CN105087581A - Rice seed specific expression promoter and application thereof - Google Patents

Abstract

The invention discloses a rice seed specific expression promoter and application thereof. The sequence of the promoter is as shown in any one of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, or the promoter has DNA molecule which is hybridized with the DNA sequence as shown in any one of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 and has the functions of the promoter. The three promoters screened by the invention can be used for promoting the specific expression of target genes (such as cinH gene) in rice seeds, so that expression products of the target genes are accumulated in a specific special region, and the constitutive expression of the target genes in all tissues of rice is prevented to avoid influence on the growth of plants. The novel promoter disclosed by the invention can be extensive in application and market prospect in agricultural field.

Description

A kind of rice paddy seed specific expressing promoter and application thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of rice paddy seed specific expressing promoter and application thereof.

Background technology

Promotor is one section of DNA sequence dna being positioned at upstream region of gene district, participates in the expression regulation of downstream gene, determines the transcription initiation of gene, transcriptional efficiency and Space-time speciality.Promotor can be divided into three major types according to the mode of action of promotor and function: constitutive promoter, inducible promoter and tissue-specific promoter.Current constitutive promoter is applied comparatively extensive in plant genetic engineering, but expose some problems gradually in the application, such as it makes foreign gene express in whole plant, produce a large amount of exogenous protein or meta-bolites, break the original metabolic balance of plant, increased the weight of the burden of plant, have impact on the form of plant and grow normally, plant even can be caused dead, cause the waste that resource is unnecessary.Therefore, people are striving to find the distinctive tissue-specific promoter of some plants self, for the structure of transgene carrier.

Tissue-specific promoter only at some specific organ or tissue position regulate gene expression, and shows the characteristic of Growth adjustment.Its feature is, in plant development process, effectively can regulate and control the expression of goal gene over time and space, the expression product of goal gene is accumulated at specific area of space, pathways metabolism is improved according to the wish of people, improve and organize Middle nutrition substances content, obtain industrial product innovation and medical new compound etc. more expediently.Therefore, tissue-specific promoter has become the starting element of the foreign gene that most is promising in transgenic research.

Paddy rice is annual gramineae monocotyledons, is one of most important food crop in the world.Seed is the topmost vegetative storage organ of paddy rice, has important economic worth, and therefore, seed specific promoters, as a kind of important cis-acting elements, has important practice significance and using value.

Summary of the invention

One object of the present invention is to provide a kind of rice paddy seed specific expressing promoter.

Another object of the present invention is the recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that provide containing above-mentioned promoter sequence.

Another object of the present invention is to be provided for increase the primer pair of above-mentioned rice paddy seed specific expressing promoter sequence.

Another object of the present invention is the application providing above-mentioned rice paddy seed specific expressing promoter.

The technical solution used in the present invention is:

A kind of DNA fragmentation, its containing, for example under the DNA molecular shown in any one of (1)-(4):

(1) DNA molecular shown in SEQIDNO.1;

(2) DNA molecular shown in SEQIDNO.2;

(3) DNA molecular shown in SEQIDNO.3;

(4) DNA sequence dna limited with (1) or (2) or (3) is under strict conditions hybridized and has the DNA molecular of promoter function.

Recombinant vectors containing above-described DNA fragmentation, expression cassette, transgenic cell line or recombinant bacterium.

For the total length of the above DNA fragmentation that increases or the primer pair of its any fragment.

Above-described DNA fragmentation is starting the application in destination gene expression.

Described destination gene expression is tissue specific expression, described in be organized as plant seed.

Described plant is monocotyledons or dicotyledons.

Described monocotyledons is paddy rice.

As preferably, described goal gene is cinHgene.

The invention has the beneficial effects as follows: the promotor that new discovery of the present invention three can be specific expressed in rice paddy seed, its can be used for starting target gene (as cinHgene) specifically expressing in rice paddy seed, the expression product of target gene is accumulated at specific area of space, can avoid target gene paddy rice institute in a organized way in a constitutive expression affect plant strain growth.New promotor of the present invention will have wide application and market outlook at agriculture field.

Accompanying drawing explanation

Fig. 1 is main carriers structural representation;

Fig. 2 is that transgenic paddy rice detects primer location schematic diagram;

Fig. 3 is each strain RT-PCR result of paddy rice.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated, but be not limited thereto.

