CN102731634B - Pleiotropic gene associated protein from wheat, encoding gene thereof and application - Google Patents

Abstract

The invention discloses a pleiotropic gene associated protein from wheat, an encoding gene thereof and an application. The pleiotropic gene associated protein from wheat is a protein as the following a) or b): a) a protein formed by amino acid sequence as shown in Sequence 3 in a sequence table; and b) a protein which is obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence as shown in the Sequence 3 in the sequence table, is derived from (a) and is correlated with any of the following properties: plant height, preharvest sprouting, plant type, heading date and fecundity of wheat. The encoding gene of the protein influences plant height, preharvest sprouting, plant type, fecundity and heading date of wheat. As the gene has strong pleiotropic effects, the gen is of great significance in wheat breeding and production.

Description

Derive from pleiotropic gene associated protein and encoding gene and the application of wheat

Technical field

The present invention relates to a kind of pleiotropic gene associated protein and encoding gene and application that derives from wheat.

Background technology

Downgrade, the effect of half dwarfed plant, not only improved the resistance to fertilizer of crop, effect resistant to lodging, the more important thing is and reduced the assimilation of stem stalk, more photosynthate is transferred to fringe portion, thereby has improved harvest index, increased significantly crop yield.Betide the agriculture Green Revolution in last century five, the sixties, just because of the extensive popularization of wheat, paddy rice Semi-dwarf cultivar and the employing of corresponding cultivation technique, wheat, rice yield are had increased significantly.Up to now, identified and the wheat of Unified number falls stalk gene one and has 21, but in producing at present, the dwarf gene of widespread use popularization only has Rht1, Rht2, Rht8 and Rht9, and other most of dwarf gene is not effectively applied to the breeding production of wheat.The serious breeding that is restricting wheat of simplification in the short source of wheat is produced, and therefore excavates, studies other important dwarf gene in wheat and have very important significance for the output that improves wheat.

Summary of the invention

The object of this invention is to provide a kind of pleiotropic gene associated protein that derives from wheat.This protein is relevant to plant height, fringe germination, plant type, heading stage and the fecundity of wheat.

Protein provided by the present invention, name be called Rht-Blc (Rht3), derive from wheat, be following a) or b) protein:

A) protein that the aminoacid sequence shown in sequence 3 forms in sequence table;

B) by the aminoacid sequence of sequence in sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to following arbitrary proterties by (a) derivative protein: the plant height of wheat, ear germinating resistance, plant type, heading stage and fecundity.

Sequence 3 in sequence table is comprised of 651 amino acid.

Albumen in above-mentioned in order to make (a) is convenient to purifying, and the N-terminal of the protein that can form at the aminoacid sequence shown in sequence in sequence table 3 or C-terminal connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tagII	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Albumen in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.

The encoding gene of above-mentioned protein also belongs to protection scope of the present invention.

The encoding gene of described protein specifically can be following 1) or 2) or 3) gene:

1) its nucleotide sequence is the DNA molecular shown in sequence 1 or sequence 2 in sequence table;

2) DNA sequence dna limiting with (1) at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have a DNA molecular of 99% homology and coding Rht-Blc;

3) under stringent condition with 1) or 2) the DNA sequence dna hybridization limiting and the DNA molecular of coding Rht-Blc.

Sequence 1 in sequence table is comprised of 3892 deoxyribonucleotides, is the genomic gene of the Rht-Blc of wheat, and the sequence 2 in sequence table is comprised of 1956 deoxyribonucleotides, is the cDNA gene of the Rht-Blc of wheat.

Described stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M Na ₃pO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 2 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃pO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃pO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.5 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃pO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃pO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 65 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, under 65C, hybridize, then use 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively washes film once.

The primer pair of above-mentioned arbitrary described full length gene or its any fragment of increasing also belongs to protection scope of the present invention.

Described primer pair specifically can extension increasing sequence table in the pair of primers pair of the DNA molecular shown in sequence 1, a primer sequence is wherein 5, another primer sequence of-ATG CCG TCT ACA ACT ACT ACG CTG-3 ' is 5 ,-AGT CCGGCC CGT GCT TAT TTT G-3 ' the genomic dna of Rht-Blc gene (this primer be used for increasing).

Described primer pair specifically can extension increasing sequence table in the pair of primers pair of a fragment of the DNA molecular shown in sequence 2, a primer sequence is wherein, 5 '-ATG AAG CGC GAG TAC CAG GA-3 ', another primer sequence is: 5 '-TCA CGG CGC GGC CAG GCG CCA TGC-3 ' the cDNA sequence of Rht-Blc gene (this primer be used for increasing).

The recombinant vectors that contains described gene, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus also belong to protection scope of the present invention.

