Application of gene NtTRY1 in regulation and control of plant glandular hair development

Abstract

The invention relates to an application of a gene NtTRY1 in regulation and control of plant glandular hair development. The nucleotide sequence of the gene NtTRY1 is shown as SEQ ID No.1. The invention also relates to a preparation method of the gene NtTRY1. According to the NtTRY1 transgenic tobacco obtained after the NtTRY1 gene is transformed into tobacco, the tobacco leaf surface glandular hair density is remarkably reduced, and it is proved that the gene can effectively regulate and control growth and development of glandular hair of plants. Therefore, the functional identification of the tobacco NtTRY1 gene not only enriches key breeding gene resources of tobacco for regulating and controlling glandular hair development and quality breeding, but also is beneficial to analyzing the diversity function of tobacco MYB transcription factors, and has important theoretical significance and application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a gene NtTRY1 in regulating the development of plant glandular hairs. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an annual herb belonging to the Solanaceae family and the genus Nicotiana L., and is one of the important economic crops.

[0003] However, the current tobacco planting field has problems such as the long planting time of the main varieties and soil degradation leading to reduced adaptability and efficiency. As a result, the problems of reduced tobacco leaf quality and insufficient characteristics that the cigarette industry is concerned about are becoming increasingly prominent, restricting the further development of the tobacco industry. Therefore, improving tobacco production and quality has become one of the current urgent issues that need to be addressed.

[0004] The surface of the plant is covered with a layer of trichomes that are specialized from epidermal cells. They are divided into glandular trichomes and protective trichomes according to whether the trichomes have the ability to secrete. Tobacco glandular trichomes can be divided into long-stalked glandular trichomes, short-stalked glandular trichomes and branched glandular trichomes according to their different shapes. Tobacco glandular trichomes are the main place for the synthesis and secretion of tobacco aroma substances such as cypermethrin, lysine and sugar esters. The secretions of glandular trichomes play an important role in the formation of tobacco aroma quality and the prevention of diseases and pests.

[0005] Related studies have shown that the development of plant trichomes is not only affected by environmental factors such as light and temperature, but also MYB transcription factors have a certain regulatory effect on it. According to the number of repetitions of the conserved domain, MYB transcription factors can be divided into four types: 1R-MYB, R2R3-MYB, 3R-MYB (R1R2R3-MYB) and 4R-MYB. GL1, MYB23 and MYB5 of the R2R3-MYB family in Arabidopsis positively regulate the development of trichomes. SI TRY of the R3MYB family in tomato negatively regulates the density of epidermal hairs and has a positive regulatory effect on the differentiation of root hairs. There is no report on the effect of MYB transcription factors on glandular hairs in tobacco.

[0006] Tobacco glandular trichome secretions are closely related to the aroma quality of tobacco leaves, and the development of tobacco glandular trichomes has a direct impact on the content and composition of leaf secretions. Existing research mainly focuses on the morphological observation of tobacco glandular trichomes and the identification of secretions. There are few studies on the molecular mechanism of glandular trichome development, and the identification of genes related to glandular trichome development in tobacco and the study of molecular regulatory mechanisms are still in the initial stage. Therefore, it is urgent to explore and utilize the key genes for glandular trichome development in tobacco, which is of great practical significance for cultivating new tobacco varieties that are key to regulating glandular trichome development and ensuring tobacco quality and yield. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an application of a gene NtTRY1 in regulating the development of glandular hairs in plants. The present invention transforms the NtTRY1 gene into tobacco to obtain transgenic tobacco, and the density of glandular hairs on the surface of tobacco leaves is significantly reduced, which proves that the gene can effectively regulate the growth and development of glandular hairs in plants.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides the use of the gene NtTRY1 and / or its expression reagent in regulating the development of plant glandular hairs. The nucleic acid sequence of the gene NtTRY1 is shown in SEQ ID No.1.

[0010] SEQ ID No.1:

[0011] ATGGATCAAAATCTCCATCATCAGCCCAAGCTCATGCACCACCGATGTTGCAGCCATGAAGAGGTTAATAGTATGGAGTGGGAGTTCATCAGCATGAGCAAGCAAGAAGAAGATCTTATTTACAGAATGCACAA GCTTGTTGGAGACAGGTGGGGACTGATAGCAGGGAGAATACCAGGGAGAACAGCAGAAGAAATAGAAAGGTTTTGGATAATGAAACACAGTGATGGCTTTGCAAACAAGAGACGACAATTAAGAAAAGTGTAG.

