CN110964740B

CN110964740B - Preparation method and application of tobacco with high flavonol content

Info

Publication number: CN110964740B
Application number: CN201911257021.0A
Authority: CN
Inventors: 贾宏昉; 宋朝鹏; 张洪映; 罗勇
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2021-11-23
Anticipated expiration: 2039-12-10
Also published as: CN110964740A

Abstract

The invention belongs to the technical field of plant genetic engineering, in particular to a preparation method and application of high flavonol tobacco. The steps are: constructing a plant expression vector of tobacco gene NtMYB12, infecting wild-type tobacco through Agrobacterium-mediated transformation, and obtaining NtMYB12-Oe transgenic homozygous line through cultivation and screening, which is high flavonol tobacco. The present invention uses tobacco Yunyan 87 and NtMYB12 overexpression transgenic plants to analyze the flavonol content of plants and the expression levels of genes involved in flavonol synthesis under low phosphorus stress conditions. The analysis shows that the NtMYB12 gene belongs to the MYB transcription factor PFG type member, and can pass the flavonoids. The transcriptional regulation of the alcohol metabolism pathway gene achieves its positive regulation of the pathway, thereby significantly increasing the content of flavonols and improving the plant's ability to tolerate low phosphorus stress.

Description

Preparation method and application of tobacco with high flavonol content

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a preparation method and application of high flavonol tobacco.

Background

Flavones are widely distributed secondary metabolites in plants, and generally have a C6-C3-C6 carbon skeleton. The most common flavonoids in plants are anthocyanins and flavonols. The flavone biosynthesis genes are transcriptionally regulated by Transcription Factors (TFs) of the MYB family. Several flavonoid-related MYB TFs are currently found in arabidopsis thaliana and woody plants such as grapes, apples and pears. Studies have shown that three subgroups in the MYB family are typical activators of the anthocyanin, flavanol and flavonol pathways, respectively. MYBs OF the protein OF ANTHOCYANIN PIGMENT (PAP) type are positive regulators OF ANTHOCYANIN synthesis; MYBs of type TRANSPARENT TESTA 2 (TT2) are positive regulators of the flavanol pathway; MYB TFs OF the protein OF FLAVONOL GLYCOSIDES (PFG) type, identified in Arabidopsis as positive regulators OF the FLAVONOL pathway. These PFG-type TFs activate the expression of genes encoding CHS, CHI and FLS.

Flavonols in plants have multiple biological functions, of which antioxidant capacity is a common feature of flavonols and most other flavonoids, and can help plants to resist various irritants of the environment. Phosphorus is an important plant nutrient element, but the effectiveness of the phosphorus in soil is very low, so that the concentration of the available phosphorus absorbable by the root system of the plant in the soil solution is usually only 2-10 mu M, and the growth requirement of the plant can not be met. Although the application of phosphate fertilizer can improve the utilization rate in the season, the phosphate rock resource is not renewable, and the application of excessive phosphate fertilizer causes soil hardening and environmental pollution. Therefore, improving the absorption and utilization of phosphorus by plants is an important research direction in the development of modern agriculture. In recent years, studies on the participation of MYB transcription factors in plant flavone metabolism have been reported, but key genes and control mechanisms for regulating flavone synthesis and nutrient metabolism in plants are still unclear.

The tobacco is an important leaf economic crop, and the high flavonol tobacco can improve the low phosphorus stress resistance of the tobacco, thereby reducing the use of phosphate fertilizer, saving production cost and protecting the environment; meanwhile, the flavonol also has the health-care effects of removing free radicals, enhancing the oxidation resistance of the human body and delaying the aging of the human body in the human body, so that a new thought and a new method are provided for the multipurpose development of the tobacco leaves by extracting the flavonol in the tobacco leaves for the biological pharmacy. In summary, the cloning, functional analysis and application research of key genes for regulating and controlling flavonol synthesis and nutrition metabolism in tobacco leaves are particularly urgent.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method and application of high flavonol tobacco.

