Tobacco NtGA3ox1 gene and its application in regulating epidermal glandular hair density

Abstract

The present invention discloses a method for cultivating multi-glandular hair tobacco using gene editing technology. The present invention belongs to the field of plant genetic engineering technology, and specifically relates to a tobacco gibberellin 3-β-dioxidase gene NtGA3ox1 gene and its application in regulating tobacco glandular hair density. The tobacco NtGA3ox1 gene includes 1643bp, and the base sequence is shown in SEQ ID NO.1. The tobacco NtGA3ox1 gene, the amino acid sequence encoded by it is shown in SEQ ID NO.2. The glandular hair density of the tobacco NtGA3ox1 gene-edited plant of the present invention is significantly increased compared with the wild type. Targeting NtGA3ox1 using gene editing technology has great potential in the molecular regulation of tobacco glandular hair density and the directional improvement of glandular hair.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a tobacco NtGA3ox1 gene and an application thereof in regulating the density of epidermal glandular hairs. Background Art

[0002] Plant glandular hairs are specialized structures of plant epidermal cells. They can be divided into two types: non-secretory and secretory, according to their secretory behaviors. Non-secretory epidermal hairs are single-cell or multi-cell epidermal hairs that do not have the ability to secrete, while secretory glandular hairs have the ability to synthesize and secrete a variety of compounds. The compounds synthesized and secreted by secretory glandular hairs, represented by Artemisia annua and mint, include polysaccharides, organic acids, proteins, terpenes, alkaloids, and polyphenols. These compounds give plants special smells, uses, and considerable economic value. In essence, whether they are secretory or non-secretory glandular hairs, their existence is an evolutionary result of the plant's own adaptation to external environmental stimuli and a way for plants to communicate with the external environment.

[0003] Tobacco glandular hairs are specialized structures of tobacco epidermal cells, which have an important influence on tobacco plant resistance and tobacco leaf quality. Tobacco glandular hairs are abundant and diverse. Ceramidine diterpenes and sugar ester compounds are the main components of tobacco glandular hair secretions. These components are not only important aroma precursors, but also have obvious repellent effects on aphids. Therefore, increasing the density of tobacco glandular hairs is one of the important ways to improve tobacco resistance and improve tobacco leaf quality. The density of tobacco glandular hairs is affected by many conditions such as light, temperature, moisture, soil, fertilizer and ecological environment, but genetic factors still play a decisive role in the density of tobacco glandular hairs. The density of glandular hairs and the content and components of secretions in different tobacco types, varieties, leaf positions and leaf development periods are quite different, which affects the quality and style of tobacco leaves to a certain extent. Cultivating tobacco varieties with high glandular hair density and high secretion is one of the important goals of conventional tobacco breeding. Therefore, how to effectively regulate the factors affecting the development of glandular hairs and make effective use of them has very important research theoretical significance and practical value for improving tobacco quality. Summary of the invention

[0004] The purpose of the present application is to provide a tobacco gibberellin 3-β-dioxygenase gene, tobacco NtGA3ox1 gene, and the tobacco NtGA3ox1 protein encoded by it, as well as its application in regulating the density of epidermal glandular hairs, which has very important application value.

[0005] The technical solution of the present invention is as follows:

[0006] The first aspect of the present invention discloses a tobacco NtGA3ox1 gene, which comprises 1643 bp and a base sequence as shown in SEQ ID NO.1.

[0007] Preferably, the tobacco NtGA3ox1 gene encodes the NtGA3ox1 protein.

[0008] Preferably, the amino acid sequence of the encoded NtGA3ox1 protein is as shown in SEQ ID NO.2.

[0009] The second aspect of the present invention discloses the application of the tobacco NtGA3ox1 gene in tobacco.

[0010] Preferably, the plants after gene editing of the NtGA3ox1 gene have more epidermal glandular hairs and a higher density of glandular hairs than the control plants.

[0011] Preferably, the gene-edited plant is a tobacco plant in which the NtGA3ox1 gene is edited, after genetic transformation, by constructing a CRISPR / Cas9 editing vector for knocking out the NtGA3ox1 gene through CRISPR / Cas9-mediated gene editing technology.

[0012] Preferably, the recombinant CRISPR / Cas9 expression vector used to knock out the tobacco NtGA3ox1 gene significantly reduces the expression level of tobacco NtGA3ox1 in the transformed plants or eliminates the expression.

[0013] Preferably, the tobacco plants after gene editing have a significantly delayed flowering period and a significantly reduced plant height compared to the control tobacco.