[0012] In the present invention, the gene is transferred into a recombinant strain, and then transformed into a plant to obtain a transgenic plant. Compared with the wild type, the density of glandular hairs on the surface of the transgenic plant is significantly reduced. This proves that the NtTRY1 gene is a key gene for regulating the development of glandular hairs in plants (such as tobacco). In the field of scientific research, the gene can be used to explore the molecular regulatory mechanism of glandular hair development, the identification of genes related to glandular hair development, and the breeding of key tobacco varieties that regulate glandular hair development. It can be understood that the expression reagent refers to a reagent that can express the gene NtTRY1 in plants. Such as an expression vector containing the gene NtTRY1, and Agrobacterium containing the corresponding vector, etc.

[0013] Preferably, the amino acid sequence of the gene NtTRY1 is as shown in SEQ ID No.2.

[0014] SEQ ID No.2:

[0015] MDQNLHHQPKLMHHRCCSHEEVNSMEWEFISMSKQEEDLIYRMHKLV GDRWGLIAGRIPGRTAEEIERFWIMKHSDGFANKRRQLRKV.

[0016] In a second aspect, the present invention provides a method for regulating the development of plant glandular hairs, the method comprising the following steps:

[0017] The nucleic acid sequence of the gene NtTRY1 described in the first aspect is amplified, connected to an expression vector, transferred into Agrobacterium, and transformed into plants to obtain plants over-expressing the gene.

[0018] Preferably, the method comprises the following steps:

[0019] (1) amplifying the gene NtTRY1 whose nucleotide sequence is shown in SEQ ID No. 1;

[0020] (2) cloning the key gene NtTRY1, ligating it into an expression vector after enzyme digestion and purification, and obtaining an overexpression recombinant vector after identification;

[0021] (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0022] (4) Transforming the overexpression recombinant strain into plants, screening and obtaining NtTRY1 transgenic strains, so as to regulate the development of glandular hairs in plants.

[0023] Preferably, in step (1), the amplification primers include SEQ ID No. 3 and SEQ ID No. 4;

[0024] SEQ ID No. 3: GGATCCATGGATCAAAATCTCCATCATCAGC.

[0025] SEQ ID No. 4: GAGCTCCTACACTTTTCTTAATTGTCGTCTCT.

[0026] Preferably, in step (1), the amplification procedure includes:

[0027] Pre-denaturation: 95-99°C, 20-40s. The 95-99°C may be, for example, 95°C, 96°C, 97°C, 98°C or 99°C. The 20-40s may be, for example, 20s, 22s, 24s, 26s, 28s, 30s, 32s, 34s, 36s, 38s or 40s.

[0028] Denaturation: 95-99°C, 10-20s. The 95-99°C may be, for example, 95°C, 96°C, 97°C, 98°C or 99°C. The 10-20s may be, for example, 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s or 20s.

[0029] Annealing extension: 52-66°C, 5-15s; 65-80°C, 40-60s; cycle 25-35 times. The 52-66°C may be, for example, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C or 66°C. The 5-15s may be, for example, 5s, 6s, 7s, 8s, 9s, 10s, 11s, 12s, 13s, 14s or 15s. The 65-80°C may be, for example, 65°C, 66°C, 68°C, 70°C, 72°C, 74°C, 76°C, 78°C or 80°C. The 40-60s may be, for example, 40s, 42s, 44s, 46s, 48s, 50s, 52s, 54s, 56s, 58s or 60s, etc. The 25-35 times may be, for example, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times or 35 times, etc.

[0030] Extension: 65-80°C, 5-15 min. The 65-80°C may be, for example, 65°C, 66°C, 68°C, 70°C, 72°C, 74°C, 76°C, 78°C or 80°C, etc. The 5-15 min may be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.

[0031] Preferably, the cloning vector comprises pEASY-Blunt simple.

[0032] Preferably, in step (2), the restriction enzyme cleavage site includes any one of BamH I, Sac I, EcoR I, Nde I, Nco I, Pac I, Pst I, Sca I, Spe I, Xba I or Xho I, or a combination of at least two of them.

[0033] Preferably, the cloning vector comprises a promoter and a terminator.

[0034] Preferably, in step (2), the expression vector includes pCambia1300.

[0035] Preferably, in step (2), the purification method comprises silica gel chromatography and / or magnetic bead method.

[0036] Preferably, in step (2), the identification method includes whole-genome sequencing and / or PCR amplification.

