CN111533794B - Tobacco NtDREB-1BL1 transcription factor and application thereof - Google Patents

Abstract

The application belongs to the technical field of tobacco genome analysis, and particularly relates to a tobacco NtDREB transcription factor and an application patent application thereof. The tobacco NtDREB transcription factor comprises three NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3, and the corresponding CDS base sequence is shown in SEQ ID No. 1-3. The functional research of the DREB transcription factors in other crops at present considers that the transcription factors are closely related to the stress resistance of plants. In the application, the inventor discovers that the NtDREB genes in the tobacco are closely related to the content of pigment substances in the tobacco through the function research of three NtDREB transcription factors in the tobacco, and realizes the synchronous adjustment of the content of the pigment substances in the tobacco leaves by regulating the expression of the NtPSY genes.

Description

Tobacco NtDREB-1BL1 transcription factor and application thereof

Technical Field

The application belongs to the technical field of tobacco genome analysis, and particularly relates to a tobacco NtDREB transcription factor and an application patent application thereof.

Background

Carotenoids are important terpenoids in plants, are important fragrance precursors, have content closely related to various properties of the plants such as quality, stress resistance, yield and appearance, and can form a series of fragrance substances through degradation and transformation. Therefore, in the tobacco leaves, the tobacco leaf aroma quality and the tobacco leaf aroma amount are closely related to the content of the terpenoid, and the accumulation of aroma precursors such as carotenoid and diterpene can improve the tobacco leaf aroma amount, improve the tobacco leaf aroma quality and determine the appearance quality of the baked tobacco leaves. Therefore, the method for improving the content of terpenoids such as carotenoid in tobacco leaves by a genetic engineering means or performing directed genetic improvement on the synthetic character of the carotenoid in the tobacco leaves is an effective measure for improving the total amount of tobacco leaf flavor precursors and improving the quality of the tobacco leaves from the root.

In the existing gene research on the influence of the carotenoid content, a tobacco embrittlement Virus Induced Gene Silencing (VIGS) technology is utilized, and a corresponding target gene is silenced, so that part of the gene is found to have obvious influence on the carotenoid content (tobacco carotenoid isomerase gene and application thereof, application No. 201410405272. X; tobacco epsilon-lycopene cyclase gene and application No. 201410405714.0). However, when VIGS is used for researching related functional genes, the technology has some defects: (1) the silencing feature of the target gene caused by VIGS is not hereditary, so that the function of the gene cannot be completely disclosed by the technology; (2) how to obtain systemic integral silencing of target genes is still a problem to be solved by the VIGS technology at present; (3) the time problem of the duration of the silencing; (4) different inoculation modes can generate different silencing efficiency, so how to effectively inoculate the virus vector into the plant body and generate stronger silencing effect is a key step for developing the VIGS technology.

The use of Overexpression (OE) and RNAi techniques is also a common research method for studying the function of target genes, and the patent application for tobacco lycopeneβCyclase Gene and use thereof (application No. 201410405713.6), a process for preparing the sameNtβ- LCYThe gene researches the growth and development conditions of tobacco plants and the influence of pigment content under the condition of the change of expression quantity. But using Overexpression (OE) andin the case of RNAi technology, there are technical defects mainly: the nature and mechanism of action of the gene cannot be sufficiently explained, for example, the regulation of the gene expression level by OE and RNAi as described above cannot be sufficiently explained and demonstratedβThe cyclase gene is the basic property of the gene on the carotenoid synthesis pathway, and other experiments and analysis are needed to be combined to further explain the lycopeneβThe mechanism of cyclase regulation of carotenoid content.

The phytoene synthetase gene (PSY) is the first key gene in the upstream of carotenoid synthesizing path, and the change of the expression level of the gene can regulate the carotenoid content, photosynthetic efficiency and stress resistance of plant. PSY gene is finely regulated in plant body, and the regulation of various factors is required to be on the specific genetic mechanism, namely transcription and post-transcription regulation, so that the transcription factor or interaction protein capable of regulating PSY is searched by utilizing different transgenic technologies (VIGS, OE, RNAi and the like) and the regulation mechanism is analyzed, and the PSY gene is an important way and a way for disclosing a new plant carotene content genetic regulation mechanism.

Disclosure of Invention

Through preliminary research and analysis on DREB transcription factors in tobacco, the application aims to provide a series of DREB transcription factors with a regulating effect on carotenoid content in tobacco, thereby laying a certain technical foundation for improving the quality of tobacco leaves.

The technical solution adopted in the present application is detailed as follows.

The tobacco NtDREB transcription factor comprises three genes of NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3, wherein the base sequences of CDS corresponding to the three genes are shown in SEQ ID No. 1-3, and the specific sequences are as follows:

NtDREB-1BL1CDS sequence of gene, 660bp, specifically (SEQ ID No. 1):

ATGGATATCTTTAGAAGCTATTATTCGGACCCACTTGCTGAATATTCATCAATTTCTGACAGTAGTAGCAGCTCCTGTAATAGAGCTAACCATTCTGATGAGGAAGTGATGTTAGCTTCGAATAACCCCAAGAAGCGAGCAGGGAGAAAGAAGTTTAGAGAAACTCGACACCCAGTATACAGGGGAGTGAGGAAGAGGAATTCAGACAAGTGGGTTTGTGAACTCAGAGAACCAAACAAGAAATCAAGAATATGGCTGGGCACTTTCCCTTCTGCAGAAATGGCGGCTAGAGCTCATGACGTGGCGGCTATTGCATTAAGGGGCCGTTCTGCTTGCTTGAACTTTGCTGACTCTGCTTGGAAGTTGCCTATTCCAGCTTCAACCGACGCCAAGGATATTCAGAAAGCGGCGGCGGAGGCCGCGGAGGCATTCCGGTCATCGGAGGCCGAAAACATGCCGGAATACTCAGGAGAAGATACGAAGGAAGTGAACAGTACTCCTGAAAATATGTTTTATATGGATGAGGAGGCGCTATTCTTCATGCCTGGATTACTAGTGAATATGGCAGAAGGACTAATGTTACCTCCACCTCAGTGTTCACAAATTGGAGATCATATGGAAGCTGATGTTGACATGCCTTTGTGGAGTTATTCTATCTAA。

NtDREB-1BL2the CDS sequence of the gene has 657bp, and is specifically (SEQ ID No. 2):

ATGGATATCTTTCGTAGCTTTTACTCGGACCCACTTGCTGATTCTTCATCACTTTCTGATAGTAGCAGCTCCTGTAATAGAGCTAACCTTTCTGATGAAGAAGTTATGTTAGCTTCAAATAACCCCAAGAAGCGGGCAGGGAGGAAGAAGTTTCGAGAAACTCGACACCCAGTATACAGGGGAGTGAGAAAGAGGAATTCAGGCAAGTGGGTTTCTGAAGTCAGAGAACCAAACAAGAAATCAAGAATATGGCTTGGCACTTTCCCTTCTGCAGAAATGGCGGCTAGAGCGCATGACGTGGCGGCTATTGCATTAAGGGGCCGTTCTGCTTGCTTGAACTTCGCAGACTCTGCTTGGAAGTTGCCTATTCCTGCCTCAACCGACGCCAAGGATATTCAGAAAGCGGCGGCTGAGGCCGCGGAGGCATTCCGGTCATCGGAGGCCGAAAAAATGCCGGAATACACAGGAGAAGATTCAAAGGAAGTGAACACTACTCCTGAAAATATGTTTTATATGGATGAGGAGACGCTATTCTGCATGCCGGGATTACTAGCAAATATGGCTGAAGGATTAATGTTACCTCCACCTCAGTGTTCACAAATTGGAGATCATTTGGAAGCTGATGTTGACATGCCTTTGTGGAGTTATTCTATTTAA。

NtDREB-1BL3the CDS sequence of the gene has 654bp, and is specifically (SEQ ID No. 3):

ATGGAAATGTGTCGAAGCTATTATTCGGACCCACTTGCTGATTCTTCATCACTGTCTGATAGTAGCAGCTCCTGTAATAGAGCTATCCGTTCTAATGAAGAAGTTATGTTAGCTTCGAATAACCCCAAGAAGCGAGCAGGGAGGAAGAAGTTTCGAGAAACTCGACACCCAGTATACAGGGGAGTGAGAAAGAGGAATTCAGGCAAGTGGGTTTCTGAAGTCAGAGAACCAAACAAGAAATCAAGAATATGGCTTGGCACTTTCCCTTCTGCAGAAATGGCGGCTAGAGCGCATGACGTGGCGGCTATTGCATTAAGGGGCCGTTCTGCTTGCTTGAACTTCGCAGACTCTGCTTGGAAGTTGCCTGTTCCTGCTTCCTCCGACGCCAAGAATATTCAGAAGGCGGCTGCCGAGGCCGCCGAGGCTTTCCGGTCATCGGAGGCCGAAAACATGCCGGAATACACAGGAGAAGATTCAAAGGAAGTGAACACTACTCCTGAAAATATGTTTTATATGGATGAGGAGGCGCTATTCTGCATGCCGGGATTACTTGCGAATATGGCAGAAGGATTAATGTTACCTCCACCTCAGTGTTCCCAAATTGGAGATGATCATATGGAGGCTGATATGCCTTTGTGGAGTTATTCAATTTAA。

amino acid sequences of proteins encoded by three transcription factor genes of NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3 of proteins encoded by the tobacco NtDREB transcription factors are shown in SEQ ID Nos. 4-6, and are as follows:

NtDREB-1BL1the gene encodes an amino acid sequence (219 amino acids, SEQ ID No. 4) that is:

MDIFRSYYSDPLAEYSSISDSSSSSCNRANHSDEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSDKWVCELREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPIPASTDAKDIQKAAAEAAEAFRSSEAENMPEYSGEDTKEVNSTPENMFYMDEEALFFMPGLLVNMAEGLMLPPPQCSQIGDHMEADVDMPLWSYSI。

NtDREB-1BL2the gene-encoded amino acid sequence (218 amino acids, SEQ ID No. 5) is:

MDIFRSFYSDPLADSSSLSDSSSSCNRANLSDEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSGKWVSEVREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPIPASTDAKDIQKAAAEAAEAFRSSEAEKMPEYTGEDSKEVNTTPENMFYMDEETLFCMPGLLANMAEGLMLPPPQCSQIGDHLEADVDMPLWSYSI。

NtDREB-1BL3the gene-encoded amino acid sequence (217 amino acids, SEQ ID No. 6) is:

MEMCRSYYSDPLADSSSLSDSSSSCNRAIRSNEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSGKWVSEVREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPVPASSDAKNIQKAAAEAAEAFRSSEAENMPEYTGEDSKEVNTTPENMFYMDEEALFCMPGLLANMAEGLMLPPPQCSQIGDDHMEADMPLWSYSI。

the recombinant expression vector constructed by the tobacco NtDREB transcription factor comprises gene silencing vectors pTRV2-DREBL1 and pTRV2-DREBL2 constructed by VISG technology; super-expression recombinant vectors Sup1300-DREBL1-OE, Sup1300-DREBL2-OE and Sup1300-DREBL3-OE which are constructed by combining a Sup1300-GFP vector by adopting a super-expression technology; RNAi-DREBL1, RNAi-DREBL2 and RNAi-DREBL3 which are constructed by using RNAi technology; when the concrete construction is carried out, the concrete structure,

when gene silencing vectors pTRV2-DREBL1 and pTRV2-DREBL2 are constructed and conserved regions are amplified, specific primer sequences are as follows:

TRV-DREB1-F：5’-CGGGATCCGGACCCACTTGCTGAATATT-3’，

TRV-DREB1-R：5’- GCGGTACCTAACATTAGTCCTTCTGCCAT-3’；

the amplification length is 586 bp;

TRV-DREB2-F：5’-CGGGATCCGACCCACTTGCTGATTCTTC-3’，

TRV-DREB2-R：5’-GCGGTACCGATCTCCAATTTGTGAACACT-3’；

the amplification length is 585 bp;

when the overexpression recombinant vectors Sup1300-DREBL1-OE, Sup1300-DREBL2-OE and Sup1300-DREBL3-OE are constructed, the primer sequences are designed as follows:

DREBL1-OE-F：5‘-GCTCTAGAATGGATATCTTTAGAAGCTATTATTC-3’，

DREBL1-OE-R：5‘-GGTACCGATAGAATAACTCCACAAAGGC-3’；

DREBL2-OE-F：5‘-GCTCTAGAATGGATATCTTTCGTAGCTTTTAC-3’，

DREBL2-OE-R：5‘-GGTACCAATAGAATAACTCCACAAAGGC-3’；

DREBL3-OE-F：5‘-GCTCTAGAATGGAAATGTGTCGAAGCTATTAT-3’，

DREBL3-OE-R：5‘-GGTACCAATTGAATAACTCCACAAAGGCA-3’；

when constructing RNAi vectors RNAi-DREBL1, RNAi-DREBL2 and RNAi-DREBL3, the RNAi vector primer sequences are designed as follows:

DREBL1-RNAi-attB-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCTATTATTCGGACCCACTTGC-3’，

DREB2-RNAi-attB-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCTTTTACTCGGACCCACTTGC-3’，

DREB3-RiattB-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGGACCCACTTGCTGATTCTTC-3’，

common RNAi-attB-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAGAACGGCCCCTTAATGCAAT-3' are provided.

According to the method for cultivating the new plant variety by using the recombinant expression vector, the content of the pigment substances of the new plant variety is obviously changed, and the pigment substances are chlorophyll a, chlorophyll b, total chlorophyll and carotenoid.

The tobacco NtDREB transcription factor is applied to phytochrome regulation, the NtDREB transcription factor is positioned in the nucleus of a tobacco leaf cell, and the regulation and control of the expression quantity of the NtPSY gene in tobacco are realized by combining with the upstream promoter sequence of the NtPSY gene in tobacco, so that the content change of the chromochrome substances in the leaf is influenced; the expression quantity of the NtDREB transcription factors and the content of the pigments show positive correlation, namely, the expression quantity of the NtDREB transcription factors is high, the content of the pigments is increased, the expression quantity of the NtDREB transcription factors is low, and the content of the pigments is reduced; the pigment substances include chlorophyll a, chlorophyll b, total chlorophyll and carotenoid.

For the function research of the DREB transcription factors in other crops, the transcription factors are considered to be closely related to the stress resistance of plants. In the application, the inventor discovers that the NtDREB genes in the tobacco are closely related to the content of pigment substances in the tobacco through the function research of three NtDREB transcription factors in the tobacco, and realizes the synchronous adjustment of the content of the pigment substances in the tobacco leaves by regulating the expression of the NtPSY genes.

Drawings

FIG. 1 shows the homology analysis of DREB protein in different species, wherein the right side is rice, Arabidopsis, potato, tomato, forest tobacco, hairy tobacco, common tobacco and Bungarus nicotiana in sequence from top to bottom;

FIG. 2 is a phylogenetic analysis of the NtDREB gene family of Nicotiana tabacum;

FIG. 3 is a diagram of NtDREB-1BL1, NtDREB-1BL2, NtDREB-1BL3 and their homologous genes;

FIG. 4 shows the relative expression amounts of the NtDREB genes in the common tobacco K326, wherein A, B, C is the relative expression amounts of NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3 in the common tobacco K326 respectively; wherein: l is leaf, S is stem, R is fibrous root, F is flower; 5th L refers to the 5th blade from bottom to top, 10th L refers to the 10th blade from bottom to top, and 15th L refers to the 15th blade from bottom to top;

FIG. 5 is a subcellular map of a Nicotiana tabacum NtDREB protein;

FIG. 6 shows the TRV-mediated plant phenotype after DREB gene silencing in Nicotiana benthamiana;

FIG. 7 shows TRV-mediated silencing effect of DREB gene in Nicotiana benthamiana; wherein WT is a non-inoculated control tobacco strain, TRV is an inoculated empty virus vector control tobacco strain without gene segments, DREB1-V is an inoculated virus vector tobacco strain carrying NtDREB-1BL1 segments, and DREB2-V is an inoculated virus vector tobacco strain carrying NtDREB-1BL2 segments;

FIG. 8 is TRV-mediated Nicotiana benthamianaDREBThe carotenoid and chlorophyll content in the gene-silenced plant; wherein CK is inoculated with an empty virus vector control tobacco strain without gene segments, DREB1-vigs is inoculated with a virus vector tobacco strain carrying NtDREB-1BL1 segments, and DREB2-vigs is inoculated with a virus vector tobacco strain carrying NtDREB-1BL2 segments;

fig. 9 is an electrophoretogram for detecting DNA of transgenic seedling of T0 generation overexpressed by NtDREB, wherein, M: DNA Marker; 1-4: NtDREB-1BL1 transgenic seedlings; 5-8: NtDREB-1BL2 transgenic seedlings; 9-12: NtDREB-1BL3 transgenic seedlings;

fig. 10 is an electrophoretogram for detecting T0 generation transgenic seedling DNA of NtDREB-RNAi, wherein M: DNA Marker; 1-4: NtDREB-1BL1 transgenic seedlings; 5-8: NtDREB-1BL2 transgenic seedlings; 9-11: NtDREB-1BL3 transgenic seedlings;

FIG. 11 shows the results of partial identification of transgenic tobacco and detection of gene expression level; wherein: a is a target gene detection result in the transgenic tobacco, wherein, P: amplifying by using a vector plasmid as a template, wherein the amplification ratio of L1-L3: 3 transgenic lines of tobacco DNA are taken as templates for amplification; b is the result of hygromycin resistance test, wherein, WT: amplifying by using non-transgenic tobacco DNA as a template, wherein the DNA sequence of the tobacco comprises L1-10, L2-2 and L3-9: 3 transgenic lines of tobacco DNA are taken as templates for amplification; c is the relative expression quantity of the NtDREB gene in the overexpression transgenic plant, wherein K326: non-transgenic tobacco, OEL3-9, OEL2-2 and OEL1-10 are transgenic tobacco strains NtDREB-1BL3, NtDREB-1BL2 and NtDREB-1BL1 respectively;

FIG. 12 shows the relative expression levels of NtPSY in different transgenic plants; wherein, A is the relative expression quantity of NtPSY in the NtDREB overexpression transgenic plant, wherein, CON: non-transgenic tobacco; OE1-10, OE2-2 and OE3-12 are NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3 transgenic tobacco strains respectively; b is the relative expression quantity of NtPSY in the NtDREB RNAi transgenic plant, wherein K326: non-transgenic tobacco; r1-7, R2-4 and R3-2 are NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3 transgenic tobacco strains respectively;

FIG. 13 shows the results of pigment content in different transgenic plants; wherein A is an NtDREB overexpression pigment content graph, and B is an NtDREB-RNAi pigment content graph;

FIG. 14 shows the results of CHIP-PCR experiments.