The recombinant expression vector that available existing plant expression vector construction contains described gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.As pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of transcribing as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean stores protein gene) 3 ' end all has similar functions.While using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor (as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn), constitutive promoter or organizing specific expression promotor (as the promotor of seed specific expression), they can be used alone or are combined with other plant promoter; In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene (gus gene of luminophor, luciferase genes etc.), antibiotic marker gene (as is given the nptII gene to kantlex and associated antibiotic resistance, give the bar gene to weedicide phosphinothricin resistance, give the hph gene to microbiotic hygromycin resistance, with the dhfr gene of giving methatrexate resistance, give the EPSPS gene to glyphosate resistance) or anti-chemical reagent marker gene etc. (as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.

The recombinant vectors that contains Rht-Blc gene specifically can be for starting by 35s promotor the recombinant expression vector p35s:Rht-Blc that Rht-Blc transcribes.P35s:Rht-Blc be in pCambia1300, insert that the Rht-Blc gene shown in the 1-3892 position Nucleotide of 35s promotor and sequence 1 obtains by 35s promotor, start the recombinant expression vector of Rht-Blc genetic transcription.

The recombinant vectors that contains Rht-Blc gene specifically also can be pRht-Blc:Rht-Blc.PRht-Blc:Rht-Blc is the recombinant expression vector that in insertion sequence table, the Rht-Blc gene shown in the promotor of Rht-Blc gene shown in the 1-4213 position nucleotide sequence of sequence 4 and the 1-3892 position Nucleotide of sequence 1 obtains in pCambia1300, in this recombinant expression vector, Rht-Blc gene is started and is transcribed by Rht-Blc promotor.

Another object of the present invention is to provide a kind of method of cultivating transgenic plant.

The method of cultivation transgenic plant provided by the present invention, is that Rht-Blc gene is imported in object plant, obtains having the transgenic plant of following at least one proterties: 1) plant height is lower than described object plant; 2) heading is bloomed and is later than described object plant: 3) tillering angle is greater than described object plant; 4) ear germinating resistance is higher than described object plant.

Wherein, Rht-Blc gene can import by the recombinant expression vector that contains Rht-Blc gene in object plant, in described recombinant expression vector, the promotor that starts Rht-Blc genetic transcription be 35s promotor or following a) or b) promotor: a) its nucleotide sequence is the DNA molecular shown in sequence 4 in sequence table; B) its nucleotide sequence is the DNA molecular shown in sequence 4 1-4213 positions in sequence table.

The recombinant expression vector that contains Rht-Blc gene specifically can be above-mentioned p35s:Rht-Blc or pRht-Blc:Rht-Blc.

Described object plant specifically can be dicotyledons or monocotyledons, and described monocotyledons specifically can be paddy rice.

Described transgenic plant are interpreted as and not only comprise the first-generation transgenic plant that described gene transformation object plant is obtained, 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 gene can first be modified as follows, then imports in host, to reach better expression effect:

1) modify according to actual needs and optimize, so that gene efficient expression; For example, the codon that can have a preference for according to recipient plant, is keeping nucleotide sequence coded amino acid whose its codon that simultaneously changes of the present invention to meet plant-preference; In optimizing process, preferably can make to keep certain GC content in the encoding sequence after optimizing, to realize best the high level expression of quiding gene in plant, wherein GC content can be 35%, be preferably more than 45%, more preferably more than 50%, most preferably more than approximately 60%;

2) modify the gene order of contiguous initial methionine, so that translation is effectively initial; For example, utilize known effective sequence in plant to modify;

3) be connected with the promotor of various expression of plants, be beneficial to its expression in plant; Described promotor can comprise that composing type, induction type, sequential regulate, grow adjusting, Chemical Regulation, tissue preferably and tissue-specific promoter; The selection of promotor will be along with expression time and space requirement and is changed, and depends on target species; For example tissue or the specific expressing promoter of organ, acceptor in what period of growing is determined as required; Although proved that the many promotors that derive from dicotyledons are operational in monocotyledons, vice versa, but ideally, select dicotyledons promotor for the expression of dicotyledons, monocotyledonous promotor is for the expression of monocotyledons;

4), with applicable Transcription Termination sub-connection, also can improve the expression efficiency of gene of the present invention; For example derive from the tml of CaMV, derive from the E9 of rbcS; Any known available terminator working in plant can be connected with gene of the present invention;

5) introduce enhancer sequence, for example, for example, as intron sequences (deriving from Adhl and bronzel) and virus leader sequence (deriving from TMV, MCMV and AMV);

In actually operating, also gene of the present invention can be carried out to cell-targeting location.Can utilize the existing technology in this area to realize.For example, the target-gene sequence and the gene order of the present invention that derive from targeted cells device are merged, then import in vegetable cell, just can locate.

