CN113774059A - Dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof - Google Patents

Abstract

The invention discloses a dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof, belonging to the technical field of molecular biology. The invention provides a promoter ProDoWOX4, which has a length of 2003bp and can induce a corresponding target gene to be preferentially expressed in sepals and petals of flower organs, and the preferential expression not only can reduce the consumption of other resources and energy of plants, but also provides a proper regulatory element for the construction of a plant genetic engineering expression vector; has important significance for improving and improving excellent characters of salt tolerance or cold tolerance and the like of plants, thereby accelerating the breeding process of excellent varieties. The invention also provides a nucleic acid construct containing the promoter ProDoWOX4, a recombinant expression vector, a host cell, a genetic engineering cell, an amplification primer and application of the nucleic acid construct and the recombinant expression vector.

Description

Dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof.

Background

In eukaryotes, transcription of gene expression is regulated by promoters (Wray et al, 2003). The promoter contains a specific DNA sequence that contains a binding site for the RNA polymerase protein complex, and is critical for the regulation of the native gene or for the proper expression of the transgene. With the research of the promoter, the promoter is rapidly developed in the application of genetic engineering. Genetic transformation is an important approach in plant breeding, from basic research to crop improvement, the use of suitable promoters is a key factor ensuring the success of genetic transformation (Potenza et al, 2004, Porto et al, 2014). Therefore, the development and utilization of a high-efficiency and/or specific promoter have extremely high application value and important research significance in the fields of plant genetic engineering breeding and the like.

Tissue-preferred promoters are expressed predominantly in a particular tissue or organ. Researches show that the specific or preferential promoter has the biological characteristic of avoiding unnecessary waste of plant nutrition biologically, and can be widely applied to genetic engineering, such as molecular breeding of crops in bioreactors, stress resistance, variety improvement and the like. In addition, this type of promoter can also be induced by hormonal or abiotic stress. For example, in tobacco, the promoter NgORF13 p can be induced by an exogenous methyl jasmonate, thereby activating the activity of NgORF13 p (Udagawa et al, 2004). In poplar, the promoter PtDrlo2 p was found to be expressed specifically in tissues such as vein, petiole, stem and stem pith, and different fragments of the promoter were affected by different induction conditions such as injury, NaCl and abscisic acid (ABA), etc. (Zheng et al, 2010). However, unlike the case in dicotyledonous plant systems, the current promoter development and application in monocotyledonous plants is limited (Damaj et al, 2010); at present, constitutive promoters with high expression are widely applied to monocotyledons (McElroy et al, 1990, He et al, 2009), and other types of promoters such as tissue-preferred promoters are less applied.

The flower is the key of the successful reproduction and multiplication of the plant and is also an important ornamental organ, and the flower forming process is very complicated. Dendrobium officinale (Dendrobium officinale) is an orchid herbaceous monocotyledon, has high medicinal value, has strict requirements on the growth environment, is often attached to cliff, trunk or rock, and is easily influenced by abiotic stress factors such as drought and salinization. Its flower is highly specialized in structure, including 3 valvular sepals, 2 petals, 1 labial valve and 1 synanthus column, and this unique flower type gives it rich value. However, few studies on flowering regulation of dendrobium officinale are carried out so far, and no report on studies on the promoter of the DoWOX4 gene of dendrobium officinale is found. Therefore, the research on the expression and regulation of the ProDoWOX4 promoter on the dendrobium officinale DoWOX4 gene and the development and utilization of a high-efficiency specific promoter in genetic engineering have very important application and value.

Disclosure of Invention

According to the invention, a ProDoWOX4 promoter is transferred into arabidopsis thaliana by an agrobacterium-mediated and inflorescence infection method, and the GUS result of a T3 generation arabidopsis thaliana plant transferred with a ProDoWOX4 promoter vector shows that the GUS gene started by the promoter ProDoWOX4 is highly expressed in flowers, especially petals and sepals of arabidopsis thaliana, and responds to salt induction and drought induction at the same time, which indicates that the ProDoWOX4 promoter is a tissue preference and inducible promoter.

