CN1164606C - Gene of 26S proteinase alpha subunit for regulating degradation of mouseearcress' abnormal protein - Google Patents

Abstract

The present invention provides a genome sequence of alpha ylidene of 26s proteasome and a coded protein sequence thereof. The function of the gene is closely related to the development and the differentiation of plant cells, the programmed death of cells, the signal transduction, the period regulation of cells, etc. The present invention has great application in production practice of agriculture, forestry, horticulture, etc.

Description

The gene of the 26S proteasome Alpha subunit of regulation and control Arabidopis thaliana paraprotein degraded

Technical field:

The invention belongs to the plant gene engineering technology field, be specifically related to a kind of gene of regulating and control the main factor 26S proteasome Alpha subunit of Arabidopis thaliana paraprotein degraded.

Background technology:

The Selected degradation of proteins proteic turnover of regulatory function effectively is one of vital process important step of carrying out the regulation and control of accurate space-time.General peptide and 26s proteasome dependence protein degradation pathway (Ubiquitin-dependent proteolytic pathway) are at present known most important, proteolytic pathway that high selectivity is arranged.It realizes adjusting to multiple metabolic process by proteinic turnover of regulatory function (turn over) and degraded abnormal protein.Along with to the understanding in depth of proteolytic degradation process, for new thinking has been opened up in the application of proteolytic degradation approach on biotechnology.By operation proteolytic degradation system, not only can impel the accumulation of useful foreign protein, also can suppress the accumulation of unwanted intrinsic protein, also can carry out regulation and control to some functional protein.

(26S Proteasomes is the distinctive proteolytic enzyme complex body of proteolytic degradation approach that relies on Ub protease) to the 26S proteasome, is Ub-albumen composition degraded place.Molecular weight is very big, and about 1.5MD contains about 30 polypeptide, when lacking ATP, can be broken into two inferior complex bodys of 20S and 19S, molecular weight each about 700kD (Rechsteiner etc., 1993; Rivett, 1993).20S particle (20Sproteasome, multicatalytic protease, or macropain) constitutes the catalytic core of proteasome, its structure is the cylinder bodily form of hollow, being arranged above and below by four ring texturees forms, and each ring comprises 7 polypeptide (Vierstra, 1996).The molecular composition of 20S particulate is α ₇β ₇β ₇α ₇(Jentsch etc., 1995).Two rings of outside are made up of identical α-subunit, and two of the inside form (Lowe etc., 1995) by identical β-subunit.

(α-like) the subunit class β subunit different with 14 formed, and molecular weight subunit is between 22～35kD by 14 different class α in the animal and plant.In yeast, 3 different β subunits are undergone mutation and are caused the cell growth slack-off, extremely sensitive to coercing, the Ub-albumen composition of can not degrading, in Arabidopis thaliana, the encoding gene of two α subunits and 1 β class subunit identified and yeast and various animal in counterpart homology more than 50% is arranged.20S granule crystal structure from Thermoplasma shows that particle contains 3 " chambeies " (cavity), and center cavity is formed by connecting from the centre by two β subunit rings, and there is the thr avtive spot the inside.Outside two chambeies lay respectively at the intersection of α subunit ring and β subunit ring, form a narrow passage, protein is entered lumen provide constraints.By this method, enzymolysis site subcellular area in the compatriot on space structure is kept apart, only allow to separate folding protein and enter output (Lowe etc., 1995) with amino acid and small peptide.

No matter this shows conservative like this structure, be in prokaryotic organism or eukaryote, and proteolytic pathway all is the important component part of cellular metabolism.It plays an important role to the normal activities of keeping cell.By improper, unnecessary in the degradation of cell and denatured protein, can eliminate the harm that their pair cells may cause, also can be synthetic new protein amino acid is provided; Part degraded by to proenzyme and hormone precursor is activated; Regulate albumen by the key enzyme in some metabolic process of degrading and some, control the level of these protein in cell, and then regulate cellular metabolism and biological g and D.

Recent studies show that, degraded needs the participation of ubiquitin in the born of the same parents of multiple proteins, as in zooblast, and p50 (transcription factor NF- _κB) precursor, some cell division factors, multiple transcription regulaton factor, histone H ₂A and H ₂Proteinic degraded such as B and oncogene protein is mediated by ubiquitin all.The degraded of the phytochrome A of vegetable cell is also relevant with ubiquitin.So research 26s proteasome and protein degradation have broad application prospects.