Another object of the present invention is to provide a kind of method of whether carrying Rht-Blc gene in auxiliary detection wheat.

The method of whether carrying Rht-Blc gene in auxiliary detection wheat provided by the present invention, that to take the genomic dna of wheat to be measured be template, with primer, carry out pcr amplification, as obtain the amplified fragments of 489bp, wheat to be measured is not for carrying candidate's wheat of Rht-Blc gene; As obtain the amplified fragments of 2515bp, wheat to be measured is the candidate's wheat that carries Rht-Blc gene; Described primer is that nucleotide sequence is respectively sequence 6 and two single stranded DNAs of 7 in sequence table.

In addition, the transposon that name is called CAAS-TRIM-6B also belongs to protection scope of the present invention.

Wherein, the transposon of CAAS-TRIM-6B be following a) or b) DNA molecular:

A) its nucleotide sequence is the DNA molecular shown in sequence 5 in sequence table;

B) and a) DNA sequence dna limiting at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have 99% homology and have the DNA molecular of transposon activity.

Rht-Blc gene promoter be following a) or b) DNA molecular:

A) its nucleotide sequence is the DNA molecular shown in sequence 4 in sequence table;

B) and a) DNA sequence dna limiting at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have 99% homology and have the DNA molecular of promoter activity.

In auxiliary detection wheat of the present invention, whether carry the method for Rht-Blc gene accurately, reliably, can be used for detecting the early stage Rht-Blc proterties that pleiotropic gene is controlled of Wheat Development, to cultivating the wheat of semi-short-stalked, the germination of resistance to fringe, high yield, there is important guiding effect, thereby optimize cross combination, accelerate breeding speed, reduce breeding cost, have simple to operate, with low cost, the advantage such as the cycle is short, be very suitable for applying, the genetic breeding of wheat is had to important meaning.The present inventor finds that the short entrained Rht-Blc gene of thumb is the insensitive complete dominance gene of Plant hormones regulators,gibberellins being positioned on wheat 4BS the short arm of a chromosome, the plant height of major effect wheat, fringe germination, plant type, fecundity and heading stage.Just because of this extremely strong pleiotropy of Rht-Blc gene, this gene has great importance in the breeding of wheat is produced.

Accompanying drawing explanation

Fig. 1 is the Phenotypic Observation of Rht-Blc near isogenic line;

In Fig. 1, c is the plant height mean+SD of 20 strains;

D is the mean+SD that the fringe of 20 strains germinates;

E is the mean+SD of the tillering angle of 20 strains;

F is the mean+SD at the heading stage of 20 strains;

Fig. 2 is clone and the gene structure analysis of Rht-Blc gene;

Fig. 3 is the checking of Rht-Blc gene function;

Wherein, pRht-Blc:Rht-Blc and p35s:Rht-Blc represent respectively to proceed to the plant of respective carrier

Fig. 4 is α-amylase assay and yeast two-hybrid experimental result;

Embodiment

The experimental technique using 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.

The structure of embodiment 1, Rht-Blc near isogenic line and phenotype analytical

First we open up (Henan local variety with laying down, hereinafter referred to as NIL-Rht-Bla, represent) as recurrent parent, thumb is short as donor parents 7 generations that backcrossed, structure obtains the near isogenic line (representing with NIL-Rht-Blc) of short bar gene Rht-Blc, then planted in long day area-Hebei, with short-day regions-Yunnan, carry out respectively the observation of field phenotype, investigate the proterties (Fig. 1 such as its plant height, tillering angle, heading stage, fringe germination, grain plumpness, A is NIL-Rht-Bla, and B is NIL-Rht-Blc).Field investigation result shows that the average plant height of near isogenic line NIL-Rht-Blc of the short bar gene that structure obtains is only 30cm, be starkly lower than the plant height (Fig. 1 a, c) of NIL-Rht-Bla.After results, observe seed and also have comparatively significantly difference, the seed of NIL-Rht-Blc is partially shrivelled, and the seed of NIL-Rht-Bla is wanted full many (Fig. 1 b).The fringe percentage of germination of same NIL-Rht-Blc in Hebei and Yunnan is respectively 13.7% and 17.8%, be starkly lower than NIL-Rht-Bla at the fringe percentage of germination (64.7% and 66.7%) (Fig. 1 d) of these two places; Relatively in tillering angle, finding that the plant of NIL-Rht-Blc obviously presents the shape of crawling, tillering angle is obviously greater than the plant (Fig. 1 e) of NIL-Rht-Bla.Also there is notable difference the heading stage of NIL-Rht-Bla and NIL-Rht-Blc plant, long day area Hebei NIL-Rht-Blc than NIL-Rht-Bla late about 5 days, and more obvious in Yunnan, short-day regions difference, late about 10 days (Fig. 1 f).In the F2 segregating population of Rht-Blc RIL (RIL-Rht-Blc) and Henan wheat 18, also find, shorter plant be all attended by anti growing out, shrivelled seed, plant type crawl and heading stage more late.These results all confirm that these proterties are controlled by same short bar gene Rht-Blc (Rht3) above.