[0037] Preferably, in step (3), the conversion method comprises a freeze-thaw method.

[0038] Preferably, the Agrobacterium comprises one or a combination of at least two of GV3101, EHA101, EHA105 or LBA4404.

[0039] Preferably, in step (4), the transformation method comprises Agrobacterium-mediated leaf disc transformation;

[0040] In a third aspect, the present invention provides the use of the gene NtTRY1 described in the first aspect in breeding key tobacco varieties for regulating the development of glandular hairs.

[0041] Compared with the prior art, the present invention has at least the following beneficial effects:

[0042] The present invention clones a MYB transcription factor NtTRY1 related to the development of tobacco glandular hairs, and then transforms the gene into tobacco after constructing an overexpression vector and an overexpression recombinant strain to obtain a transgenic tobacco plant NtTRY1-OE. Compared with the wild type, the density of glandular hairs on the tobacco leaf surface is significantly reduced.

[0043] This proves that the NtTRY1 gene is a key gene regulating the development of tobacco glandular hairs. Therefore, the functional identification of the tobacco NtTRY1 gene not only enriches the key breeding gene resources for tobacco regulation of glandular hair development and quality breeding, but also helps to analyze the diverse functions of tobacco MYB transcription factors, which has important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the electrophoresis diagram of NtTRY1 gene amplification.

[0045] Figure 2 This is the electrophoresis diagram for the identification of transgenic plants.

[0046] Figure 3 It is a bar graph showing the expression level of NtTRY1 gene in transgenic and wild-type plants.

[0047] Figure 4 These are microscope images of glandular hairs in transgenic and wild-type plants.

[0048] Figure 5 It is a bar graph showing the number of glandular hairs in transgenic and wild-type plants. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0050] Example 1

[0051] This example is used to obtain the gene NtTRY1

[0052] Extract the mRNA from the seedling leaves of the tobacco cultivar "Honghua Dajinyuan" and reverse transcribe it into cDNA, which is used as a template for PCR amplification. The PCR amplification system is: 31 μL ddH2O, 10 μL Mg 2+ 5× HF buffer ( HF BufferPack, NEB), 2 μL of 2.5 mM dNTP, 2 μL of 5 μM upstream primer of SEQ ID No.3, 2 μL of 5 μM downstream primer of SEQ ID No.4, 0.6 μL of DMSO, 0.5 μL of DNA polymerase, and 2 μL of template.

[0053] The primers used were:

[0054] SEQ ID No. 3-NtTRY1-F: GGATCCATGGATCAAAATCTCCATCATCAGC.

[0055] SEQ ID No. 4-NtTRY1-R:GAGCTCCTACACTTTTCTTAATTGTCGTCTCT.

[0056] The PCR reaction conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 58°C for 10 s, extension at 72°C for 50 s, 30 cycles; post-extension at 72°C for 10 min.

[0057] like Figure 1 As shown, after electrophoresis, it can be seen that the NtTRY1 gene has been successfully amplified. After first-generation sequencing verification, the nucleic acid sequence of the gene NtTRY1 is SEQ ID No.1.

[0058] Example 2

[0059] This example constructs an expression vector

[0060] The PCR amplified fragment obtained in Example 2 was connected to the cloning vector pEASY-Blunt simple. After the sequencing was correct, the plasmid was extracted, double-digested with BamH I and Sac I, purified, and connected to the expression vector digested with BamH I and Sac I, which was the pCambia1300 vector containing the 35S promoter and the NOS terminator. After the sequencing was correct, it was transformed into Agrobacterium GV3101.

[0061] The common tobacco Honghua Dajinyuan was genetically transformed by Agrobacterium-mediated leaf disc method to obtain the transgenic plant NtTRY1-OE. The specific steps are as follows: The transgenic tobacco plant was obtained by Agrobacterium-mediated leaf disc transformation, and the specific steps are as follows:

[0062] Sterile seedling culture: Take an appropriate amount of safflower Dajinyuan seeds, disinfect the surface with 75% alcohol for 30 seconds, wash with sterile water 3 times, soak in 15% hydrogen peroxide for 8 minutes, wash with sterile water 3 times and soak in sterile water for 24 hours. Sow the sterilized seeds on the MS culture dish, transfer the seedlings to the tissue culture bottle containing MS after growing 3 leaflets, culture in the artificial climate room for about 45 days, and select the leaves with strong growth for Agrobacterium infection.