The first aspect of the invention provides a flower tissue preference and stress inducible promoter ProDoWOX4, the nucleotide sequence of which has more than 95% homology with SEQ ID NO.1, or more than 95% homology with the complementary sequence of SEQ ID NO.1, or has the function of a promoter by replacing, adding or deleting one or more bases in SEQ ID NO.1 or the complementary sequence thereof;

preferably, the nucleotide sequence of the promoter is shown as SEQ ID NO.1, or the nucleotide sequence of the promoter is complementary to the nucleotide sequence shown as SEQ ID NO. 1.

In a second aspect, the present invention provides a nucleic acid construct comprising the promoter of the first aspect, ProDoWOX4, and a foreign gene operably linked to the promoter ProDoWOX 4;

preferably, the exogenous gene may include, but is not limited to, a selection marker gene, a reporter gene, a regulatory gene, a plant quality-related gene, a stress-resistance gene;

more preferably, the stress resistance gene may include, but is not limited to, an antiviral gene, a cold resistance gene, a high temperature resistance gene, a drought resistance gene, a waterlogging resistance gene, an insect resistance gene, a high salt protein resistance gene.

In a third aspect, the present invention provides a recombinant expression vector comprising the promoter of the first aspect, ProDoWOX4, or the nucleic acid construct of the second aspect;

preferably, the recombinant expression vector is pCAMBIA1301: ProDoWOX4: GUS;

more preferably, the pCAMBIA1301: ProDoWOX4: GUS is the dendrobium officinale promoter ProDoWOX4 cloned on pCAMBIA1301 plasmid containing GUS gene.

In a fourth aspect of the present invention, there is provided a host cell into which the promoter of the first aspect, ProDoWOX4, or the nucleic acid construct of the second aspect, or the vector of the third aspect, has been introduced;

preferably, the host cell is Agrobacterium or Escherichia coli;

more preferably, the host cell is Agrobacterium EHA 105.

In a fifth aspect, the present invention provides a recombinant cell having integrated into its genome the promoter of the first aspect, ProDoWOX4, or the nucleic acid construct of the second aspect;

preferably, the genetically engineered cell is a plant cell.

The sixth aspect of the present invention provides the use of the promoter ProDoWOX4 according to the first aspect, the nucleic acid construct according to the second aspect, the vector according to the third aspect, or the cell according to the fourth aspect, for promoting the efficient expression of a target gene;

preferably, the promoter ProDoWOX4 of the invention can promote the high-efficiency expression of the target gene in plant floral tissue.

The seventh aspect of the present invention provides the use of the promoter of the first aspect, ProDoWOX4, the nucleic acid construct of the second aspect, the vector of the third aspect or the cell of the fourth aspect for growing improved plant varieties;

preferably, the improvement is to improve the nutritional quality and content, yield, stability, stress resistance and the like of the plants, such as improving the fiber quality of cotton, such as warmth retention, color, strength, length and the like, improving the potato processability, reducing harmful ingredients in crops, such as toxicity, allergens and the like, improving the nutritional ingredients and digestibility of feed crops and pasture and the like;

more preferably, the improvement is an improvement in salt tolerance or cold tolerance of the plant.

In an eighth aspect, the invention provides a set of primers for amplifying a nucleotide sequence shown as SEQ ID NO.1, wherein the primers are:

ProDoWOX4-FP2：5'-tttcttatgatctatgctcc-3'，ProDoWOX4-RP2：5'-atggaacttgaggcatcttcc-3'；

preferably, the primers are used for cloning or identifying the promoter ProDoWOX4 of the invention, wherein the PCR reaction system is 50 μ L (KODFX) and the reaction program is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 40 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

The ninth aspect of the present invention provides a method for efficiently expressing a target gene, comprising the steps of: a. cloning the promoter ProDoWOX4 to pCAMBIA1301 plasmid containing target genes, and then transferring the plasmid into agrobacterium tumefaciens EHA105 to obtain a recombinant plant expression vector through screening; b. the recombinant plant expression vector is transformed into a plant cell of interest.