Detailed Description

The present application is further illustrated by the following examples.

Example 1

Based on the screening result of the previous yeast single hybridization experiment, the inventor preliminarily considers that the NtDREB transcription factor in the tobacco has interaction with the key region of the tobacco PSY gene promoter, so that the NtDREB transcription factor in the tobacco is presumed to regulate the expression of the tobacco phytoene synthase gene (NtPSY) and further influence the change of the carotenoid content in the tobacco leaf. For further research and determination, the inventors firstly cloned the NtDREB transcription factor in nicotiana tabacum and obtained three NtDREB transcription factor genes: NtDREB-1BL1, NtDREB-1BL2, and NtDREB-1BL 3. For this purpose, the present example is first summarized below in terms of the process of obtaining clones of the three NtDREB transcription factor genes.

(one) extracting total RNA of tobacco and reverse transcribing into cDNA for standby

With reference to the instructions of the Plant Total RNA Isolation Kit (Shanghai bioengineering Co., Ltd.), the Total RNA of the tobacco tissue can be extracted as follows:

taking 50 mg of tobacco leaf (common tobacco K326) tissue, fully grinding the tissue under the protection of liquid nitrogen, transferring the tissue into an Eppendorf tube, adding 600 mu l of Buffer Rlysis-P, vibrating and uniformly mixing; carrying out water bath at 65 ℃ for 5min to fully crack the sample, then adding 60 muL of Buffer PCA into the cracked sample, fully and uniformly mixing, centrifuging at 12000 rpm at 4 ℃ for 5min, and taking the supernatant; adding equal volume of phenol and chloroform (volume ratio of 25:24, pH 4.5) into the supernatant, mixing, centrifuging at 12000 rpm for 5min at 4 deg.C, and collecting supernatant; 1/3 volumes of anhydrous water were addedMixing with ethanol, standing at room temperature for 3 min, centrifuging at 12000 rpm at 4 deg.C for 5min, and carefully pouring off the supernatant; using 75% ethanol (DEPC-treated ddH) of 700 μ L₂O preparation), washing the precipitate, centrifuging at 4 ℃ and 12000 rpm for 3 min, carefully pouring off the supernatant, and repeating the step once; standing at room temperature for 10 min to completely volatilize ethanol remained in the centrifuge tube as much as possible; subsequently adding 50 mu L of DEPC-treated ddH₂The pellet was dissolved O and samples were taken to determine RNA concentration and quality to determine if the application was satisfactory and used immediately or stored at-70 ℃ until use.

During specific reverse transcription, the total RNA extracted above is used as a template to synthesize a first cDNA chain through reverse transcription, and a 20 mu l reverse transcription system is designed as follows:

Total RNA，1 mg；5× Reaction Buffer，4µl；10 mM dNTP Mix，2µl；RiboLock RNase Inhibitor (20 U/µL)，1µl；

Oligo(dT)，1µl；RevertAid M-MuLV RT (200 U/µL)，1µl；ddH₂o, adding to 20 mu l;

after the reverse transcription system is uniformly mixed, the mixture is placed in a PCR instrument for reaction according to the following reaction program: 42 ℃, 1h, 70 ℃, 5min, 4 ℃ pause; after the reaction is finished, the reverse transcription product is diluted by 10 times, and the concentration is measured by a spectrophotometer and then directly applied or stored at the temperature of minus 20 ℃ for later use.

(II) designing primers and carrying out PCR amplification

By means of homologous comparison, with reference to the DREB gene sequence in the existing plants such as arabidopsis, tomatoes, potatoes and the like, the primer sequence for PCR amplification is designed as follows:

DREBL1-F：5‘-ATGGATATCTTTAGAAGCTATTATTCG-3’，

DREBL1-R：5‘-TTAGATAGAATAACTCCACAAAGGCATG-3’；

DREBL2-F：5‘-ATGGATATCTTTCGTAGCTTTTAC-3’，

DREBL2-R：5‘-TTAAATAGAATAACTCCACAAAGGC-3’；

DREBL3-F：5‘-ATGGAAATGTGTCGAAGCTATTAT-3’，

DREBL3-R：5’-TTAAATTGAATAACTCCACAAAGGCA-3’；

and (2) performing PCR amplification by using the cDNA prepared in the step (I) as a template and using the primers, wherein the reference design of a 50 mu l reaction system is as follows:

template cDNA, 2 μ l;

f primer, 2 mul;

r primer, 2 mu l;

2× HiFi-PCR Master，25µl；

ddH₂O，19µl；

the reaction procedure is as follows: pre-denaturing at 94 ℃ for 3 min; 94 ℃, 30 s, 60 ℃, 30 s, 72 ℃, 90s, 35 cycles; stretching at 72 deg.C for 10 min; the amplification product was stored at 4 ℃ until use.

The PCR amplification product was detected by electrophoresis in 1.5% agarose gel (TAE as the electrophoresis buffer, electrophoresis for about 16 min at 120V, DL2000plus DNA maker, 10 × Loading buffer was used for electrophoresis), and the band was observed by UV scanner.

And (3) recovering the PCR product by using a glue recovery kit (Shanghai bioengineering Co., Ltd.) for the target strip, wherein the specific operation is referred to the instruction or the following:

cutting a target DNA band into an Eppendorf tube, weighing, adding a solution Buffer B2 with the glue weight being 3 times of that of the target DNA band, and melting the glue until the glue is completely melted;

transferring the melted solution into a purple column for column loading, centrifuging at 8000g for 1min, and discarding the waste liquid; adding 500 mu L of Wash buffer, centrifuging 8000g for 1min, and removing waste liquid;

centrifuging the empty tube at high speed for 2 min, placing the adsorption column in a new Eppendorf tube, adding 30 μ L of precipitation Buffer in the center of the column, standing at room temperature for 1min, centrifuging for 1min, and eluting DNA; the eluted DNA was used for subsequent experiments.

(III) sequencing analysis after ligation with T vector

Connecting the PCR product recovered in the step (II) with pEASY-T1 carriers (a connection system is 5 mu L, wherein the PCR product is 4 mu L, pEASY^®-T1, 1 μ L; reacting at room temperature for 5 min);

and further adopting heat shock to transform E.coli cells, screening to ensure that the recombination plasmid is correctly recombined, and sequencing to obtain a specific DREB gene sequence.

For the specific heat shock transformation and screening process, the following operations can be referred to:

placing the ligation solution in thawed 100 μ L DH5 α competence, and standing on ice for 30 min; then thermally shocking for 90s at 42 ℃, and standing on ice for 2 min; adding 500 μ L LB liquid culture medium, and shaking at 37 deg.C for 1 h; sucking 150 μ L of bacterial liquid, spreading on LB plate containing ampicillin (100 μ g/mL), and culturing in 37 deg.C incubator for overnight;

picking single colony in LB liquid culture medium containing ampicillin (100 mug/mL), shaking and culturing overnight at 37 ℃, carrying out bacteria liquid PCR identification to ensure correct recombination, and then extracting positive clone plasmid which is correctly identified and sending to Shanghai bioengineering limited company for DNA sequence sequencing.