The technical scheme of the invention is realized as follows:

a preparation method of tobacco with high flavonol comprises the following steps: constructing a plant expression vector of a tobacco gene NtMYB12, infecting wild tobacco through agrobacterium-mediated transformation, and obtaining a NtMYB12-Oe (NtMYB 12 overexpression) transgenic homozygous line, namely the high flavonol tobacco, through cultivation and screening.

The sequence of the NtMYB12 is shown in SEQ ID No. 1.

The plant expression vector is provided with a 35S strong promoter.

The wild type tobacco is Yunyan 87.

The high flavonol tobacco prepared by the method is applied to culturing low phosphorus stress resistant tobacco plants.

The invention has the following beneficial effects:

according to the invention, the plant flavonol content and the gene expression level participating in flavonol synthesis under the condition of low phosphorus stress are analyzed by utilizing tobacco yunyan 87 and NtMYB12-Oe transgenic plants, and the NtMYB12 gene is known to belong to a MYB transcription factor PFG type member through analysis, so that the forward regulation of the pathway can be realized through the transcription regulation of flavonol metabolic pathway genes, the content of the flavonol is obviously increased, and the low phosphorus stress resistance of the plant is improved.

The NtMYB12-Oe transgenic homozygous line tobacco obtained by the transgenic overexpression method has obviously improved tolerance to low phosphorus stress. Through the analysis, the invention shows that the expression levels of NtMYB12 overexpression tobacco plants can be increased by up-regulating the expression levels of NtCHS, NtCHI, NtF3H and NtFLS, so that the content of flavonol in tobacco leaves is obviously increased by 12 times compared with that of Wild Tobacco (WT), the low-phosphorus stress tolerance of plants is improved, and the tobacco plants can maintain normal growth and development under the condition of low phosphorus or even no phosphorus.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a sequence arrangement and phylogenetic analysis of NtMYB 12.

FIG. 2 is the subcellular localization of NtMYB12 in tobacco epidermal cells.

FIG. 3 is the expression pattern of NtMYB12 in different parts of tobacco under phosphorus stress and the effect on expression of NtMYB12 provided by examples of the invention.

FIG. 4 is a phenotypic characteristic and NtMYB12 expression level of wild type tobacco (WT) and NtMYB12-Oe transgenic tobacco under phosphorus stress as provided by an embodiment of the invention.

FIG. 5 is a graph of the effect of wild type tobacco (WT) and NtMYB12-Oe transgenic tobacco antioxidant enzymes under phosphorus stress as provided by an embodiment of the invention.

FIG. 6 is a graph showing the effect of total phosphorus content, Pi content and expression of two members of the tobacco Pht1 gene family in wild type tobacco (WT), NtMYB12-Oe transgenic tobacco under phosphorus stress as provided by the examples of the present invention.

FIG. 7 is a study of the flavonoid content and the expression levels of major genes encoding flavonoid biosynthetic enzymes in WT, NtMYB12-Oe plants under phosphorus stress as provided in the examples of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Examples

Aiming at the problems that NtMYB12 is not cloned and expressed in the prior art, and the specific regulation and control function of the NtMYB12 gene is not clear; the invention clones NtMYB12 gene from tobacco yunyan 87, constructs an overexpression vector of the NtMYB12 gene and carries out genetic transformation to obtain an NtMYB12 overexpression transgenic plant, analyzes the flavonol content of the plant and the expression level of the gene participating in the synthesis of the flavonol under the low-phosphorus stress condition by utilizing the tobacco yunyan 87 and NtMYB12 overexpression transgenic plants, and can know that the NtMYB12 gene belongs to a MYB transcription factor PFG type member through analysis, realize the forward regulation of the flavonol metabolic pathway through the transcription regulation of the flavonol metabolic pathway gene, and improve the low-phosphorus stress resistance of the plant.