Compared with the prior art, the invention has the following beneficial effects: the invention discovers and positions a new tissue preference and stress inducible promoter ProDoWOX4 of dendrobium officinale flowers for the first time. The promoter ProDoWOX4 can induce the preferential expression of the corresponding target gene in sepals and petals of flower organs, and the preferential expression can reduce the consumption of other resources and energy of plants and provide a proper regulatory element for the construction of plant genetic engineering expression vectors. Another advantage of ProDoWOX4 is that transgene expression can be induced by one or more abiotic stresses (such as salt stress and drought stress) in leaves, which is significant for improving and improving excellent traits such as salt tolerance or cold tolerance of plants, thereby accelerating the breeding process of excellent varieties.

Drawings

FIG. 1 is an electrophoretogram of PCR products of ProDoWOX 4.

FIG. 2 shows the result of GUS staining of different tissues (root, stem, leaf, inflorescence and fruit pod) in the maturation phase of a pCAMBIA1301: ProDoWOX4: GUS Arabidopsis transgenic line.

FIG. 3 shows the result of GUS staining in drought stress and salt stress at the seedling stage of pCAMBIA1301, ProDoWOX4 and GUS Arabidopsis thaliana.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The experimental methods not specifically mentioned in the following examples can be carried out according to conventional methods. Such as those described in molecular cloning guidelines, sambrook et al, f, osiber et al, fine compiled guidelines for molecular biology, or according to the instructions of the manufacturer of the product used.

Example 1 obtaining and structural analysis of Dendrobium officinale promoter ProDoWOX4 sequence

Dendrobium officinale (Dendrobium officinale Kimura et Migo) tissue culture seedlings were grown on a culture medium of 1/2MS + 0.1% activated carbon + 2% sucrose + 0.6% agar powder (pH5.4) and placed in a tissue culture room for culture (conditions of 24 + -2 ℃ C., 12 hours of illumination).

And (4) extracting the dendrobium officinale genome DNA. The experiment adopts a hand-held method to extract the genome DNA of the dendrobium officinale, and comprises the following experimental steps:

(1) grinding Dendrobium officinale protocorm into powder in liquid nitrogen, quickly transferring into a 2mL centrifuge tube, adding 700 μ L of 2 × CTAB extract (preheated 65 ℃ in advance), and fully reversing and mixing for several times. The mixture was then placed in a 65 ℃ water bath for 15min, during which time it was shaken every 5 min.

(2) Taking out the mixed solution, cooling at room temperature, adding equal volume of phenol chloroform, mixing with vortex mixer (SCILOGEX, USA) under shaking, and centrifuging at 12000r/min for 15 min.

(3) Taking the supernatant, adding 700 mu L of chloroform for extraction once again, transferring the supernatant to a new 1.5mL centrifuge tube, adding 0.6 time volume of isopropanol, and shaking and mixing uniformly. Standing in a refrigerator at-20 deg.C for 10 min.

(4) Centrifuging: centrifuging at 12000r/min for 10 min. The supernatant was carefully aspirated, and 500. mu.L of 70% ethanol was added to the DNA pellet in a 1.5mL centrifuge tube to wash the DNA pellet. Centrifuging at 12000r/min for 30sec, and removing the alcohol in the centrifuge tube as thoroughly as possible by using a gun head. Repeat the steps once and wash the DNA pellet twice.

(5) The tube was then opened and air-dried at room temperature for 5min, 100. mu.L of 1 XTE (containing RNaseA, 20. mu.g/mL) was added to dissolve the DNA, and the reaction was carried out at 37 ℃ for 30min to remove RNA.

(6) Detection of concentration and purity of DNA: the OD260/OD280 and OD260/OD230 values were measured using a NanoDrop 2000 spectrophotometer to determine the purity of the DNA, and the concentration of the DNA was recorded.

