CN102485895B

CN102485895B - Arabidopis thaliana flower and embryo specific promoters EL5 and EL6, and application thereof

Info

Publication number: CN102485895B
Application number: CN 201110045893
Authority: CN
Inventors: 傅永福; 江颖; 柳霖坡; 张晓玫
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2011-02-24
Filing date: 2011-02-24
Publication date: 2013-07-10
Anticipated expiration: 2031-02-24
Also published as: CN102485895A

Abstract

The invention provides Arabidopis thaliana flower and embryo specific promoters EL5 and EL6. The nucleotide sequences of the promoters are respectively represented by SEQ ID No.1 and SEQ ID No.2. According to results of overexpression of Arabidopis thaliana and soybean GUS genes by using the promoters EL5 and EL6, the promoters EL5 and EL6 can obviously promote the expression level of the GUS genes in plant floral organs and embryos; therefore, the promoters EL5 and EL6 have specificity in plant floral organs and embryos and are applicable to specific expression of a target gene in flowers and embryos of the plants like a variety of crops, forest trees, vegetable, flowers, pasture, etc.

Description

Arabidopis thaliana flower and embryo's specific promoter EL5 and EL6 and application thereof

Technical field

The invention belongs to molecular biology, gene engineering technology field, specifically, relate to a kind of Arabidopis thaliana flower and embryo's specific promoter EL5 and EL6 and application thereof.

Background technology

Promotor is cis-acting elements important in the gene, generally be positioned at tens base places, transcription initiation site upstream, its core texture comprises TATA-box and CAAT-box, except core texture, also comprise transcription initiation site, organizing specific expression element and other controlling elements, coerce the element of inducing with abiotic stress as biology, this has important effect to research expression of gene and function, the promotor of being induced simultaneously can be controlled this gene at specific time and specific space expression by inducing, and brings into play specific function.

Promotor can be divided into three kinds according to the transcriptional profile difference: constitutive promoter, tissue or organ specific promoters and inducible promoter.The activity of tissue or organ specific promoters is subjected to specific histocyte structure and chemistry, physical signalling to induce adjusting, i.e. the expression of this class promotor has specific space-time.Under the driving of this class promotor, expression of gene often is only limited to some specific organ or tissue position or specific developmental stage.Tissue or organ specific promoters can not only make the expression product of goal gene in the position accumulation of certain organ or tissue, increase regional expression amount, also can avoid target gene to express the disadvantageous effect that causes in other histoorgans simultaneously.

Up to the present, existing a large amount of tissue or organ specific promoters is separated, comprising flower specific promoter and seed-specific expression promoter.As existing reports that studies in great detail such as the relevant tissue-specific promoter of pollen development such as TA29, Lat52, Osg6B, Zm13, S1; UBC6 promotor (Watts, 1994) is mainly being spent middle expression; Tsp1 is tapetum specific promoter in the paddy rice (Ma Qinqin, 2005); DefH9 is ovary specific promoter (Rotino, 1997); PtNIP1 is cypress fetal development specificity promoter (Vincent, 2002).These promotors can both special startup target gene expression, change the growth of specific tissue's organ.But the energy while promotor of specifically expressing in flower and embryo rarely has report.

ESD4 is a kind of SUMO enzyme in the Arabidopis thaliana, and it regulates the protein s UMOization decorating state in the plant.EL5 and EL6 (ESD4like 5 and 6) are the homologous genes of ESD4 in the Arabidopis thaliana, and on activity, EL5 and EL6 and ESD4 have common SUMO enzymic activity, but physiological mechanism is incomplete same.ESD4 mainly influences blooming of plant, and EL5 and EL6 then pass through to regulate floral organ, microgametophyte, megagametophyte and embryo's growth, thereby influences the fertility of plant.

Summary of the invention

The purpose of this invention is to provide a kind of from Arabidopis thaliana (Arabidopsis thaliana) isolated flower and embryo's specific promoter EL5 (ESD4Like 5) and EL6 (ESD4Like 6).

Another object of the present invention provides promotor EL5 and the application of EL6 in plant flowers and fetal development adjusting.

In order to realize the object of the invention, a kind of Arabidopis thaliana of the present invention is spent and embryo's specific promoter EL5, and it has: a) nucleotide sequence shown in the SEQ ID NO.1; Or b) nucleotide sequence shown in the SEQ ID No.1 be substituted, lack and/or add one or several Nucleotide and same function by the nucleotide sequence of a) deriving.As under the situation that does not change promoter function, the 28th C is substituted by T, the 222nd C is substituted by T, the 2369th G is substituted by A, the 2473rd T is substituted by C.