Sequencing results show that three NtDREB transcription factor genes are obtained: the CDS base sequences of the corresponding genes are shown as SEQ ID No. 1-3, and are specifically as follows:

NtDREB-1BL1gene CDS sequence (660 bp):

ATGGATATCTTTAGAAGCTATTATTCGGACCCACTTGCTGAATATTCATCAATTTCTGACAGTAGTAGCAGCTCCTGTAATAGAGCTAACCATTCTGATGAGGAAGTGATGTTAGCTTCGAATAACCCCAAGAAGCGAGCAGGGAGAAAGAAGTTTAGAGAAACTCGACACCCAGTATACAGGGGAGTGAGGAAGAGGAATTCAGACAAGTGGGTTTGTGAACTCAGAGAACCAAACAAGAAATCAAGAATATGGCTGGGCACTTTCCCTTCTGCAGAAATGGCGGCTAGAGCTCATGACGTGGCGGCTATTGCATTAAGGGGCCGTTCTGCTTGCTTGAACTTTGCTGACTCTGCTTGGAAGTTGCCTATTCCAGCTTCAACCGACGCCAAGGATATTCAGAAAGCGGCGGCGGAGGCCGCGGAGGCATTCCGGTCATCGGAGGCCGAAAACATGCCGGAATACTCAGGAGAAGATACGAAGGAAGTGAACAGTACTCCTGAAAATATGTTTTATATGGATGAGGAGGCGCTATTCTTCATGCCTGGATTACTAGTGAATATGGCAGAAGGACTAATGTTACCTCCACCTCAGTGTTCACAAATTGGAGATCATATGGAAGCTGATGTTGACATGCCTTTGTGGAGTTATTCTATCTAA。

NtDREB-1BL1the gene encodes the amino acid sequence:

MDIFRSYYSDPLAEYSSISDSSSSSCNRANHSDEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSDKWVCELREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPIPASTDAKDIQKAAAEAAEAFRSSEAENMPEYSGEDTKEVNSTPENMFYMDEEALFFMPGLLVNMAEGLMLPPPQCSQIGDHMEADVDMPLWSYSI。

NtDREB-1BL2gene CDS sequence (657 bp):

ATGGATATCTTTCGTAGCTTTTACTCGGACCCACTTGCTGATTCTTCATCACTTTCTGATAGTAGCAGCTCCTGTAATAGAGCTAACCTTTCTGATGAAGAAGTTATGTTAGCTTCAAATAACCCCAAGAAGCGGGCAGGGAGGAAGAAGTTTCGAGAAACTCGACACCCAGTATACAGGGGAGTGAGAAAGAGGAATTCAGGCAAGTGGGTTTCTGAAGTCAGAGAACCAAACAAGAAATCAAGAATATGGCTTGGCACTTTCCCTTCTGCAGAAATGGCGGCTAGAGCGCATGACGTGGCGGCTATTGCATTAAGGGGCCGTTCTGCTTGCTTGAACTTCGCAGACTCTGCTTGGAAGTTGCCTATTCCTGCCTCAACCGACGCCAAGGATATTCAGAAAGCGGCGGCTGAGGCCGCGGAGGCATTCCGGTCATCGGAGGCCGAAAAAATGCCGGAATACACAGGAGAAGATTCAAAGGAAGTGAACACTACTCCTGAAAATATGTTTTATATGGATGAGGAGACGCTATTCTGCATGCCGGGATTACTAGCAAATATGGCTGAAGGATTAATGTTACCTCCACCTCAGTGTTCACAAATTGGAGATCATTTGGAAGCTGATGTTGACATGCCTTTGTGGAGTTATTCTATTTAA。

NtDREB-1BL2the gene encodes the amino acid sequence:

MDIFRSFYSDPLADSSSLSDSSSSCNRANLSDEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSGKWVSEVREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPIPASTDAKDIQKAAAEAAEAFRSSEAEKMPEYTGEDSKEVNTTPENMFYMDEETLFCMPGLLANMAEGLMLPPPQCSQIGDHLEADVDMPLWSYSI。

NtDREB-1BL3gene CDS sequence (654 bp):

ATGGAAATGTGTCGAAGCTATTATTCGGACCCACTTGCTGATTCTTCATCACTGTCTGATAGTAGCAGCTCCTGTAATAGAGCTATCCGTTCTAATGAAGAAGTTATGTTAGCTTCGAATAACCCCAAGAAGCGAGCAGGGAGGAAGAAGTTTCGAGAAACTCGACACCCAGTATACAGGGGAGTGAGAAAGAGGAATTCAGGCAAGTGGGTTTCTGAAGTCAGAGAACCAAACAAGAAATCAAGAATATGGCTTGGCACTTTCCCTTCTGCAGAAATGGCGGCTAGAGCGCATGACGTGGCGGCTATTGCATTAAGGGGCCGTTCTGCTTGCTTGAACTTCGCAGACTCTGCTTGGAAGTTGCCTGTTCCTGCTTCCTCCGACGCCAAGAATATTCAGAAGGCGGCTGCCGAGGCCGCCGAGGCTTTCCGGTCATCGGAGGCCGAAAACATGCCGGAATACACAGGAGAAGATTCAAAGGAAGTGAACACTACTCCTGAAAATATGTTTTATATGGATGAGGAGGCGCTATTCTGCATGCCGGGATTACTTGCGAATATGGCAGAAGGATTAATGTTACCTCCACCTCAGTGTTCCCAAATTGGAGATGATCATATGGAGGCTGATATGCCTTTGTGGAGTTATTCAATTTAA。

NtDREB-1BL3geneThe encoded amino acid sequence:

MEMCRSYYSDPLADSSSLSDSSSSCNRAIRSNEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSGKWVSEVREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPVPASSDAKNIQKAAAEAAEAFRSSEAENMPEYTGEDSKEVNTTPENMFYMDEEALFCMPGLLANMAEGLMLPPPQCSQIGDDHMEADMPLWSYSI。

(IV) genetic evolution and analysis of Gene Structure

Based on the related sequencing sequence in the existing tobacco database and the existing genome sequence of crops such as rice, tomato, potato and the like, the inventor utilizes MEGA 7.0 to compare and analyze the DREB gene obtained by the application with the existing gene sequence, and adopts a maximum likelihood method to construct an evolutionary tree model (Bootstrap Replication is set as 1000).

The results of the genetic evolution analysis are shown in FIGS. 1 and 2. Analysis can see that: the similarity between NtDREB-1BL1 (Ntab 0136830) and the forest tobacco is 99.54%, which indicates that NtDREB-1BL1 may be derived from forest tobacco in evolution; the similarity between NtDREB-1BL2 (Ntab 0217720) and NtDREB-1BL3 (Ntab 0217680) and the similarity between the tobacco and the hairy tobacco are respectively 99.09% and 91.82%, which indicates that NtDREB-1BL2 and NtDREB-1BL3 are probably from the hairy tobacco in evolution. And as can be seen from the phylogenetic analysis results of fig. 2: the NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3 genes are gathered in a subclass, wherein the NtDREB-1BL2 and NtDREB-1BL3 genes are gathered together, and the sequence similarity between the NtDREB-1BL1 genes is higher than that between the NtDREB-1BL1 genes.

The structural analysis result of the DREB gene provided by the present application is shown in fig. 3, and it can be seen that: ntab0136830 (NtDREB-1 BL 1), Ntab0217720 (NtDREB-1 BL 2) and Ntab0217680 (NtDREB-1 BL 3) have no intron, and the total length of gDNA (CDS) is 660bp (coding 219 amino acids), 657bp and 654bp in sequence. The analysis result of the physical properties such as molecular weight, isoelectric point and the like of the protein coded by the gene by using the existing related software further shows that: the molecular weight of the protein coded by NtDREB-1BL1 is 24.58 kDa, and the isoelectric point is 5.35; NtDREBL2 encodes 218 amino acids, has a protein molecular weight of 24.25 kDa and an isoelectric point of 5.53; NtDREBL3 encodes 217 amino acids, has a protein molecular weight of 24.14 kDa and an isoelectric point of 6.00; the similarity of the amino acid sequences of the three genes is 95.73 percent, and the similarity of the coding sequences is 93.79 percent.

(V) tissue expression of genes

Further, 26S rRNA is used as an internal standard gene, a SYBR Green dye method is adopted, a BIO-RAD fluorescence quantitative PCR instrument is utilized, and the inventor carries out detection and analysis on the tissue expression mode of the DREB gene in the tobacco K326.

In the fluorescent quantitative PCR, the primers are designed as follows:

DREBL1-Q-F：5‘-ACCGACGCCAAGGATATTCAG-3’，

DREBL1-Q-R：5‘-ACAAAGGCATGTCAACATCAGC-3’；

DREBL2-Q-F：5‘-CTGCTTGCTTGAACTTCGC-3’，

DREBL2-Q-R：5‘-TCAGCTTCCAAATGATCTCCAA-3’；

DREBL3-Q-F：5‘-CGGACCCACTTGCTGATTC-3’，

DREBL3-Q-R：5‘-ATTCTTGGCGTCGGAGGAAG-3’。

during fluorescent quantitative PCR, a 20 mu l reaction system is designed as follows:

template cDNA, 2 μ l;

SYBR Green，2µl；

forward primer (F primer), 0.4. mu.L (10. mu.M);

the reverse primer (R primer), 0.4. mu.L (10. mu.M);

ddH₂and O is supplemented to 20 mu L.

The qPCR reaction conditions were: 95 deg.C for 5 min; at 95 deg.C, 15 s, 60 deg.C, 30 s, 40 cycles; 4 ℃ for 10 min. Each sample was tested in triplicate and the three Cp values averaged and calculated using the 2- Δ Cp method.