(7) Taking 2 microliter of DNA solution for agarose gel electrophoresis detection, and storing the rest in a refrigerator at-20 ℃ for later use.

The promoter ProDoWOX4 sequence was cloned using nested PCR technology. First, corresponding primers were designed and synthesized. Obtaining two pairs of PCR specific primers by using the dendrobium officinale according to a whole genome sequencing result:

ProDoWOX4-FP1：5'-TGAACTCTAGAGCATAATAC-3'，

ProDoWOX4-RP1：5'-GAGTATCATAAGTTGCTCTG-3'；

ProDoWOX4-FP2：5'-TTTCTTATGATCTATGCTCC-3'，

ProDoWOX4-RP2：5'-ATGGAACTTGAGGCATCTTCC-3'。

then, using the genomic DNA of Dendrobium officinale as a template, and using a pair of primers ProDoWOX4-FP1/ProDoWOX4-RP1 to perform a first round of PCR amplification. The enzyme used is Toyo Boseki high fidelity DNA polymerase Kit (KODFX), the reaction system is 20 μ L, and the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 36 cycles; extension at 72 ℃ for 10 min. Then, the PCR product of the first round of amplification was diluted 50 times and used as a template, and the second round of PCR amplification was performed using a pair of primers ProDoWOX4-FP2/ProDoWOX4-RP 2. The high fidelity enzyme used for amplification was the same as the first PCR, with a reaction system of 50. mu.L, and the reaction procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 40 cycles; extension at 72 ℃ for 10 min. The specific operation is shown in the specification. The PCR amplification is carried out to obtain a fragment with the size of 2003bp, the PCR electrophoresis picture is shown in figure 1, and the nucleotide sequence is shown in SEQ ID NO. 1.

The promoter structure and cis-element prediction were carried out on the obtained promoter ProDoWOX4 sequence fragment using the online bioinformatics software PlantCARE (http:// bioinformatics. psb. element. be/webtools/plantare/html /) (Lescot et al, 2002). Further, we analyzed the ProDoWOX4 cis-acting element (Table 1).

TABLE 1

The results show that the ProDoWOX4 promoter was identified to contain multiple light-responsive elements, such as ATCT-motif, Box 4, and GT1-motif, among others. Meanwhile, hormone response-related elements include Gibberellin (GA) and auxin (auxin), etc. A number of cis-acting elements in response to abiotic stress, namely anaerobically induced AREs, drought response elements MBS and defense and stress response elements TC-rich repeats, were also predicted (Table 1). In addition, ProDoWOX4 contains 29 common cis-acting elements for promoter and enhancer regions (CAAT-box), and about 59-30 core promoter transcription initiation elements (TATA-box). The presence of these elements not only indicates that the fragment has the complete promoter structure (including promoter and enhancer regions, common cis-acting elements of TATA-box and other promoter cis-elements), but also indicates that DoWOX4 has numerous cis-regulatory elements in the promoter region, is an important regulator of plant biological processes, and may play an important role in plant growth and development, hormone response and stress response.

Example 2 construction of a plant expression vector containing promoter ProDoWOX4 sequence and transformation of the vector into Agrobacterium

The expression vector of interest used in this example was the binary expression vector pCAMBIA1301 (which contains a 35S promoter element as well as a GUS site, Kana resistance).

Firstly, a primer is designed to amplify a promoter ProDoWOX4 with a pCAMBIA1301 vector joint, a template is extracted dendrobium officinale DNA, a high-fidelity enzyme for amplification is a Toyobo high-fidelity DNA polymerase kit (KOD FX), a reaction system is 50 mu L, and the specific reaction system and operation steps are referred to the specification. The cloning primers of the ProDoWOX4 promoter with the carrier 1301 joint are as follows:

ProDoWOX4-1301-FP1：5'-CGGTACCCGGGGATCCTTTCTTATGATCTATGCTCC-3'，

ProDoWOX4-1301-RP1：5'-CCTCAGATCTACCATGGATGGAACTTGAGGCATCTTCC-3', the linkers used to construct the vector are underlined. The PCR product was identified by 1% agarose gel electrophoresis and recovered by cutting gel using gel DNA kit (Magen, Guangzhou, China) to obtain the recovered ProDoWOX4 product with 1301 vector linker, the detailed procedure was as described.

