CN103865929B

CN103865929B - A kind of promotor relevant to phosphorus absorption and transport and application thereof

Info

Publication number: CN103865929B
Application number: CN201410080522.7A
Authority: CN
Inventors: 童依平; 赵艳艳; 赵学强; 何雪; 马文英
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2014-03-06
Filing date: 2014-03-06
Publication date: 2016-03-02
Anticipated expiration: 2034-03-06
Also published as: CN103865929A

Abstract

The invention discloses a kind of promotor relevant to phosphorus absorption and transport and application thereof.The invention provides 1, a kind of DNA fragmentation, be following 1) or 2) or 3) DNA molecular: the 1) DNA molecular shown in sequence 1 in sequence table; 2) under strict conditions with 1) DNA sequence dna that limits hybridizes and has the DNA molecular of promoter function; 3) with 1) DNA sequence dna that limits has more than 70% homology, and has the DNA molecular of promoter function.Experiment of the present invention proves, the present invention has cloned a ProTaPHT1.6 promotor all expressed in wheat root cauline leaf and fringe from wheat, and demonstrates goal gene GUS can be driven to express in wheat, proves that it is the promotor of tissue specific expression.

Description

A kind of promotor relevant to phosphorus absorption and transport and application thereof

Technical field

The present invention relates to biological technical field, particularly relate to a kind of promotor relevant to phosphorus absorption and transport and application thereof.

Background technology

Promotor is an important component part of gene, one section of thymus nucleic acid (DNA) sequence that gene can be made to carry out transcribing is referred in genetics, also be the most important factor in Factor of gene expression, it determines substantially, and whether a gene expresses, when express and where expression.By the mode of action and function, promotor can be divided into constitutive promoter, specificity promoter and inducible promoter three major types substantially.The feature of constitutive promoter is, the expression by its structure gene controlled has persistence, but does not have Space-time speciality; RNA and protein expression amount relative constancy, not by the induction of extraneous factor.Such as: the Actinl promotor of corn Ubiqultin promotor and paddy rice.The feature of organ or tissue's specificity promoter, is that the genetic expression controlling by it or regulate has significantly spatiotemporal, and often shows the characteristic of Growth adjustment.Such as, seed specific promoters, fruit-specific promoter, mesophyll cell specificity promoter, root-specific promoter.The feature of inducible promoter, the gene controlled for the type promotor is not having do not express under inducible factor existent condition or only have low-down expression, but once be subject to the induction of inducible factor, the expression amount of gene rapidly and increase considerably.Such as: hormone induction promotor, chemically inducible promoter, photoinduction promoter etc.

Phosphorus is one of necessary macronutrient of plant-growth, and it is not only ATP in vegetable cell, the important composition composition of Nucleotide and phosphatide, and plays very important regulating effect in energy trasfer, protein activation and C N metabolism.In soil, absolute phosphorus content usually very high (>1000 μM), but phosphorus is very easily fixed with organic and inorganic form in soil, and thus the concentration of available phosphorus is very low, greatly about 2-10 μM, the phosphorus being diffused into root table face is lower, is difficult to the needs meeting growth and development of plants.Many crops in agriculture production are monocotyledons, and therefore, the clone of promotor that can be relevant to phosphorus absorption and transport in monocotyledons and utilizing has great importance for the genetic improvement of farm crop and molecular breeding.

Summary of the invention

An object of the present invention is to provide a kind of promotor relevant to phosphorus absorption and transport.

The invention provides a kind of DNA fragmentation, is following 1) or 2) or 3) DNA molecular:

1) DNA molecular shown in sequence 1 in sequence table;

2) under strict conditions with 1) DNA sequence dna that limits hybridizes and has the DNA molecular of promoter function;

3) with 1) DNA sequence dna that limits has more than 70% homology, and has the DNA molecular of promoter function.

Recombinant vectors containing above-mentioned DNA fragmentation, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.

The total length of above-mentioned DNA fragmentation that increases or the primer pair of its any fragment are also the scope of protection of the invention.

Above-mentioned primer pair is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.