[0063] Agrobacterium infection: Take out the Agrobacterium liquid that has been correctly preserved and after it is completely thawed, take 500uL and put it into 50mL YEP liquid medium of corresponding resistance, and culture it at 28℃ and 220rpm until OD 600 0.6. Take 50 mL of bacterial solution, centrifuge at 4000 rpm for 10 min, remove the supernatant. Collect the bacteria and resuspend to OD 600 The concentration of acetosyringone was 0.6, and then 20 mg / L of acetosyringone was added as the Agrobacterium infection solution. Cut off the edges of the leaves of the sterile seedlings and cut the leaves into 1 cm along the main veins. 2 The leaf discs were placed in the Agrobacterium infection solution and infected for 5 minutes.

[0064] Co-culture and subculture: remove the infected leaves, use filter paper to absorb the Agrobacterium, lay the leaves face down on the co-culture medium, put them in the artificial climate chamber, and culture them in the dark for 3 days. S1 subculture: transfer the leaves that have been co-cultured for 3 days with the leaves face up to the S1 differentiation medium, culture them in the dark in the artificial climate chamber for about 1 week, and then transfer them to the light for further culture until clusters of about 0.5 cm grow on the edge of the leaves. S2 subculture: transfer the clusters of buds grown on S1 to the S2 differentiation medium, remove the leaves that have not grown clusters, and culture them in the light for 2 weeks until the clusters grow into seedlings. S3 subculture: transfer the seedlings on S2 to the S3 differentiation medium and culture them in the light for 2 weeks. Rooting culture: remove the swollen part and yellow leaves at the bottom of the seedlings, transfer the seedlings to the tissue culture bottle containing the rooting medium, and culture them in the light for 2 weeks. The culture media used in co-culture and subculture are shown in Table 1.

[0065] Table 1

[0066]

[0067] Obtaining transgenic tobacco: When the seedlings have grown about 8 roots about 3 cm long, open the lid of the culture bottle to harden the seedlings. After 3 days, transfer the seedlings to a flower pot filled with sterile soil and cover with plastic film to keep warm. After a week, remove the plastic film and let it grow rapidly under natural conditions.

[0068] Example 3

[0069] This example is used to identify positive transgenic tobacco plants

[0070] After the transgenic plants obtained in Example 2 were grown under natural conditions for one month, young tobacco leaves were selected for sampling, the DNA of the tobacco leaves was extracted by the CTAB method, and positive transgenic plants were identified by PCR for subsequent test analysis. The PCR amplification system is: 7 μL of ddH2O, 1 μL of 5 μM upstream primer of SEQ ID No.3, 1 μL of 5 μM downstream primer of SEQ ID No.5, 10 μL of 2×Rapid Taq Master Mix, and 2 μL of template. The DNA sequence amplified by the PCR is the gene NtTRY1 and a partial plasmid fragment connected to the gene, and the length of the amplified sequence fragment is 1049 bp. The primer sequences identified by PCR are shown in Table 1 below.

[0071] Table 1

[0072] name serial number Primer sequence (5'-3') NtTRY1-F SEQ ID No.3 GGATCCATGGATCAAAATCTCCATCATCAGC NtTRY1-P9R SEQ ID No.5 ATTCTGGTGTGTGCGCAATG

[0073] The PCR reaction conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 58°C for 10 s, extension at 72°C for 50 s, 30 cycles; post-extension at 72°C for 10 min.

[0074] like Figure 2 As shown, the PCR amplification bands were clear after electrophoresis, proving that the transgenic plants obtained were all positive and could be used for subsequent experimental analysis.

[0075] Example 4

[0076] This example is a key identification of transgenic tobacco regulating glandular hair development

[0077] The T1 generation seeds of the transgenic plant NtTRY1-OE were sterilized and grown on normal MS solid culture medium for one week. Then, tobacco seedlings with basically the same growth potential were selected and transferred to new MS solid culture medium for one week to identify the key role of regulating glandular trichome development in the seedling stage. At the same time, wild-type tobacco seeds (WT) were used as a control.

[0078] (1) Detection of NtTRY1 gene expression

[0079] Five transgenic tobacco seedlings, 1-13-2, 1-13-3, 1-13-1, 1-31-2, 1-31-3 and wild-type tobacco seedlings, were selected to extract mRNA, which was reverse transcribed into cDNA and used as a template for fluorescence quantitative PCR detection. The primer sequences of fluorescence quantitative PCR are shown in Table 2 below.

