] The results are as follows Figure 3 As shown in Figure 2, all the five transgenic lines obtained were positive transformants, and the four lines OE2 to OE5 were positive. GHF1 The expression level was more than 2 times that of the control group. GHF1 Overexpression line (K326-OE).

[0083] Example 3: GHF1 Obtaining gene knockout plants

[0084] 1. Construction of knockout vector and genetic transformation

[0085] Tobacco obtained by sequencing GHF1 The genome sequence of gRNA Target sequence (SEQ ID NO.11): CAGTTGTCTATATTCTATGG. Vector construction and tobacco transformation were completed by Weimi Biotechnology (Jiangsu) Co., Ltd.

[0086] 2. GHF1 Screening of homozygous mutant plants

[0087] To detect the mutation of the target site, the following detection primers (see SEQ ID NO. 12-13) were designed on both sides of the target site and PCR amplified on the genomic DNA of the positively transformed plants:

[0088] F: 5'-GGACCAATTGCAAGACATAGAT-3',

[0089] R: 5'-GTGAGGGCTTCCTTTGCTAGCT-3'.

[0090] The amplified bands were PCR purified and the products were sequenced and screened. GHF1 The mutant plant was obtained as K326-M.

[0091] The results are as follows Figure 4 As shown, two K326-M mutants were obtained, in which a T base was inserted into the first SG sequence of K326-M1, and two bases were deleted in the second SG sequence; and a T base was inserted into the first SG sequence of K326-M2, and two bases were deleted in the second SG sequence, and a base substitution occurred (T was replaced by G).

[0092] Example 4: Phenotypic identification and physiological characteristics detection of each strain

[0093] The common cultivated tobacco variety K326 was used as a control, the tobacco overexpression strain constructed in Example 2 was labeled K326-OE, and the tobacco overexpression strain constructed in Example 3 was labeled GHF1 The mutant plant (gene knockout line) was labeled K326-M, and the epidermal hair morphology and leaf chemical composition were observed. The results were as follows: Figure 5 、 Figure 6 shown.

[0094] Phenotypic identification results ( Figure 5 ) showed that compared with K326, the heads of the long-stalked glandular hairs of the K326-OE strain were basically gone, and the main types of epidermal hairs were short-stalked glandular hairs and guard hairs; the heads of the long-stalked glandular hairs of the K326-M plant were enlarged, and the main types of epidermal hairs were short-stalked glandular hairs and long-stalked glandular hairs. Leaf chemical composition analysis showed that ( Figure 6 ), the glandular hair secretion components of K326, K326-OE and K326-M are the same, all containing cedarwood diterpenes and sucrose esters; compared with K326, the content in K326-M is significantly increased, while the content in K326-OE is significantly reduced.

[0095] The above results further prove that GHF1 The gene can negatively regulate the development of the head of long-stalked glandular hairs in plants and plays a key regulatory role in regulating the type of plant epidermal hairs. It is of great significance to the study of the regulation of glandular hair tissue development and the formation of glandular hair-specific synthetic substances.

[0096] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the inventive concept. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present application is intended to include such changes and modifications.

Claims

1. The nucleotide sequence of the glandular hair head gene is shown in SEQ ID NO.1 GHF1 The invention is used in regulating the occurrence of glandular hair heads and / or the content of glandular hair secretions in Solanaceae plants, wherein the glandular hair secretions are cepine diterpenes and / or sucrose esters.

2. The use according to claim 1, characterized in that The following steps are involved: (1) Cloning the glandular hair head gene GHF1 , and insert the strong promoter 35S into the upstream of the vector to obtain the overexpression vector; (2) The overexpression vector is transformed into a recipient plant and cultivated to obtain a transgenic plant in which the heads of the long-stalked glandular hairs have disappeared.

3. The use according to claim 1, characterized in that The following steps are involved: (1) Design based on conserved sequences sgRNA Primers, construct the glandular hair head gene GHF1 Gene editing vectors; (2) The gene editing vector is transformed into a recipient plant and cultivated to obtain a transgenic plant with enlarged heads of long-stalked glandular hairs.

4. The use according to claim 1, characterized in that The glandular hair head gene GHF1 The encoded protein sequence is shown in SEQ ID NO.2.

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