Wherein, fringe percentage of germination is measured as follows: adopt the de-direct germination test method of seed, every kind of drawing to repeat to get at random 50, mixed de-seed, put and in culture dish, directly carry out germination test, respectively repeat by grain germination inspection every day to every kind, until one week, calculate its percentage of germination.

Tillering angle is measured as follows: tillering angle is when harvesting time, to investigate 20 strains, the measuring method of maximum angle is 20 centimetres, wheelbase ground eminence in plant, with different directions, measure axis and peripheral horizontal throw of tillering 3 times, calculate mean value, obtain again wide/high ratio, and with arctan function, convert thereof into the maximum angle of tillering of this strain.

The acquisition of embodiment 2, wheat protein Rht-Blc and encoding gene Rht-Blc thereof

One, the acquisition of Rht-Blc genomic dna sequence in wheat

First the primer of initiator codon and terminator codon amplification gene total length is crossed in design: Rht-Blcp 5 '-ATGCCG TCT ACA ACT ACT ACG CTG-3 ' and 5 '-AGT CCG GCC CGT GCT TAT TTT G-3 '.Take China spring (Rht-Bla), agricultural No. 10 (Rht-Blb), thumb short (Rht-Blc) genomic dnas is template, with primer Rht-Blcp, carry out pcr amplification, PCR product detects with agarose gel electrophoresis, detected result shows in No. 10, China spring and agricultural and all obtained the 2Kb amplified fragments of expection, and obtain one in thumb is short, than No. 10, China spring and agricultural, obviously the new amplified fragments about 2Kb are (in Fig. 2 a) greatly.The genomic dna sequence that this fragment cloning and sequencing is obtained to the short middle Rht-Blc gene of thumb, forms (sequence 1) by 3892bp.

Two, the acquisition of Rht-Blc cDNA sequence in wheat

Also design primer Rht-Blc-eDNAp (5 '-ATG AAG CGC GAG TAC CAG GA-3 ' and 5 '-TCACGG CGC GGC CAG GCG CCA TGC-3 ') simultaneously, the cDNA that amplification China spring (Rht-Bla), agricultural No. 10 (Rht-Blb), total mRNA reverse transcriptions of thumb short (Rht-Blc) become is template, PCR product detects with agarose gel electrophoresis, and gel electrophoresis figure is as a in Fig. 2.The PCR product that amplification is obtained carries out cloning and sequencing, obtains the eDNA sequence of the short middle Rht-Blc gene of thumb, by 1956 Nucleotide, formed, its nucleotide sequence as shown in sequence in sequence table 2, the protein of sequence 3 in code sequence list.

Three, the acquisition of Rht-Blc gene promoter sequence in wheat

We design the promotor of primer amplification gene: Rht-Bp (5 '-GCG GCG CTT TTC CTG CTT TGT C-3 ' and 5 '-GAT CTC GCC TCT CGC CTC CCT ACC-3 ').Take China spring (Rht-B1a), agricultural No. 10 (Rht-Blb), thumb short (Rht-Blc) genomic dnas is template, with primer Rht-Bp, carry out pcr amplification, PCR product detects with agarose gel electrophoresis, gel electrophoresis figure has all obtained the amplified fragments 4kb expecting as b detected result in Fig. 2 shows China spring and No. 10, agricultural and thumb in short, this fragment cloning and sequencing is obtained to the promoter DNA sequence of Rht-Blc gene in wheat, its nucleotide sequence, as shown in sequence in sequence table 4, is comprised of 4213 Nucleotide.

Four, the comparison of Rht-Blc genomic dna and cDNA sequence in wheat

Resulting sequence in above-mentioned one, two is compared to analysis, and result shows that Rht-Blc full length gene is 3892bp, comprises 2 exons and 1 intron, Exon1=1～147bp; Intronl=148～2083bp; Exon2=2084～3892bp; Its opening code-reading frame is 1956bp, 651 amino acid of encoding, and theoretical molecular is 68.0kD, iso-electric point pI value is 4.94.Sequence is more also discovery, and Rht-Blc genomic dna sequence is that 147bp place inserts 2026 Nucleotide (c in Fig. 2) after the ATG of Rht-Bla genomic dna sequence.