A kind of Arabidopis thaliana flower of the present invention and embryo's specific promoter EL6, it has: the c) nucleotide sequence shown in the SEQID NO.2; Or d) nucleotide sequence shown in the SEQ ID No.2 be substituted, lack and/or add one or several Nucleotide and same function by c) nucleotide sequence of deriving.As under the situation that does not change promoter function, increased C after the 3rd, the 952nd A is substituted by T, the 1099th A is substituted by G, the 2481st T is substituted by C.

The present invention also provides and cloned the used primer of EL5 sequence that obtains 2500bp from the arabidopsis gene group, and it comprises forward primer 5 '-CTCCCTTATCGTGTTGACA-3 ' and reverse primer 5 '-CGCTAGGGAAATCTGAGGAA-3 '.

The present invention also provides and cloned the used primer of EL6 sequence that obtains 2501bp from the arabidopsis gene group, and it comprises forward primer 5 '-CTGCTGATTATCTCGTTC-3 ' and reverse primer 5 '-CCAGTATTATTCTGGCGCCG-3 '.

The present invention also provides the carrier that contains promotor EL5 and EL6 and the host cell that contains described carrier.

The present invention also provides the transformed plant cells that contains promotor EL5 and EL6.

The present invention also provides promotor EL5 and the application of EL6 in the regulation and control downstream gene expression, and preferred downstream gene is gus gene.

In the present invention, can select various carrier known in the art for use, as commercially available carrier and plasmid.

The present invention has separated Arabidopis thaliana flower and embryo's specific promoter EL5 and EL6 first, utilize promotor EL5 and EL6 to cross expression Arabidopis thaliana, soybean gus gene, the result shows that promotor EL5 and EL6 can obviously promote the expression amount of gus gene among plant flower organ and the embryo, thereby, EL5 and EL6 promotor have specificity in plant flower organ and embryo, be adapted at specifically expressing target gene among the flowers of plants such as various crops, forest, vegetables, flowers, herbage and the embryo.

Description of drawings

Fig. 1 is the structural representation of plasmid pGreen-GW-GUS;

Fig. 2 is the expression of results of Arabidopis thaliana EL5-GUS in floral organ (gynoecium);

Fig. 3 is the expression of results of Arabidopis thaliana EL5-GUS in floral organ (stamen, pollen);

Fig. 4 is the expression of results of Arabidopis thaliana EL5-GUS in floral organ (pollen);

Fig. 5 is the expression of results of Arabidopis thaliana EL5-GUS in floral organ;

Fig. 6 is the expression of results of Arabidopis thaliana EL5-GUS in the embryo;

Fig. 7 is the expression of results of Arabidopis thaliana EL5-GUS in the embryo;

Fig. 8 is the expression of results of Arabidopis thaliana EL5-GUS in the embryo;

Fig. 9 is the expression of results of Arabidopis thaliana EL5-GUS in the embryo;

Figure 10 is the expression of results of Arabidopis thaliana EL6-GUS in floral organ;

Figure 11 is the expression of results of Arabidopis thaliana EL6-GUS in the embryo;

Figure 12 is the expression of results of Arabidopis thaliana EL6-GUS in the embryo;

Figure 13 is the expression of results of Arabidopis thaliana EL6-GUS in the embryo;

Figure 14 is the expression of results of Arabidopis thaliana EL6-GUS in the embryo;

Figure 15 A is the expression of results of Arabidopis thaliana EL5-GUS in inflorescence, and B is the expression of results of Arabidopis thaliana EL6-GUS in inflorescence;

Figure 16 is the PCR detected result of Arabidopis thaliana EL5-GUS and EL6-GUS.

Embodiment

Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.

Unreceipted concrete experimental technique in the following example all can carry out or according to the operation instruction of making production firm according to ordinary method.

The clone of embodiment 1 Arabidopis thaliana EL5 and EL6 promotor

Utilizing forward primer 5 '-CTCCCTTATCGTGTTGACA-3 ' and reverse primer 5 '-CGCTAGGGAAATCTGAGGAA-3 ' pcr amplification from the arabidopsis gene group also to check order and obtaining length is the EL5 promotor of 2500bp, and its nucleotide sequence is shown in SEQ ID NO.1.

Utilizing forward primer 5 '-CTGCTGATTATCTCGTTC-3 ' and reverse primer 5 '-CCAGTATTATTCTGGCGCCG-3 ' pcr amplification clone and order-checking from the arabidopsis gene group to obtain length is the EL6 promotor of 2501bp, and its nucleotide sequence is shown in SEQ IDNO.2.

