CN1298021A - One-purpose expression promoter in rice endosperm tissue and its application - Google Patents

Abstract

The present invention relates to one separated DNA sequence, which encodes the rice waxy gene 5' controlling sequence to direct the one-purpose expression of rice endosperm tissue and includes the nucleotide sequence expressed by SEQ ID No.1. The present invention provides the expression carrier including the sequence and the method of utilizing the expression carrier in directing the one-purpose expression of exogenous gene in endosperm or blocking the expression of natural rice waxy gene.

Description

Single-minded expression promoter and application thereof in the paddy endosperm tissue

The present invention relates to the genetically engineered field, relate to a kind of plant gene regulating and controlling sequence particularly.More particularly, the present invention relates to 5 ' regulating and controlling sequence of single-minded expression in the paddy endosperm tissue and the application in genetically engineered thereof.

Paddy rice is one of topmost food crop, and the population more than 1/3rd all is staple food with rice in the world.Along with expanding economy, in the global range more and more higher requirement has been proposed for yield of brown rice and quality.Thereby, utilize the whole bag of tricks to improve rice yield, improvement rice quality, extremely important meaning is arranged.Modern biotechnology is applied to can solve some and use the insoluble problem of ordinary method in the rice varieties improvement.Since the nineties, the rice biological technology has obtained very big development, (the Song WY etc. of separation in succession of some control Main Agronomic Characters genes, nineteen ninety-five, Science, 270:1804-1806) and constantly perfect (the Hiei Y of paddy rice transgenic technology, Otha etc., 1994, Plant J., 6:271-282; Liu Qiaoquan etc. 1998, plant physiology journal, 24 (3): 259-271) have laid a good foundation for improveing rice varieties with genetic engineering technique.

The big area of high-yield rice kind is promoted and is brought increasing substantially of China's rice yield.But these high-yield rice kinds existing common problem at present are the food flavor inferior qualitys, far can not adapt to the requirement of the raising of living standards of urban and rural residents level to rice quality.Rice quality is poor, and price is relative also lower, and volume increase can not increase income, thereby dampens peasant's kind grain enthusiasm.Cause that the inferior major cause of these high-yield variety food flavors is that the amylose content of the grain of rice is higher.Starch in the rice paddy seed endosperm can be divided into two kinds of amylose starch and amylopectin, and the amylose content in the different rice varieties rice endosperm is different, and rice variety is generally 20%～30%, and japonica rice variety is 15%～22%, and glutinous rice is 0～2%.Amylose content is higher to tend to then make that the meal qualitative change is hard, mouthfeel is poor.

Khush equals 1984 at Genetics, reports among the 107:141-167, and the paddy rice waxy gene (waxy gene) on No. 3 karyomit(e) of paddy rice is being controlled the synthetic of amylose starch in the endosperm.This genes encoding is incorporated into amylosynthease (the Granule-bound starch synthase on the starch small grain, GBSS) [EC.2.4.1.11], thereby synthetic (Okagaki and the Wessler of amylose starch in control paddy pollen, endosperm and the blastular, 1988, Genetics, 120:1137-1143), be to influence the key gene that starch is formed in the paddy endosperm.Because this gene only efficiently expresses in pollen, endosperm and the blastular of paddy rice, so it is space-time specificity expression's a gene.

At present, 35S promoter commonly used makes up engineering plasmid in the plant genetic engineering, and this promotor can make foreign gene obtain higher expression in dicotyledons.Yet 35S promoter drives the indifferent of genetic expression in monocotyledons, and the expression that it drives does not have tangible tissue and organ specificity.

Therefore, still wish from the paddy rice waxy gene, to isolate can be in paddy endosperm and pollen specificity expression promoter and expression regulation element, thereby can be in monocotyledons, particularly comprise in the food crop of paddy rice and instruct exogenous gene expression.

An object of the present invention is to provide one section separated DNA sequence that instructs the paddy rice waxy gene 5 ' regulating and controlling sequence of single-minded expression in the paddy endosperm tissue.

Another object of the present invention provides the expression vector that contains paddy rice waxy gene 5 ' regulating and controlling sequence of the present invention.

Another object of the present invention is to utilize paddy rice waxy gene 5 ' regulating and controlling sequence specificity expression alien gene in paddy endosperm.

A further object of the invention is to utilize paddy rice waxy gene 5 ' regulating and controlling sequence to suppress the expression of paddy rice waxy gene.

On the one hand, the invention provides a kind of separated DNA sequence, it is one section paddy rice waxy gene 5 ' regulating and controlling sequence that instructs the paddy endosperm tissue specificity to express, and this sequence comprises the nucleotide sequence shown in the SEQ ID NO:1.In a preferable example, this paddy rice waxy gene 5 ' regulating and controlling sequence comprises the nucleotide sequence shown in the SEQ ID NO:2.