Summary of the invention:

The genome sequence that the purpose of this invention is to provide coding 26s proteasome Alpha subunit, and it is carried out the research of aspects such as growth and development of plants and old and feeble, environment-stress reaction, susceptible and intracellular signal transduction, in the hope of huge pushing effect is played in the production practice of agricultural, forestry and gardening etc.

Technical scheme of the present invention is as follows:

Do not have endogenous tagging element in the Arabidopis thaliana, T-DNA integrates becomes main insertion mutafacient system.Arabidopis thaliana has obtained thousands of mutant by the T-DNA label.Through TAIL-PCR (ThermalAsymmetric Interlaced PCR, Yaoguang Liu et al, 1998, Plant Molecular BiologyReporter 16:175-181, hot asymmetric interlaced polymerase chain reaction) technology amplification and mensuration label insert two terminal sequences in site, the whole genome sequence that to measure sequence and Arabidopis thaliana with Blast2.0 software compares, and finds out the mutational site, the plantation mutant.Wherein pass through the screening of phenotype analytical and various envrionment conditions, we have been separated to the gene of the main factor 26s proteasome Alpha subunit of regulation and control Arabidopis thaliana paraprotein degraded, T-DNA has inserted Arabidopis thaliana the 3rd chromosomal the 18909932nd base place, has inserted the tail end of gene A t3g51260.The cDNA sequence of AT3G51260 gene is seen sequence table 1 (SEQ ID No.1), and At3g51260 encoded protein matter sequence is seen sequence table 2 (SEQ ID No.2).

One, material

The Arabidopis thaliana ecotype (Columbia); Plasmid pSKI015 (Weigel, D., et al 2000); Various common antibiotics.Crown gall soil Agrobacterium (Agrobacterium tumefaciens) is GV3101.

Two, method

1. plant-growth and conversion

Arabidopis thaliana is 22 ℃ of growths in the greenhouse, illumination every day 16 hours, dark 8 hours.The Agrobacterium GV3101 that contains pSKI015 is earlier with four the CaMV35S enhansers complete the existence (Weigel, D., et al 2000) in the PCR reaction evaluation plasmid.

The Ti-plasmids that we adopt is pSKI015, and BAR gene, conferring herbicide resistance are contained in the T-DNA zone in the plasmid.Also contain and in prokaryotic system, be used for the Amp resistant gene that screens, placed in-line 4 35S enhansers are arranged near the T-DNA right hand edge, might after inserting the arabidopsis gene group, strengthen flank or near certain expression of gene, thereby obtain certain function, as one that inserts gene inactivation extra replenishing.

The method of arabidopsis thaliana transformation mainly adopts agriculture bacillus mediated method for transformation.More than ten years previous conviction scholar has invented seed and has contaminated conversion method, adopting Agrobacterium to cultivate dip-dye plant stem apex afterwards becomes whole strain plant to carry out vacuum filtration, these methods have all replaced former tissue culture and plant regeneration method, shortened the step of tissue culture, the method for transformation that we test use is a flower dip method (flaral dip), more easier than vacuum filtration, transformation efficiency can not reduce yet, wild Arabidopis thaliana (Columbia kind) is long to certain phase, some immature petals are arranged, the flower shears of having opened is fallen, as plant to be transformed.The Agrobacterium that has a pSKI015 after identifying 28 ℃ shake stationary phase (0.06 ≈ 2.0) and be suspended in again after centrifugal and contaminate in the substratum.Requisite raising transformation efficiency is sucrose and two kinds of materials of Surfactant in the dip-dye substratum.To contain in the soak solution of dip-dye substratum that the Arabidopis thalianas of not blooming in a large number are upside down in Agrobacterium 15 minutes, and continue to bloom after secretly cultivating certain hour then, set seeds,, transgenic line just be arranged wherein seed collection.