The results of measuring the transcription levels of the tissue organs of 5th, 10th, 15th, root, stem and flower 6 of K326 senna at full-bloom stage are shown in FIG. 4. It can be seen that the expression sites of the NtDREB transcription factors are similar, and at the full-bloom stage, the NtDREB genes have the highest expression level in leaves, the expression level in lower leaves (or young leaves) is higher than that in higher leaves (or relatively mature leaves), and the expression level in flowers is the lowest.

(VI) subcellular localization

Because the gene function exertion and the intracellular localization have close relevance, the protein transient expression system for infecting tobacco leaves by using the existing agrobacterium is used, the NtDREB-1BL1 gene (because NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3 belong to homologous genes, and the localization should be consistent) is taken as an example, agrobacterium containing recombinant plasmid Sup1300-NtDREB-GFP (the specific plasmid recombination method is referred to as example 2) is used for infecting Nicotiana benthamiana (the tobacco plant injected with the Sup1300-GFP empty vector is taken as a control, and the specific infection operation is referred to as follows:

adopting tobacco plants which are sown and cultivated for about one month as samples to carry out experiments; transferring the recombinant plasmid vector into agrobacterium tumefaciens (GV 3101) by an electro-transformation method, culturing for 2 days at 28 ℃, transferring into 10mL LB liquid culture medium, culturing for 1h at 170 rpm/min, centrifuging at 4000 rpm/min for 4 min, removing supernatant, and collecting thalli; followed by reaction with MgCl containing 10 mmol/L₂(containing 120. mu. mol/L AS) suspension the cells were resuspended and OD adjusted₆₀₀To about 0.6; selecting tobacco plants with good growth conditions, injecting the tobacco plants from the lower epidermis of tobacco leaves by using a 1 mL injector with a pipette tip, and marking (during co-positioning, agrobacterium is transformed by Marker plasmids, and suspended together with agrobacterium serving as a recombinant plasmid vector, mixed according to the proportion of 1:1 before injection, and then the tobacco leaves are injected). (ii) a The tobacco plants after injection can be observed after 2 days of low light culture; during observation, the marked tobacco leaves are taken to be made into a glass slide, observed under a laser confocal microscope, and photographed.

The results are shown in FIG. 5. The result of early prediction by using PSORT software shows that the NtDREB protein is possibly positioned in the nucleus of the tobacco cell. From the actual identification result of fig. 5, it can be seen that the green fluorescence emitted by NtDREB-GFP and the red fluorescence emitted by the leaf cell nuclear pigment are distributed alternately, and the green fluorescence is gathered in the cell nucleus to be shown, which indicates that the NtDREB mature protein is located in the cell nucleus of the tobacco leaf cell, which is consistent with the regulation and control function exerted by the NtDREB mature protein.

Example 2

On the basis of example 1, in order to further determine the function of the NtDREB transcription factor, different recombinant expression vectors are constructed by using VIGS, OE overexpression and RNAi technologies respectively, so as to further carry out transformation for further research and determination on the function of the related gene. This example is briefly described below with respect to the relevant vector construction process.

Recombinant vector constructed based on virus-induced gene silencing (VIGS) technology

Before the vector is constructed, a conserved functional region is found out by comparing the NtDREB gene sequence of the application with the NibenDREB gene sequence in the existing NibenDREB gene sequence of the NibenDREB, then a primer for a TRV-VIGS vector is designed, then the T plasmid containing the NtDREB gene sequence constructed in the embodiment 1 is used as a template, the designed TRV primer is used for amplifying the conserved region, and the recombinant vector is further constructed and obtained.

It should be noted that, in the sequence alignment process, a sequence highly homologous to the NtDREB-1BL3 gene is not aligned in the nicotiana benthamiana genome, so that a recombinant vector is constructed only for the two genes, NtDREB-1BL1 and NtDREB-1BL 2.

When the conserved region is amplified, specific primer sequences are as follows:

TRV-DREB 1-F: 5'-CGGGATCCGGACCCACTTGCTGAATATT-3', (cleavage site)BamHI）

TRV-DREB 1-R: 5'-GCGGTACCTAACATTAGTCCTTCTGCCAT-3', respectively; (cleavage site)KpnI）

The amplification length is 586 bp;

TRV-DREB 2-F: 5'-CGGGATCCGACCCACTTGCTGATTCTTC-3', (cleavage site)BamHI）

TRV-DREB 2-R: 5'-GCGGTACCGATCTCCAATTTGTGAACACT-3', respectively; (cleavage site)KpnI）

The amplification length is 585 bp;

after PCR amplification is finished, recovering an NtDREB-1BL1 target fragment carrying a VIGS enzyme cutting site by glue, and carrying out double enzyme cutting by using restriction enzymes KpnI and BamHI; simultaneously carrying out KpnI and BamHI double enzyme digestion on the pTRV2 (pYL 156) vector; the reference design of a 50 mu l enzyme digestion system is as follows:

the product (or pTRV2 vector) was recovered by PCR, 2. mu.g;

KpnI（10 U/μL），2.5 μL；

BamHI（10 U/μL），2.5 μL；

10× M Buffer，5 μL；

ddH₂supplementing O to 50 μ L system;

after digestion at 4 ℃ overnight, detection was carried out by 1% agarose gel electrophoresis.

Subsequently, the enzyme digestion products are recovered and purified, T4 DNA ligase is used for connection, the connection products are transformed into escherichia coli competent cells, positive plasmids are further screened for colony PCR identification and double enzyme digestion (KpnI and BamHI) identification, and the correctly identified plasmids are further sequenced and identified (provided by Beijing engine science and New technology Co., Ltd.) to ensure that the plasmid recombination is correct. The recombinant correct plasmid vectors are named as pTRV2-DREBL1 and pTRV2-DREBL2 and are directly applied or stored at the temperature of 20 ℃ below zero for later use.

(II) construction of OE overexpression vector

Sup1300-GFP vector containing 35S strong promoter is used as vector to recombine into NtDREB gene, so that the overexpression of NtDREB gene can be realized, and the specific process is briefly described as follows.

First, primer sequences were designed as follows (to add XbaI and KpnI cleavage sites):

DREBL1-OE-F：5‘-GCTCTAGAATGGATATCTTTAGAAGCTATTATTC-3’，

DREBL1-OE-R：5‘-GGTACCGATAGAATAACTCCACAAAGGC-3’；

DREBL2-OE-F：5‘-GCTCTAGAATGGATATCTTTCGTAGCTTTTAC-3’，

DREBL2-OE-R：5‘-GGTACCAATAGAATAACTCCACAAAGGC-3’；

DREBL3-OE-F：5‘-GCTCTAGAATGGAAATGTGTCGAAGCTATTAT-3’，

DREBL3-OE-R：5‘-GGTACCAATTGAATAACTCCACAAAGGCA-3’；

secondly, the T vector containing the NtDREB gene in the embodiment 1 is used as a template, and the primers are used for PCR amplification;

then, carrying out electrophoresis detection on the amplification product, and recovering gel;

and then, carrying out double enzyme digestion on the PCR amplification product and the Sup1300-GFP vector respectively by utilizing Xba I and Kpn I restriction enzyme digestion, wherein the reference design of a 50 mu l enzyme digestion system is as follows:

PCR recovery product (or sup 1300-GFP), 15. mu.L;

XbaI（10 U/μL），2.5 μL；

KpnI（10 U/μL），2.5 μL；

Buffer Y，5 μL；

ddH₂supplementing O to 50 μ L system;

and finally, after the enzyme digestion products are converged, connecting the enzyme digestion products by using T4 DNA, converting escherichia coli competent cells, further screening positive plasmids to carry out colony PCR identification and double enzyme digestion (Xba I and Kpn I) identification, and further carrying out sequencing identification on correctly identified plasmids to ensure that the plasmid recombination is correct. The recombinant correct plasmid vectors are named as Sup1300-DREBL1-OE, Sup1300-DREBL2-OE and Sup1300-DREBL3-OE, and are directly applied or stored at the temperature of 20 ℃ below zero for later use.

(III) construction of RNAi vector

When constructing the RNAi vector, the 200-and 300-BP fragment is generally inserted into the pHellsgate2-RNAi silencing vector by using BP recombination reaction technology, and the specific construction process is briefly described as follows.

Firstly, after finding a conserved functional region according to the alignment of related sequences, designing RNAi vector primer sequences as follows:

DREBL1-RNAi-attB-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCTATTATTCGGACCCACTTGC-3’，

DREB2-RNAi-attB-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCTTTTACTCGGACCCACTTGC-3’，

DREB3-RiattB-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGGACCCACTTGCTGATTCTTC-3’，

common RNAi-attB-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAGAACGGCCCCTTAATGCAAT-3', respectively;

subsequently, the T vector containing the NtDREB gene in example 1 was used as a template, and PCR amplification was performed using the above primers; carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and recovering an amplification fragment;

and then, carrying out BP recombination reaction on the recovered PCR amplification fragment and a pHellsgate2 vector, wherein a 10 mu l reaction system is designed as follows:

PCR recovery product (50 ng/. mu.L), 3. mu.L;

pHellsgate2 vector（150 ng/μL），1μL；

TE Buffer，4μL；

BP Clonase II enzyme mix，2μL；

reacting for 2 hours at 25 ℃;

and finally, transforming the reaction solution into escherichia coli DH5 alpha competent cells, further screening positive plasmids by using a plate containing Spectinomycin (Spe) to perform colony PCR identification and double enzyme digestion (Xba I and XhoI) identification, and further performing sequencing identification on correctly identified plasmids to ensure that the plasmid recombination is correct. The recombinant correct plasmid vectors are named as RNAi-DREBL1, RNAi-DREBL2 and RNAi-DREBL3 and are directly applied or stored at the temperature of 20 ℃ below zero for later use.