On the other hand, the invention provides an expression vector, this expression vector comprises paddy rice waxy gene 5 ' regulating and controlling sequence and heterologous gene encoding sequence that links to each other with this control region operability or natural paddy rice gene antisense sequence.In a preferable example, the described natural paddy rice gene antisense sequence that links to each other with paddy rice waxy gene 5 ' regulating and controlling sequence operability is a paddy rice waxy gene antisense sequences.

The present invention also provides the method for a kind of production rice plant of efficiently expressing exogenous gene in paddy endosperm, this method is with expression vector rice transformation of the present invention, the foreign gene encoding sequence that this expression vector contains paddy rice waxy gene 5 ' regulating and controlling sequence of the present invention and links to each other with this 5 ' regulating and controlling sequence operability.In a preferable example, described foreign gene is selected from the rice protein gene.

The present invention also provides a kind of method of producing the rice plant of paddy rice natural gene expression by inhibitation system, this method is with expression vector rice transformation of the present invention, the natural paddy rice gene antisense sequence that this expression vector contains paddy rice waxy gene 5 ' regulating and controlling sequence of the present invention and links to each other with this 5 ' regulating and controlling sequence operability.In a preferable example, the antisense encoding sequence that described natural paddy rice gene antisense sequence is the paddy rice waxy gene.

Term used herein " 5 ' regulating and controlling sequence " refers to the sequence in the control texture genetic expression of gene translation initiator codon (ATG) 5 ' upstream.In a preferred embodiments of the present invention, paddy rice waxy gene 5 ' regulating and controlling sequence comprises the Nucleotide-2100 of Fig. 1 to+1300 (SEQ ID NO:2).The modification that keeping of being done of paddy rice waxy gene 5 ' regulating and controlling sequence shown in the SEQ ID NO:2 instructed paddy endosperm specificity expression characteristic also within the scope of the present invention.These modifications comprise insertion, the disappearance of one or more Nucleotide and substitute.

Term used herein " isolating " nucleic acid is meant that this dna sequence dna has been arranged in the sequence of its both sides and has separated under native state.The isolating nucleic acid of coding paddy rice waxy gene 5 ' regulating and controlling sequence available following several modes usually obtains:

(1) from the genome dna library that contains sequence, isolates dna sequence dna;

(2) synthetic with chemical process or enzyme method; With

(3) with polymerase chain reaction synthetic DNA sequence.

Term used herein " operability links to each other " is meant that some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.

Paddy rice waxy gene 5 ' regulating and controlling sequence and modification thereof or deletion fragment instruct endosperm specificity expression's activity to identify like this: make the encoding sequence of this regulating and controlling sequence and heterologous gene transcribe fusion, this fusion gene is transferred in the suitable host, and detected expression of heterologous genes.For example, use reporter gene (being typically E.C. 2.3.1.28 and beta-Glucuronidase (GUS)) to estimate transcribing and translation ability of chimeric construct thing usually.Reporter available standards in the transgenic organism is tested delicately and is detected.The beta-Glucuronidase gene can be used as the promoter activity reporter in the transgenic plant, because this enzyme is stable at the vegetable cell camber, do not have intrinsic beta-Glucuronidase activity in the higher plant, this enzyme can be measured with quantitative fluorescence test and Histochemical localization technology.People such as Jerfferson (1987 EMBO J 6:3901) have set up the active biological chemistry of GUS and histological chemistry's standard program in the detection plant tissue.

The technology that makes up expression vector of the present invention is well known to those of ordinary skill in the art, and can find in the reference such as people such as Sambrook (1989, " molecular cloning experiment guide ").Can adopt various strategies to connect dna fragmentation, the character of dna fragmentation end is depended in the selection of strategy.

Those of ordinary skills know that in order to make allogeneic gene expression, construction needs promoter element and makes the signal of the effective polyadenylation of transcript.Therefore, the paddy rice waxy gene 5 ' regulating and controlling sequence that contains total promoter sequence (TATA box) can directly be connected with no promotor allogeneic coding sequence.