Above-mentioned DNA fragmentation is also the scope of protection of the invention starting the application in destination gene expression.

In above-mentioned application, described destination gene expression is organizing specific expression.

In above-mentioned application, described in be organized as plant vasular bundle, pulvinus and/or cob.

In above-mentioned application, described plant vasular bundle is roots of plants and/or cauline bundle;

Described plant is monocotyledons or dicotyledons; Described dicotyledons is specially barley.

In above-mentioned application, described goal gene is gus gene.

Experiment of the present invention proves, the present invention has cloned a ProTaPHT1.6 promotor all expressed in wheat root cauline leaf and fringe from wheat, and demonstrate goal gene GUS can be driven to express in wheat, prove that it is the promotor of tissue specific expression, can express in vascular bundle, pulvinus and/or cob.

Accompanying drawing explanation

Fig. 1 is PACH25-ProTaPHT1.6-GUS carrier schematic diagram

Fig. 2 is the GUS coloration result of transgenic line root

Fig. 3 is the GUS coloration result of the parts such as transgenic line stem

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.

PACH25 carrier: the public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute, be documented in as in Publication about Document: ChristensenAH, QuailPH, 1996, Ubiquitinpromoter-basedvectorsforhigh-levelexpressionofs electableand/orscreenablemarkergenesinmonocotyledonouspl ants.TransgenicRes5:213 – 218, this carrier has gus gene, but is not activated son before it.

The acquisition of embodiment 1, wheat phosphate transpoter gene ProTaPHT1.6 promoter sequence

According to HvPHT1 (AAN37900 in barley, AY187020, AY187025, AAO72435, AAN37901, AAO72436, AAO72440, AM904733) OsPHT1(AAN39042 and in paddy rice, AAN39043, AAN39044, AAN39045, AAN39046, AAN39047, AAN39048, AAN39049, AAN39050, AAN39051, AAN39052) over-designed primer, with the BAC storehouse of wheat (wheat breed is little lays down 54) for template, screening has the mono-clonal of amplification object fragment, after this BAC cloning and sequencing, obtain TaPHT1.6 coding region and upstream sequence, the 2047bp choosing this upstream sequence studies as promoter sequence, called after ProTaPHT1.6, its nucleotides sequence is classified as the sequence 1 in sequence.

Also can sequence 1 in artificial synthesized sequence table.

Embodiment 2, wheat phosphate transpoter gene ProTaPHT1.6 promoter function are analyzed

One, the structure of recombinant vectors

1, using the sequence 1 in the sequence table of synthetic as template, the primers according to promotor ProTaPHT1.6 carries out pcr amplification,

PCR primer (all introducing PstI restriction enzyme site in forward primer and reverse primer) sequence is as follows:

Forward primer: 5 '-TGACTGCAG cTGCAGGATTTATCGTCTGG-3 ';

Reverse primer: 5 '-TGACTGCAG gGATCCGCCGGCGATGACGA-3 '.

PCR reaction system: ultrapure water 39.5 μ l, 10 × PCRbuffer5 μ l, template 2 μ l, concentration is each 1 μ l of the forward and reverse primer of 10 μMs, Pfu enzyme (5u/ μ l) 0.5 μ l, dNTPs (10mM) 1 μ 1.

PCR reaction conditions: 94 DEG C 4 minutes; 94 DEG C 1 minute, 56 DEG C 1 minute, 72 DEG C 3 minutes, 42 circulations; 72 DEG C 8 minutes.

Obtain the pcr amplification product of about 2kb, be ProTaPHT1.6.

Reclaim kits pcr amplification product with QIAquick glue, at 16 DEG C, connect 8 hours with pMD18-T carrier (TAKARA, production number D101A), obtain recombinant plasmid pMD18-ProTaPHT1.6.

Use 2mm pole cup, 2500V is by recombinant plasmid pMD18-ProTaPHT1.6 transformation of E. coli DH5 α (Quan Shijin, production number CD201), and conversion product, growing containing on the LB plate culture medium of penbritin, selects positive colony.

