CN102994502B - Promoter from malus sieversii and application thereof - Google Patents

Abstract

The invention discloses a promoter from malus sieversii and application thereof. The promoter is DNA (deoxyribonucleic acid) molecules of a), b), c) or d): a) DNA molecules at the 9th-1325th sites with a nucleotide sequence SEQ ID No.1; b) DNA molecules with a nucleotide sequence SEQ ID No.1; c) a DNA fragment of which the nucleotide sequence has consistency of 75% or higher limited by a) or b) and having a promoter function; and d) a DNA fragment hybridized with the nucleotide sequence limited by a) or b) under strict conditions and having a promoter function. The promoter is a tissue-specific and adversity-induced promoter. A gene can be expressed under specific tissue organ and induction conditions by use of an MsDREB2C gene promoter, thus a negative effect caused by over-expression of a constitutive promoter is avoided to generate a great quantity of heterologous proteins and toxic substances, and then a new way is provided for genetic engineering breeding.

Description

Derive from promotor and the application thereof of Malus sieversii

Technical field

The present invention relates to derive from promotor and the application thereof of Malus sieversii.

Background technology

Promotor (Promoter) is an important component part of genomic gene, and it is as " switch ", and whether the encoding gene on transcriptional level under its control of basic decision is expressed, when expresses, where expressed and expression intensity.Generally according to the promotor mode of action and function, it can be divided into three major types substantially: constitutive promoter, specificity promoter and inducible promoter (Wang Guanlin, Fang Hongjun, 2002, plant genetic engineering philosophy and technique (second edition), Beijing, science tech publishing house).But this classification is not absolute, in some cases, the promotor of a type often has the characteristic of other type promotor concurrently, for example, and inducing reinforced composing type promoter (Xiao Xingguo etc., patent of invention number 200710177962.4).

Constitutive promoter (Constitutive promoter) refers to that can order about it controls lower encoding gene in a class promotor of Different Organs and/or the constant expression of tissue cardinal principle.Its feature is: be subject to the expression of the encoding gene of its control to have persistence, but do not have Space-time speciality; RNA is relative with protein expression amount constant, is not subject to the induction of extraneous factor.For example: cauliflower mosaic virus 35 S promoter CaMV35S(Odell et al., Nature, 1985,313:810-812), corn Ubiquitin promotor (Holtorfet al., Plant Mol.Biol., 1995,29:637-646) and Actinl promotor (the McElroy et al of paddy rice, 1990, Plant Cell, 2:163-171).

Inducible promoter (Inducible promoter) refers to and can respond some specific physics, chemistry and bio signal (being referred to as " elicitor " or " inducible factor ") and drive the encoding gene of its control to increase significantly a class promotor of transcriptional level.Inducible promoter is usually classified and names according to its inducement signal, such as: fungal induction promotor, symbiotic bacterium evoked promoter, chemically inducible promoter, metal ion evoked promoter, photoinduction promoter, heat shock promoter and wound-induced promotor etc.

Organ and/or tissue-specific promoter (Organ-and/or Tissue-specific promoter), can order about it and control lower encoding gene only at a certain of organism or some specific organ and/or tissue, or only at a genoid of a certain or some specified phase expression of growing.Being characterized as of it, is subject to the genetic expression of its control or adjusting to have obvious space-time, and often shows the characteristic of growing adjusting.For example, the root-specific promoter on plant, blade specific promoter, flower specific promoter, pore specificity promoter, pore tapetum specific efficient promoter, pollen specific promoter, fruit-specific promoter, seed specific promoters, endosperm specificity promoter, cotton fiber specific promotor and phloem specific promoter etc.

In physical environment, plant-growth, in open system, often can run into the impact of the poor environments such as hot and cold, non-irrigated, flooded, saline and alkaline, topsoil.Poor environment acts on plant, will cause in plant materials a series of physiological metabolism reaction occurs, and shows as the reversible inhibition of metabolism and growth, when serious, even causes irreversible injury, causes whole plant dead.In various environment-stress, the abiotic stress such as arid, low temperature, Gao Re and high salt are particularly outstanding on the impact of plant, show as the impact on water regime in plant materials to some extent, therefore becoming again water stress, is the main inanimate adverse circumstance factor of restriction plant-growth and crop yield.Plant, in long-term evolution, has formed a series of physiology, metabolism and systems of defense of replying environment stress gradually.From plant, clone abiotic stress evoked promoter and will the resistance of abiotic stress be established to basic substance for improving plant.

Summary of the invention

Technical problem to be solved by this invention is to provide plant tissue specificity and abiotic stress evoked promoter.