In order to make heterologous gene or natural gene specificity expression in paddy endosperm, available fusion gene construction of the present invention transforms plant.The method of transgenosis is to know in this area.Can transform-reproducer (as described in people such as Horsch (1985) Science 227:1229) by leaf disk imports fusion gene in the plant.Also can adopt other method for transformation, as PEG method, particle bombardment, electrization (people such as Horsch, 1984, Scienc223:496, people such as Barton, 1983, Cell 32:1033, people such as Liu, Acta Phytophysiol Sin, 21:195-205), this also within the scope of the present invention.(Potrykus 1990, Bio/Technol, 8:535-542 but these methods have complicated operation, the multiple copied of foreign gene insertion and the shortcomings such as sterile plant of imperfection and higher proportion; Finnegan and McElroy, 1994, Bio/Technol.12:883-888).In a preferable example, according to people (1994) Plant J such as for example Hiei, the method for describing among the 6271-6282 utilizes agrobacterium tumefaciens to transform plant.When method for transformation needed, fusion gene of the present invention can insert in the plant conversion carrier, for example in the T-DNA district of binary vector pCAMBIAI300 (professor Jefferson provides) in.

Paddy rice waxy gene 5 ' regulating and controlling sequence of the present invention is to make foreign gene tissue specificity, regulating and controlling sequence of efficiently expressing in paddy endosperm and pollen.It has following potential using value in plant genetic engineering:

(1) nutrition of vitamin A in the increase rice: modern medicine study shows that the A that is deficient in vitamin is the important factor that influences the healthy growth of infant.Precursor one carotene of vitamin A is a kind of pigment that all exists in the green plants photosynthetical system, and its biosynthetic pathway is studied in great detail.Can not synthesize carotene in the rice paddy seed and may be because gene some or several enzymes is not opened or similar to the situation in some corn in the route of synthesis, be because certain gene that plays regulating and controlling effect loses activity.Therefore can pass through gene engineering method, utilize the waxy gene promotor to introduce deactivated gene, overcome this natural obstacle, just can cultivate the new rice variety that is rich in carotene in the rice.

(2) improve Protein content in the rice: paddy rice is the staple food crop of China, though the contained proteinic quality of rice is preferably in cereal crop, but the content of Methionin and Threonine is still on the low side in the indispensable amino acid, and the protein content that compares in the rice with wheat, corn is also lower.In order to improve the protein content in the rice, utilize the waxy gene promotor to introduce some high-quality storage protein genes, to improve the content of high-quality protein in the rice with gene engineering method.

(3) cultivate the hybrid rice new lines: the paddy rice waxy gene also is the gene of expressing in the pollen granule tissue, constitute chimeric plasmid with waxy gene initiating sequence and some virulent gene, just can cultivate the rice varieties of may command fertility by gene engineering method, set up new hybridisation rice strain.

(4) improve the eating quality of rice: running into a problem in the rice breeding work at present is, some high-yield new variety because their eating quality and boiling processing characteristics are undesirable, thereby is not subjected to people's welcome.The content that studies show that amylose starch is the principal element that influences the rice eating quality.Therefore, utilize the antisense clone technology to reduce the expression of waxy gene in these high-yield varieties by paddy rice waxy gene initiating sequence of the present invention, just can be issued to the purpose that reduces amylose content, thereby improve the eating quality of rice in the situation that does not influence other cultivation feature.

(5) increase paddy rice setting percentage and circularity: with the waxy gene initiating sequence key gene of certain plants hormone in synthetic expressed in rice paddy seed, regulate the grouting of rice paddy seed, this will have far-reaching influence to the raising yield of brown rice.

In the accompanying drawings, Fig. 1 is long-grained nonglutinous rice 232 (O.sativaindica a 232) waxy gene-2100 to the 3.4kb dna sequence dna of+1300bp.

Fig. 2 is the physical map of waxy gene in the long-grained nonglutinous rice 232, wherein: TSS, transcription initiation site; AATAAA adds poly A site; ATG, translation initiation codon; TGA, translation stop codon; B, the BamH I; Bg, the Bgl II; E, the EcoR I; H, the Hinf I; P, the Pst I; R, the Rsa I; S, the Sai I; X, the Xho I.

Fig. 3 is the agarose gel electrophoresis analysis of paddy rice waxy gene 5 ' upstream regulatory region deletion clone after restriction enzyme EcoR I enzyme is cut.

Fig. 4 is the structural representation and the relative enzyme activity of their GUS of a series of waxy-gus fusion genes.

Fig. 5 is the structure of binary expression vector p13W4T-DNA.Wherein: Xh, Xho I; E, the EcoR I; The Sa-Sac I, H, Hind III; B, Bam H I; LB, left margin; RB, right margin; TSS, transcription initiation site; ATG, translation initiation site; Wx-pro, 5 ' upstream of the 3.1kb of paddy rice waxy gene; Wx-frag, antisense waxy gene fragment; HYGr, hygromycin gene; Kmr, kalamycin resistance gene.

