Plant glandular hair head generation gene GHF1 as well as expression product, expression vector and application thereof

Abstract

The invention discloses a plant glandular hair head generation gene GHF1 as well as an expression product, an expression vector and application thereof, and aims to solve the technical problems of insufficient research on the glandular hair head generation gene and unclear glandular hair generation molecular regulation network at present. According to the invention, a gene GHF1 capable of negatively regulating plant glandular hair head generation is identified, an overexpression vector and a gene editing vector of the gene GHF1 are constructed, and tobacco transformation is respectively carried out to obtain a corresponding overexpression strain and a gene knockout plant; epidermal hair morphological identification and foliar chemical component analysis show that the GHF1 gene can negatively regulate and control plant glandular hair head generation and development, and has important application value in glandular hair development regulation and control, glandular hair specific substance synthesis and the like.

Description

Technical Field

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

[0002] The structure and function of Arabidopsis epidermal hairs are relatively simple. They are single-celled, glandless structures that do not have secretory capabilities, and the molecular network of their morphological construction has been analyzed. Unlike Arabidopsis, most terrestrial plants such as Solanaceae, Leguminosae, Asteraceae, Cannabaceae, and Lamiaceae have more complex and diverse epidermal hair structures. Their epidermal hairs are multicellular structures that include not only glandless hairs (protective hairs) but also glandular hairs with secretory functions, and the morphological construction process is complex. The glandular hairs of plants of different families and genera vary in morphology, but their basic structures are similar, all consisting of a base, a stalk, and a glandular head, of which only the glandular head has synthetic and secretory functions.

[0003] As a physical barrier between plants and the environment, epidermal hairs can reduce water and heat loss, buffer direct sunlight, and resist pathogens and insects. Glandular hairs, as secretory organs, can promote the excretion of heavy metals and reduce their accumulation in plants. In addition, glandular hairs are known as "plant chemical factories" and can specifically synthesize and release a large number of secondary metabolites. These metabolites not only play an important role in plant adaptation to environmental stress, but can also be used as pharmaceutical precursors, flavors and fragrances, food additives and pesticides, etc., and have important commercial value. For example, artemisinin specifically synthesized in the head of Artemisia annua glandular hairs belongs to sesquiterpene lactones and is currently the most effective drug for treating malaria; menthol monoterpenes in peppermint glandular hairs have anti-inflammatory, analgesic and bactericidal effects; pyrethrins specifically synthesized in pyrethrum glandular hairs are commonly used broad-spectrum insecticides; cypermethrin diterpenes specifically synthesized in tobacco glandular hairs have antifungal and anti-aphid functions; phenylpropanoids are the main components of glandular hairs in Lamiaceae plants and are mainly involved in defense reactions and insect pollination processes. 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] As a model plant for molecular biology and genetic engineering research, tobacco 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. It is 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 4-6 cell glandular stalks and 1-6 cell glandular heads. The glandular heads are rich in chloroplasts and specifically synthesize cypermethrin diterpenes, lisdactyl diterpenes and sucrose esters; (2) short-stalked glandular hairs, which are composed of 1 stalk cell and 8-16 cell glandular heads. The glandular heads do not contain chloroplasts and specifically synthesize leaf resistance proteins; (3) guard hairs, which have no glandular cells and no synthesis and secretion capabilities.

[0005] At present, there are more and more reports on the genes regulating glandular hair development, but there are no reports on the genes regulating the development of glandular hair heads, and the molecular regulatory network of glandular hair development is not clear. Therefore, exploring the genes regulating the development of plant glandular hair heads and studying the molecular regulatory network of plant glandular hair development are of great significance for the study of the development regulation of glandular hair tissue and the formation of glandular hair-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 acknowledging or suggesting in any form 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 for glandular hair formation, laying the foundation for research on 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 1 stop codon, and has a typical TIFY conserved domain.

[0009] According to a 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, there is provided a method comprising: GHF1 or an expression vector containing a fragment thereof.

[0011] According to a fourth aspect of the present disclosure, the plant glandular hair head gene GHF1 , the encoded 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: (1) Cloning the plant glandular hair head gene GHF1 , and after inserting the strong promoter 35S upstream, an overexpression vector was obtained; (2) Transforming the overexpression vector into a recipient plant and cultivating the plant to obtain a transgenic plant in which the heads of the long-stalked glandular hairs have disappeared.

[0013] In some embodiments of the present disclosure, the application includes the following steps: (1) Design based on conservative sequences sgRNA Primers, construct the plant glandular hair head gene GHF1 Gene editing vectors; (2) Transforming the gene editing vector into a recipient plant and cultivating the plant to obtain a transgenic plant with enlarged heads of long-stalked glandular hairs.

[0014] In some embodiments of the present disclosure, the recipient plant comprises a plant of the Solanaceae family.

[0015] 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 in regulating the type of plant epidermal hairs.

[0016] 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.