Promotor provided by the present invention, name is called MsDREB2C gene promoter, derives from Malus sieversii (Malus sieversii (Ledeb.) Roem.), be following a), b), c) or DNA molecular d):

A) nucleotide sequence is the DNA molecular of the 9-1325 position of SEQ ID No.1;

B) nucleotide sequence is the DNA molecular of SEQ ID No.1;

C)) and a) or b) nucleotide sequence that limits has 75% or 75% above consistence, and has the DNA fragmentation of promoter function;

D) under stringent condition with a) or b) nucleotide sequence hybridization that limits, and there is the DNA fragmentation of promoter function.

Above-mentioned 75% or 75% above consistence, can be 80%, 85%, 90%, more than 95% consistence.

Above-mentioned stringent condition can be with 6 * SSC, the solution of 0.5%SDS, and at 65 ℃, hybridization, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively washes film once.

Wherein, SEQ ID No.1 is comprised of 1339 Nucleotide; 1-8 position is EcoR I recognition site and protection base, and 1326-1330 position is front 5 bases in GRD beta-glucuronidase gene (GUS) encoding sequence, and 1331-1339 position is Bgl II recognition site and protection base.

The expression cassette that contains MsDREB2C gene promoter, recombinant vectors, recombinant microorganism or transgenic cell line also belong to protection scope of the present invention.

The described expression cassette that contains MsDREB2C gene promoter, refer to the DNA that can express goal gene in host cell, this DNA not only comprises the MsDREB2C gene promoter that starts described goal gene, also can comprise and stop the terminator that described goal gene is transcribed.Further, described expression cassette also can comprise enhancer sequence.Described transcription terminator includes but not limited to: Agrobacterium rouge alkali synthetase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (referring to, such as: the people (I such as Odell ⁹⁸⁵) Nature 313:810; The people such as Rosenberg (1987) Gene, 56:125; The people such as Guerineau (1991) Mol.Gen.Genet, 262:141; Proudfoot (1991) Cell, 64:671; The people Genes Dev. such as Sanfacon, 5:141; The people such as Mogen (1990) Plant Cell, 2:1261; The people such as Munroe (1990) Gene, 91:151; The people such as Ballad (1989) Nucleic Acids Res.17:7891; The people such as Joshi (1987) Nucleic Acid Res., 15:9627).

The EcoR I that the described recombinant vectors that contains MsDREB2C gene promoter specifically can be at pCAMBIA1301 and Bgl II are inserted the recombinant vectors that MsDREB2C gene promoter obtains.Described recombinant microorganism specifically can be bacterium, yeast, algae and fungi.Wherein, bacterium can be from Escherichia (Escherichia), Erwinia (Erwinia), agrobacterium tumefaciens belongs to (Agrobacterium), Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Rhodopseudomonas (Pseudomonas), Bacillus (Bacillus) etc.Described transgenic cell line does not comprise the reproductive material of plant.

The primer pair of amplification MsDREB2C gene promoter total length or its arbitrary fragment also belongs to protection scope of the present invention.

Experiment showed, that MsDREB2C gene promoter can start goal gene specifically expressing in root, leaf and/or the seed of plant.

Described startup goal gene specifically expressing in the root of plant specifically can be and starts goal gene specifically expressing in the vascular tissue of root, and described startup goal gene specifically expressing in the leaf of plant specifically can be and starts goal gene specifically expressing in the vascular tissue of leaf.

Experiment showed, MsDREB2C gene promoter can under abiotic stress induction, start leaf, root and/or the seed of goal gene plant in specifically expressing; Described abiotic stress is heat stress, coldly coerce, drought stress and/or ABA coerce.

MsDREB2C gene promoter can be used for cultivating transgenic plant, these transgenic plant can be in root, leaf and/or seed specifically expressing goal gene, also can under abiotic stress induction, increase the expression amount of goal gene.

In above-mentioned application, described plant can be monocotyledons or dicotyledons, as tobacco.

Described transgenic plant are interpreted as not only to comprise goal gene are transformed to the first-generation transgenic plant that object plant obtains, also comprise its filial generation.For transgenic plant, can in these species, breed this gene, also available traditional breeding method enters this transgenosis other kind of same species, in commercial variety.Described transgenic plant comprise seed, callus, whole plant and cell.

MsDREB2C gene promoter of the present invention is organizing specific and adverse circumstance inducible promoter.Utilize MsDREB2C gene promoter can make gene express under particular organization's organ and inductive condition, thereby avoided the negative interaction that constitutive promoter overexpression brings-produce a large amount of heterologous proteins and toxic substance, for genetic engineering breeding provides new approach.