[0014] The application of the tobacco NtGA3ox1 gene of the present invention in regulating the density of epidermal glandular hairs comprises the following steps: cloning the tobacco NtGA3ox1 gene to obtain a tobacco epidermal glandular hair density regulating gene; using gene editing technology to obtain a gene editing vector with a functional deletion of the tobacco NtGA3ox1 gene, thereby increasing the number of epidermal glandular hairs and the density of glandular hairs; and at the same time, the plant height of the gene-edited plant is significantly shortened and the flowering time is significantly prolonged.

[0015] Compared with the prior art, the beneficial effect of the present invention is that the tobacco NtGA3ox1 gene is used to effectively regulate the density of epidermal glandular hairs of tobacco. Using gene editing technology to target NtGA3ox1 has great potential in the molecular regulation of tobacco glandular hair density and the directional improvement of glandular hairs, and has very important research theoretical significance and practical value for cultivating high glandular hair density and improving tobacco quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the electrophoresis pattern of the NtGA3ox1 gene clone provided in the embodiment of the present invention.

[0017] Figure 2 Comparison of glandular hair density between the control sample of non-gene-edited tobacco plants (left) and the gene-edited tobacco plants (right)

[0018] Figure 3 Microscope photos of marginal glandular hairs of a non-gene-edited tobacco plant control (left) and a gene-edited tobacco plant (right)

[0019] Figure 4 Overall phenotypic observation of the control tobacco plants without gene editing (left) and the gene-edited tobacco plants (right). DETAILED DESCRIPTION

[0020] The present application is further explained below with reference to the embodiments.

[0021] Before introducing specific embodiments, some biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments are briefly introduced as follows.

[0022] Biomaterials:

[0023] Tobacco variety: Honghua Dajinyuan, a commercial tobacco. The seeds used in the examples were provided by the National Tobacco Gene Research Center.

[0024] Vector: pEASY-T1 Simple vector, purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0025] The CRISPR / Cas9 vector was kindly provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University.

[0026] Strains:

[0027] Trans1-T1 chemically competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0028] LBA4404 Agrobacterium strain, a commonly used strain in biological experiments, is publicly available;

[0029] Primer synthesis and DNA sequencing were provided by Beijing Liuhe BGI Genomics Co., Ltd.;

[0030] Experimental reagents:

[0031] RNA extraction kit, SuperPure Plant polyRNA Kit;

[0032] Fluorescence quantitative PCR enzyme (SYBR qPCR kit) was purchased from Zhengzhou Ansai Biotechnology Co., Ltd.;

[0033] Reverse transcription kit and T4 ligase were purchased from Takara Biotechnology (Dalian) Co., Ltd.;

[0034] Restriction endonuclease BsaI was purchased from NEB;

[0035] DNA amplification enzyme was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0036] Plant genome extraction kit and DNA purification kit were purchased from QIAGEN.

[0037] Experimental equipment:

[0038] PCR synthesizer TProfessional Thermocycler, Biometra;

[0039] Quantitative PCR instrument CFX96, Bio-Rad;

[0040] UV gel imaging system BioSpectrum, UVP company.

[0041] Example 1

[0042] This example briefly introduces the cloning process of the NtGA3ox1 gene as follows.

[0043] (1) Preparation of cDNA as cloning template

[0044] Take 100 mg of stems of tobacco (Honghua Dajinyuan) in vigorous growth period as samples, grind them thoroughly in liquid nitrogen, extract total RNA according to the instructions of RNA extraction kit, and then reverse transcribe it into cDNA for later use;

[0045] (2) Design primers and perform PCR amplification

[0046] The primer sequences designed for amplifying the tobacco NtGA3ox1 gene are as follows:

[0047] NtGA3ox1-F:5'-ATGCCTTCAAGAATCTCAGA-3' (SEQ ID No. 3),

[0048] NtGA3ox1-R:5′-TTAGCCAACTTGGACTCTATTA-3′ (SEQ ID No. 4);

[0049] The cDNA prepared in step (1) was used as a template and PCR amplification was performed using the above primers. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 30 s, 35 cycles, and complete extension at 72°C for 10 min after the cycle. The PCR amplification was stored at 4°C for future use, or directly subjected to electrophoresis detection and analysis.

[0050] Purify the PCR amplification product according to the instructions of the gel recovery kit, and then connect the purified product to the pEASY-T1 vector. The connection system is as follows:

[0051] DNA amplification product, 6 μL;

[0052] pEASY-T1 vector, 1 μL;

[0053] After mixing, connect at 25°C for 25 min.