The molecular biology experiment technology adopted in following examples comprises pcr amplification, plasmid extraction, Plastid transformation, DNA fragmentation connects, enzyme cuts, gel electrophoresis etc., if no special instructions, usually conventionally operate, specifically can see " Molecular Cloning: A Laboratory guide " (third edition) (SambrookJ, RussellDW, JanssenK, ArgentineJ. yellow training hall etc. are translated, 2002, Beijing: Science Press), or according to the condition that manufacturer advises.

Embodiment

one, experimental technique and step

1, the screening of rice paddy seed different expression gene

By retrieving RNA-seq data in the paddy gene expression database in " rice genome mark project (RiceGenomeAnnotationProject; RGAP) " website (http://rice.plantbiology.msu.edu/), screening organizing specific expression gene alternatively seed specific expression gene.RGAP website is subsidized by American National foundation, provides genome sequence and 12 chromosomal markup informations of rice subspecies " Japan is fine ".Alternate promoters is numbered, and candidate gene is numbered, and RNA-seq data, encode as shown in table 1.According to rice genome database information, choose gene start codon ATG upstream and be about promotor and 5 ' the UTR district of 2kb as gene.Wherein LOC_Os02g25640 gene is endosperm specific expression (JExpBot, 2008,59 (9): 2417-2424).

Table 1 candidate gene RNA-seq result in a database

2, the fine extracting genome DNA of wild rice Japan

Extract the fine genomic dna of wild rice Japan according to a conventional method.

3, the pcr amplification of alternate promoters and purifying

According to rice genome data, design corresponding primer (table 2), obtain respective segments from rice genome amplification.

Table 2 increases the primer of promotor

The Phusion high-fidelity enzyme of NEB company is adopted to carry out pcr amplification, the fine genomic dna 0.25 μ g of Japan, forward primer and each 2.5 μ L(final concentrations of reverse primer are each 0.5 μm of ol/L), 10mMdNTPs1 μ L, 5 × HF damping fluid 10 μ L, PhusionDNA polysaccharase 0.5 μ L, adds water to 50 μ L.PCR reaction conditions is as table 3:

Table 3Phusion high-fidelity enzymatic amplification parameter

PCR primer detects through agarose gel electrophoresis, and reclaim test kit with the gel of Mei Ji company and reclaim DNA fragmentation ,-20 DEG C save backup.

4, the structure of paddy rice Agrobacterium binary conversion carrier

With hindiII and spei enzyme cuts p35sCinH and pZH041 plasmid (Fig. 1), reclaims corresponding p35s- cinHand vector backbone segment, T4 ligase enzyme 16 DEG C connects 3 hours, will connect product conversion DH5 α competent escherichia coli cell.Utilize conventional PCR method to detect screening positive clone, obtain pZH051.With hindiII and asci cut vector pZH051, reclaims large fragment vector backbone sequence, will 35spromotor replaces with organizing specific expression alternate promoters (referred to as P-ts).

Table 4 conversion carrier is numbered with corresponding promotor and gene

5, binary vector is transformed in Agrobacterium

Adopt electric method for transformation will to be used for the genetic transformation of paddy rice in vector introduction Agrobacterium EHA105.The checking of Agrobacterium adopts PCR method, and the primer is in table 5.

Table 5 Agrobacterium bacterium colony PCR detects primer

6, the detection of transgenic rice plant

According to a conventional method the above-mentioned Agrobacterium prepared is infected Rice Callus and obtain transfer-gen plant, after obtaining regrowth, utilize PCR method determination regeneration plant to be transfer-gen plant.PCR the primer sequence is:

05x-test-F：5’-ACTTGATTCGTTTTGGTGCT-3’（SEQIDNO.23），

05x-test-R：5’-GTGCCACCTTCCTTTTCTAC-3’（SEQIDNO.24）。

7, recombinase gene CinH is in the expression of rice paddy seed and leaf tissue

Plant tissue total RNA extraction reagent box (Guangzhou Mei Ji company) is adopted to extract the total serum IgE of blade and seed.Liquid nitrogen flash freezer, puts into-80 DEG C of Refrigerator stores for subsequent use.

The PrimeScript RTreagentKitWithgDNAEraser test kit reverse transcription of TaKaRa company is adopted to produce cDNA Article 1 chain, concrete steps reference reagent box specification sheets.

Use sxemiquantitative in rice leaf and seed cinHexpression amount measure.Goal gene cinHhouse-keeping gene endogenous with paddy rice ubiquitinrT-PCR primer sequence as follows:

cinh-rt-f:AGGGCTTCCGGCAAGAATAC（SEQIDNO.25），

cinh-rt-r:TGGATATGCCGAACGCTTTG（SEQIDNO.26），

ubi-rt-f:ACGGACGCACCCTGGCTGACTAC（SEQIDNO.27），

ubi-rt-r:CTGCCAATTACCATATACCACGAC（SEQIDNO.28）。

At 1%(m/V after PCR has reacted) agarose gel electrophoresis detect.