CDNA sequence relatively finds that Rht-Blc has the insertion of 90bp in DELLA district, and this 90bp is just in time consistent with the 90bp sequence of 3 ' end of the insertion of 2026-bp.Rht-Bla (Rht-Blb) is all genes of intronless, still, due to the insertion of 2026-bp sequence, gene structure is changed, and Rht-Blc gene includes two exons and an intron.At intron and exon joining place calling sequence, also meet " GT-AG " rule completely, when the insertion of transcriptional level 90-bp nucleotide sequence has also caused at Rht-Blc gene translation protein, in DELLA region, have 30 amino acid whose insertions (d in Fig. 2) simultaneously.

Five, the identification of a new TRIMs transposon

Near the Nucleotide AG of the 5bp insertion sequence of 2026bp and its insertion point (being positioned at insertion sequence 5 ' end) GTG (being positioned at insertion sequence 3 ' end) forms a complete intact Terminal repeat retro-transposonsin miniature (TRIMs).This transposon sequence length is 2031bp (sequence 5 in sequence table), wherein comprises the terminal direct repeats (TDRs) of two 503bp, and very conservative between these two TDRs, without any base difference.Sequence between these external two TDRs any albumen of not encoding, and at the two ends of intermediate sequence, there is the primer binding site (PBS) of 22bp (CGA ACC TTG TGG CTC TGA ATA C), and the polypurine tract (PPT) of 15bp (TTT CCC CCC TTA ATC), and at the two ends of sequence, there is the target site duplications (TSD) of 5bp (AGGTG), and the sequence of this 5bp forms behind this position at TRIMs swivel base, this sequence possesses the basic structure that has TRIMs element, can assert that this insertion sequence is a TRIMs element (e in Fig. 2).Comparison wheat tumor-necrosis factor glycoproteins database TREP Release 10 (theTriticeae Repeat Sequence Database:http: //wheat.pw.usda.gov/ITMI/Repeats/) result shows, without any similarity, we think that this is a new TRIMs accordingly with known tumor-necrosis factor glycoproteins.

In order further to study the origin of this new TRIMs, we compare China spring sequencing data storehouse (http://www.cerealsdb.uk.net/search_reads.htm.) through electronic splicing sequence, we have obtained including insertion sequence in the sequence of interior about 5Kb, and according to the sequence of this 5Kb, we have designed the primer pTRIMs (5 '-CCG GAC AAA TGG AGG AGA-3 ' and 5 '-CGC GCA TCA TCC CGA GTAT-3 ') of pair for amplification 2026bp sequence.

For this sequence is positioned in wheat cdna group, first we use this mark to increase to AA genome (T.urartu) AABB genome (Langdon) the DD genome (aegilops tauschii Ae.tauchii) of wheat, pcr amplification result shows only can amplify target fragment in Longdon material, this result also illustrates that this insertion sequence is arranged in B genome (Fig. 2 f of wheat, A is T.urartu, AB is Langdon, D is aegilops tauschii Ae.tauchii, CS is China spring, H10 is that drought is selected No. 10, and L14 is Shandong wheat 14).For more detailed to its locate we increased drought select No. 10 with Shandong wheat 14 (parent of DH mapping population), in Shandong wheat 14, amplification obtains the fragment 2878bp of expection, and amplified fragments is approximately 800bp in drought is selected No. 10, sequencing result shows, drought is selected No. 10 than Shandong wheat 14 disappearance 2026bp, just with consistent at the fragment sequence of the short middle insertion of thumb.This insertion/deletion the diversity mark drought that is used for increasing select 150 strains of the DH colony of No. 10 * Shandong wheat 14, the diagram data of doing in conjunction with this colony, utilize Mapmaker3.0 software that this insertion sequence is positioned between wheat 6B chromosomal SSR mark Wmc341 and Wmc182, genetic distance is respectively 7.9 and 7.5cM (g in Fig. 2).Accordingly, we are by this insertion sequence called after CAAS-TRIM-6B (sequence 5 in sequence table).In order to detect the distribution of CAAS-TRIM-6B in wheat cdna group, we have detected the material that a set of diversity is very abundant, detected result shows only to have 7 parts of materials CAAS-TRIM-6B to be detected on the 6B of wheat karyomit(e), comprises in China spring, Shandong wheat 14, platform 23, Shanxi wheat 2148, large grain half awns, Sunstar, Wenmai 6, Buddhist monk wheat, white shell shaven head.And other most of material does not detect and contains CAAS-TRIM-6B on the 6B of wheat karyomit(e).Comprise that thumb is short, Langdon, 96S-226, safflower wheat, red winter wheat, middle peasant 28, 96S-223, Chengdu shaven head, AM3, drought is selected No. 10, lay down and open up No. 1, little Bai awns, product drought 328, red No. 5 of capital, interior township 188, A Bo, Bai Pu, No. 9, new gram of drought, Ou Rou, fringe 30 early, the square wheat of long tribute, interior wheat 11, Kangding wheat, the peaceful spring 13, W7984, the red awns that curls up, little lipstick, Bai Maizi, spend wheat in vain, Opata85, GIAVA, Wangshuibai, June is yellow, Ka Jiemu F71, Henan wheat 18, Zheng draws No. 4, upper woods wheat, the tall and erect loud, high-pitched sound of wood ancestor, short Meng ox, Atlas 66, half awns, change fruital, the Tibetan winter No. 4, ocean wheat, agricultural university 311, white oil wheat, fireball, No. 10, rust-proofing, salty agriculture 39, Ningchun4.This explanation transposon CAAS-TRIM-6B is other position from 6B chromosome transfer to wheat cdna group, such as in thumb is short from the 6B chromosome transfer of wheat to the 4B karyomit(e) of wheat.These experimental result explanations CAAS-TRIM-6B enlivens in wheat cdna group very much above, and the jump of this transposon has randomness.