Then, a overexpression vector was constructed. The target fragment (the recovered product of ProDoWOX4 with 1301 vector linker) was connected to the target expression vector (the linearized vector pCAMBIA1301 after double digestion) by using a seamless cloning technique, the kit used was In-Fusion (TaKaRa, Kyoto, Japan), and the specific operation was performed according to the homologous recombination reaction system In the instruction. The homologous recombination reaction system is as follows: 5 XIn-Fusion HD Enzyme Premix 2. mu.L; linear Vector 50-200 ng; purification PCR Fragment 400 ng; ddH2O was supplemented to a total volume of 10. mu.L. The added sample is placed in a PCR instrument (reaction is carried out for 60min at 50 ℃), and immediately placed on ice after the reaction is finished, so that the subsequent transformation experiment can be carried out. Coli used for transformation experiments was Tiangen DH5 alpha (Tiangen Biochemical technology Co., Ltd., Beijing, China)), and the specific transformation procedures were described in the specification. The transformed sample was then spread on a solid plate containing LB of Kana (50mg/mL) and cultured in an inverted incubator at 37 ℃ for about 12 hours. After the bacterial colony grows out of the culture plate, selecting a single bacterial colony in a liquid LB culture medium containing Kana (50mg/mL), culturing the single bacterial colony in a shaking table (37 ℃, 220rpm) until the bacterial liquid is turbid (about 14-16 h), and then carrying out bacterial liquid PCR to verify the positive recon. The positive clones were sent to Beijing Ongzhike Biotech Co., Ltd for sequencing. Finally, the ProDoWOX4 sequence cloned from dendrobium officinale successfully replaces the CaMV 35S promoter sequence in the pCAMBIA1301 expression vector to obtain the pCAMBIA1301-ProDoWOX4-GUS overexpression vector, and the pCAMBIA1301-ProDoWOX4-GUS overexpression vector is stored at the temperature of-20 ℃ for later use. The plasmid of the positive recombinant pCAMBIA1301: ProDoWOX4: GUS overexpression vector is transformed into the agrobacterium strain EHA105 by a freeze-thaw method, the specific operation is described in the specification, and the pCAMBIA1301: CaMV 35S: GUS expression vector containing 35S promoter is used as a control. And (3) selecting the agrobacterium tumefaciens single colony on the culture plate which is successfully transformed, and verifying by adopting a bacterial liquid PCR method (PCR primers are ProDoWOX4-FP2 and ProDoWOX4-RP2) to screen positive clones, wherein the target expression vector is determined to be successful if the size of a glue running strip is consistent with that of a target strip. The positive agrobacterium liquid is preserved as glycerol (the final concentration of glycerol is 30%) by glycerol, and the glycerol is placed in an ultra-low temperature refrigerator at minus 80 ℃ for standby.

Example 3 pCAMBIA1301: ProDoWOX4: GUS for Arabidopsis genetic transformation and molecular characterization

The agrobacterium containing the plant expression vector obtained in example 2 is infected by inflorescence, and the expression vector containing the target sequence is introduced into arabidopsis thaliana to realize the genetic transformation of arabidopsis thaliana. The steps of the arabidopsis inflorescence infection method are as follows:

(1) the target agrobacterium liquid stored at-80 ℃ is subjected to plate cutting activation, a single colony is picked up in an LB liquid medium (Kana resistance), and is placed in a shaking table (28 ℃,200 rpm) for shaking culture overnight (about 16h) until the bacterial liquid OD600 is about 0.8.