Fig. 6 is the Molecular Identification of transgenic rice plant.A is the pcr analysis result; B is the Southern engram analysis, and all DNA digest with Eco R I; C is the Southern engram analysis, and all DNA digest with the Hind III.Swimming lane 1 contains the p13W4 plasmid DNA; Swimming lane 2 contains the DNA of unconverted plant; Swimming lane 3 to 8 contains the DNA of 6 strain transgenic plant respectively.Dna molecular amount mark (kb) is presented at the left side.

Below in conjunction with accompanying drawing embodiments of the invention are described.

The molecular cloning of embodiment 1 paddy rice waxy gene

Long-grained nonglutinous rice (Oryza sativa subsp.indica) 232 (Wx ⁺) (Yunnan Province's local variety, press people Cereal Chemistry such as Williams, 47 (4): 410-415,1983 described methods record and contain amylose starch 21%) seed germination after, three weeks of growth are collected leaf texture's liquid nitrogen flash freezer under 25 ℃ of artificial lighting conditions, grind to form fine powder in liquid nitrogen, press people's (" The EMBO J " 3 (6): 1021-1028,1984) such as Schwarz-Sommer method extracting rice total dna then.The method of pressing people such as Maniatis (molecular cloning, 1989) reclaims the 15-20kb dna fragmentation with the total DNA of Mbo I part enzymolysis with the 10-40% sucrose density gradient ultra-centrifugation.

The method extracting of pressing people such as Maniatis (molecular cloning, 1989) goes out the DNA of phage EMBL3.With reference to people's such as Frishauf (J.Mol.Biol., 170:827-842,1983) method EcoR I and BamH I double enzymolysis carrier DNA, isopropanol precipitating is removed the DNA small segment between EcoR I and the BamH I.

Above-mentioned dna fragmentation is mixed (1: 1) with the λ EMBL3 carrier DNA of double enzymolysis, and DNA concentration is not less than 200 mcg/ml.Adding magnesium chloride to 10 mmole/liter, 42 ℃ are incubated 60 minutes, slowly are cooled to 12 ℃ again.Add damping fluid and T4DNA ligase enzyme (BRL company), 12 ℃ of connections are spent the night.The DNA phenol that connects: chloroform (1: 1) extracting once, the chloroform extracting once, the ether extracting is once.Ethanol sedimentation DNA, it is standby to be dissolved in a small amount of TE damping fluid (10 mmoles/rise Tris, 1 mmole/rise EDTA) at last.Get 3 microlitres and carry out external packing by the external packing box specification sheets of Gigpack company, the lysogen Q359 mensuration with the P2 phage contains 4 * 10 in the external packing mixt ⁵Individual recombinant phage.Calculating the F value according to the calculation formula (the P value selects 99%) of Clarke (Cell 9:91-93,1976) gene library fraction of coverage is 15/ (3 * 10 ⁵), when promptly the cloned sequence mean length is 15kb, as long as have 1 * 10 ⁵Individual recombinant phage can cover whole rice genome.

Contain pWx plasmid DNA people such as (, Cell, 25:225-230,1983) Dr.N.Fedoroff of corn Wx gene with EcoR I enzymolysis, from low melting point sepharose, reclaim the 10.6kb dna fragmentation that has the Wx gene.With the nick translation reaction box of BRL company to specifications slightly modified come mark to become the probe of radioactive activity is arranged.Each reaction volume is 30 microlitres, include dna probe 0.1-0.2 microgram, isotropic substance α- ³²P-dATP 30 μ Ci, 3 microlitre reaction box reagent (contain dCTP, dGTP, each 0.2 mmole of dTTP/liter), 2.5 microlitre enzymes (Pol I and DNase), 15 ℃ of insulations 60 minutes.Add 3 microlitres, 0.5 mol EDTA termination reaction, collect first radioactivity elution peak by Sephadex G-50 post.

Press the plaque in-situ hybridization method of people such as Maniatis (molecular cloning, 1989), plaque is transferred on the nitrocellulose filter, carry out molecular hybridization and radioautograph, find out positive colony.Each positive colony is all through three single plaque purifications.In order to confirm that gained is the Wx gene of paddy rice, one of them positive colony λ Wx25 is carried out Southern analyze, the result shows that itself and corn Wx gene coding region hybridize.To have BamH I/Sal I fragment cloning of corn Wx dna homolog 0.4kb in proper order among the λ Wx25 on phage M13mp18 and M13mp19, press Sanger, Proc.Acad.Sci.USA, the terminal cessation method of two deoxidations described in the 74:5466-5468 (1977), with the dna sequence analysis instrument of isotopic labeling and ABI company measure its sequence and with the respective regions of corn gene relatively, the result shows that both have the homology of height.