Example 3

Using the different vectors constructed in example 2, tobacco plants were further transformed for further analysis of different gene functions, and the specific experimental conditions are briefly described below.

(I) infection of Nicotiana benthamiana by Agrobacterium-mediated VIGS vector

Phytoene Dehydrogenase (PDS) is a key gene in a carotenoid synthesis pathway, has the effect of protecting chlorophyll from photobleaching, can generate photobleaching after being silenced, and is often used as a reference gene of various virus VIGS systems to verify the infection efficiency of a TRV vector. For this reason, the inventors constructed pTRV2-PDS vector simultaneously for comparison with reference to the vector construction in example 2.

The specific infection and transformation process of the tobacco plants comprises the following steps:

colonies containing plasmids pTRV1, pTRV2, pTRV2-DREBL1, pTRV2-DREBL2 and pTRV2-PDS were picked up and inoculated into 5mL of LB medium (50 mg/L Kan, 25 mg/L Rif), and cultured overnight with shaking at 28 ℃;

then, 0.2 mL of overnight-cultured bacteria was inoculated into 20 mL of LB medium (containing 10 mM 2-N-morpholinoethanesulfonic acid (MES) and 20. mu.M Acetosyringone (AS)), and cultured overnight at 28 ℃;

the cells were collected by centrifugation at 5000 rpm at room temperature and then resuspended in LB (containing 10 mM MgCl)₂+10 mM MES +200 mM acetosyringone) to adjust OD₆₀₀About 1.0, mixing with pTRV1 at a ratio of 1:1, standing at room temperature for 3h, and performing injection experiment.

In a specific experiment, a blank control group is not injected, pTRV1+ pTRV2 (TRV), pTRV1+ pTRV2-PDS (TRV-PDS) are respectively used as a negative control group and a positive control group, and pTRV1+ pTRV2-DREBL1 and pTRV1+ pTRV2-DREBL2 are used as experimental groups; selecting four leaf-aged Benshi tobacco seedlings for injection (three leaves are injected for each tobacco), respectively placing infected tobaccos according to gene categories, and carrying out dark treatment for one night;

the culture conditions of the infected tobacco are as follows: observing the phenotype change condition after 18 h of illumination and 6 h of darkness, controlling the culture temperature at 22 ℃ and the humidity at about 60 percent for about 30 d, simultaneously collecting leaf samples, and detecting the silencing effect of the DREB gene by Real-time PCR (specific operation refers to example 1).

Observation results show that after the TRV-PDS group agrobacterium is infected for 10 days, the fresh tobacco leaves begin to generate a photobleaching phenomenon, which shows that the PDS gene of the tobacco is successfully silenced, and the efficiency of the TRV vector infecting the tobacco by transforming the agrobacterium is close to 100%. The phenotypic results after 30 days are shown in figure 6. It can be seen that:

the phenotype of the plant infected by TRV-PDS agrobacterium for one month has a serious photobleaching phenomenon, and the tobacco plant can not generate obvious growth and development difference with a control tobacco plant (WT) when the empty viral vector (TRV) of the system is inoculated into tobacco; however, compared with the control, the tobacco plants infected with TRV-DREB have no visual phenotype, which indicates that the inhibition of the expression of DREB-1BL1 and DREB-1BL2 genes has no adverse effect on the growth and development of tobacco.

FIG. 7 shows TRV-mediated silencing effect of DREB gene in Nicotiana benthamiana; wherein WT is a non-inoculated control tobacco strain, TRV is an inoculated empty virus vector control tobacco strain without gene segments, DREB1-V is an inoculated virus vector tobacco strain carrying NtDREB-1BL1 segments, and DREB2-V is an inoculated virus vector tobacco strain carrying NtDREB-1BL2 segments;

the results of further testing the TRV-mediated silencing efficiency of the DREB gene by using q-PCR are shown in FIG. 7. It can be seen that the DREB gene expression level of the TRV empty carrier injected plant is not obviously reduced compared with that of the WT non-injected plant, and the DREB transcription expression level is reduced in the TRV-DREB injected plant, which shows that the DREB gene effect of the TRV-VIGS system for silencing Nicotiana benthamiana is obvious, and can lay a material foundation for further functional research.

FIG. 8 shows the content of carotenoids and chlorophyll in TRV-mediated DREB gene silencing plants of Nicotiana benthamiana; wherein CK is inoculated with an empty virus vector control tobacco strain without gene segments, DREB1-vigs is inoculated with a virus vector tobacco strain carrying NtDREB-1BL1 segments, and DREB2-vigs is inoculated with a virus vector tobacco strain carrying NtDREB-1BL2 segments;

furthermore, the inventor determines the content of chlorophyll and carotenoid in different tobaccos, and the specific determination method refers to the following steps:

taking 50 mg of tobacco leaf sample, adding the tobacco leaf sample into a centrifuge tube filled with 1.5 mL of precooled 80% acetone (which is used as a preparation in situ), and shaking the tobacco leaf sample at 4 ℃ in a dark place until the tobacco leaf sample is completely decolorized; centrifuging at 9000 rpm at 4 deg.C for 2 min, and collecting supernatant; respectively measuring A with a microplate reader₆₆₃、A₆₄₇、A₄₇₀Absorbance at wavelength; the calculation formula of the contents of chlorophyll and carotenoid is as follows:

chlorophyll a =12.25 × a₆₆₃ - 2.79×A₆₄₇；

Chlorophyll b =21.50 × a₆₄₇ - 5.10×A₆₆₃；

Total chlorophyll =7.15 xa₆₆₃ + 18.71×A₆₄₇；

Carotenoid = (1000 × a)₄₇₀ -1.82 × chlorophyll a-85.02 × chlorophyll b)/198.

The measurement results are shown in FIG. 8. It can be seen that: in the Nicotiana benthamiana, after virus-induced gene silencing, the carotenoid and chlorophyll contents of metabolites are remarkably reduced, and the carotenoid and chlorophyll contents in tobacco plants with NtDREB-1BL1 and NtDREB-1BL2 gene silencing are reduced more, which indicates that the carotenoid and pigment contents are influenced by the expression quantity change of NtDREB in transgenic plants.

(II) Agrobacterium-mediated overexpression and RNAi vector transformation

When the tobacco plant is transformed by overexpression and RNAi, the specific process is as follows: firstly, DREBL1-RNAi, RNAi-DREBL2, RNAi-DREBL3 and Sup1300-DREBL1-OE, Sup1300-DREBL2-OE and Sup1300-DREBL3-OE plasmids are respectively transformed into GV3101 agrobacterium tumefaciens, and specifically:

adding 100 μ L GV3101 Agrobacterium infected cells into 2 μ L plasmid, standing on ice for 30 min, quickly freezing with liquid nitrogen for 1min, and bathing at 37 deg.C for 5 min; then 200. mu.L of LB liquid medium was added, shaking cultured at 28 ℃ and 180 rpm for 3 hours, centrifuged at 4000 rpm for 30 seconds, the supernatant was discarded, and plated for screening.

After agrobacterium is transformed, tobacco leaves (growing for about one month after seeds are sown) are used as transgenic receptor materials, agrobacterium carrying a target gene vector is used for infecting the receptor materials, so that T-DNA is inserted into a genome, independent resistant callus is obtained after screening of antibiotics with corresponding resistance, and further differentiation and regeneration are carried out to obtain transgenic strains, namely the experimental process is as follows: culturing a tobacco sterile seedling → infecting with agrobacterium → screening → differentiating → rooting → detecting to obtain a T0 generation transgenic positive seedling,

the specific operation is performed with reference to the prior art, and is not described in detail. Only some matters of screening and identification will be described below.

The overexpression transgenic strain has hygromycin plant resistance, so when the hygromycin resistance gene is identified based on PCR, the sequence of the primer of the hygromycin resistance gene is designed as follows:

Hyg-F：5’-GTGCTTTCAGCTTCGATG-3’，

Hyg-R： 5’-AACCAAGCTCTGATAGAG-3’。

on the other hand, since the overexpression vector contains a GFP tag protein, verification was carried out by using the aforementioned DREBL-OE-F (DREBL 1-OE-F, DREBL2-OE-F, DREBL 3-OE-F) and GFP-R (5'-ACGAACTCCAGCAGGACCATGTGAT-3') for detection and identification by PCR.