(2) And (4) centrifuging the cultured agrobacterium liquid (5000rpm for 10min) to collect target bacteria.

(3) 100mL of the permeate medium (1/2MS + 5% sucrose) was prepared and 20. mu.L of the surfactant Silwet L-77 was added to resuspend the Agrobacterium.

(4) Taking arabidopsis thaliana with proper growth vigor (about 4-5 weeks, bolting to about 8cm, and no pod growth), immersing all arabidopsis thaliana inflorescences in the agrobacterium heavy suspension for 1min, and obliquely airing. Culturing the above Arabidopsis thaliana in the dark for 2 days, taking out, and culturing in a plant temperature-controlled culture chamber under the conditions of 22 deg.C, 16 hr light, and 8 hr dark circadian rhythm. One week later, the infection was performed again according to the previous procedure.

And (3) watering, fertilizing and deinsectizing the infected arabidopsis thaliana regularly until the seeds are mature, and collecting T0 generation seeds. And carrying out the next positive transgenic plant screening work on the seeds.

The hygromycin screening method comprises the following specific steps of: the seeds of the T0 generation obtained above were screened with medium (1/2MS + 25. mu.g/mL hygromycin), and after about two weeks, the growth of Arabidopsis seedlings was examined. Arabidopsis seedlings, which were preliminarily determined to be positive (two true leaves were grown and roots were long), were carefully removed from the medium, then transferred to a substrate (peat soil: vermiculite: 2: 1, V/V) for culture, and finally harvested to obtain T1 seeds. Repeating the steps and continuously screening for 2 weeks to obtain T2 generation seeds; the above steps are repeated again for 2 weeks to obtain T3 generation seeds. And (3) sowing seeds of T3 generations, taking down leaves of all strains (numbered) to extract genome DNA when the arabidopsis seedlings grow for about one month (rosette leaves appear), and designing primers in a promoter region and a GUS gene coding region for PCR verification, so that the promoter sequence of ProDoWOX4 is successfully integrated into the arabidopsis genome DNA. The PCR identification primers were ProDoWOX4-FP2 and ProDoWOX4-RP 2. Thereby obtaining a transgenic Arabidopsis line.

Example 4 GUS staining analysis of Arabidopsis transgenic plants

To verify the expression pattern of the ProDoWOX4 promoter, transgenic Arabidopsis lines were subjected to GUS staining. Taking a proper amount of transgenic arabidopsis thaliana T3 generation seeds to sterilize in a sterilized 2mL centrifuge tube, and the specific operation steps are as follows: the Arabidopsis seeds were sterilized with 75% alcohol containing 0.01% TritonX-100 in situ for 10min, while shaking and mixing. Centrifuging slightly, removing the supernatant, washing with anhydrous ethanol for 1 time, centrifuging, and removing the supernatant; washing with 70% ethanol, centrifuging, and collecting supernatant; finally, washing the mixture for 1 time by using absolute ethyl alcohol, and centrifuging the mixture to discard the supernatant. Subsequently, absolute ethanol was added to the centrifuge tube and the arabidopsis seeds were transferred to sterilized filter paper. After the seeds are dried, the seeds are carefully spotted on a flat plate of 1/2MS culture medium (1.5 percent of cane sugar and 0.8 percent of agar powder, pH5.7) by using sterilized sterile tweezers on a clean bench, the seeds grow after being synchronized in the dark in a refrigerator at 4 ℃ for 2 days, and then the seeds are moved to a culture room and cultured under the conditions of 22 ℃ plus or minus 2 ℃ and 16h of illumination/8 h of dark.