The checking of embodiment 3Rht-Blc gene function

In order to verify the exactness of our resulting gene order, we are according to the Insert Fragment design primer Rht-BlcM of 2026 Nucleotide in Rht-Blc genome sequence (5 '-ATG CCG TCT ACA ACT ACT ACG CTG-3 ' 5 '-TAG TGC ACG GTG TCC GTG GCG A-3 '), and this primer can be directly used in special detection Rht-Blc gene.When containing Rht-Blc gene, amplified fragments is 2515bp, otherwise amplified fragments is 489bp.Here we by primer Rht-BlcM the near isogenic line (representing with NIL-Rht-Blc) for detection of the short bar gene building in embodiment 1, and the F2 segregating population of RIL-Rht-Blc.Detected result all shows that all short bar plant all contain Rht-Blc gene, can't detect on the contrary Rht-Blc gene (a in Fig. 3, Rht-Blc is short bar plant, Rht-bla is high bar plant) in high bar material.This detected result has illustrated Rht-Blc gene and plant height, fringe germination, tillering angle, heading stage and fecundity direct correlation.

In order further to confirm the function in plant of Rht-Blc gene, we have built transgene expression vector rice transformation, embody vector construction and conversion process is as follows:

Wherein, pCambia1300-35s builds as follows: according to 35s promoter primer primers F:5 '-CCG gAA TTCaTG GTG GAG CAC GAC ACT CTC GTC T-3 ' (underscore sequence is that EcoRI is restriction enzyme site) and R:5 '-CGG gGT ACCcTG TCC TCT CCA AAT GAA ATG A-3 ' (underscore sequence is KpnI restriction enzyme site), from plasmid pCambia1300 amplification 35s promotor, the restriction enzyme EcoRI and the KpnI recognition site that this 35s promotor are inserted to pCambia1300 obtain pCambia1300-35s.

The structure of 35s promoter expression vector p35s:Rht-Blc: specifically according to cDNA primers F:5 '-CGG of Rht-Blc gGT ACCaTG AAG CGC GAG TAC CAG-3 ' (underscore sequence is KpnI restriction enzyme site) and R:5 '-CTA G tC TAG AtC ACG GCG CGG CCA GGC G-3 ' (underscore sequence is XbaI enzyme cutting site), the ORF (the 1-3892 position nucleotide sequence with sequence 1) that amplification obtains the genomic dna of Rht-Blc, after KpnI/XbaI double digestion, passes through T ₄kpnI and XbaI site that DNA ligase is inserted into pCambia1300-35s carrier by gene obtain recombinant vectors p35s:Rht-Blc.

The structure of self promoter expression vector pRht-Blc:Rht-Blc: specifically according to promoter region primers F:5 '-GCA T of Rht-Blc gene cA ATT GgC GGC GCT TTT CCT GCT TTG TC-3 ' (underscore sequence is MfeI site) and R:5 '-GGC gGT ACCgAT CTC GCC TCT CGC CTC CCT ACC-3 ' (underscore sequence is KpnI restriction enzyme site), amplification obtains the promotor (the 1-4213 position nucleotide sequence with sequence 4 in sequence table) of Rht-Blc, after MfeI/KpnI double digestion, by T4 ligase enzyme, promotor is inserted into the MfeI of p35s:Rht-Blc and KpnI site to replace 35s promotor, obtains recombinant vectors pRht-Blc:Rht-Blc.