[0017] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: Screening identified GHF1 The gene encodes 242 amino acids and 1 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 the overexpression and knockout plants of this gene, the results showed that compared with the control plants, GHF1 The long-stalked glandular hairs of the overexpression 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 secretion content of glandular hairs in the overexpressed strains was significantly reduced. This gene can negatively regulate the occurrence of long-stalked glandular hair heads, and therefore has important significance for the study of the regulation of glandular hair tissue development and the formation of glandular hair-specific synthetic substances. It lays a foundation for further research on the molecular mechanism of plant glandular hair occurrence and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the prediction result of the conserved domain of tobacco GHF1 protein in one embodiment of the present application.

[0019] Figure 2 In one embodiment of the present application, tobacco GHF1 Analysis diagram of gene tissue expression characteristics.

[0020] Figure 3In one embodiment of the present application, tobacco GHF1 Detection diagram of gene overexpression strains; A: DNA-PCR gel electrophoresis diagram; B: GHF1 Expression analysis of overexpression lines.

[0021] Figure 4 In one embodiment of the present application, tobacco GHF1 Analysis diagram of gene mutant sequencing; K326: control strain; K326-M: GHF1 Gene mutant plants.

[0022] 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.

[0023] 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

[0024] The specific implementation modes of the present invention are described below in conjunction with the accompanying drawings and examples. However, the following examples are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way.

[0025] 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.

[0026] Example 1: Genes Predominantly Expressed in Glandular Hairs GHF1 Cloning and tissue expression analysis This example is based on the common tobacco ( Nicotiana tobacum ) glandular hair cDNA library (Cui et al. BMC Plant Biology 2011, 11:76), screened out a tobacco glandular hair dominant gene, which was named GHF1 .

[0027] 1. Tobacco Gene GHF1 Clone (1) Take 2 g of common tobacco leaves, add liquid nitrogen and grind thoroughly, and follow the steps of 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 RNA into cDNA.

[0028] (2) Gene cloning: Design amplification primers and perform PCR amplification using cDNA as a template.

[0029] PCR system (10 μL): 5 μL Mix; 0.25 μL F / R each; 0.5 μL cDNA; 4 μL ddH2O.

[0030] 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.

[0031] The amplification primers are (SEQ ID NO.3~4): F:5'-ATGTCAAGTTTGCAACTTTCTTC-3'; R:5'-CTATAACGTGAAGTTGAGATCA-3'.

[0032] (3) After PCR amplification, gel electrophoresis was performed, and the results showed a clear band at approximately 700 bp.

[0033] (4) Cut out the electrophoresis band in the gel and recover the PCR fragment using a DNA recovery kit.

[0034] (5) The recovered PCR fragment was ligated with the pMD19-T vector (Takara).

[0035] (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.

[0036] 2. Prediction of sequence conserved domains 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 GHF1 The gene has a conserved TIFY domain, and it is speculated that the gene belongs to the plant-specific TIFY family member.

[0037] 3. Analysis of tissue expression characteristics 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 freezing and brushing. The total RNA of the samples was extracted and reverse transcribed to synthesize cDNA. LqCy 5 as an internal reference gene, and semi-quantitative and qRT-PCR methods were used to analyze tobacco GHF1Tissue expression characteristics of genes.

[0038] The amplification primers are (SEQ ID NO.5~8): GHF1 -F: 5'-GGAAGAACAATCAACCAAGACAATG-3'; GHF1 -R: 5'-GGTATGGCGCTCTGGCCGTGTCTC-3'; LqCy 5-F: 5'-CCCCTCACCACAGAGTCTGC-3'; LqCy 5-R: 5'-TTCTAACTCCTGTTGTTGTGGGAA-3'.

[0039] PCR system for semi-quantitative gene analysis (10 μL): 5 μL Mix; 0.25 μL F / R each; 0.5 μL cDNA; 4 μL ddH2O.

[0040] The PCR reaction program for semi-quantitative analysis of genes was as follows: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, for 28 cycles; and 72°C for 10 min.

[0041] 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.

[0042] qRT-PCR reaction program: 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, 40 cycles.

[0043] 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.

[0044] Embodiment 2 GHF1 Obtaining gene overexpression strains 1. Construction of overexpression vector tobacco GHF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the expression vector used is pCAMBIA-NPT vector: (1) Selection Spe I and Nr I Two restriction sites are ligase sites, designed as follows GHF1 Add cloning primers with restriction sites (see SEQ ID NO.9-10) for PCR amplification: F: 5'-AGGACTAGTATGTCAAGTTTGCAACTTTCTTC-3', R: 5'- GAGATCGCGACTATAACGTGAAGTTGAGATCA-3'; The restriction sites marked in bold are Spe I and Nr I.

[0045] The PCR reaction system was: 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.

[0046] 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.

[0047] (2) Gel electrophoresis was used to detect the PCR product, and the results showed a clear band at approximately 750 bp.

[0048] (3) Cut the gel to recover the target fragment and recover the DNA fragment.

[0049] (4) Use Spe I and Nr I respectively digested the recovered DNA fragments and pCAMBIA-NPT vector.