RNAi transgenic seedlings have kanamycin plant resistance, and kanamycin resistance gene primer sequences are designed based on PCR detection and identification as follows:

hptII-F：5’-TCGCCGCCAAGCTCTTCAGCAAT-3’，

hptII-R：5’-GTGGAGAGGCTATTCGGC TATGACT-3’。

it should be further noted that, when analyzing the NtDREB overexpression and NtPSY gene expression change condition in RNAi plant based on qPCR, the primer sequence is designed as follows:

PSY-Q-F：5’-TGTTGGAGAAGATGCCAGAAGAG-3’，

PSY-Q-R：5’-ATAAGCAATAGGTAAGGAAATTAGCTTC-3’。

the electrophoresis images of the T0 generation tobacco NtDREB overexpression and RNAi transgenic tobacco seedlings are shown in figures 9 and 10, wherein the plants with bright bands are positive transformation plants.

Further carrying out self-pollination on the positive tobacco strain obtained from the T0 generation, harvesting seeds (seeds of the T0 generation), screening resistant seedlings on a hygromycin (overexpression) or kanamycin (RNAi) culture medium, and culturing to obtain T1 generation transgenic seedlings, wherein one part of the T1 generation transgenic seedlings is used for a stress resistance experiment, and the other part of the T1 generation transgenic seedlings continuously bear seeds. During the cultivation process, no obvious dysplasia is found no matter over-expression or RNAi transgenic tobacco seedlings, which indicates that the normal growth and development of plants are not influenced by the NtDREB transgene.

The T1 transgenic seedlings were verified by PCR, and some results are shown in FIG. 11. The GFP gene fragment and the amplified fragment in the plasmid control are equivalent in size (FIG. 11A), and the specific hygromycin resistance gene band can be amplified in the transgenic line, but the specific hygromycin resistance gene band is not amplified in the wild tobacco (FIG. 11B), which indicates that the construction of the transgenic line is successful.

Further, the results of analysis of gene expression in transgenic lines by qPCR technology showed that the transcription expression level of NtDREB gene in over-expressed transgenic plants was significantly increased (fig. 11C). In the transgenic tobacco plants with over-expression of NtDREB, the expression amount of NtPSY genes is obviously improved (figure 12A), which shows that the over-expression of NtDREB stimulates the expression of NtPSY genes. In the NtDREB RNAi transgenic tobacco strain (FIG. 12B), the NtPSY gene expression level is constantIndicates that silencing of NtDREB affectsNtPSYThe expression level of the gene. These results confirmNtDREBTo pairNtPSYThe gene has positive regulation function.

The results of further detecting the carotenoid and chlorophyll contents in the transgenic plants are shown in fig. 13. It can be seen that:NtDREBthe contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid in the over-expressed transgenic tobacco are increased (fig. 13A); whileNtDREBGene silencing resulted in a decrease in the metabolite carotenoid and chlorophyll content, with a significant decrease in carotenoid and chlorophyll content in NtDREB1BL1-RNAi tobacco strain (R1-7) (fig. 13B). This is shown inNtDREBIn transgenic tobacco, the change of the gene expression level affects the content of carotenoid and pigment.

Example 4

The ChIP (chromosome immunoprecipitation) chromatin co-immunoprecipitation technology can detect the binding condition of a specific transcription factor and a known gene promoter region, and searches the binding site of the specific transcription factor in the known promoter region. The ChIP-qPCR combines a ChIP technology and a real-time quantitative PCR technology, wherein the ChIP technology utilizes the specificity of antibodies to enrich DNA fragments combined with the modification of specific transcription factors/histones; the qPCR technology uses SYBR fluorescent dye, non-specifically mixes into DNA double chain, emits fluorescence, ensures the increase of fluorescent signal and PCR product to be completely synchronous, and finally uses Ct value to do quantitative analysis. Therefore, ChIP-qPCR can specifically, sensitively, rapidly and repeatedly quantify the combination of specific transcription factor/histone modification and known gene promoter in a biological sample. Therefore, the inventors further performed CHIP-qPCR experiments to preliminarily investigate the mechanism of action of NtDREB.

Based on the preliminary analysis of the cis-acting element of the PSY gene upstream promoter region (divided into 8 segments), the inventor carries out preliminary research by designing a fluorescent quantitative PCR primer and a qPCR experiment, and the experimental process comprises the following steps: the cross-linking-de-cross-linking-ultrasonic breaking-immunoprecipitation-washing-DNA purification-PCR detection and the like are summarized as follows:

(1) sample preparation: infecting Nicotiana benthamiana with Agrobacterium containing recombinant plasmid (DREBL 1-OE overexpression vector), and using WT and Sup 1300-overexpression empty-vector-injected tobacco plants as controls; collecting infected leaves after two weeks, treating the infected leaves with formaldehyde solution, and performing vacuum infiltration on tissues to perform a crosslinking reaction;

(2) preparing a chromatin solution: firstly, proteins bound on DNA are cross-linked on the bound sites in a covalent mode to avoid separation and loss in the subsequent operation steps, and chromatin particles are suspended in a mixture containing a protease inhibitor to form a chromatin solution after the cross-linking is removed;

(3) ultrasonic smashing: carrying out ultrasonic treatment on the chromatid solution on ice by using an ultrasonic water bath kettle so as to break the long-chain DNA into 200-and 1000-bp DNA fragments; after the cell suspension becomes clear and transparent after the ultrasonic treatment, 5 mu L of ultrasonic cell lysate can be taken for agarose gel analysis after the centrifugation;

(4) protein immunoprecipitation: after dilution, the sonicated product was divided into two portions, one was Input DNA and the other was transferred to a Strip well bound to the antibody for purification;

(5) DNA purification: the eluted product was purified with a DNA purification kit to obtain 50. mu.L of purified product for detection of ChIP results.

The results are shown in FIG. 14. Analysis shows that: the relative expression of the second segment is obviously higher than that of other segments, the promoters distributed in the second segment comprise CAAT-box, O2-site and MYB, wherein CAAT-box is a common constitutive element in promoter and enhancer regions, O2-site is involved in the metabolic regulation of zein, and MYB binding sites are important elements involved in drought induction, which is consistent with the function of DREB transcription factors. In summary, the NtDREB transcription factors can be combined with and regulate the promoter region of the PSY gene, and can regulate the expression of the tobacco NtPSY gene, but the combination of the NtDREB-1BL1 transcription factors is more typical.

