CN1236061C - Promotor for controlling plant root system development and use thereof - Google Patents

Abstract

The present invention discloses a promoter for controlling development genes of a plant root system and the application thereof. The present invention aims to provide the promoter for controlling the development genes of the plant root system. The promoter for controlling the development genes of the plant root system comprises one of the following nucleotide sequences: a DNA sequence in a sequence 1 in a sequence table, and a DNA sequence with more than 80% of homology in a DNA sequence limited in the sequence 1 in the sequence table. An expression vector and a cell line containing the promoter belong to the protective range of the present invention. The discovery of the promoter and the explanation of the function of the promoter have important meaning to breed plants, particularly paddy rice.

Description

The promotor and the application thereof of controlling plant root system development gene

Technical field

The present invention relates to plant promoter and application thereof, particularly relate to the promotor and the application thereof of control rice root development gene.

Background technology

The growth of farm crop all needs to rely on root system with growth and absorb moisture and nutrient inorganic salt from soil.Therefore, the growth of root plays crucial effects for the output of farm crop.The root system of paddy rice can be divided into two classes according to position and the etap that they take place, and a class is that embryo is taken root, and a class is the root of postembryonal development.Embryo is taken root and is sprouted the back growth by radicle, and it has two kinds of forms, a primary root and several seminal roots.The root of postembryonal development also has two types, and a class is the adventive root that produces from the plant stipes, and another kind of is on all types root, the axillary root that all produces.

In the model plant Arabidopis thaliana, it has been found that the gene of a lot of participation growth hormone signal transductions, regulating and control the growth of root.For example be subjected to the member of the AUX/IAA gene of growth hormone rapid induction, comprise SHY2/IAA3, SLR1/IAA14, IAA28, MSG2/IAA19, if undergo mutation all can cause axillary root to reduce or do not have (Reed2001, Trends Plant Sci.6,420-425).Opposite some gene such as AUX1, TIR1 or NAC1 overexpression then can promote formation (Marchant et al., 2002, Plant Cell.14, the 589-597. of axillary root; Gray et al., 1999, Genes Dev.13,1678-1691; Xie et al., 2000, Genes Dev.14,3024-3036).(indolylacetic acid IAA) plays an important role in the root development many aspects plant hormone, and they comprise the elongation that suppresses primary root, promotes adventive root, and the root-tip cells differentiation state is kept in the formation of lateral root and Gen Mao.As an important adjusting hormone, growth hormone is being regulated and control cell fission, the differentiation of cell expansion and cell thereof.This just determines growth hormone to influence development of plants is many-sided, and it is determining the elongation of hypocotyl and stem end, is mediating the geotropism of root and stem, is keeping apical dominance, and promotes fascicular formation (Thimann, 1977; Sachs, 1991; Estelle and Klee, 1994; Hobbie, 1998).

Much being subjected to all has one section common sequences-auxin response element AuRE (Auxin Response Element), 5 '-TGTCTC-3 ' in the promoter sequence of gene of growth hormone regulation and control, this sequence is proved to be auxin Response Factor ARF (auxin response factor) in conjunction with necessary (Guilfoyle et al., 1998, PlantPhysiol.118,341-347; Liu et al., 1994, Plant Cell.6,645-57; Ulmasov etal., 1999, Plant J 19,309-319).Spire original hase, spire and tip of a root meristematic tissue may be growth hormone synthetic positions.Spire synthetic growth hormone, polarity is transported to root downwards; And the growth hormone that the tip of a root produces, its transportation that can in tip of a root cortex, make progress.The transportation of this polarity can cause the gradient of intercellular auxin concentration, and this concentration gradient has very likely determined embryonic cell differentiation form, and the merismatic cytodifferentiation form of the fascicular differentiation and the tip of a root is set up.Nearest research also shows, growth hormone not only can induced growth the expression of positive regulator protein of plain signal transduction, negative regulation albumen that also can the plain signal transduction of induced growth simultaneously, as the AUX/IAA family protein, (del Pozo andEstelle, 1999 and the AUX/IAA family protein can grown plain inductive SCFTIR1 proteolytic degradation complex body be degraded; Gray et al., 1999,2001).Plant is by the feedback inhibition and the regulating and controlling effect of separating the next meticulous adjusting growth hormone of inhibition system of an exquisiteness like this.The signal transduction pathway of growth hormone has higher conservative property in dicotyledonous and monocotyledons.At first found 11 genes in paddy rice, the ARF albumen of encoded protein sequence and Arabidopis thaliana has homology (Sato et al., 2001).And in paddy rice isolating OsIAA1 gene, as the member of a unifacial leaf AUX/IAA gene family, be proved it can grown element and light induce (Thakuret al., 2001).Compare with dicotyledons, the research of monocotyledons root development Regulation Mechanism relatively lags behind.The mutant that the several and root development of only finding in corn is relevant can supply research: rtcs does not produce adventive root fully; Rt1 only forms seldom or does not have crown and a prop root; Asr1, seminal root forms defective; Slr1 and slr2, only form very short axillary root (Hetz et al., 1996, Plant J.10,845-857), but regrettably the corresponding gene of these mutant not the clone come out.And in paddy rice, the gene studies relevant with root development then still less now only confirmed formation (Vergere et al., 1976 of water logging and ethene processing energy inducing adventitious root; Suge, 1985; Bleecker et al., 1986,1987).