In a search for cis-acting elements of the ProDoWOX4 promoter, elements associated with abiotic stress were found (table 1). To verify their predicted function, we studied the inducible expression of the GUS gene driven by the ProDoWOX4 promoter in arabidopsis transgenic plants. Thus, after germination, we took 7d, 14d and 14d arabidopsis seedlings after different treatments (salt stress and drought stress) 7d for whole GUS staining analysis (Jefferson et al, 1987); the other part of the seedlings of the positive transgenic arabidopsis thaliana T3 generation about 7d is removed from the 1/2 culture medium, the roots are carefully cleaned, the culture medium is planted in a matrix (peat soil: vermiculite: 2: 1, V/V), and after the plants enter reproductive growth, roots, stems, leaves, flowers (florets and full blooms) and pods of the plants in the mature period are respectively taken for GUS staining analysis. In this example, transgenic Arabidopsis thaliana material was stained with a Biosharp BL622A GUS staining kit (Biosharp, Anhui, China), and the specific procedure for staining was performed with reference to the instruction manual. The specific part of the expression of the dyed material was observed with a super depth-of-field digital stereoscope (Leica DVM6) and photographed.

The results of the staining of different tissues (roots, stems, leaves, inflorescences and pods) of the ProDoWOX 4-driven GUS Arabidopsis transgenic line at maturity are shown in FIG. 2. The result shows that the dendrobium officinale promoter ProDoWOX4 sequence has promoter activity and can drive GUS gene to express in Arabidopsis; and the transgenic pCAMBIA1301: ProDoWOX4: GUS Arabidopsis thaliana is mainly expressed in sepals of flowers (shaded darker in inflorescence in FIG. 2) and is also expressed in petals but is weaker (shaded lighter in inflorescence in FIG. 2), which suggests that the gene has the highest expression level in the early stage of bud development and has a reduced expression level after full bloom. In addition, weak GUS staining was observed in pCAMBIA1301: ProDoWOX4: GUS Arabidopsis leaves, and no color development was observed in roots, stems and pods. It was demonstrated above that pCAMBIA1301: ProDoWOX4: GUS has tissue specificity and is expressed only in flowers and leaves. In addition, the ProDoWOX4 promoter can be applied to plant genetic engineering, such as inducing the expression of corresponding target genes in flower organs, thereby improving the characters of the flower organs of plants.

GUS staining was performed on ProDoWOX 4-driven GUS Arabidopsis transgenic 7d seedlings after treatment for 7d with salt stress (150mM NaCl) and drought stress (15% PEG), respectively (i.e., 14d Arabidopsis transgenic seedlings). The results show (fig. 3), that transgenic lines become more shaded at drought and salt stress relative to controls, indicating that ProDoWOX4 drives GUS gene expression at both drought and salt induction.

Sequence listing

<110> south China plant garden of Chinese academy of sciences

<120> dendrobium officinale flower tissue preference and stress induction type promoter ProDoWOX4 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2003

<212> DNA

<213> Dendrobium officinale (Dendrobium officinale)