Embodiment 2: the clone and the order-checking of a series of deletion fragments of paddy rice waxy gene 5 ' upstream regulatory region

To increase the paddy rice waxy gene translation initiation codon upstream 3.4kb fragment subclone that obtains in the pUC18 of BamH I and the digestion of Hind III by PCR method, obtain plasmid pWx3.4.Digest this plasmid DNA with kpn I and BamH I, use phenol: chloroform (1: 1) and each extracting of equal-volume chloroform are once, in the supernatant dna solution, add 0.1 volume NaAc3 mol and 2 volume dehydrated alcohols, placed 30 minutes for-70 ℃, centrifugal 15 minutes of 10000 * g, the precipitation drain with 70% washing with alcohol final vacuum, with DNA be dissolved in 1 * Exo III damping fluid (the Tris-HCl66 mmole/liter, pH8.0, MgCl ₂0.66 mmole/liter) in, ultimate density is 0.1 microgram/microlitre, 37 ℃ of insulations are after 5 minutes, add exonuclease Exo III (reaction density is 5 units/microlitre), respectively in reaction 0.25,0.5,1,2,4,8,16, after 32 and 48 minutes, take out 10 microlitre reaction solutions and be added to the 25 microlitre dehydrated alcohols that are placed in advance on the ice bath, 0.5 microlitre EDTA 0.2 mol, termination reaction and deposit D NA in the mixed solution of 1 microlitre NaAc3 mol, behind the centrifugal drying DNA is dissolved in 50 microlitres * S1 nuclease damping fluid (the NaAc16 mmole/liter, pH4.6, the NaCl400 mmole/liter, ZnSO ₄1.6 mmole/liter, 8% glycerine) in, add s1 nuclease (reaction density 0.2 unit/microlitre) room temperature reaction 30 minutes, and added 1 microlitre S1 nuclease stop buffer (Tris alkali 0.3 mol, EDTA50 mmole/liter), place in 70 ℃ and made the s1 nuclease inactivation in 10 minutes, DNA is behind ethanol sedimentation, and vacuum is drained, and is dissolved in TE damping fluid (Tris-HCl10 mmole/rise pH8.0, the EDTA1 mmole/liter) in, ultimate density is 0.1 microgram/microlitre.Get 0.2 micrograms of DNA sample after the T4DNA polysaccharase is repaired into tack, connect cyclisation through the T4DNA ligase enzyme again, transformed into escherichia coli DH5 α competent cell, identify the molecular weight of plasmid DNA in the transformant, select and lacked 0.3,0.6,0.7,0.8,0.95,1.3,1.5,1.6,1.8,2.1 and the clone (Fig. 3) of 2.4kb respectively.These deletion clones are called after pWx3.1, pWx2.8, pWx2.7, pWx2.6, pWx2.45, pWx2.1, pWx1.9, pWx1.8, pWx1.6, pWx1.3 and pWx1.0 respectively.The clone pWx1.4 of another one disappearance excises to mend after the 2.0kb fragment to put down from connecting between BamH I and the Sal I from the pWx3.4 plasmid DNA to obtain.

In order to determine the exact position of dna sequence dna disappearance in some pWx plasmids, these plasmid DNA are inserted the corresponding site of carrier M13mp19 after Sac I and the digestion of EcoR I, connect back transfection Escherichia coli MV1190 competent cell, enzyme cuts digestion and electrophoresis evaluation back is extracted single stranded DNA out from transformant, carry out dna sequence analysis (with AB I company 373 type dna sequencing instrument) with terminal fluorescent marker method, the result shows that pWx2.1 lacks-the 861bp place, pWx1.9 lacks-the 640bp place, pWx1.4 lacks-the 119bp place, and pWx1.3 lacks-the 27bp place.DNA linear measure result is very identical in sequencing analysis and the gel electrophoresis.

Embodiment 3: the structure of paddy rice waxy gene 5 ' upstream regulatory region and gus gene coding region fusion plasmid

For ease of making up fusion plasmid, with restriction enzyme EcoR I and Hind III from plasmid pB I 101.1DNA (Jefferson 1987 Plant Molecular Biology Report, cut out the fragment that contains gus gene coding region and NOS 3 ' end region 5:387), insert in the corresponding site of pUC18 carrier, constitute plasmid pGN.Digest this plasmid with the EcoR I, the sticky end of otch adds Hind III joint after klenow fragment benefit is flat, use the T4 ligase enzyme from concatemerization again, transforms the host bacterium, obtains plasmid pGNH.Plasmid pGNH can obtain containing the dna fragmentation of gus gene coding region and NOS end region after Hind III enzyme is cut, it is inserted in the Hind III site of embodiment 2 described a series of pWx deletion clones, cut evaluation through enzyme, choose a series of pWG plasmids (Fig. 4) that forward connect.