Summary of the invention

The promotor that the purpose of this invention is to provide control rice root development gene.

The present inventor adopts the method for reverse genetics, separating clone in the paddy rice with Arabidopis thaliana AtFPF1 (flowering promoting factor 1) gene (Kania et al., 1997) homologous OsRAA1 (Oryzasativa Root Architecture Associated 1) gene, and preliminary study its function in development of plants.Its promoter sequence is based on that existing EST in the database (expression sequence tag) and genome sequence obtain.

The promotor of control rice root development gene provided by the invention is the promotor of rice Os RAA1, is the dna sequence dna shown in the sequence 1 in the sequence table, by 1987 based compositions.

The expression vector, the transgenic cell line that contain promotor of the present invention all belong to protection scope of the present invention.

Illustrating of the discovery of promotor of the present invention and its function particularly has great importance in the rice breeding plant.

Description of drawings

Fig. 1 is the electrophoretogram from oryza sativa genomic dna amplification OsRAA1 promotor.

Fig. 2 is the Plant hormones regulators,gibberellins and the analysis of growth hormone cis regulation and control response element of promoter sequence of the present invention.

Fig. 3 is the collection of illustrative plates that the promotor of rice transformation OsRAA1 gene merges the plant expression vector of gus gene.

Fig. 3 is the collection of illustrative plates that the promotor of rice transformation OsRAA1 gene merges the plant expression vector of gus gene.

Fig. 4 cuts the evaluation bands of a spectrum for the enzyme of expression vector.

Fig. 5 is the GUS active coloring of OsRAA1::GUS gene in transgenic paddy rice.

Fig. 6 is the expression of growth hormone induction OsRAA1.

Embodiment

In the following embodiments, used rice varieties spend in being No. 10 (Oryza sativa L. cv Zhonghua 10) and in spend (Oryza sativa L.cv Zhonghua 11) No. 11.

The separation of embodiment 1, rice Os RAA1 gene promoter sequence

1, promoter primer design

Choose before the ORF 1987bp as target sequence, according to PAC (AP002525) sequences Design 5 ' end primer 5 '-TGCA GGA TCCATA GAT TTG TCA AGA AAA CAT TTC GA-3 ' (the underscore sequence is BamH I site).Consider simultaneously and need link to each other its reporter gene GUS, and on the reporter gene GUS of pCAMBIA1301 initiator codon, a Nco I (CC is arranged with expression of plants binary vector pCAMBIA1301 ATGG) therefore site on the initiator codon of target gene, also is modified to Nco I (CC ATGG) site, 3 ' end primer is: 5 '-CCTG CC A TGG CTT AGA TCT CTC TCA AAC TC-3 '.

2, the promoter sequence of pcr amplification OsRAA1 gene

Ordinary method separating rice genomic dna, primer such as preceding design, response procedures is: 97 ℃ of 5min, 1 cycle adds the Taq enzyme; 94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 3min, 30 cycles; 72 ℃ of 7min, 1 cycle.

As template, obtained the target stripe that needs with the fine genomic dna of Japan, as shown in Figure 1, M3 among the figure: the product that λ DNA digests with Hind III and EcoR I; LP: single forward (forward) primer extension product; DP: two primer extension products; RP: single oppositely (reverse) primer extension product.Show the fragment that a special 2.0kb is arranged in two primer extension products among the figure.The promoter sequence 1987bp that sequencing result obtains is the dna sequence dna of sequence in the sequence table 1.

In resulting promoter sequence, contain a large amount of pyrimidine boxes, at least three GARE, two TATCCA boxes, and auxin response elements T GTCTC, it distributes as shown in Figure 2, and wherein B is 3 components of location Plant hormones regulators,gibberellins response element in promoter region, the pyrimidine box, TAACAAA and TATCCA box, (using the DNAMAN software processes).C is the core sequence TGTCTC (arrow is depicted as the potential response element) of location auxin response element in promoter region, and the core sequence of these response elements is in the position of the about 150bp in transcripting start point upstream.The existence of these conserved sequences may hint that this gene is subjected to the regulation and control of Plant hormones regulators,gibberellins and growth hormone.

