阅读说明：本技术 烟草NtDREB-1BL1转录因子及其应用 (Tobacco NtDREB-1BL1 transcription factor and application thereof ) 是由 王燃 魏攀 王宇博 董臣 金立锋 李锋 张梅 谢小东 李泽锋 郑庆霞 王晨 于 2020-06-04 设计创作，主要内容包括：本申请属于烟草基因组解析技术领域,具体涉及烟草NtDREB类转录因子及其应用专利申请事宜。烟草NtDREB类转录因子,包括NtDREB-1BL1、NtDREB-1BL2、NtDREB-1BL3三个,相应CDS碱基序列如SEQ ID No.1~3所示。对于现有其他作物中DREB类转录因子功能研究认为,这类转录因子与植物抗逆性紧密关联。而本申请中,发明人通过对烟草中三个NtDREB转录因子功能研究,发现烟草中NtDREB类基因与烟草中色素类物质含量紧密相关,通过调节NtPSY基因表达来实现烟叶中色素类物质含量的同步调整,基于此结果,可为烟草遗传定向改良和烟叶品质提升奠定良好的理论基础和技术基础。(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.)

1. The tobacco NtDREB-1BL1 transcription factor is characterized in that the CDS base sequence of the gene is shown as SEQ ID No.1, and has 660bp, which is as follows:

ATGGATATCTTTAGAAGCTATTATTCGGACCCACTTGCTGAATATTCATCAATTTCTGACAGTAGTAGCAGCTCCTGTAATAGAGCTAACCATTCTGATGAGGAAGTGATGTTAGCTTCGAATAACCCCAAGAAGCGAGCAGGGAGAAAGAAGTTTAGAGAAACTCGACACCCAGTATACAGGGGAGTGAGGAAGAGGAATTCAGACAAGTGGGTTTGTGAACTCAGAGAACCAAACAAGAAATCAAGAATATGGCTGGGCACTTTCCCTTCTGCAGAAATGGCGGCTAGAGCTCATGACGTGGCGGCTATTGCATTAAGGGGCCGTTCTGCTTGCTTGAACTTTGCTGACTCTGCTTGGAAGTTGCCTATTCCAGCTTCAACCGACGCCAAGGATATTCAGAAAGCGGCGGCGGAGGCCGCGGAGGCATTCCGGTCATCGGAGGCCGAAAACATGCCGGAATACTCAGGAGAAGATACGAAGGAAGTGAACAGTACTCCTGAAAATATGTTTTATATGGATGAGGAGGCGCTATTCTTCATGCCTGGATTACTAGTGAATATGGCAGAAGGACTAATGTTACCTCCACCTCAGTGTTCACAAATTGGAGATCATATGGAAGCTGATGTTGACATGCCTTTGTGGAGTTATTCTATCTAA。

2. the protein encoded by the NtDREB-1BL1 transcription factor encoding gene of claim 1, wherein the specific amino acid sequence is shown in SEQ ID No.4, and specifically as follows:

MDIFRSYYSDPLAEYSSISDSSSSSCNRANHSDEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSDKWVCELREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPIPASTDAKDIQKAAAEAAEAFRSSEAENMPEYSGEDTKEVNSTPENMFYMDEEALFFMPGLLVNMAEGLMLPPPQCSQIGDHMEADVDMPLWSYSI。

3. the recombinant expression vector constructed by the NtDREB-1BL1 transcription factor of claim 1, which is characterized by comprising a gene silencing vector pTRV2-DREBL1 constructed by VISG 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’；

the amplification length is 586 bp;

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, the RNAi vector primer sequence is designed as follows:

DREBL1-RNAi-attB-F：

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCTATTATTCGGACCCACTTGC-3’，

common RNAi-attB-R:

5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGAACGGCCCCTTAATGCAAT-3’。

4. a method for breeding a new variety of plants using the recombinant expression vector of claim 3.

5. The use of the tobacco NtDREB-1BL1 transcription factor in phytochrome regulation as claimed in claim 1, wherein the transcription factor is located in the nucleus of tobacco lamina cell, and is combined with the upstream promoter sequence of ntpsay gene in tobacco to realize the regulation and control of the expression level of ntpsay gene in tobacco, thereby affecting the content change of pigment substances in lamina; the expression quantity of the NtDREB transcription factor and the content of the pigments show positive correlation; the pigment substances include chlorophyll a, chlorophyll b, total chlorophyll and carotenoid.

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 carotenoid content, a tobacco friable Virus Induced Gene Silencing (VIGS) technology is utilized, and a part of genes are found to have obvious influence on the carotenoid content by silencing corresponding target genes (tobacco carotenoid isomerase gene and application thereof, application No. 201410405272. X; tobacco-lycopene cyclase gene and application thereof, 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), namely, a process for preparing a cyclaseNtβ- 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. However, the technical defects existing when over-expression (OE) and RNAi techniques are used are mainly as follows: 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 regulatory mechanism of cyclase for 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 landed on a specific genetic mechanism, namely, transcription and post-transcription regulation, so that transcription factors or interacting proteins capable of regulating PSY are 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 mode and 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 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 transcription factors 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, which are encoded by the tobacco NtDREB transcription factors, are shown in SEQ ID Nos. 4-6, and are specifically as follows:

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

MDIFRSYYSDPLAEYSSISDSSSSSCNRANHSDEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSDKWVCELREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPIPASTDAKDIQKAAAEAAEAFRSSEAENMPEYSGEDTKEVNSTPENMFYMDEEALFFMPGLLVNMAEGLMLPPPQCSQIGDHMEADVDMPLWSYSI。

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

MDIFRSFYSDPLADSSSLSDSSSSCNRANLSDEEVMLASNNPKKRAGRKKFRETRHPVYRGVRKRNSGKWVSEVREPNKKSRIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLPIPASTDAKDIQKAAAEAAEAFRSSEAEKMPEYTGEDSKEVNTTPENMFYMDEETLFCMPGLLANMAEGLMLPPPQCSQIGDHLEADVDMPLWSYSI。

NtDREB-1BL3amino acid sequence of gene codingColumn (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 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, when a conserved region is 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 super-expression 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, 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 utilizing 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 plant pigment 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 the tobacco are realized by combining with the upstream promoter sequence of the NtPSY gene in the tobacco, so that the content change of pigment 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.

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.

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 showing the structure of NtDREB-1BL1, NtDREB-1BL2, NtDREB-1BL3 and their homologous genes;

FIG. 4 shows the relative expression levels of the NtDREB genes in Nicotiana tabacum K326, wherein A, B, C is the relative expression levels of NtDREB-1BL1, NtDREB-1BL2 and NtDREB-1BL3 in Nicotiana tabacum 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 an unvaccinated control tobacco strain, TRV is an inoculated empty viral vector control tobacco strain without gene segments, DREB1-V is an inoculated viral vector tobacco strain carrying NtDREB-1BL1 segments, and DREB2-V is an inoculated viral 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 an empty virus vector control tobacco strain inoculated with no gene fragment, DREB1-vigs is a virus vector tobacco strain inoculated with NtDREB-1BL1 fragment, and DREB2-vigs is a virus vector tobacco strain inoculated with NtDREB-1BL2 fragment;

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 of 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 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 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.