2. screening of transgenic plant and PCR detect

The filial generation that transforms plant is come out with label screenings such as microbiotic.That we use is weedicide PPT, adopts certain density PPT (100mg/ml) to add in the substratum, make not contain seed that T-DNA inserts and do not sprout or do not grow, and contain seed that T-DNA inserts because containing complete BAR gene normal growth.In these plants, T-DNA inserts and is generally monoploid, is recessive mutation, so phenotype generally is difficult for finding.

With total DNA of CATB method extraction plant leaf, utilize the primer of BAR gene on the pSKI015 carrier to carry out the PCR detection:

5 ' primer: 5 '-TCGACTCTAGCGAATTCCTC-3 ',

3 ' primer: 5 '-ATAGGCGTCTCGCATATCTC-3 ';

And carry out PCR simultaneously as over against photograph with the primer of COP1 in the Arabidopis thaliana:

5 ' primer: 5 '-TGACTATGCTCTGTTTCAGCT-3 ',

3 ' primer: 5 '-TTAGTAAACCAAGGAAACACCA-3 '

Reaction conditions is: 94 ℃ of 50s, and 60 ℃ of 60s, 72 ℃ of 90s, 35 circulations, the PCR product carries out electrophoresis detection with 1% sepharose.

3.T-DNA insert the amplification and the order-checking of flanking sequence

We adopt TAIL-PCR, the instant heating asymmetry PCR (Thermal Asymmetry InterlacePCR) that interlocks, TAIL-PCR comprises the random primer on the little flanking sequence nearby of annealing of three-wheel successive half nested type insertion sequence special primer and, be easy to amplify insertion site flanking sequence, this method need not carried out a lot of complicated operations before PCR, and produce highly purified special product, can directly be used as hybridization probe and sequencing template, promptly have efficient, sensitive, easy, special advantage.

The plant that will determine contains the BAR gene inserts flanking sequence (Liu, 1995) with TAIL-PCR amplification T-DNA, and three used special primers are according near the sequences Design the left hand edge of T-DNA, are respectively:

DL1：5’-GACAACATGTCGAGGCTCAGCAGG-3’；

DL2：5’-TGGACGTGAATGTAGACACGTCGA-3’；

DL3：5’-GCTTTCGCCTATAAATACGACGG-3’。

Used any degenerate primer has two:

AD2：5’-NGTCGA(G/C)(A/T)GANA(A/T)GAA-3’；

AD2-2：5’-NGTGCA(G/C)(A/T)GTNT(A/T)GAA-3’。

A large amount of amplification third round PCR products reclaim every rule band with low melting-point agarose, and the water that adds two volumes makes the blob of viscose fusing 65 ℃ of temperature baths, with phenol, each extracting of chloroform one time.Again with 2-2.5 times of volume ethanol and 1/10 volume sodium-acetate (NaAC) precipitation.12,000 change, and centrifugal 20 minutes postprecipitations are respectively washed one time with 70%, 100% ethanol, drain.Soluble in water, can be used to order-checking after quantitatively.

Also can with third round PCR product directly with the T-vector T of pBS generation _4-Dna ligase connects, and filters out the positive colony of insertion.The order-checking of upgrading grain.

4. the comparison of sequence and analysis

Obtain a large amount of insertion site flanking sequences, just need carry out sequential analysis, at first use BLAST (BasicLocal Alignment Search Tool) and the sequence in the database to compare, compare out the sequence of coupling in EMBL or the GENBANK, thereby determine that T-DNA inserts the site actually at chromosomal what position of which bar, near opening code-reading frame (Open Reading Frame, ORF), translated proteinic possibility function, with the homology of known protein relatively or the like, obtain the mutant of the 26s proteasome gene A T3G51260 sudden change that we seek thus.

5. phenotype analytical

The plantation mutant is planted the wild-type Arabidopis thaliana simultaneously and is contrast.As shown in Figures 1 and 2, Fig. 1 is the form photo of arabidopsis mutant body plant in the fruiting period of blooming; Fig. 2 is the form photo of wild Arabidopis thaliana in the fruiting period of blooming.As can be seen from Figure, mutant shows obvious growth speed and slows down with plant short and small blooming fruiting period and wild-type relatively.

