CN111349634B

CN111349634B - Betula platyphylla BpNAC100 gene and amino acid sequence and application thereof

Info

Publication number: CN111349634B
Application number: CN202010226165.6A
Authority: CN
Inventors: 方功桂; 李天芳; 姜静; 刘桂丰
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2020-03-27
Filing date: 2020-03-27
Publication date: 2021-04-06
Anticipated expiration: 2040-03-27
Also published as: CN111349634A

Abstract

The invention discloses a birch BpNAC100 gene and an amino acid sequence and application thereof, wherein the application of the BpNAC100 gene in the response of salt stress of the birch is realized, the nucleotide sequence of the BpNAC100 gene is shown as SEQ ID NO 1 in a sequence table, and the sequence table of amino acid is shown as SEQ ID NO 2 in the sequence table; according to the invention, an agrobacterium-mediated method is utilized to respectively overexpress and inhibit and express a BpNAC100 gene and a promoter thereof in white birch, proBpNAC100, a Luc transgenic white birch plant and BpNAC100, GFP and BpNAC100-RNAi transgenic white birch plants are obtained, the promoter of the BpNAC100 gene has spatial expression specificity in different tissue parts of the white birch, and the BpNAC100, GFP transgenic white birch plant has salt damage resistance.

Description

Betula platyphylla BpNAC100 gene and amino acid sequence and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a birch BpNAC100 gene and an amino acid sequence and application thereof.

Background

NAC (NAM, ATAF1, ATAF2 and CUC2) transcription factors are plant-specific transcription factors, widely present in various terrestrial plants and having various biological functions, 117 and 151 NAC genes in Arabidopsis (Arabidopsis thaliana) and rice (Oryzasative), respectively, and many NAC genes in other species (Pereira-Santana et al, 2015). the N-terminal region of NAC protein is highly conserved, having 150-160 amino acid NAC domains, and the C-terminal region is a highly diverse transcription control region (TRR). the NAC transcription factors have a wide range of biological functions, including apical meristem formation and organ differentiation, cell division, hormone signal transduction, leaf senescence, flowering, lateral root formation, stress response and xylem formation.

White birch (Betulaplatyphulla) is one of the important broad-leaf timber species in China, and the white birch grows rapidly, is easy to reproduce and cultivate, has wide application and also has certain medicinal and ornamental values. Because the NAC transcription factor is a plant specific transcription factor with multiple biological functions, the research on the function and molecular mechanism of the NAC transcription factor in the white birch is very little, and the research on the function of the NAC100 gene in the white birch has important significance. Therefore, the invention provides a birch BpNAC100 gene, an amino acid sequence and application thereof, aiming at solving the defects in the prior art.

Disclosure of Invention

Aiming at the problems, the invention provides a birch BpNAC100 gene, an amino acid sequence and application thereof, the invention obtains a proppNAC 100 gene promoter by expressing the BpNAC100 gene promoter in the birch by utilizing an agrobacterium-mediated method, the promoter of the BpNAC100 gene has space expression specificity in different tissue parts of the birch, and the proppNAC 100 gene promoter has response to salt; the BpNAC100 gene is overexpressed and inhibited to express in the white birch by utilizing an agrobacterium-mediated method, a BpNAC100 GFP and BpNAC100-RNAi transgenic white birch plant is obtained, and the BpNAC100 gene is obtained, wherein the GFP transgenic white birch plant has salt damage resistance.

In order to realize the purpose of the invention, the invention is realized by the following technical scheme:

the invention provides an application of a BpNAC100 gene in the response of white birch to salt stress.

The application is the application on the salt resistance of the white birch, and the nucleotide sequence of the BpNAC100 gene is shown as SEQ ID NO. 1 in a sequence table.

The sequence table of the amino acid of the BpNAC100 gene is shown as SEQ ID NO:2 in the sequence table.

The sequence table of the promoter of the BpNAC100 gene is shown as SEQ ID NO. 3 in the sequence table.

The invention also provides a birch BpNAC100 gene obtaining and specificity expression analysis method, which comprises the following steps:

taking clone white birch of 1 year as a material, extracting DNA of the white birch as a cloning vector, and obtaining a BpNAC100 gene upstream 2000bp sequence as a target sequence according to a white birch genome;

designing a primer for cloning, connecting the target sequence to a cloning vector after tapping and recovering the target sequence, then transforming competent cells, and culturing;

selecting 3 monoclonal shake bacteria for sequencing, treating the 3 monoclonal shake bacteria by using restriction enzymes Eco RI and Bam HI, and then connecting a target band into a pCMBIA1300-Luc plant expression vector;