[0050] Enzyme digestion reaction system: 10 ul DNA (100 ng / μL); 1 ul restriction endonuclease 1 (15 U / μL); 1μL restriction endonuclease 2 (15 U / μL); 5μL restriction endonuclease reaction 10× Buffer; total volume is 50μL; enzyme digestion at 37℃ for 5h.

[0051] (5) Perform gel electrophoresis and cut the gel to recover the gene and vector fragments respectively.

[0052] (6) Ligating the recovered gene and vector fragments; Ligation reaction system: 1μL vector fragment; 3μL GHF1; 1μL T4 ligase; 1μL 10×T4 ligase buffer; 4μL water; total volume is 10μL; ligation is carried out at 16℃ overnight.

[0053] (7) Heat shock transformation of Escherichia coli, and screening with Kan resistance, select positive clones to extract the recombinant vector, and name it 35S:GHF1 , and store at -20℃ for future use.

[0054] 2. Recombinant vector 35S:GHF1 Transformation of Agrobacterium (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.

[0055] (2) After heat shock, add 500 μL of resistance-free YEB liquid culture medium and activate the cells in a shaker at 28°C and 160 rpm for 2 h.

[0056] (3) Spread the bacterial solution on YEB solid medium containing Kan and Rif and culture it at 28°C in a dark place for 3 days. Pick a single colony for PCR detection to obtain a positive colony.

[0057] 3. Obtaining transgenic plants The leaves of common tobacco cultivar K326 were transformed by 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:GHF1 The positive transformed strains were initially determined. Then the transgenic strains were analyzed by qRT-PCR. GHF1 The expression level of the gene.

[0058] 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 GHF1 The expression level was more than 2 times that of the control group. GHF1 Overexpression line (K326-OE).

[0059] Embodiment 3 GHF1 Obtaining gene knockout plants 1. Construction of knockout vector and genetic transformation Tobacco obtained by sequencing GHF1 The genome sequence of gRNATarget sequence (SEQ ID NO.11): CAGTTGTCTATATTCTATGG. Vector construction and tobacco transformation were completed by Weimi Biotechnology (Jiangsu) Co., Ltd.

[0060] 2. GHF1 Screening of homozygous mutant plants In order 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 amplification was performed on the genomic DNA of the positively transformed plants: F: 5'-GGACCAATTGCAAGACATAGAT-3', R: 5'-GTGAGGGCTTCCTTTGCTAGCT-3'.

[0061] The amplified bands were purified by PCR and the products were sequenced and screened. GHF1 The mutant plant was obtained as K326-M.

[0062] 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).

[0063] Example 4: Phenotypic identification and physiological characteristics detection of each strain The common cultivated tobacco variety K326 was used as a control, the tobacco overexpression strain constructed in Example 2 was labeled as K326-OE, and the tobacco overexpression strain constructed in Example 3 was labeled as K326-OE. GHF1 The mutant plant (gene knockout line) was labeled K326-M, and the epidermal hair morphology and leaf surface chemical composition were observed. The results were as follows: Figure 5 , Figure 6 shown.

[0064] Phenotypic identification results ( Figure 5 ) showed that compared with K326, the heads of the long-stalked glandular hairs of the K326-OE strain basically disappeared, 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 became larger, and the main types of epidermal hairs were short-stalked glandular hairs and long-stalked glandular hairs. Leaf surface 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.

[0065] The above results further prove that GHF1 The gene can negatively regulate the occurrence of long-stalked glandular hair heads in plants and plays a key role in regulating the types 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.

[0066] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other 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.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A gene for the development of plant glandular hairs GHF1 , whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The plant glandular hair head gene according to claim 1 GHF1 The encoded protein has an amino acid sequence as shown in SEQ ID NO.

2.

3. A plant containing the plant glandular hair head gene according to claim 1 GHF1 or an expression vector containing a fragment thereof.

4. The plant glandular hair head gene of claim 1 GHF1 Or use of the expression vector according to claim 3 in the construction or breeding of transgenic plants.

5. The plant glandular hair head gene of claim 1 GHF1 , Use of the encoded protein according to claim 2 or the expression vector according to claim 3 in regulating the type of plant epidermal hairs.

6. The plant glandular hair head gene of claim 1 GHF1 , use of the coding protein described in claim 2 or the expression vector described in claim 3 in regulating the occurrence of plant glandular hair heads and / or the content of glandular hair secretions.

7. The use according to claim 6, characterized in that: The following steps are involved: (1) Cloning the plant glandular hair head gene described in claim 1 GHF1 , and insert the strong promoter 35S upstream to obtain the overexpression vector; (2) Transforming the overexpression vector into a recipient plant and cultivating the plant to obtain a transgenic plant in which the heads of the long-stalked glandular hairs have disappeared.

8. The use according to claim 6, characterized in that: The following steps are involved: (1) Design based on conservative sequences sgRNA Primers for constructing the plant glandular hair head generation gene according to claim 1 GHF1 Gene editing vectors; (2) Transforming the gene editing vector into a recipient plant and cultivating the plant to obtain a transgenic plant with enlarged heads of long-stalked glandular hairs.

9. The use according to claim 7 or 8, characterized in that: The recipient plants include plants of the Solanaceae family.

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