Accompanying drawing explanation

Fig. 1 is that pCAMBIA1301-MsDREB2CP enzyme is cut checking electrophoretogram.

M is DNA Marker III, and 1,2,3,4 cut result for pCAMBIA1301-MsDREB2CP enzyme.

Fig. 2 is that the PCR of pCAMBIA1301-MsDREB2CP transfer-gen plant identifies collection of illustrative plates.

M is DNAMarker III, and 1,2 is pCAMBIA1301-MsDREB2CP transgenic line.

Fig. 3 is the GUS histochemical stain of pCAMBIA1301-MsDREB2CP transgene tobacco.

Root (A, B), stem (C, D), leaf (E, F), seed (G), fruit (H, J), flower (I), pistil (K).PCAMBIA1301-MsDREB2CP transgene tobacco is used for staining analysis (A, B, C, D, E, F) grow to high approximately 20 centimetres in Nutrition Soil after.After plant blossom result, further GUS staining analysis (G, H, I, J, K).Scale=0.2 centimetre (A, B); 0.5 centimetre (C, D, E, F, G, H, I, J); 150 microns (K).

Fig. 4 is the GUS relative reactivity of pCAMBIA1301-MsDREB2CP transgene tobacco under abiotic stress.

Error line represents the standard deviation repeating 3 times.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.Wherein,

Prybest archaeal dna polymerase, dNTP Mixture, RNase Inhibitor, RNase-Free DNase I, restriction enzyme EcoR I and Bgl II, T4DNA ligase enzyme, purchased from TAKARA company.DNAMarker is purchased from middle Ke Ruitai Beijing Bioisystech Co., Ltd.Penbritin, sulfuric acid card sodium enzyme element, Rifampin, Totomycin, GRD beta-glucuronidase, the acid of the chloro-3-indoles of the bromo-4-of 5--beta-glucoside, 4-methyl umbelliferone is purchased from Beijing Bioisystech Co., Ltd of fresh warp thread section.DNA rapid extraction test kit, centrifugal column type sepharose DNA reclaims test kit and purchases, and centrifugal column type plasmid extracts test kit in a small amount purchased from Beijing vast Tai Heng Bioisystech Co., Ltd.Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 is purchased from Beijing vast Tai Heng Bioisystech Co., Ltd.Double base plant conversion carrier pCAMBIA1301(is purchased from Beijing vast Tai Heng Bioisystech Co., Ltd) (carrier accession number: AF234297.The western cigarette of coral (Nicotiana tabacum var Xanthi nc) (Sun Feng Cheng Leixin cloud. the resistance that the western cigarette of resistance to virus induction agent 88-D induction coral produces PR albumen and TMV is infected. the 04th phase of Plant Pathology .1995) acquisition of public Ke Cong China Agricultural University, to repeat the application's experiment.

The clone of embodiment 1, MsDREB2C gene promoter and the structure of transgene carrier

One, the clone of MsDREB2C gene promoter

Malus sieversii (Malus sieversii (Ledeb.) Roem.) Rooted Cuttings of take is material, extracts its leaf DNA, take DNA as template, at 5 ' non-coding region design special primer amplification Malus sieversii MsDREB2C gene promoter.

Primer sequence is as follows:

PCR reaction system is as follows:

In 200ul centrifuge tube, add following component (25ul system):

PCR response procedures: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30sec, 62 ℃ of annealing 30sec, 72 ℃ are extended 2min, 30 circulations of increasing; 72 ℃ are extended 10min.0.8% agarose gel electrophoresis, reclaims object fragment, adds after being connected with pMD18-T cloning vector after " A " purifying and checks order.

Add A reaction system as follows:

In 200ul centrifuge tube, add following component (10ul system):

Temperature program(me): 70 ℃ of reaction 30min.

The recombinant vectors called after pMD18-T-MsDREB2CP of the DNA molecular that sequencing result is shown contain SEQ ID No.1.Wherein, SEQ ID No.1 is comprised of 1339 Nucleotide; 1-8 position is EcoR I recognition site and protection base; 1326-1330 position is front 5 bases in GRD beta-glucuronidase gene (GUS) encoding sequence; 1331-1339 position is Bal II recognition site and protection base, and 9-1325 position is MsDREB2C promotor.