The present invention adopts agrobacterium mediation method, and the T-DNA of Agrobacterium is inserted in the middle of the genomic dna of Arabidopis thaliana, inserts mutant thereby produce.The seed that transforms plant is containing the enterprising row filter of MS substratum of PPT (100mg/ml).Total DNA with CATB method extraction plant leaf utilizes the primer of BAR gene on the pSKI015 carrier to carry out PCR.To determine that the plant that contains the BAR gene inserts flanking sequence with TAIL-PCR amplification T-DNA.A large amount of amplification third round PCR products reclaim every rule band with low melting-point agarose.Carry out comparison and the analysis of BLAST after the order-checking, find the insertion site of T-DNA thus, the insertion point of finding this T-DNA is just on the dna sequence dna of 26s proteasome gene α subunit, be positioned at Arabidopis thaliana trisomic long-armed on.This gene and the 26s proteasome gene Rpn1 that has cloned, Rpn2, Rpn6 and Rpn10 have higher homology.The growth of its function and vegetable cell, differentiation and apoptosis and signal transduction, cell cycle regulating etc. all have confidential relation.

Description of drawings:

Fig. 1 is the form photo of arabidopsis mutant body plant in the fruiting period of blooming;

Fig. 2 is the form photo of wild Arabidopis thaliana in the fruiting period of blooming.

Sequence table 1 (SEQ ID No.1)

1 ATGGCGAGAT?ACGATCGAGC?AATCACTGTA?TTCTCCCCCG?ATGGTCACCT?CTTCCAAGTA

61 GAGTATGCCC?TAGAAGCCGT?CCGAAAGGGT?AACGCCGCCG?TCGGAGTCCG?CGGCACAGAT

121?ACTGTCGTCC?TCGCCGTCGA?AAAGAAATCC?ACCCCTAAGC?TCCAGGATTC?CAGATCAGCA

181?AGGAAGATAG?TGAGCCTTGA?TAATCACATT?GCATTGGCCT?GTGCTGGACT?GAAAGCAGAT

241?GCTCGTGTCT?TGATAAACAA?GGCGAGGATA?GAGTGTCAAA?GCCACAGGCT?TACGTTGGAG

301?GACCCAGTTA?CTGTTGAGTA?CATTACTCGG?TACATAGCAG?GGCTTCAACA?GAAGTATACT

361?CAAAGTGGTG?GTGTGAGGCC?TTTTGGTCTT?TCCACTCTTA?TTGTTGGGTT?TGATCCCTAC

421?ACTCGTATAC?CCGCGCTTTA?TCAGACCGAT?CCATCTGGTA?CATTCTCTGC?TTGGAAAGCT

481?AATGCAACTG?GGAGAAACTC?TAACTCAATT?AGGGAGTTTC?TGGAGAAAAA?CTACAAAGAA

541?AGCGCTGGCC?AAGAAACTGT?TAAGCTTGCT?ATCCGTGCTC?TGCTTGAGGT?TGTTGAGAGT

601?GGGGGAAAGA?ACATTGAGGT?TGCTGTAATG?ACACGAGAGG?AAGGTGTTCT?GAAGCAACTA

661?GAAGAAGAAG?AAATTGATAT?CATTGTGGCT?GAGATCGAAG?CAGAGAAGGC?TGCAGCTGAA

721?GCAGCCAAGA?AAGGCCCTGC?GAAGGAAACA?TGA

Sequence table 2 (SEQ ID No.2)

MARYDRAITV?FSPDGHLFQV?EYALEAVRKG?NAAVGVRGTD?TVVLAVEKKS?TPKLQDSRSA

RKIVSLDNHI?ALACAGLKAD?ARVLINKARI?ECQSHRLTLE?DPVTVEYITR?YIAGLQQKYT

QSGGVRPFGL?STLIVGFDPY?TRIPALYQTD?PSGTFSAWKA?NATGRNSNSI?REFLEKNYKE

SAGQETVKLA?IRALLEVVES?GGKNIEVAVM?TREEGVLKQL?EEEEIDIIVA?EIEAEKAAAE

AAKKGPAKET*

Claims (3)

1. polynucleotide have the nucleotide sequence shown in SEQ ID No.1.

2. a protein has the aminoacid sequence shown in SEQ ID No.2.

3. polynucleotide shown in claim 1, it is the gene of Arabidopis thaliana 26S proteasome Alpha subunit.

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