SEQUENCE LISTING

<110> Zhengzhou tobacco institute of China tobacco general Co

<120> tobacco NtDREB-1BL1 transcription factor and application thereof

<130> none

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 660

<212> DNA

<213> Nicotiana tabacum

<400> 1

atggatatct ttagaagcta ttattcggac ccacttgctg aatattcatc aatttctgac 60

agtagtagca gctcctgtaa tagagctaac cattctgatg aggaagtgat gttagcttcg 120

aataacccca agaagcgagc agggagaaag aagtttagag aaactcgaca cccagtatac 180

aggggagtga ggaagaggaa ttcagacaag tgggtttgtg aactcagaga accaaacaag 240

aaatcaagaa tatggctggg cactttccct tctgcagaaa tggcggctag agctcatgac 300

gtggcggcta ttgcattaag gggccgttct gcttgcttga actttgctga ctctgcttgg 360

aagttgccta ttccagcttc aaccgacgcc aaggatattc agaaagcggc ggcggaggcc 420

gcggaggcat tccggtcatc ggaggccgaa aacatgccgg aatactcagg agaagatacg 480

aaggaagtga acagtactcc tgaaaatatg ttttatatgg atgaggaggc gctattcttc 540

atgcctggat tactagtgaa tatggcagaa ggactaatgt tacctccacc tcagtgttca 600

caaattggag atcatatgga agctgatgtt gacatgcctt tgtggagtta ttctatctaa 660

<210> 2

<211> 657

<212> DNA

<213> Nicotiana tabacum

<400> 2

atggatatct ttcgtagctt ttactcggac ccacttgctg attcttcatc actttctgat 60

agtagcagct cctgtaatag agctaacctt tctgatgaag aagttatgtt agcttcaaat 120

aaccccaaga agcgggcagg gaggaagaag tttcgagaaa ctcgacaccc agtatacagg 180

ggagtgagaa agaggaattc aggcaagtgg gtttctgaag tcagagaacc aaacaagaaa 240

tcaagaatat ggcttggcac tttcccttct gcagaaatgg cggctagagc gcatgacgtg 300

gcggctattg cattaagggg ccgttctgct tgcttgaact tcgcagactc tgcttggaag 360

ttgcctattc ctgcctcaac cgacgccaag gatattcaga aagcggcggc tgaggccgcg 420

gaggcattcc ggtcatcgga ggccgaaaaa atgccggaat acacaggaga agattcaaag 480

gaagtgaaca ctactcctga aaatatgttt tatatggatg aggagacgct attctgcatg 540

ccgggattac tagcaaatat ggctgaagga ttaatgttac ctccacctca gtgttcacaa 600

attggagatc atttggaagc tgatgttgac atgcctttgt ggagttattc tatttaa 657

<210> 3

<211> 654

<212> DNA

<213> Nicotiana tabacum

<400> 3

atggaaatgt gtcgaagcta ttattcggac ccacttgctg attcttcatc actgtctgat 60

agtagcagct cctgtaatag agctatccgt tctaatgaag aagttatgtt agcttcgaat 120

aaccccaaga agcgagcagg gaggaagaag tttcgagaaa ctcgacaccc agtatacagg 180

ggagtgagaa agaggaattc aggcaagtgg gtttctgaag tcagagaacc aaacaagaaa 240

tcaagaatat ggcttggcac tttcccttct gcagaaatgg cggctagagc gcatgacgtg 300

gcggctattg cattaagggg ccgttctgct tgcttgaact tcgcagactc tgcttggaag 360

ttgcctgttc ctgcttcctc cgacgccaag aatattcaga aggcggctgc cgaggccgcc 420

gaggctttcc ggtcatcgga ggccgaaaac atgccggaat acacaggaga agattcaaag 480

gaagtgaaca ctactcctga aaatatgttt tatatggatg aggaggcgct attctgcatg 540

ccgggattac ttgcgaatat ggcagaagga ttaatgttac ctccacctca gtgttcccaa 600

attggagatg atcatatgga ggctgatatg cctttgtgga gttattcaat ttaa 654

<210> 4

<211> 219

<212> PRT

<213> Nicotiana tabacum

<400> 4

Met Asp Ile Phe Arg Ser Tyr Tyr Ser Asp Pro Leu Ala Glu Tyr Ser

1 5 10 15

Ser Ile Ser Asp Ser Ser Ser Ser Ser Cys Asn Arg Ala Asn His Ser

20 25 30

Asp Glu Glu Val Met Leu Ala Ser Asn Asn Pro Lys Lys Arg Ala Gly

35 40 45

Arg Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg

50 55 60

Lys Arg Asn Ser Asp Lys Trp Val Cys Glu Leu Arg Glu Pro Asn Lys

65 70 75 80

Lys Ser Arg Ile Trp Leu Gly Thr Phe Pro Ser Ala Glu Met Ala Ala

85 90 95

Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly Arg Ser Ala Cys

100 105 110

Leu Asn Phe Ala Asp Ser Ala Trp Lys Leu Pro Ile Pro Ala Ser Thr

115 120 125

Asp Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu Ala Ala Glu Ala Phe

130 135 140

Arg Ser Ser Glu Ala Glu Asn Met Pro Glu Tyr Ser Gly Glu Asp Thr

145 150 155 160

Lys Glu Val Asn Ser Thr Pro Glu Asn Met Phe Tyr Met Asp Glu Glu

165 170 175

Ala Leu Phe Phe Met Pro Gly Leu Leu Val Asn Met Ala Glu Gly Leu

180 185 190

Met Leu Pro Pro Pro Gln Cys Ser Gln Ile Gly Asp His Met Glu Ala

195 200 205

Asp Val Asp Met Pro Leu Trp Ser Tyr Ser Ile

210 215

<210> 5

<211> 218

<212> PRT

<213> Nicotiana tabacum

<400> 5

Met Asp Ile Phe Arg Ser Phe Tyr Ser Asp Pro Leu Ala Asp Ser Ser

1 5 10 15

Ser Leu Ser Asp Ser Ser Ser Ser Cys Asn Arg Ala Asn Leu Ser Asp

20 25 30

Glu Glu Val Met Leu Ala Ser Asn Asn Pro Lys Lys Arg Ala Gly Arg

35 40 45

Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys

50 55 60

Arg Asn Ser Gly Lys Trp Val Ser Glu Val Arg Glu Pro Asn Lys Lys

65 70 75 80

Ser Arg Ile Trp Leu Gly Thr Phe Pro Ser Ala Glu Met Ala Ala Arg

85 90 95

Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly Arg Ser Ala Cys Leu

100 105 110

Asn Phe Ala Asp Ser Ala Trp Lys Leu Pro Ile Pro Ala Ser Thr Asp

115 120 125

Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu Ala Ala Glu Ala Phe Arg

130 135 140

Ser Ser Glu Ala Glu Lys Met Pro Glu Tyr Thr Gly Glu Asp Ser Lys

145 150 155 160

Glu Val Asn Thr Thr Pro Glu Asn Met Phe Tyr Met Asp Glu Glu Thr

165 170 175

Leu Phe Cys Met Pro Gly Leu Leu Ala Asn Met Ala Glu Gly Leu Met

180 185 190

Leu Pro Pro Pro Gln Cys Ser Gln Ile Gly Asp His Leu Glu Ala Asp

195 200 205

Val Asp Met Pro Leu Trp Ser Tyr Ser Ile

210 215

<210> 6

<211> 217

<212> PRT

<213> Nicotiana tabacum

<400> 6

Met Glu Met Cys Arg Ser Tyr Tyr Ser Asp Pro Leu Ala Asp Ser Ser

1 5 10 15

Ser Leu Ser Asp Ser Ser Ser Ser Cys Asn Arg Ala Ile Arg Ser Asn

20 25 30

Glu Glu Val Met Leu Ala Ser Asn Asn Pro Lys Lys Arg Ala Gly Arg

35 40 45

Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg Gly Val Arg Lys

50 55 60

Arg Asn Ser Gly Lys Trp Val Ser Glu Val Arg Glu Pro Asn Lys Lys

65 70 75 80

Ser Arg Ile Trp Leu Gly Thr Phe Pro Ser Ala Glu Met Ala Ala Arg

85 90 95

Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly Arg Ser Ala Cys Leu

100 105 110

Asn Phe Ala Asp Ser Ala Trp Lys Leu Pro Val Pro Ala Ser Ser Asp

115 120 125

Ala Lys Asn Ile Gln Lys Ala Ala Ala Glu Ala Ala Glu Ala Phe Arg

130 135 140

Ser Ser Glu Ala Glu Asn Met Pro Glu Tyr Thr Gly Glu Asp Ser Lys

145 150 155 160

Glu Val Asn Thr Thr Pro Glu Asn Met Phe Tyr Met Asp Glu Glu Ala

165 170 175

Leu Phe Cys Met Pro Gly Leu Leu Ala Asn Met Ala Glu Gly Leu Met

180 185 190

Leu Pro Pro Pro Gln Cys Ser Gln Ile Gly Asp Asp His Met Glu Ala

195 200 205

Asp Met Pro Leu Trp Ser Tyr Ser Ile

210 215

Claims (2)

1. The application of the tobacco NtDREB-1BL1 transcription factor in tobacco pigment regulation is characterized in that the transcription factor is positioned in the nucleus of a tobacco lamina cell and is combined with the upstream promoter sequence of the NtPSY gene in tobacco to realize the regulation and control of the expression quantity of the NtPSY gene in tobacco, thereby influencing the content change of pigment substances in lamina; the expression quantity of the tobacco NtDREB-1BL1 transcription factor is in positive correlation with the content of pigment substances;

the pigment substances are chlorophyll a, chlorophyll b, total chlorophyll and carotenoid;

the CDS base sequence of the tobacco NtDREB-1BL1 transcription factor is shown in SEQ ID No. 1.

2. The method for cultivating the new tobacco variety by utilizing the tobacco NtDREB-1BL1 transcription factor is characterized in that the new tobacco variety with obviously changed pigment substance content is obtained by constructing a recombinant vector, transforming tobacco and screening;

the tobacco NtDREB-1BL1 transcription factor expression level and the content of pigment substances present a positive correlation;

the pigment substances are chlorophyll a, chlorophyll b, total chlorophyll and carotenoid;

the CDS base sequence of the tobacco NtDREB-1BL1 transcription factor is shown in SEQ ID No. 1;

the recombinant vector comprises a gene silencing vector pTRV2-DREBL1 constructed by the VIGS technology; a super-expression recombinant vector Sup1300-DREBL1-OE constructed by combining a Sup1300-GFP vector by adopting a super-expression technology; RNAi-DREBL1 constructed by RNAi technology; when the concrete construction is carried out, the concrete structure,

constructing a gene silencing vector pTRV2-DREBL1, and when amplifying a conserved region, specifically obtaining the following primer sequences:

TRV-DREB1-F：5’-CGGGATCCGGACCCACTTGCTGAATATT-3’，

TRV-DREB1-R：5’- GCGGTACCTAACATTAGTCCTTCTGCCAT-3’；

when constructing the over-expression recombinant vector Sup1300-DREBL1-OE, the primer sequence is designed as follows:

DREBL1-OE-F：5‘-GCTCTAGAATGGATATCTTTAGAAGCTATTATTC-3’，

DREBL1-OE-R：5‘-GGTACCGATAGAATAACTCCACAAAGGC-3’；

when constructing RNAi vector RNAi-DREBL1, RNAi vector primer sequences are designed as follows:

DREBL1-RNAi-attB-F：

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCTATTATTCGGACCCACTTGC-3’，

common RNAi-attB-R:

5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGAACGGCCCCTTAATGCAAT-3’。

Images