The sequence of the tobacco MYB transcription factor NtMYB12 is SEQ ID NO. 1.

The cloning method of the tobacco MYB transcription factor NtMYB12 specifically comprises the following steps:

the first step is as follows: extracting total RNA of tobacco leaves; extracting total RNA by taking young leaves of Yunyan 87 as a material according to a method provided by a plant total RNA extraction kit of Tiangen Biochemical technology (Beijing) Co., Ltd;

the second step is that: obtaining tobacco leaf total cDNA and genome total DNA; 1ug of a total RNA sample of the young leaf of the Yunyan 87 is taken as a template, and Oligo dT-Adaptor Primer is adopted for reverse transcription, and a product after the reverse transcription is total cDNA; extracting total DNA of the tobacco genome by adopting EasyPure Plant Genomic DNA Kit of Beijing all-purpose gold biotechnology limited according to a reference instruction;

in a preferred embodiment of the invention, the reverse transcription conditions are: 40min at 42 ℃, 30min at 50 ℃, 5min at 99 ℃ and 5min at 5 ℃.

The third step: designing tobacco NtMYB12 gene cloning primers; obtaining a coding region sequence and a genome DNA sequence of a NtMYB12 gene through phylogenetic analysis of a tobacco MYB gene family, comparing polypeptide sequences by using DNAMAN V6.0 software, selecting a specific site of the NtMYB12 gene for primer design, and designing a primer NtMYB12-F sequence which is shown as SEQ ID No.2 and is constructed by amplification of a full-length cDNA and a genome DNA sequence of the NtMYB12 gene and a eukaryotic expression vector; the sequence of NtMYB12-R is shown as SEQ ID No. 3;

in a preferred embodiment of the invention, the eukaryotic expression vector is NtMYB12-pCAMBIA 1305.

The fourth step: PCR amplification of the tobacco NtMYB12 gene. 1 mu L of each of the first strand of the cDNA of the young tobacco leaves and the total DNA of the genome is taken as a template, and a standard 50 uLPCR reaction system is adopted to amplify the full-length CDNA and the genome DNA sequence of the NtMYB12 gene by Phusion ultra-fidelity DNA polymerase;

in a preferred embodiment of the invention, the parameters for PCR amplification of cDNA are as follows: pre-denaturation at 94 ℃ for 4min, 35 cycles, and heat preservation at 72 ℃ for 10 min. When the genome DNA is amplified, the extension time is increased to 3 min;

the cycle process comprises denaturation at 94 deg.C for 1min, annealing at 58 deg.C for 1min, and extension at 72 deg.C for 2 min.

The construction method of the over-expression vector provided by the embodiment of the invention specifically comprises the following steps:

and (3) constructing and testing an overexpression vector. To construct the over-expression vector NTMYB12-pCAMBIA1305, the entire coding sequence (CDS) of NTMYB12 was amplified using primers. The PCR product was digested with SacI/KpnI and cloned into SacI/KpnI digested pCAMBIA1305 plasmid under the control of CaMV35S promoter. Selecting positive clones to carry out PCR identification on bacteria liquid, sending positive clones to sequence, verifying that the sequence is correct, and successfully constructing the vector;

the real-time fluorescent quantitative PCR detection primers for NtCHS, NtCHI, NtF3H and NtFLS are designed and shown in Table 1, and the expression changes of the four genes of NtCHS, NtCHI, NtF3H and NtFLS in the transgenic plants are detected and shown in FIG. 7.

TABLE 1

Tobacco (Nicotianatabacum cv, Yunyan 87) leaf RNA extraction and cDNA synthesis

Extracting tobacco Yunnan 87 leaf RNA by TRIZOL Plus, and taking 1 mu g of total RNA to perform reverse transcription to obtain a cDNA template.