Moment expression and the quantitative assay of embodiment 4:waxy-gus fusion gene in rice protoplast

Collect about 2 grams of rice suspension cell of logarithmic phase growth, add 20 milliliters of protoplastis enzymolysis solutions [CPW solution (Zhang and Wu, 1988, Theor Appl Genet, add 1% cellulase RS 78:835), 0.1% polygalacturonase Y-23], 26 ℃ of lucifuge enzymolysis 2 hours filter enzymolysis solution, filtrate centrifugal 5 minutes through 100 * g with 300 eye mesh screens, supernatant discarded, protoplastis be suspended in 10 milliliters of MaMg solution (N.F,USP MANNITOL 0.4 mol, magnesium chloride 15 mmoles/liter, MES0.1%pH5.6), centrifugal 5 minutes of 80 * g, protoplastis is resuspended in the MaMg solution and is adjusted to 1 * 10 ⁶Protoplastis/milliliter, get 0.3 milliliter of protoplastis suspension, add 10 microgram pWG plasmid DNA, 10 microgram pMON 772i plasmid DNA [contain the CaMV 35S promoter, corn Adh first intron and luciferase (LUC) gene, provide by professor N.Olszewski], 0.3 milliliter of MaMg solution that contains 30%PEG, slowly mixing.26 ℃ of following lucifuges were placed 30 minutes, slowly added the CPW solution of 15 times of volumes, the centrifugal supernatant that goes of 80 * g, the protoplastis precipitation is suspended in 5 milliliters of KPR nutrient solutions (Zhang and Wu,, Theor Appl Genet in 1988,78:835), 26 ℃ of lucifuges were cultivated 48 hours.

Protoplastis culture after the conversion, through 100 * g centrifugal 2 minutes, supernatant discarded added 300 microlitre GUS/LUC extraction buffer (KPO ₄100 mmoles/rise pH7.8, DTT 1 mmole/liter, PMSF 0.8 mmole/liter) the suspension protoplastis, (handled 30 seconds at interval 3 seconds with the broken protoplastis of ultrasonic wave (Soniprep 150 types, MES company), repeat 3 times), centrifugal 5 minutes of 5000 * g collects supernatant liquor, i.e. zymoprotein crude extract.For negative contrast, is internal reference with pMON 772i with pGN, and quantitative assay GUS enzyme activity and luciferase vigor, the GUS after the correction compare vigor with GUS/LUC ratio pmol MU h ^-1/ U (10s) ^-1Expression, the result is presented among Fig. 4.

The mensuration of GUS enzyme activity is as follows: get 100 microlitre zymoprotein crude extracts, add 4-methyl umbellulone β-D-glucuronide (4-MUG) to 1 mmole/liter, adding methyl alcohol to concentration is 20%, 37 ℃ were reacted 1 hour, at fluorophotometer HITACHI 650-10LC (excitation wavelength 455nm, absorbing wavelength 365nm) in, the fluorescence intensity of product 4-methyl umbellulone (MU) in the assaying reaction liquid, and converse the productive rate (Jefferson of MU according to the typical curve of MU, 1987, Plant Molecular Biology Report 5:387), calculates the relative reactivity of GUS.

The mensuration of LUC enzyme activity is as follows: get 100 microlitre zymoprotein crude extracts, add 100 microlitre reaction buffers (Tris-HCl 20 mmoles/liter, pH7.8, magnesium chloride 15 mmoles/liter), adding ATP to 5 mmole in the reaction solution/liter, put into TG-300 type luminometer, add 100 microgram fluoresceins, measure luminous intensity, calculate the LUC enzyme activity.

Fig. 4 result shows, lack 824bp to transcription initiation site upstream-1294bp place since 5 ' end, the relative vigor of GUS enzyme still maintains 85.6%, and this shows in the protoplastis system, in waxy gene 5 ' upstream-2100 to-the 1294bp section is little to the genetic expression influence.Further disappearance 433bp is to-861bp place, and the relative vigor of GUS enzyme drops to 62.9%, shows that existence influences the order of genetic expression-1294 to-861bp zone.When continuing disappearance 221bp to-640bp place, significantly 1 times of the decline of the relative vigor of GUS enzyme drops to 31.6% from 62.9%.When continuing to lack transcriptional start point upstream-119bp place, owing to still kept the minimal promoter district of TATA box and upstream thereof, GUS is relative, and enzyme activity still remains on about 25.4%.When lacking to transcription initiation site upstream-27bp place, when the TATA box was destroyed, the relative enzyme activity of GUS dropped to and bears control level consistent (the relative vigor 11.5% of detected GUS has been represented the background level of system in the negative contrast).