From positive colony, extract plasmid, use AbIPRISM3700DNA analyser (Perkin-Elmer/AppliedBiosystem) to check order.Sequencing result shows, pMD18-ProTaPHT1.6 is for inserting the carrier obtained in pMD18-T carrier from the ProTaPHT1.6 promotor shown in 5 ' end the 1 to 2047 Nucleotide by the sequence 1 of sequence table.

2, cut pMD18-ProTaPHT1.6 with restriction enzyme PstI enzyme, reclaim the small segment of about 2kb.

3, cut pACH25 carrier with restriction enzyme PstI enzyme, reclaim large fragment carrier framework.

4, the carrier framework that small segment step 2 reclaimed and step 3 reclaim is connected, and obtains recombinant plasmid pACH25-ProTaPHT1.6(Fig. 1).

Through order-checking, recombinant plasmid pACH25-ProTaPHT1.6 for the sequence 1 of sequence table to be inserted the carrier obtained in pACH25 carrier from the ProTaPHT1.6 promotor shown in 5 ' end the 1 to 2047 Nucleotide, and inserts the upstream of gus gene.

Two, recombinant vectors transforms

The method of the wheat expression vector pACH25-ProTaPHT1.6 particle gun built is proceeded to wheat Gansu Province spring 23(Yuan pretty, Yang Wenxiong, high yield wide suitable high-quality New Spring Wheat Variety-Gansu Province spring No. 23, wheat crops journal, 2009,29 (4): 740.The public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute.) in, obtain T0 for turning ProTaPHT1.6 wheat.

Extracting T0 for turning the genomic dna of ProTaPHT1.6 wheat leaf blade as template, carrying out pcr amplification with forward primer and reverse primer, obtaining the fragment of about 2kb, being positive T0 for turning ProTaPHT1.6 wheat.

In the above-mentioned T0 generation being accredited as the positive, is turned ProTaPHT1.6 wheat and is cultured to T1 generation, results seed.

Being adopted by empty carrier pACH25 uses the same method proceeds in wheat Gansu Province spring 23, cultivates, and obtains T1 for turning empty carrier wheat.

Three, the detection of GUS expression

After T1 generation is turned ProTaPHT1.6 wheat seeds sprouting, obtaining T1 for turning ProTaPHT1.6 wheat, proceeding to normal Hydroponic culture, and analysis carries out GUS dyeing at different times to different tissues, observe GUS expression.Empty carrier wheat is turned for contrast with T1 generation.

As shown in Figure 2, A is the GUS coloration result of root for root and sectional view thereof, and B is the crosscut GUS coloration result of root, can find out, T1 generation turns ProTaPHT1.6 wheat root GUS signal, proves mainly in main root vascular bundle, have GUS to express further in sectional view.

As shown in Figure 3, A is the sectional view GUS coloration result of stem to the result of stem, stalk and cob, and B is pulvinus GUS coloration result, and C is cob GUS coloration result, can find out, all has obvious GUS signal at the vascular bundle of stem, pulvinus and cob.

In a word, ProTaPHT1.6 mainly expresses in root, cauline bundle tissue, pulvinus and cob.

And T1 generation turns empty carrier wheat, any GUS signal do not detected.

The above results shows, ProTaPHT1.6 is promotor, and goal gene GUS can be driven to express in the vascular tissue at the position such as root, stem.

Claims

1. a DNA fragmentation is the DNA molecular shown in sequence in sequence table 1.

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

3. the primer pair of the total length of DNA fragmentation described in claim 1 that increases.

4. primer pair according to claim 3, is characterized in that: described primer pair is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.

5. DNA fragmentation described in claim 1 is starting the application in destination gene expression.

6. apply as claimed in claim 5, it is characterized in that: described destination gene expression is organizing specific expression.

7. apply as claimed in claim 6, it is characterized in that: described in be organized as plant vasular bundle, pulvinus and/or cob; Described plant is wheat.

8. apply as claimed in claim 7, it is characterized in that: described plant vasular bundle is roots of plants and/or cauline bundle; Described plant is wheat.

9., as the application as described in arbitrary in claim 5-8, it is characterized in that: described goal gene is gus gene.