After expression vector establishment completes, through enzyme cut with its exactness of sequence verification after, getting expression vector plasmid electric shock that 1 μ l builds transforms and enters in 20 μ l EHA105 Agrobacterium competent cells, then use 28 ℃ of 250rpm renewal cultivation 3h of 1mlYEB liquid nutrient medium, get 10 μ l bacterium liquid and coat (kantlex 50mg/L in YEB resistant panel, Rifampin Rif50mg/L), in 28 ℃, cultivate 2～3 days.The plasmid that extracts Agrobacterium carries out the PCR detection of target gene, and positive bacteria liquid is stored in-70 ℃ of Ultralow Temperature Freezers standby.By positive bacteria liquid enlarged culturing, then infect the callus of rice varieties Japan fine (high stalk and upright), with hygromycin resistance, screen callus, and further cultivate and be divided into seedling, then transplant land for growing field crops.

Whether the primer of designed specific detection Rht-Blc base: Rht-BlcM in the detection of pRht-Blc:Rht-Blc transfer-gen plant: embodiment 3 (5 '-ATG CCG TCT ACA ACT ACT ACG CTG-3 ' and 5 '-TAG TGC ACG GTGTCC GTG GCG A-3 ') can be used for detecting pRht-Blc:Rht-Blc and proceed to Japanese fine, as proceeded to, there is amplification, as there is no amplified fragments without proceeding to.

The detection of p35s:Rht-Blc transfer-gen plant: the primer is 5 '-ACA GCA CCA GAC GCT CAC C-3 ' and 5 '-TTG GTA TTC AGA GCC ACA AGG-3 '), institute's amplified fragments size is 606bp.If any amplified fragments, illustrate that this plant has proceeding to of target gene, without amplified fragments, think that this gene does not have proceeding to of target gene.Establish the fine contrast of transgenosis Japan that proceeds to pCambia1300-Rht-Blc (genomic dna of the Rht-Blc in pRht-Blc:Rht-Blc being removed to the recombinant vectors obtaining) simultaneously, proceed to the fine contrast of transgenosis Japan of pCambia1300-35s.

Through PCR, detect, the plant that 72 strains turn T0 generation of pRht-Blc:Rht-Blc detected altogether, its average plant height is 8.6 ± 0.8cm, and falling stalk amplitude is 80.9%; T0 that 27 strains turn p35s:Rht-Blc detected for plant, its average plant height is 4.6 ± 0.4cm, and it falls stalk amplitude is 89.8%.The average plant height of 25 strain Japan fine (Nipponbare) is 45 ± 1.2cm.The average plant height that 25 strains turn on the plant in T0 generation of pCambia1300-35s is 43.5 ± 1.1cm; 25 strains turn pCambia1300-Rht-Blc (by the genomic dna of the Rht-Blc in pRht-Blc:Rht-Blc) and remove the recombinant vectors obtain) the average plant height of plant in T0 generation be 44.5 ± 2.1cm.Observe the transfer-gen plant of p35s:Rht-Blc and pRht-Blc:Rht-Blc obviously than empty map Japan fine crawling (c in Fig. 3) simultaneously.Fine and contrast than Japan at the heading stage that simultaneously field investigation it can also be seen that p35s:Rht-Blc and pRht-Blc:Rht-Blc transfer-gen plant heading stage late more than 10 days.Field investigation result has all confirmed that Rht-Blc gene has reduction plant height, postpones the effect that tillering angle is bloomed, increased in heading.RT-PCR analytical results illustrates that these transfer-gen plants are all single copy genes, and has identical expression amount at transcriptional level, the result shows that these T0 are due to Insert Fragment caused (b in Fig. 3) for the phenotype of plant.It is that Actin primer is that RT-PCR analyzes the primer: 5 '-TGT GCC CCG TGC TGTTCT TAT G-3 ' and 5 '-CCC TTG GCC CAG TTG TTA CCC-3 '.Detecting Rht-Blc expression amount the primer is 5 '-ATC TGG GGC GGC TGC TGC TCC TG-3 ' and 5 '-GTG TGC CAC CCC AGC GTC AGG CAG-3 '.

The mensuration of α-amylase content in embodiment 4 thumb Tom Thumb

Because the content of aleurone layer α-amylase directly affects the germination degree of seed, so we measure the content of the α-amylase of Rht-Bla and Rht-Blc seed.Concrete grammar is: after wheat seed peeling, remove half that has embryo, without half of embryo with 1.5%NaCl0 surface sterilization 30 minutes, then with after aqua sterilisa cleaning seed three times, be placed on and contain 0.2% starch, 2% agar powder, 10mM sodium-acetate and 2mM CaCl ₂, on the flat board that pH value is 5.3, cultivate, wherein add 1 μ M GA ₃, not add GA ₃as positive control.When flat board is placed after about 60-72h in 30 ℃ of dark, taking-up is placed in iodine steam and detects alpha-amylase activity.Material therefor is: China spring (Rht-Bla) and thumb short (Rht-Blc), detected result is as shown in a in Fig. 4: do not containing GA ₃flat board in Rht-Bla and Rht-Blc all there is no the release of α-amylase, and containing 1 μ M GA ₃flat board in observe Rht-Bla and have significantly compared with Great White Spot and occur, illustrate that Rht-Bla is at additional GA ₃condition under have the release of more α-amylase, and Rht-Blc almost can't see the appearance of hickie, illustrates that Rht-Blc only discharges micro-α-amylase.The result shows in Rht-Blc seed that α-amylase content will be starkly lower than Rht-Bla, thereby Rht-Blc shows the stronger ability of supporting anti growing out.