[0054] The ligation product was transformed into E. coli competent cells. The specific transformation process is briefly described as follows:

[0055] Take out the competent cells from the -80℃ refrigerator, place them on ice to dissolve, add the ligation product to 50μL of Trans1-T1 competent cells, flick to mix, and place on ice for 30min;

[0056] Heat shock in a 42°C water bath for 30 seconds, and immediately place on ice for 2 minutes; add 250 μL of LB (without antibiotics) equilibrated to room temperature, and culture at 37°C with shaking for 1 hour;

[0057] Take 8 μL of 500 mM IPTG and 40 μL of 20 mg / mL X-gal, mix them and evenly spread them on a LB solid plate (containing 60 μg / μL ampicillin), invert the culture dish, and culture at 37°C overnight.

[0058] After the white spots were picked and amplified and cultured, the plasmid DNA was extracted, the recombinant plasmid was identified by plasmid PCR amplification, and the corresponding positive clones were sent for sequencing to obtain the NtGA3ox1 gene sequence.

[0059] The sequencing analysis results showed that the length of the coding region of the NtGA3ox1 gene was 1643 bp nucleotides, as specifically shown in SEQ ID NO.1; after analyzing the gene, it was found that the amino acid sequence of the NtGA3ox1 protein encoded by it was shown in SEQ ID NO.2. Figure 1 The electrophoresis pattern of the NtGA3ox1 gene clone provided in this example. Figure 1 It can be seen that the PCR product was detected by agarose gel electrophoresis, and a PCR band of about 1000 bp was obtained. The sequencing results showed that the full length of the protein coding region (CDS) of the tobacco NtGA3ox1 gene was 1092 bp.

[0060] Example 2

[0061] Using the tobacco NtGA3ox1 gene obtained in Example 1, a CRISPR / Cas9 vector was further constructed, and gene-edited plants were obtained by leaf disc transformation.

[0062] A more specific 23nt nucleotide sequence (SEQ ID No. 5) in the NtGA3ox1 gene was selected as the guide sequence of CRISPR / Cas9, and the knockout primer sequences GA3ox1-KF and GA3ox1-KR were designed as follows:

[0063] GA3OX1-KF: 5'-GATTGAATTACCCGAATCCCATGCA-3' (SEQ ID No. 6), GA3OX1-KR: 5'-AAATGCATGGGATTCGGGTAATT-3' (SEQ ID No. 7);

[0064] The above primer sequences were used to obtain the double-stranded DNA (annealed) of the target site. The obtained double-stranded DNA (annealed product) of the target site was connected to the CRISPR / Cas9 vector after BsaⅠ digestion, and transformed, screened, and PCR amplified and tested. The PCR-positive clones were sent to a sequencing company for sequencing confirmation, and finally the CRISPR / Cas9-NtGA3ox1 editing vector was obtained.

[0065] Using the CRISPR / Cas9-NtGA3ox1 editing vector plasmid constructed in the previous step, taking Honghua Dajinyuan as an example, genetic transformation and tissue culture were performed to obtain plants with knockout editing of the tobacco flowering time-related gene NtGA3ox1. The relevant experimental process is briefly described as follows:

[0066] After the surface of tobacco seeds is disinfected, they are planted on MS medium. When they grow to 4 cotyledons (15-20 days), they are transferred to culture bottles containing MS solid medium and kept at 25±1℃ and light intensity of 30-50μmol / (m 2 ·s), and continue culturing for 35-40 days under the condition of 16h / d light intensity, and set aside.

[0067] Take out the LBA4404 electroporation competent Agrobacterium cells stored at -80℃ and freeze-thaw on ice. When the competent cells are just thawed, add 2μL of CRISPR / Cas9-NtGA3ox1 editing vector plasmid, mix well, and place on ice. Then transfer the mixed competent cells to the pre-cooled electroporation cup, place the electroporation cup in the electroporator for transformation, add 1mL of YEB liquid medium and mix with the transformation liquid after the transformation is completed, and then place it on a shaker at 28℃ and 200rpm for 1.5-2h. Centrifuge the cells at 8000rpm and discard the supernatant medium, then suspend the cells with 200μL of YEB liquid medium, apply it on YEB solid medium containing 50mg / L rifampicin, 50mg / L streptomycin and 50mg / L kanamycin, and culture it in the dark at 28℃ for 2-3d.