Two, the structure of MsDREB2C gene promoter transgene carrier

EcoR I and Bgl II double digestion pMD18-T-MsDREB2CP, reclaim MsDREB2C promoter gene fragment and cut with Bgl II enzyme with EcoR I after pCAMBIA1301 plant expression vector be connected, as shown in Figure 1, pCAMBIA1301-MsDREB2CP obtains the MsDREB2C gene promoter about 1300bp after EcoR I and Bgl II double digestion for the EcoR I that obtains starting by MsDREB2C gene promoter the expression vector pCAMBIA1301-MsDREB2CP that gus gene expresses and Bgl II double digestion qualification result.

Embodiment 2, utilize MsDREB2C gene promoter to cultivate transgene tobacco

1, MsDREB2C gene promoter starts goal gene specifically expressing

PCAMBIA1301-MsDREB2CP is transformed to agrobacterium tumefaciens EHA105 competent cell, adopt Ye Panfa (Horsch RB etc., 1985, Science, 227:1229-1231) (the western cigarette of coral (Nicotiana tabacumvar Xanthi nc), and use hygromycin selection, extracts the resistance seedling DNA of taking root and carries out PCR Molecular with Pf and Pr transformation of tobacco, result as shown in Figure 2, shows that pCAMBIA1301-MsDREB2CP transfer-gen plant obtains the PCR product of MsDREB2C gene promoter.

PCAMBIA1301-MsDREB2CP transfer-gen plant is cultured in Nutrition Soil to 20 centimetres of left and right of height of seedling under normal condition according to Jefferson RA[1987, Plant Mol Biol Rep, 5 (4): 387-405] method detects with the histological chemistry that X-GLuc solution carries out gus gene expression, and the histological chemistry that carries out again gus gene expression after plant blossom result is detected.Result shows that gus gene in pCAMBIA1301-MsDREB2CP transfer-gen plant mainly expresses in the vascular tissue of root and leaf and seed, presents tissue specificity (Fig. 3).

2, MsDREB2C gene promoter is changed by adverse circumstance abduction delivering

PCAMBIA1301-MsDREB2CP transfer-gen plant is cultured in Nutrition Soil to 20 centimetres of left and right of height of seedling under normal condition, carries out respectively following five kinds of processing: contrast, in whole experimentation, all under normal growth condition, cultivating; Heat stress, 45 ℃ of illumination boxs are placed sampling in 6 hours; Drought stress, plant does not sample after within 7 days, not watering; Cold coercing, plant is placed sampling in 6 hours at 0 ℃ of illumination box; ABA coerces, and plant sprays 200 μ M ABA, sampling after 6 hours.Choose the tobacco leaf that growing way is consistent and be GUS enzyme biopsy survey (Jefferson, R.A., Burgess, S.M., and Hirsh, D. (1986) .Pglucuronidase from Escherichia coli as a gene-fusion marker.EMBOJ.83,8447-8451).Result as shown in Figure 4, show under arid, hot and cold and ABA coerce, GUS enzyme is lived and all higher than the enzyme under collating condition, is lived, wherein the GUS enzyme work under heat stress be under normal condition more than 2 times, GUS enzyme work under cold coercing is 2.5 times under normal condition, ABA coerce GUS enzyme work with drought stress be under normal condition more than 1.5 times.

The above-mentioned MsDREB2C of experimental results show that gene promoter is organizing specific and adverse circumstance inducible promoter.

Claims (10)

1. Following a) or b) DNA molecular:

   A) nucleotide sequence is the DNA molecular of the 9-1325 position of SEQ ID No.1;

   B) nucleotide sequence is the DNA molecular of SEQ ID No.1.
2. 2. the expression cassette that contains DNA molecular described in claim 1.
3. 3. the recombinant vectors that contains DNA molecular described in claim 1.
4. 4. the recombinant microorganism that contains DNA molecular described in claim 1.
5. 5. the primer pair of the DNA molecular total length claimed in claim 1 that increases.
6. 6. DNA molecular claimed in claim 1 is as the application of promotor.
7. 7. DNA molecular claimed in claim 1 starts the application of goal gene specifically expressing in root, leaf and/or the seed of plant; Described plant is tobacco.
8. 8. application according to claim 7, it is characterized in that: specifically expressing is for starting specifically expressing in the vascular tissue of goal gene at root in the root of plant for described startup goal gene, and described startup goal gene specifically expressing in the leaf of plant is specifically expressing in the vascular tissue of startup goal gene at leaf.
9. 9. the application that the lower startup of DNA molecular abiotic stress induction claimed in claim 1 goal gene is expressed in plant; Described abiotic stress is heat stress, coldly coerce, drought stress and/or ABA coerce; Described plant is tobacco.
10. 10. the application of DNA molecular claimed in claim 1 in cultivating transgenic plant; Described plant is tobacco.

Images