Embodiment 5: with antisense waxy gene transformation paddy rice

In the Bam H I site in the reverse back insertion of dna fragmentation (from the 2333rd bit base to the of waxy gene in the rice variety 232 3087 bit bases) the pWG3.1 fusion gene that one section 0.75kb in the waxy gene coding region is long between waxy gene 5 ' upstream and the gus gene coding region, be built into plasmid pW4.This antisense fusion gene cloning in the middle T-DNA district of binary vector pCAMBIA I 300 (professor Jefferson provides), is formed binary expression vector p13W4 (Fig. 5).

This test adopted 2 japonica rice variety: 95-2 and in spend 11, both are the kind of large scale application.With 95-22 is example, and it is planted in phytotron or land for growing field crops, and the water intaking rice blooms back about 15 days immature embryo at N ₆D ₂Substratum [N ₆(Chu, 1978, Proc Symp Plant Tissue Culture, 43-50), casamino acids 500 mg/litre, sucrose 30 grams per liters, 2, the 4-D2 mg/litre, phytagel 2.5 grams per liters, pH8.5] go up in advance and cultivated 4 to 7 days, cultivate (Hiei etc. altogether with Agrobacterium EHAl05/p13W4 again, 1994, Nucl AcidRes, 16:9877).Selecting substratum N ₆D ₂S ₁(N ₆D ₂, Totomycin 50 mg/litre) and go up to select cultivated for two generations (2 week/generation).As seen the result has produced eugonic resistant calli from more rataria.By table 1 as seen, rataria through cultivate altogether and screening and culturing after, have on 38% the rataria to produce well-grown resistant calli.The transformation efficiency of different tests is different, but can both be stabilized in the higher transformation frequency about 30%.Change thus obtained resistant calli over to division culture medium MSRCH (MS (Murashige and Skoog, 1962, Physiol Plant, 15:473-479), sucrose 30 grams per liters, the 6-BA2 mg/litre, NAA 0.2 mg/litre, zeatin 0.2 mg/litre, phytagel 2.5 grams per liters, Totomycin 50 mg/litre pH5.8), have renewable resistance seedling on the resistant calli in 26.3% rataria source at last.Obtained the regeneration of strain more than 100 seedling altogether.Owing to belong to the different somatic clones of conversion (resistant calli in same rataria source also might belong to the different somatic clones of conversion) each other from the resistant calli in different ratarias source, therefore, as shown in Table 1,15 independent transformation plants (regeneration) have been obtained at least in different transformants.The regeneration seedling is transplanted behind strong plantlets and rootage and is buried, and transgenic rice plant is grown normally under field conditions (factors), and most plant can be normally solid.We are 13 independent (RW1～RW13) analyze of strain system that transform to obtaining among the rice varieties 95-22.

The Agrobacterium tumefaciens mediated transformation efficiency that antisense waxy-GUS fusion gene is imported japonica rice variety of table 1

Kind	Experiment	The immature embryos number			Transformation efficiency
							Gong Pei Yang (A)	Survival also forms hygromycin resistance callus (B) *	Survival and regenerated seedling (C)	B/A(％)	C/A(％)
95-22	12 altogether	23 34 57	7 16 23	6 9 15	30.4 47.1 38.8	26.1 26.5 26.3

Note *: in the selection substratum that contains 50 mg/litre Totomycin, select after 1 month.

In order to identify whether foreign gene has been integrated into rice genome, we respectively select an individual plant to extract the total DNA of blade totally 6 independent transformants from RW1, RW2, RW5, RW7, RW8 and the RW11 that derives from rice varieties 95-22, carry out PCR and Southern hybridization analysis.