The interaction of embodiment 5 yeast two-hybrid experimental verification GIDl and DELLA albumen

According to existing research report, acceptor-GIDl of GA is at GA ₃when existing and DELLA albumen be interactional, thereby cause the degraded of DELLA albumen.In order to study Rht-Blc and the GIDl interaction situation in wheat, we are according to the experimental implementation flow process of clontech company, utilize NdeI and EcoRI restriction enzyme site to be connected to pGADT7 carrier TaGIDl, utilize equally NdeI and EcoRI restriction enzyme site that Rht-Bla and Rht-Blc are connected to pGBKT7 carrier.Then by the cotransformation that carries Rht-Bla and Rht-Blc and TaGIDl in yeast cell, finally on-Leu-Trp-Ade-His defective type substratum, observe the growing state of yeast.Yeast two-hybrid result is as Fig. 4 b: lacking GA ₃substratum on Rht-Bla-TaGIDl and Rht-Blc-TaGIDl all do not observe the growth of bacterial plaque, illustrate and lacking GA ₃situation under Rht-Bla-TaGIDl and Rht-Blc-TaGIDl all do not have to interact; And at additional GA ₃-the defective type substratum of Leu-Trp-Ade-His on Rht-Bla-TaGIDl there is obvious positive colony, illustrate that Rht-Bla-TaGIDl is at GA ₃when existing, be that generation is interactional, and Rht-Blc-TaGIDl can't see any positive colony on the substratum of similarity condition, even illustrate that Rht-Blc-TaGIDl is at GA ₃when existing, can not interact yet, then we adopt filter paper dyeing method validation this result.Above yeast two-hybrid presentation of results Rht-Blc gene is due to the insertion of the 30AA having in DELLA region, caused the interaction of Rht-Blc and TaGIDl to disappear, thereby DELLA albumen is accumulated in a large number, suppressed the synthetic of the interior GA of plant materials, DELLA albumen regulates the genetic expression in downstream, and then causes the generation of multiple phenotype.

Claims (12)

1. a protein, is the protein that the aminoacid sequence shown in sequence 3 forms in sequence table.

2. the encoding gene of protein described in claim 1.

3. gene according to claim 2, is characterized in that: the encoding gene of described protein is the DNA molecular shown in sequence 2 in sequence table.

4. the recombinant vectors that contains gene described in claim 2 or 3.

5. the expression cassette that contains gene described in claim 2 or 3.

6. the recombinant bacterium that contains gene described in claim 2 or 3.

7. the recombinant virus that contains gene described in claim 2 or 3.

8. cultivating a method for transgenic plant, is that gene described in claim 2 or 3 is imported in object plant, obtains having the transgenic plant of following at least one proterties: 1) plant height is lower than described object plant; 2) heading is bloomed and is later than described object plant: 3) tillering angle is greater than described object plant; 4) ear germinating resistance is higher than described object plant;

Described object plant is paddy rice.

9. method according to claim 8, it is characterized in that: described in claim 2 or 3, gene imports in object plant by the recombinant expression vector that contains gene described in claim 2 or 3, in described recombinant expression vector, start the promotor of genetic transcription described in claim 2 or 3 and be 35s promotor or following a) or b) promotor: a) its nucleotide sequence is the DNA molecular shown in sequence 4 in sequence table; B) its nucleotide sequence is the DNA molecular shown in sequence 4 1-4213 positions in sequence table.

10. in auxiliary detection wheat, whether carry the method for gene described in claim 2 or 3, that to take the genomic dna of wheat to be measured be template, with primer, carry out pcr amplification, if obtain the amplified fragments of 489bp, wheat to be measured is not for carrying candidate's wheat of gene described in claim 2 or 3; If obtain the amplified fragments of 2515bp, wheat to be measured is the candidate's wheat that carries gene described in claim 2 or 3; Described primer is that nucleotide sequence is respectively sequence 6 and two single stranded DNAs of 7 in sequence table.

11. 1 kinds of DNA moleculars, its nucleotide sequence is the DNA molecular shown in sequence 5 in sequence table.

12. 1 kinds of DNA moleculars, its nucleotide sequence is the DNA molecular shown in sequence 4 in sequence table.

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