<400> 1

tttcttatga tctatgctcc aaattattga gtattggaac ataactccta caaagaattc 60

attgatcatt aagaattcct cctcctcatc attccttcta tattgaatta atgataaaga 120

cttatgaaca taaaccacat taagaactag atgatgatag ttatgaagtt tcttatagaa 180

atttttatga ggaagtattt catatatgat ttgagttcaa gtattggaca aaattccata 240

catattgaat tagtaatttg ttaatctata tcatgttata ttttactaat gtgtaaaaat 300

ttgttgagga tgtaaatata atatataaaa tggatatttt taggtaaata tattatatga 360

atttcattta aaaaaacata tagtataaat tatttttagt tattaaactt aacactacag 420

gaaaaacgct gaaaaccggc ggttagtata gatttaccgg cgggttttaa agttaccggc 480

ggtctaacgg cggtttgtga accgccggaa aaaacggcgt aggaaaccat ttccgacggc 540

tatttaccgg cggtttattt ttgcggcggt tttccggcgg ccaataccgc cggtgaccac 600

cggaatttta accgtcggtc ggccggcggt taggccgccg gtattgtaac cgccggaaaa 660

ccgccggtca aattaccggc ggttatacct ggcgtcgtct cgttcattag accgccggac 720

gacgccgtta gaccgccggt cggcgtcgtt atagtccgac ggcttaaccg ccggcggagc 780

cgtcggtaat accgacggtc tataaaccgc cggttaaaac cgccggtatt tgccattttt 840

cctgtagtgt aaggttgatt aacacttatt tttgacatgt caatatagaa catataaaaa 900

catgtttttt ttaatataaa tatggaagta tactgtttaa aatatacata atttagaagt 960

taaacttttt ctatttatgt gtttttttta aaaaatactg agaaaagaaa gatgattttc 1020

ctgtgcattc tcataaatgg cttgattagg caattcaatg aaacttttag attaaatgta 1080

agagatatta ttatttgggg aataagtgtt cacttaaaat taaagagcaa taaatttatt 1140

ctcaaaaaaa taaaaaatca agtcaaaata taataaatac caaggcttcg tttggagctg 1200

ctttctaaag aaatttaatt agtacaatta aaaaactttt gaactagaaa tttttgtaaa 1260

aaaattttgt ttttgaaagc ggtaaaaaaa cagtctcaaa cgaagcctaa accacatgca 1320

actatatatg ttctatacta tgtattgatt taatattaaa cttttaatta gtttagtaaa 1380

atattatatt aaaaattcag ctcctctaag atctaagatc aagtgtagtc attagtacat 1440

atatttataa aacaatgaaa cttgcttatg aaaaaatatc tgagtccatg aaggtgataa 1500

tgtgtttgat gtcggtgaat gttgcacatc atcatcacct aattatatta aactctcccc 1560

aactgaaatg gtagatagac agagacaaac atgtcaagct gggtgtcatt ccttctcatc 1620

aatgtaatcc taactcaatc agccaacaaa catacagatt gatgtgatat ctccccactc 1680

tctctctctc tctctctctc tctctctctc tctcttcctg ttcctgcctt caaaagtgaa 1740

caaagttgag aacccaaaag gctgaaatga agcaaagaag taaaacacaa ccactgaacg 1800

ccctatttaa ttcttctcag aacagcatca gcatgactgt actgcctcac ccccactttg 1860

ctctcttctc ctttcttctc ctcctcacat cccttttgtc tcttgtgtcg tctctcttac 1920

acttcgtctc tacatataaa tattgcatta ccaatatacc aaaaaccaaa actagaatag 1980

aaggaagatg cctcaagttc cat 2003

Claims (9)

1. A promoter is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID NO.1 or the nucleotide sequence which is complementary with the nucleotide sequence shown as SEQ ID NO. 1.

2. A nucleic acid construct comprising the promoter of claim 1 and a foreign gene operably linked to the promoter.

3. A recombinant expression vector comprising the promoter of claim 1 or the nucleic acid construct of claim 2.

4. A host cell comprising the promoter of claim 1 or the nucleic acid construct of claim 2 or the vector of claim 3.

5. A recombinant cell having a genome into which the promoter of claim 1 or the nucleic acid construct of claim 2 is integrated.

6. Use of the promoter of claim 1, the nucleic acid construct of claim 2, the vector of claim 3, or the cell of claim 4 to promote efficient expression of a gene of interest.

7. Use of the promoter of claim 1, the nucleic acid construct of claim 2, the vector of claim 3, or the cell of claim 4 for growing improved plant varieties.

8. A group of primers used for amplifying a nucleotide sequence shown as SEQ ID NO.1, which is characterized in that the primers are: ProDoWOX4-FP 2: 5'-tttcttatgatctatgctcc-3', ProDoWOX4-RP 2: 5'-atggaacttgaggcatcttcc-3' are provided.

9. A method for efficiently expressing a target gene, comprising the steps of:

a. cloning the promoter of claim 1 onto pCAMBIA1301 plasmid containing target gene, then transferring into Agrobacterium EHA105 to obtain recombinant plant expression vector;

b. the recombinant plant expression vector is transformed into a plant cell of interest.

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