[0068] In the clean bench, a tobacco leaf disc was made into a square leaf disc with a side length of 1 cm, and the Agrobacterium colonies containing the CRISPR / Cas9-NtGA3ox1 editing vector were prepared into a suspended bacterial solution (OD600 = 0.6-0.8) using MS liquid. The tobacco leaf disc was soaked and infected with the suspended Agrobacterium bacterial solution for 10 minutes. The leaf disc was then placed on an MS solid medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA, 28 ° C, dark, and co-cultured for 3 days. After subculture, it was placed on a medium containing 2.0 mg / L NAA + 0.5 mg / L 6-BA + 250 mg / L Cb + 50 mg / L Kan's MS solid medium, culture conditions are: 28 ℃ light culture 16h / d, light intensity 30-50μmol / (m2·s), 25 ℃ dark culture 8h / d, culture 45-60d, until differentiation buds are formed, change the differentiation culture medium every 7-10d, change 3-4 times; culture until differentiation buds are formed; cut the callus with differentiation buds, place it on MS medium containing 500mg / L carbenicillin and 50mg / L kanamycin, and wait for the differentiation buds on the callus to grow. The plants were cultured to 2-4 cm in height, and the culture conditions were consistent with the differentiation culture conditions for 8-14 days; the regenerated plants were cultured for rooting, and the differentiated buds were cut off and inserted into MS medium containing 500 mg / L carbenicillin and 50 mg / L kanamycin for rooting culture. The culture conditions were consistent with the differentiation culture conditions for 20-30 days. The plants were regenerated and transplanted into flower pots for culture, and then the leaves of the transformed plants were sampled and sent to BGI for molecular detection to determine that the NtGA3ox1 gene-edited plants were obtained, and then the seeds of the T0-generation edited plants were harvested. The T0-generation seeds were self-pollinated and homozygous at 23 times. When the plants grew to 5-6 leaves, the leaves of the individual plants were sampled and sent to BGI for molecular detection to determine that the plants with homozygous editing of the NtGA3ox1 gene were obtained, and then the seeds of the T1-generation plants with homozygous editing of the NtGA3ox1 gene were harvested.

[0069] The application of the tobacco NtGA3ox1 gene of the present invention is to reduce the expression of the NtGA3ox1 gene in tobacco plants, and the gene-edited plants have more epidermal glandular hairs and increased glandular hair density compared to the control plants; at the same time, the gene-edited plants are significantly shorter in height and have a significantly longer flowering time. The commonly used methods for reducing gene expression or gene silencing in the prior art are applicable to the present invention.

[0070] Example 3

[0071] Using the plants identified as homozygous knockouts of the NtGA3ox1 gene by molecular detection in Example 2, the differences between the gene-edited materials and the wild type were obtained through leaf and overall phenotypic analysis. Figure 2Comparison of leaf glandular hair density between the control sample of non-gene-edited tobacco plants (left) and the gene-edited tobacco plants (right); Figure 3 Microscope photos of marginal glandular hairs of a non-gene-edited tobacco plant control (left) and a gene-edited tobacco plant (right); Figure 4 The overall phenotype of the control tobacco plant without gene editing (left) and the gene editing tobacco plant (right). Figure 2 , Figure 3 , Figure 4 It can be seen that the number of epidermal glandular hairs and the density of glandular hairs of NtGA3ox1 gene-edited tobacco plants increased; at the same time, the plant height of the gene-edited plants was significantly shortened and the flowering time was significantly prolonged.

[0072] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

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Claims

1. Use of a reagent for knocking out tobacco NtGA3ox1 gene in tobacco, characterized in that: The density of epidermal glandular hairs of plants after gene editing of NtGA3ox1 gene is increased compared with that of control plants; the plants after gene editing are constructed by CRISPR / Cas9-mediated gene editing technology, and a CRISPR / Cas9 editing vector for knocking out NtGA3ox1 gene is constructed, and tobacco plants with edited NtGA3ox1 gene are obtained after genetic transformation; the recombinant CRISPR / Cas9 expression vector for knocking out the tobacco NtGA3ox1 gene significantly reduces or eliminates the expression of tobacco NtGA3ox1 in the transformed plants; The base sequence of the tobacco NtGA3ox1 gene is shown in SEQ ID NO.1; the amino acid sequence of the NtGA3ox1 protein encoded by the tobacco NtGA3ox1 gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: Compared with the control tobacco, the flowering period of the gene-edited tobacco plants was significantly delayed and the plant height was significantly reduced.

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