Separation and sequence analysis of

NtMYB12(GenBank accession XM-016624824) and the full-length coding sequence (CDS) of the promoter were cloned from tobacco Yunyan 87, and specific primers were designed from the sequence data in the tobacco genomic database (Table 1). Polypeptide sequences were aligned using Dnaman software Version 6 and phylogenetic analysis was performed using mega v5.0 software. The results show that NtMYB12 is a MYB transcription factor of PFG type (FIG. 1), indicating that NtMYB12 may be involved in the regulation of flavonol biosynthesis in tobacco.

Subcellular localization in tobacco epidermal cells

The entire coding sequence (CDS) of NtMYB12 was amplified, and the N-terminal Green Fluorescent Protein (GFP) fusion vector pMDC43-NtMYB12 was constructed by LR reaction (Gateway R Technology with clone R II reagent), which was transiently introduced into tobacco epidermal cells by injection. An N-terminal GFP fusion vector driven by a cauliflower mosaic virus 35S promoter is constructed, and the vector is used for infecting tobacco epidermal cells as control cells. After transformation, tobacco epidermal samples were stored in a dark room at 25 ℃ for 16h, and then observed by imaging GFP fluorescence with a confocal laser scanning microscope (Nikon C2-ER). The results showed that the fusion protein was restricted to the nucleoplasm, whereas in control transgenic cells expressing p 35S:gfp, GFP signal was detected throughout the cells (fig. 2), confirming NTMYB12 as a regulator.

Determination that NTMYB12 is regulated by absence of Pi

The promoter sequence of NTMYB12 in tobacco was analyzed using plantarcae. Wild Type (WT) tobacco seeds were sterilized, sown to 1/2MS solid medium, cultured for 30 days, transferred to pots containing quartz sand, cultured for 7 days using nutrient solution supplemented with 0.02mM Pi (LP), and tested for expression of NtMYB12 in tobacco roots, stems, leaves, and flowers. The results indicate that NtMYB12 may play a regulatory role in tobacco response and adaptation to low Pi levels (fig. 3).

Obtaining NtMYB12 overexpression transgenic tobacco plants

The CDS of NtMYB12 was amplified from tobacco leaves using specific primers in figure 1 for overexpression of NtMYB 12. The PCR product was ligated into the pCAMBIA1305 vector driven by the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase terminator. The construct was transferred to Agrobacterium tumefaciens strain EHA105 by electrophoresis and transformed into tobacco (Nicotianatabacum cv, Yunyan 87). By introducing the NtMYB12 overexpression construct, 20 transgenic tobacco lines were obtained. Two independent transgenic lines (designated Oe1 and Oe2) were confirmed and used in this study (FIG. 4).

And verifying the tolerance of the NtMYB12 overexpression transgenic tobacco to low phosphorus stress. Wild type tobacco (WT) seeds and NtMYB12-Oe transgenic tobacco seeds (T2 generation) were sterilized, sown in 1/2MS solid medium, cultured for 10 days, transferred to pots containing quartz sand, and cultured for 21 days each using a nutrient solution supplemented with 1mM Pi (HP) and 0.02mM Pi (LP) by introducing the NtMYB12 overexpression structure.

The following studies were performed on wild type and transgenic line samples:

1. study of phenotypic characteristics of tobacco (fig. 4A, 4B); it was shown that under sufficient phosphorus supply conditions (HP), the phenotype of the NtMYB12-Oe transgenic plants was not significantly different from that of wild-type tobacco (WT) plants, and that under low phosphorus conditions (LP), the growth vigor of the NtMYB12-Oe transgenic plants was significantly better than that of the WT plants.

2. The expression level of NtMYB12 was determined by qRT-PCR method (fig. 4C); the relative expression level of the NtMYB12 gene of the NtMYB12-Oe transgenic plant under different phosphorus supply treatment conditions is obviously higher than that of the WT plant.