RT-PCR agarose gel electrophoresis result as shown in Figure 3.Wherein, in Fig. 3 A be whole T1 for transgenosis RT-PCR result, above two row electrophorograms be respectively cinHthe expression of gene in rice paddy seed and blade, below two row display positive controls ubiquitinthe expression of gene.In Fig. 3, B is the RT-PCR result of second batch transgenic line T0 for plant, and wherein first row electrophorogram is cinHthe expression of gene, second row is ubiquitinthe expression of gene, finally shows WT lines cinHgene and ubiquitinthe expression of gene is the negative control of all experiments.Pass through ubiquitinthe electrophorogram display of gene, the RNA quality extracted in the blade of all plant and seed is all no problem, and the amount being shown during each PCR reacts the cDNA added by the brightness of electrophoretic band is basically identical, explanation cinHthe expression amount of gene has comparability between different tissues and different strain, namely by cinHthe power of the electrophoretic band of gene expression amount contrasts the differential expression between different strain and different tissues.Wild-type negative control illustrates that the cDNA of the total serum IgE reverse transcription of rice paddy seed and blade can not be with cinHthe RT-PCR primer cinh-rt-f of gene and cinh-rt-r non-specific binding, the band amplified is exactly cinHgene fragment.

By the result of upper figure, can find out, cinHgene has very weak expression in the leaf tissue of strain ZH053 is except strain ZH053.64.1 and ZH053.64.2, and the blade of other strains is expressed hardly; In all strain seeds, expression amount is very high.In the strain of ZH058, seed group is woven with very high expression, and blade is not expressed, so this promotor is seed-specific expression promoter.In the strain of ZH059, ZH059.5.1-3 and ZH059.8.2-3 blade has faint expression, and seed tissue is not expressed.But strain ZH059.13.1-4 only expresses in seed, can use as seed-specific expression promoter.Positive control, except strain ZH070.1.1 and ZH070.4.1, is seed specific expression.

By comparing with reference gene ubiquitin expression amount, cinHthe expression amount of gene in strain ZH053 seed apparently higher than strain ZH070, the expression amount in strain ZH059 seed lower than strain ZH070, the expression in strain ZH058 seed and strain ZH070 similar.

Therefore, alternative promotor in carrier pZH053, pZH058, pZH059, corresponding gene is numbered LOC_Os07g11510(SEQIDNO.1 respectively), LOC_Os02g15090(SEQIDNO.2), LOC_Os07g11650(SEQIDNO.3) the promotor of gene can as Seeds oil-body-specific promoter, and their promotor intensity is different, can the feature of binding purposes gene, meet the needs of different experiments.

Three Seeds oil-body-specific promoter that above-mentioned screening obtains also may be used for controlling the expression of other genes, the expression product of target gene at specific tissue accumulation, may avoid in a organized way in express and cause waste, or affect vine growth and development.

Above embodiment is only introduces preferred case of the present invention, to those skilled in the art, not deviating from any apparent changes and improvements of carrying out in the scope of spirit of the present invention, all should be regarded as a part of the present invention.

<110> South China Botanical Garden Chinese Academy of Sciences

<120> rice paddy seed specific expressing promoter and application thereof

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Claims (8)

1. a DNA fragmentation, its containing, for example under the DNA molecular shown in any one of (1)-(4):

(1) DNA molecular shown in SEQIDNO.1;

(2) DNA molecular shown in SEQIDNO:2;

(3) DNA molecular shown in SEQIDNO.3;

(4) DNA sequence dna limited with (1) or (2) or (3) is under strict conditions hybridized and has the DNA molecular of promoter function.

2. the recombinant vectors containing DNA fragmentation according to claim 1, expression cassette, transgenic cell line or recombinant bacterium.

3. increase the total length of DNA fragmentation or the primer pair of its any fragment described in claim 1.

4. DNA fragmentation according to claim 1 is starting the application in destination gene expression.

5. application according to claim 4, is characterized in that, described destination gene expression is tissue specific expression, described in be organized as plant seed.

6. application according to claim 5, is characterized in that, described plant is monocotyledons or dicotyledons.

7. application according to claim 6, is characterized in that, described monocotyledons is paddy rice.

8. the application according to any one of claim 4-7, is characterized in that, described goal gene is cinHgene.