Embodiment 2, rice Os RAA1 gene promoter merge the structure of the plant expression vector of gus gene

Will be through the 2.0kb PCR segment of order-checking evaluation, with BamH I and Nco I digestion; Cut the CaM35S promotor before the gus gene in the pCAMBIA1301 plasmid with BamH I and Nco I enzyme simultaneously, connect the good 2.0kbPCR segment of digestion, promptly finished OsRAA1Pro::GUS (pCAMBIA1301) plant expression vector, its gene structure collection of illustrative plates as shown in Figure 3.Fig. 4 is that the enzyme of three expression vectors is cut evaluation, wherein is with 1 to be Marker; With 2 is that the OsRAA1 justice gene plant expression vector of rice transformation carries out the evaluation collection of illustrative plates (having cut out the target stripe of 360bp) that enzyme is cut with Kpn I and Sac I; With 3 is that the OsRAA1 inverted defined gene plant expression vector of rice transformation carries out the evaluation collection of illustrative plates (having cut out the target stripe of 360bp) that enzyme is cut with BamH I and Kpn I; With 4 is that plant expression vector that the promotor of rice transformation OsRAA1 gene merges gus gene carries out enzyme with BamH I and Nco I and cuts evaluation collection of illustrative plates (cutting out the target stripe of 2kb), as can be seen from the figure, and after gus gene has placed the promotor of OsRAA1 gene.

The tissue chemical analysis of embodiment 3, plant expression vector transformed plant identifies

With β-glucuronidase reporter gene (GUS) plant conversion carrier of OsRAA1 promoters driven, the method rice transformation that utilizes Agrobacterium to infect, and detect with the activation analysis of GUS original position histological chemistry.Method is that the resistant calli that will be after transforming through agrobacterium tumefaciens produces and the blade and the root segment of transformed plant are put into GUS staining fluid (100mmol/L NaPO respectively ₄(pH7.0); 0.1%Triton X-100; 10mmol/L EDTA; 0.5mmol/L yellow prussiate of potash; 0.5mmol/L the Tripotassium iron hexacyanide; 1mg/mL X-Gluc) in, the several minutes of bleeding places 37 ℃ to be incubated overnight then, observes blue reaction.Tissue after the dyeing 70% ethanol decolorization.The result as shown in Figure 5, among the figure, A, the X-gluc in primary root branch district dyeing, showing not only at lateral root but also in their primordial tissue all has very strong signal (shown in the arrow); B, the dyeing of the primary root tip of a root, showing in the mitogenetic and elongation zone of the tip of a root all has the center pillar of GUS active signal, particularly root that very strong signal is arranged; C, the ripe Xiao Hua before being pollinated shows that flower glume also has the GUS activity, particularly vascular bundle is stronger; D, there are some signals in the dyeing of seedling in some part of spire, but instability and no regularity may be owing to change the leakage expression of gus gene and cause; E, the floral organ after the dyeing, after the stamen maturation, also there is stable very strong signal (shown in the arrow) connection portion of style and filigree.

From the expression pattern of OsRAA1 as can be seen OsRAA1 always be present in the cell of quick growth, as, lateral-root primordia, lateral root and adventive root center pillar.For the cell division and the elongation fast of these tissues, growth hormone plays an important role.

The NAA of embodiment 4, OsRAA1Pro::GUS transgenic paddy rice handles and the active histochemical stain of GUS

OsRAA1Pro::GUS transgenic paddy rice T1 in the greenhouse growth after two months, carefully extracts from soil for seed, and flush away soil was cultivated 3 days in 1/2MS inorganic salt nutrient solution, then paddy rice was transferred in the 1/2MS inorganic salt nutrient solution that contains 2.5 μ MNAA.The adventive root (about 1mm of diameter and 0.5mm) of choosing two kinds of new lives is as the dyeing object.At the difference clip tip of a root of different time period (0,1,2,4 hours), carry out the GUS histochemical stain.The result as shown in Figure 6, A wherein, the RNA Northern hybridization of paddy rice young root shows that transcribing of OsRAA1 is subjected to inducing of growth hormone (to handle wild-type seedling 0,1/4,1/2,1,2,4 respectively with 10nM IAA, 8hours), contrast the painted rRNA into EB.B, the root of OsRAA1::GUS transgenic paddy rice NAA (2.5 μ M) processing different time (0,1,2, GUS coloration result 4hours).C-E, the wild-type paddy rice is in 14 days phenotype of 1/2MS substratum (contain 1 μ M NAA or do not have NAA) growth, and C shows the growth of the processing strongly inhibited plant root of 1 μ M NAA, and the right two strains are that NAA handles plant, and the left side is the plant of being untreated; D, the situation of untreated plant foundation portion; E, the situation of the plant foundation portion that handles with 1 μ M NAA, arrow is depicted as the primary root of spiral.From figure, can draw, induce one hour the tip of a root that the strongest signal is arranged at NAA.