constructing a recombinant plasmid, transforming the recombinant plasmid into escherichia coli by using a heat shock method, transforming the escherichia coli into agrobacterium EHA105 by using an electrotransformation method, infecting a white birch zygotic embryo by using an agrobacterium-mediated method, and culturing the infected white birch zygotic embryo to obtain a proBpNAC100: Luc transgenic white birch plant;

step five, detecting proBpNAC100 by adopting a PCR amplification technology by taking the Luc gene upstream and downstream sequences as primers, wherein the Luc transgenic white birch plant;

sixthly, measuring the specific expression of the BpNAC100 in the ProBpNAC100: Luc transgenic white birch plant, and observing the fluorescent reaction of the ProBpNAC100: Luc transgenic white birch plant under the excitation wavelength of 328 nm;

step seven: IAA, GA, ABA, SA and NaCl solutions are respectively applied to the Luc transgenic white birch plants in the proBpNAC100 formula, spraying aqueous solution is used as a blank control, and the response of the Luc transgenic white birch plants in the proBpNAC100 formula to hormone and salt is observed.

The further improvement is that the specific process of culturing the competent cells in the second step is to coat the competent cells by a coating plate method and then to culture the competent cells at 37 ℃ overnight for 1 d.

The further improvement is that the white birch zygotic embryo after infection in the fourth step is cultured in the second 2 days, and then the white birch zygotic embryo is placed in a culture medium containing cefamycin and hygromycin for selective culture until a green callus grows at the incision of the zygotic embryo.

The further improvement is that the concentration of the cefuroxime in the step four is 200mg/L, and the concentration of the hygromycin is 50 mg/L.

The further improvement is that when specific expression of the ProBpNAC100 in the Luc transgenic white birch plant is measured in the sixth step, spraying D-Luciferin standard working solution on the ProBpNAC100 in the Luc transgenic white birch plant, observing the fluorescent reaction of the ProBpNAC100 in the Luc transgenic white birch plant at an excitation wavelength of 328nm, imaging by using a plant living body imaging system, outputting a pseudo-color photo, and analyzing Luc tissue expression characteristics driven by the ProBpNAC 100.

The invention has the beneficial effects that the BpNAC100 gene and the promoter thereof are respectively overexpressed and inhibited in the white birch by utilizing an agrobacterium-mediated method to obtain the propNAC 100 gene, the Luc transgenic white birch plant and the BpNAC100 gene, GFP and BpNAC100-RNAi transgenic white birch plant, the promoter of the BpNAC100 gene has space expression specificity in different tissue parts of the white birch, the BpNAC100 gene responds to salt, the GFP transgenic white birch plant responds to the salt, the BpNAC100 gene has salt damage resistance, the BpNAC100 gene promoter can be obtained by the method of the invention, the white birch can be transformed, and the number of the white birch specific promoter is supplemented.

Drawings

FIG. 1 is a schematic diagram of a process for obtaining a ProBpNAC100 Luc transgenic white birch plant in the first embodiment of the present invention.

FIG. 2 is a schematic diagram of the observation results of 20d salt stress phenotype of GFP and BpNAC100-RNAi transgenic white birch plants in the first embodiment of the present invention.

FIG. 3 is a schematic diagram of the response of ProBpNAC100 Luc transgenic white birch to hormone and salt treatment in accordance with one embodiment of the present invention.

FIG. 4 is a diagram showing the observation result of the phenotype of each strain in the second embodiment of the present invention.

FIG. 5 is a schematic diagram showing the salt damage index of leaves of each strain in the second embodiment of the present invention.

FIG. 6 is a schematic diagram showing the chlorophyll fluorescence measurement results of seedlings of various strains after salt stress in the third embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious 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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example one

Referring to fig. 1 and 2, this example proposes the acquisition and specificity table analysis of the birch BpNAC100 gene, including the following steps:

taking white birch of clone 1 year old in forest genetic breeding test base of northeast forestry university as a material, extracting DNA of the white birch as a cloning vector, and obtaining a BpNAC100 gene upstream 2000bp sequence according to a white birch genome as a target sequence, wherein the nucleotide sequence of the target sequence is shown as SEQ ID NO. 3 in a sequence table;

designing a primer for cloning, connecting the target sequence to a cloning vector after tapping and recycling the target sequence, then transforming competent cells, culturing, coating the competent cells by a coating plate method, and then culturing at 37 ℃ overnight for 1 d;

constructing a recombinant plasmid, transforming the recombinant plasmid into escherichia coli by using a heat shock method, transforming the escherichia coli into agrobacterium EHA105 by using an electrotransformation method, infecting a white birch zygotic embryo by using an agrobacterium-mediated method, culturing the infected white birch zygotic embryo for 2d two days, placing the white birch zygotic embryo on a culture medium containing cefuromycin and hygromycin for selective culture, wherein the concentration of the cefuromycin is 200mg/L, and the concentration of the hygromycin is 50mg/L until a green callus grows at a zygotic embryo cut, and obtaining a proBpNAC100:: Luc transgenic white birch plant;