3. Biomass, root-to-crown ratio, longest root length were determined (fig. 4D,4E, 4F); it was shown that under low phosphorus conditions (LP), the biomass of NtMYB12-Oe transgenic tobacco was significantly higher than that of WT plants, with a difference in root-cap ratio, longest root length, and WT plants, but not significant.

4. NBT staining, determination of SOD, CAT, MDA content (figure 5); it was shown that by NBT staining, a large amount of ROS accumulation was detected in the leaves of WT tobacco plants, but not in the NtMYB12-Oe transgenic plants. Under the condition of Low Phosphorus (LP), the SOD and CAT activities of the NtMYB12-Oe transgenic tobacco are obviously higher than that of a WT plant, the MDA content is obviously lower than that of the WT plant, and the antioxidant capacity of the plant is obviously improved by the over-expression of the NtMYB12 in the tobacco.

5. Determining the total phosphorus content and the expression level of the Pht1 gene family (fig. 6); the total phosphorus (total P) content, inorganic phosphorus (Pi) content and relative expression of Pht1 gene families NtPT1 and NtPT2 of the NtMYB12-Oe transgenic tobacco are obviously higher than those of a WT plant under the condition of Low Phosphorus (LP).

6. Measuring the flavonol content and the expression levels of the genes NtCHS, NtCHI, NtF3H and NtFLS involved in flavone biosynthesis (FIG. 7); shows that: under sufficient phosphorus supply conditions (HP), the flavonol content of the WT plant is 0.0644 mg/g, the flavonol content of the NtMYB12-Oe transgenic lines Oe1 and Oe2 is 0.8001 mg/g and 0.9215 mg/g respectively, and the flavonol content of the Oe1 and Oe2 transgenic lines is 12.42 times and 14.31 times that of the WT plant respectively; under low phosphorus conditions (LP), the flavonol content of WT plants was 0.5093 mg/g, the flavonol content of NtMYB12-Oe transgenic lines Oe1 and Oe2 were 1.0391 mg/g and 1.1432 mg/g, respectively, and the flavonol content of Oe1 and Oe2 transgenic lines were 2.04 times and 2.24 times higher, respectively, than that of WT plants. Meanwhile, under the condition of sufficient phosphorus supply (HP), the relative expression amounts of related genes involved in flavone biosynthesis of Oe1 and Oe2 transgenic lines are significantly higher than that of a WT plant, wherein the relative expression amounts of NtCHS in the Oe1 and Oe2 transgenic lines are respectively 25.34 times and 27.52 times of that of the WT plant, the relative expression amounts of NtCHI are respectively 7.42 times and 6.84 times of that of the WT plant, and the relative expression amounts of NtFLS are respectively 15.25 times and 16.15 times of that of the WT plant.

The results show that NtMYB12 up-regulates the expression levels of NtCHS, NtCHI, NtF3H and NtFLS, leading to the accumulation of flavonols in tobacco leaves; overexpression of NtMYB12 improves Pi intake and enhances the tolerance of tobacco to low phosphorus stress; provides a basis for the cultivation technology of the tobacco with high flavonol and the comprehensive utilization of the tobacco leaves.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

<110> Henan university of agriculture

<120> preparation method and application of tobacco with high flavonol content

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Claims

1. A preparation method of tobacco with high flavonol content is characterized by comprising the following steps: constructing a plant expression vector of a tobacco gene NtMYB12, infecting wild tobacco through agrobacterium-mediated transformation, obtaining a NtMYB12-Oe transgenic homozygous line which is the high flavonol tobacco through cultivation and screening, wherein the sequence of the NtMYB12 is shown in SEQ ID No.1, and the wild tobacco is Yunyan 87.

2. The method of preparing tobacco high in flavonol according to claim 1, wherein the step of: the plant expression vector is provided with a 35S strong promoter.

3. Use of the high flavonol tobacco prepared by the method of claim 1 or 2 as a plant for cultivating low phosphorus stress resistant tobacco.