Above-mentioned pcr amplification system is: (20 μ l system)

LA Taq enzyme 0.2 μ l

10x damping fluid 2.0 μ l

DNTP (each 2.5mM) 1.0 μ l

Primer 1 (10uM) 1.0 μ l

Primer 2 (10uM) 1.0 μ l

Dna profiling 1.0 μ l

ddH ₂O 13.8μl

Cumulative volume 20.0 μ l

The PCR program:

The expression of embodiment 2 Arabidopis thaliana EL5 and EL6 promoter regulation downstream gene GUS

Merge with EL5 and EL6 promotor clone and with gus gene respectively, (building process of plasmid pGreen-GW-GUS is: the method (Invitrogen) that adopts the GateWay recombinant clone with the fusion gene construction of expression vector pGreen-GW-GUS that obtains then, earlier by the BP reaction with the PCR product pDONR207 that recombinates, then by LR reaction reorganization to the purpose carrier pGreen-GW-GUS that contains gus reporter gene.The structure of pGreen-GW-GUS is as shown in Figure 1), obtain binary expression vector pEL5-GUS and pEL6-GUS; With plant expression vector pEL5-GUS and pEL6-GUS be converted into expand in the bacillus coli DH 5 alpha numerous.The conversion of competent escherichia coli cell comprises: (1) preparation contains the LB solid medium of penbritin; (2) taking-up is stored in-80 ℃ competent cell, after melting in the ice bath, adds 2 μ l plasmids mixing gently, places 30 minutes in the ice bath; (3) 42 ℃ of heat shocks 30 seconds placed ice bath 3 minutes then immediately; Add 300 μ l and do not contain antibiotic LB liquid nutrient medium, 37 ℃ of shaking table shaking culture 1 hour (160rpm); (4) get an amount of nutrient solution and evenly be applied on the selective medium, be inverted for 37 ℃ and cultivated 12-20 hour, with 5-10 (or more) bacterium colony of sterilization toothpick picking, place 200 μ l selected liq LB substratum to shake bacterium and cultivate; (5) PCR detects the positive colony (Figure 16) that screens.By the agrobacterium mediation converted method, change pEL5-GUS and pEL6-GUS over to Arabidopis thaliana, Arabidopis thaliana 4 strains that obtain to transform Arabidopis thaliana 2 strains of pEL5-GUS and transform pEL6-GUS; Show by the GUS activation analysis, EL5 and EL6 promotor be specifically expressing in flower (Fig. 5,10 and 15), gynoecium (Fig. 2), pollen (Fig. 3 and 4) and the embryo (Fig. 6,7,8,9,11,12,13 and 14) of plant, can be used for comprising specifically expressing target gene among the flowers of each kind of plant of various crops, forest, vegetables, flowers, herbage etc. and the embryo.

Arabidopis thaliana GUS dyeing:

(1) tissue sample is added in 90% acetone, placed 20～30 minutes on ice;

(2) after acetone treatment, with 50mM phosphoric acid buffer (pH7.2), 2mMK ₄Fe[CN] ₆And 2mM K ₃Fe[CN] ₆Solution rinse tissue;

(3) sample is placed on (50mM phosphoric acid buffer (pH7.2), 1mM X-gluc, 2mM K in the X-Gluc staining fluid ₄Fe[CN] ₆, 2mM K ₃Fe[CN] ₆), bled 10 minutes, 37 ℃ of dyeing are spent the night then;

Used 70% ethanol decolorization in (4) second days;

(5) add several HCG solution at slide glass, the sample after the decolouring is placed in one;

(6) compressing tablet, transparent 15 minutes or spend the night transparent (according to the tissue developmental stage decide).

The GUS prescription of its dyeing liquor:

20mM X-gluc(MW＝521.8)

With DMSO or N, the dinethylformamide dissolving, 100mg X-gluc needs the dissolving with 9.58mlDMSO, and final concentration is 20mM.

50mM phosphoric acid buffer (pH7.0)

A liquid: NaH ₂PO ₄2H ₂O 3.12g is dissolved in sterile distilled water, is settled to 100ml;

B liquid: Na ₂HPO ₄12H ₂O 7.17g is dissolved in sterile distilled water, is settled to 100ml;

Getting 39mlA liquid mixes with 61mlB liquid.

Staining fluid preparation: the 5mM Tripotassium iron hexacyanide; The 5mM yellow prussiate of potash; 10mM EDTA; The 50mM phosphoric acid buffer; 1mM X-gluc.

Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims

1. Arabidopis thaliana flower and embryo's specific promoter EL5 is characterized in that its nucleotide sequence is shown in SEQ ID NO.1.

2. the carrier that contains the described promotor of claim 1.

3. the host cell that contains the described carrier of claim 2.

4. the application of the described promotor of claim 1 in the regulation and control downstream gene expression.

5. application according to claim 4 is characterized in that, downstream gene is gus gene.