sixthly, measuring the specific expression of BpNAC100 in the Luc transgenic white birch plant, observing the fluorescent reaction of the Luc transgenic white birch plant under the excitation wavelength of 328nm in the ProBpNAC100, specifically, spraying the Luc transgenic white birch plant under the excitation wavelength of 328nm by using D-Luciferin standard working solution to the ProBpNAC100, then observing the fluorescent reaction of the Luc transgenic white birch plant under the excitation wavelength of 328nm, then imaging by using a plant living body imaging system, outputting a pseudo-color photo, and analyzing the expression characteristic of the Luc tissue driven by the ProBpNAC 100;

the fluorescent reaction result shows that the ProBpNAC100 shows that the Luc transgenic white birch plant emits obvious yellow-green fluorescence (533nm) under the excitation wavelength of 328nm, the fluorescence intensity is obviously different along with different tissue parts, and the fluorescent reaction result shows that after a pseudo-color picture is output by using a plant living body imaging system for imaging, the fluorescent signals are detected in the leaves, stems and roots of the Luc transgenic white birch plant in the ProBpNAC100, then qRT-PCR detection is carried out on each tissue part of the Luc transgenic white birch plant in the ProBpNAC100, the result shows that the expression quantity of Luc in stems is the highest, and the expression quantity of the Luc in the leaves is the lowest, so that the expression of the Luc gene driven by the BpNAC100 promoter sequence in the white birch has tissue part specificity and the expression quantity in the stems is the highest, and the promoter of the BpNAC100 gene has space expression specificity in different tissue parts of the white birch.

And (3) applying 100uMIAA, 100uM GA, 50uM ABA, 50uM SA and 100uMNaCl solutions to 2-month-old Luc transgenic white birch tissue culture seedlings respectively, treating for 2 hours by taking a spraying aqueous solution as a blank control, taking white birch leaves, and quickly freezing by using liquid nitrogen. And detecting the Luc expression level in the leaves by using a qRT-PCR method, wherein the Luc expression level in the leaves applied with the NaCl solution is the highest.

Example two

According to fig. 4 and 5, the present example provides a NaCl stress treatment method of transgenic birch plants, comprising the following steps:

selecting three overexpression lines BpNAC100, namely GFP transgenic white birch plants marked as OEX1, OEX2 and OEX3, selecting three suppression expression line BpNAC100-RNAi transgenic white birch plants marked as REX1, REX2 and REX3, and finally selecting one non-transgenic white birch plant marked as WT;

transplanting tissue culture rooted seedlings of six BpNAC100 transgenic white birch plants and non-transgenic white birch plants into seedling culture cups, selecting 30 white birch plants with consistent growth vigor from each plant line after half a year, transplanting the plants into flowerpots of 21cm multiplied by 21cm, adding equivalent matrix into each flowerpot, placing a plastic tray under the pots, and then performing conventional management in a plastic greenhouse;

and step three, performing NaCl stress treatment after the conventional management is carried out for 1 year, measuring the plant height of the white birch plants before the NaCl stress treatment is started, recording the plant height as a base value, immediately performing flood irrigation by using 0.4% NaCl, stopping water supply during the stress period, supplementing 500ml of 0.4% NaCl only every 3d, investigating the salt damage condition of leaves after 12d, calculating the salt damage, measuring chlorophyll fluorescence parameters, performing phenotype observation photography on the white birch plants of each line respectively after 20d of salt stress, and measuring the plant height and investigating the high growth condition.

The salt damage grade standard of NaCl stress treatment is as follows:

grade 0, healthy and harmless leaves;

grade 1, leaf edge begins to yellow or wilting (the affected surface is less than 20% of the total area of the leaf);

level 2, yellowing and wilting symptoms appear in 50% area of the total area of the leaves;

grade 3, the yellow and wilting symptoms appear in the area which is more than 80 percent of the total area of the leaves;

at stage 4, the leaf dies.

The calculation formula of the salt damage index of each seedling leaf is as follows:

LSI＝[∑(si×Nsi)/(NsT×G smax)]×100％

wherein si represents different salt damage grades (0-4), Nsi represents the number of si-grade salt damage leaves of each seedling, NsT represents the total number of leaves of each seedling, and G smax represents the highest salt damage grade.