Sequence table

<160>1

<210>1

<211>1987

<212>DNA

＜213〉Oryza paddy rice (Oryza sativa L.)

<400>1

tggccattaa?caaataaaca?aaaataaaca?tatccataga?tttgtcaaga?aaacatttcg 60

atcatataca?ccatgatata?tattgtaggt?gctagttatc?caacaatatt?atgattttaa 120

gcattgcgat?aattttaaaa?agctggtgat?tatgtttatt?taacttagaa?cttgtggtat 180

atttatgtgt?gatttgtcac?aacacggaac?cactgtactg?aggattttaa?aacagtacta 240

tgttcggccg?gtaccttttg?cacaaacaag?cagtggcgcc?cgaaagcata?tatatgatgg 300

atgtttaggt?taagtcattg?gaacctttct?attgattaat?cggttgattg?aggagcaaga 360

ttaacgccaa?cttttagcat?atatatatcg?gccagacaaa?tttgaaaagt?aaacttttaa 420

ctctgaaaaa?catttcttaa?ttatggggcg?ttcatttttc?tgccgtacaa?cacaaccaaa 480

acaggcctcg?attaaacaaa?tgtgtgccaa?attttaggga?cagacgtgca?ccaatattat 540

cacaaaggga?acgaacacaa?taaccaaatt?aatgcgccat?tgcgtgcgca?ctatgggaat 600

aattttgtta?catatatata?taaatagatg?tggtattcta?agataaacca?aaatcttttt 660

aaattttctg?cagaagatgc?tagcttgagc?ttgtcaatca?atatagctag?agctataaaa 720

attaaagaaa?aaaattatgt?cctgccgtac?acaaaatata?tacttgacaa?tgagctagga 780

gaatcggttt?tgttcctgat?aaaaggtgat?cttagctctg?tcgatcggcc?agaattcact 840

ttcactagaa?aaaaaaaatt?gttcgaaaag?agatgggcat?gcacgtacag?ctgaaattct 900

cttgtgtgat?ggctaagtaa?agtagtaaac?tatgggaagt?gaatcatatt?cccttctttg 960

ataaatagac?cccttttgtt?ttcgagaaaa?gaaagtaggt?aggattcctc?gcacagcatc 1020

acaagttatt?ttcgttacca?tatgaaaatt?cttatctatc?tatatatcaa?tccattgtga 1080

aatcaattag?ctttatggga?tatataacac?taatttatca?ttgtggtcca?agtaagatgc 1140

atggatatta?ggatttaacc?gattaatcat?gcattttata?tacacaaccc?atatggtttt 1200

tatctctctt?tttctacgcg?tcatggtcac?acgataacat?gtgttgataa?tcgagaaccc 1260

ttgatgtcat?gcatgcatgt?atatatttca?atcgactttg?gtttgtccca?aaaattagag 1320

aataaatact?agtttatttt?tcttcttttt?agagattgta?cgatcactag?ctagctaggt 1380

atagctagct?gacggatttt?tccaattagg?tatatatcca?tgcacacaca?ccgatctttt 1440

cattcattct?tttctttctt?ttcacagata?tattccaggt?agcttatcaa?gcacataatg 1500

ttggaaaatt?aaacaaggaa?tccttaacta?acaaatgaaa?cacggggagg?gaggaagaga 1560

gggggcagag?ctagagagct?agctagctat?tgctacagca?catcttcgat?atcatgacct 1620

aacctaacta?cgaccccacg?tatactcgat?actaaactac?tatagacgaa?cgaacgatcg 1680

ttcgttcgtt?tcgcatatat?ctataaatcc?tttatgatat?cgatatctct?atttcttagt 1740

atatctaata?gcatagtagt?aggttagtat?atctgctata?gctagctagc?agtacatgca 1800

tgcatgcaca?catgtacata?ctaccatatc?tctgtcttgt?ctccctttca?catgtcacct 1860

ctgacccaca?aacaaaccct?ttccaagctc?ctaacactag?ctttaccttc?ttcttcctcc 1920

tcctctgtat?atatacaccc?cctcacctcc?ctatcttgca?cccatccatc?cacccatagc 1980

tatctct 1987

Claims (4)

1, the promotor of control rice root development gene is the dna sequence dna shown in the sequence 1 in the sequence table.

2, the expression vector that contains the described promotor of claim 1.

3, the transgenic cell line that contains the described promotor of claim 1.

4, the application of the described promotor of claim 1 in rice breeding.

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