[0080] Table 2

[0081]

[0082]

[0083] The results are as follows Figure 3 As shown, the expression level of NtTRY1 gene in transgenic plants increased significantly, by 5-8 times.

[0084] (2) Detection of glandular hairs on tobacco leaf surface

[0085] The specific detection method is to take tobacco leaves with the same growth and development and the same leaf position, add water drops on the slide, tear off the lower epidermis of the tobacco leaves, lay the side with glandular hairs flat on the slide, use absorbent paper to gently absorb excess water to avoid wrinkles on the leaf epidermis, cover with a coverslip, and use an upright and inverted integrated fluorescence microscope to observe the density and morphology of glandular hairs on the leaf surface. Six fields of view were observed for each leaf, and the results were repeated three times. The specific results are as follows: Figure 4 The density of glandular hairs was calculated using ImageJ software, and the results were as follows. Figure 5 The density of glandular hairs on the leaf surfaces of transgenic tobacco 1-31-2 and 1-31-2 was significantly reduced.

[0086] This shows that the key to regulating the development of glandular hairs in transgenic tobacco is better than that in wild type, that is, the NtTRY1 gene can regulate the development of glandular hairs in transgenic tobacco.

[0087] In summary, the present invention provides an application of a gene NtTRY1 in regulating the development of glandular hairs in plants. In the transgenic tobacco obtained by transforming the NtTRY1 gene into tobacco, the density of glandular hairs on the surface of tobacco leaves is significantly reduced, which proves that the gene can effectively regulate the growth and development of glandular hairs in plants.

[0088] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of gene NtTRY1 and / or its expression reagent in regulating the development of plant glandular hairs, characterized in that: The nucleic acid sequence of the gene NtTRY1 is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the gene NtTRY1 is shown in SEQ ID No.

2.

3. A method for regulating the development of plant glandular hairs, characterized in that: The method comprises the following steps: The nucleic acid sequence of the gene NtTRY1 according to claim 1 is amplified, connected to an expression vector, transferred into Agrobacterium, and transformed into plants to obtain plants overexpressing the gene.

4. The method for regulating the development of plant glandular hairs according to claim 3, characterized in that: The method comprises the following steps: (1) amplifying the gene NtTRY1 whose nucleotide sequence is shown in SEQ ID No. 1; (2) cloning the gene NtTRY1, ligating it into an expression vector after enzyme digestion and purification, and obtaining an overexpression recombinant vector after identification; (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) Transforming the overexpression recombinant strain into plants, screening and obtaining NtTRY1 transgenic strains, so as to regulate the development of glandular hairs in plants.

5. The method for regulating the development of plant glandular hairs according to claim 4, characterized in that: In step (1), the amplification primers include SEQ ID No. 3 and SEQ ID No. 4; Preferably, in step (1), the amplification procedure includes: Pre-denaturation: 95-99°C, 20-40s; Denaturation: 95-99°C, 10-20s; Annealing extension: 52-66°C, 5-15s; 65-80°C, 40-60s; cycle 25-35 times; Extension: 65-80℃, 5-15min.

6. The method for regulating the development of plant glandular hairs according to claim 4 or 5, characterized in that: In step (2), the cloning vector includes pEASY-Blunt simple; Preferably, in step (2), the restriction enzyme cleavage site comprises any one of BamH I, Sac I, EcoR I, Nde I, Nco I, Pac I, Pst I, Sca I, Spe I, Xba I or Xho I, or a combination of at least two thereof; Preferably, the cloning vector comprises a promoter and a terminator.

7. The method for regulating the development of plant glandular hairs according to any one of claims 4 to 6, characterized in that: In step (2), the expression vector includes pCambia1300; Preferably, in step (2), the purification method comprises a silica gel chromatography column method and / or a magnetic bead method; Preferably, in step (2), the identification method includes whole-genome sequencing and / or PCR amplification.

8. The method for regulating the development of plant glandular hairs according to any one of claims 4 to 7, characterized in that: In step (3), the transformation method includes freeze-thaw method; Preferably, the Agrobacterium comprises one or a combination of at least two of GV3101, EHA101, EHA105 or LBA4404.

9. The method for regulating the development of plant glandular hairs according to any one of claims 4 to 8, characterized in that: In step (4), the transformation method includes Agrobacterium-mediated leaf disc transformation.

10. Use of the gene NtTRY1 described in claim 1 in breeding tobacco varieties with regulated glandular hair development.

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