NaCl stress treatment results show that after 20 days of salt stress, phenotype observation and photography are respectively carried out on each strain. Phenotypically, after salt stress, the overexpression lines (OEX1, OEX2, OEX3) exhibited relatively mild stress symptoms, while the repressive expression (REX1, REX2, REX3) and non-transgenic white birch WT lines exhibited more severe salt damage symptoms, as shown in fig. 4. After being subjected to salt stress, the seedling height of each line was investigated as shown in table 1, and the relatively high growth of the OEX line was the largest (wherein, the OEX3 line was relatively high in growth up to 0.340), while the relatively high growth of the REX line was the smallest (wherein, the REX2 was relatively high in growth only 0.136). Relatively high growth represents the growth rate of each line during the same time period, thus OEX plants grew the fastest and OEX plants grew the slowest under salt stress conditions.

Table 1 investigation results of seedling height of each strain before and after NaCl stress treatment

And (4) carrying out leaf salt damage index investigation after 12d of salt stress, and calculating the leaf salt damage index of each strain according to a leaf salt damage index formula. The final calculation results show that after salt stress, the salt damage index of the OEX strain leaf is obviously lower than that of the WT and REX strain leaf, especially the OEX3 strain has the salt damage index of 25.59 percent, while the REX3 strain has the salt damage index of only 48.30 percent, as shown in FIG. 5.

EXAMPLE III

As shown in FIG. 6, this example uses a chlorophyll fluorometer (PAM2500, WALZ, Germany) to determine the maximum photochemical efficiency (F) of PS II of the NaCl-treated 12d reference line_v/F_m) Actual photochemical efficiency of PS II (phi)_PSⅡ) And photochemical quenching coefficient (qP), measuring 3 plants in each transformation strain, measuring 3 healthy functional leaves in each transformation strain, selecting a fourth functional leaf from the 3 healthy functional leaves, measuring dark adaptation of the front leaf for 20min, and measuring the photochemical light intensity of 500 mu mol photons m^-2s^-1。

The chlorophyll fluorescence parameter of the plant leaf can directly reflect the photosynthetic capacity of the plant, when the plant is subjected to stress injury, the photosynthetic capacity of the plant is reduced, and the related photosynthetic parameter is changed. According to the chlorophyll fluorescence measurement results of each strain of seedlings after salt stress (as shown in figure 6), after the seedlings are subjected to the salt stress, the photochemical efficiency (Fv/Fm) of the chloroplast PSII system of OEX strain leaves is slightly higher than that of WT and REX, but the difference is not obvious; phi PS II represents the actual photochemical efficiency and reflects the actual photosynthetic capacity under adversity stress, and the parameters of the OEX strain leaf are obviously higher than those of WT and REX, which shows that the OEX strain has the highest actual photochemical efficiency after salt stress. qP is the photochemical quenching coefficient, i.e. fluorescence quenching caused by photosynthesis, which reflects the photosynthetic activity, and OEX has the same photochemical quenching coefficient as WT and REX under salt stress, and the difference is not significant. qN reflects the ability of plants to dissipate excess light energy into heat, reflects the photoprotective ability of plants, and OEX photoprotective ability under salt stress is significantly reduced compared to WT, REX. Analysis of chlorophyll fluorescence parameter data suggests that photosynthesis of WT and REX strains is impaired by more severe salt stress, while photosynthesis of OEX strain is affected by less salt stress.

According to the invention, the promoter of the BpNAC100 gene is expressed in the white birch by utilizing an agrobacterium-mediated method, so that the proBpNAC100 is obtained, wherein Luc transgenosis white birch plants are obtained, and the promoter of the BpNAC100 gene has space expression specificity in different tissue parts of the white birch; the BpNAC100 gene is overexpressed and inhibited in the white birch by utilizing an agrobacterium-mediated method, BpNAC100, GFP and BpNAC100-RNAi transgenic white birch plants are obtained, proBpNAC100, Luc transgenic white birch plants respond to salt, BpNAC100, GFP transgenic white birch plants have the salt damage resistance, the promoter of the BpNAC100 gene can be obtained by the method, the white birch is transformed, and the number of the specific promoters of the white birch is supplemented.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims

1. The application of the BpNAC100 gene in the response of the white birch to salt stress is characterized in that: the application is the application on the salt resistance of the white birch, and the nucleotide sequence of the BpNAC100 gene is shown as SEQ ID NO. 1 in a sequence table.

2. Use of the amino acid sequence encoded by the BpNAC100 gene of claim 1 for the response of white birch to salt stress, characterized in that: the amino acid sequence is shown as SEQ ID NO. 2 in the sequence table.

3. The use of the amino acid sequence encoded by the BpNAC100 gene of claim 1 for the response of white birch to salt stress, characterized in that: the promoter sequence of the BpNAC100 gene is shown as SEQ ID NO. 3 in the sequence table.