In pcr analysis, with primer P1 (5 '-TGGCAAGAACAAGCATAGACC-3 ', be positioned at antisense waxy gene coding region fragment), primer P2 (5 '-TAACATACGGCGTACATCG-3 ', be positioned at gus gene sign indicating number district partially), from total DNA of 6 resistant plants, amplify the dna fragmentation about 626bp specifically, identical with positive control (is template with plasmid p13W4DNA); And in the total DNA of unconverted adjoining tree, do not amplify purpose fragment (Fig. 6 A).Cut total DNA with Eco R I enzyme, with [ ³²P] the gus gene coding region DNA of dATP mark is that probe carries out molecular hybridization, the result all has hybridization band (Fig. 6 B) in 6 total DNA of resistant plant that detected.Because no Eco R I restriction enzyme site between the gus gene coding region in plasmid p13W4 T-DNA district and left margin, so hybridize the integration copy number that how much can represent foreign gene of band.Therefore, by Fig. 6 B as can be known, having among 5 total DNA of transgenic rice plant is single copy insertion of this fusion gene, and has three copies among the RW2.In addition, whether complete for detecting the antisense waxy-GUS fusion gene that is inserted, cut with Hind III enzyme again and (two Hind III point of contacts are arranged in plasmid p13W4T-DNA district, can cut out dna fragmentation long about 2.8kb, comprising waxy gene antisense fragment and gus gene coding region fragment and NOS gene terminator fragment) total DNA carries out hybridization analysis, the result shows the hybridization stripe size of all transfer-gen plants identical with positive control (Fig. 6 C), illustrates that antisense waxy gene fragment and gus gene coding region are complete the connection in the transgenic paddy rice genome.

After transgenic rice plant moved into artificial chamber, at Plnat Mol.Biol.Rep., the method described in the 5:387-405 was got tissue such as blade and is detected the GUS activity with histochemical staining method in vegetative period with reference to people such as Jefferson 1987.Found that at 13 independent transformants of the RW1～RW13 that is detected in the totally 143 strain individual plants, have in the blade of 102 strains and the cane tissue to detect the GUS activity, and observe blue reaction in the cane of 41 strains and the blade.Further the endosperm of immature seed is carried out histochemical method and detect, find to detect GUS activity (blueness) in the endosperm of most transformed plants, have only in the endosperm of a few individual plant (RW6-1) not detect distinctive blueness.Detected simultaneously the separation of gus gene between the endosperm of same plant, table 2 is the active detection case of GUS in the part transgenic rice plant.

With reference to Juliano, 1968, Cereal Sci.Today, the method among the 16:334-360 is used I ₂The content of amylose starch in the-K I colorimetric method for determining paddy rice mature seed endosperm, recording this institute is about 17% with the amylose content of receptor parent 95-22.For detail knowledge antisense waxy gene imports behind the paddy rice changeing the influence of amylose content, our emphasis transforms the T1 that is tied on the individual plant to 23 of RW1-3 etc. and measures for the amylose content in the seed, and the results are shown in Table 2.In 23 different individual plants being analyzed, the changing conditions of amylose content is different, and the existing decline in various degree of 11 amylose contents in the individual plant part mature seed is arranged.The comparative analysis of the amylose content of different seeds from the same individual plant shows that antisense waxy gene in generation separation has taken place at T1.

To T ₁For tying seed (T on the plant ₂Generation) with GUS histochemical method screening conversion of pure zygote, and the seed of tying on the homozygous plants is pressed individual plant measure amylose content, the result shows that amylose content and T1 generation conforms to by (table 2), but illustrate in the antisense waxy trans-genetic hybrid rice plant after the amylose content change that genetic stability is to T2 generation.

Table 2 japonica rice 9522/p13W4 transgenic paddy rice is T partly ₀Plant and T ₁, T ₂The character analysis of seed Nt: do not detect

All compositions that this paper discloses and discloses and method can need not too much to test and make and implement by reference this paper disclosure.Although the compositions and methods of the invention are described by preferred embodiments, those skilled in the art obviously can change composition as herein described, method and method steps and order of steps in not breaking away from content of the present invention, spirit and scope.More specifically, some relevant reagent replaces reagent as herein described to obtain same or analogous result on obvious available chemistry and the physiology.Replacement that all these are similar and change will become apparent to those skilled in the art that they all are regarded as comprising in spirit of the present invention, scope and the content of accessory claim definition.

Sequence table (1) general information:

(ⅰ) applicant:

(A) name: Shanghai Inst. of Plant Physiology, Chinese Academy of Sciences

(B) street: No. 300,Fenglin Road, Shanghai City

(C) city: Shanghai City

(F) postcode (postcode): 200032

(ⅱ) denomination of invention: single-minded expression promoter and application thereof in the paddy endosperm tissue

(ⅲ) sequence number: the information of 2 (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 121 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:1:GTCGACACCG GCCGGGGACC GCGTAAAATG TGTTGCGGGA GGGAGAGGGG GAGAGAGAGA 60TCGCGCGGGC TTCACGCAAC GGCGCTACAA ATAGCCACCC ACACCACCAC CCCCTCTCTC 120A 121 (2) SEQ ID NO:2:

(ⅰ) sequence signature:

(A) length: 3400 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

( ⅹⅰ ) ：SEQ ID NO：2：TGCCTCCTTG GCCGAGGCCT TGGTGGCCAA GGTCGAGATC ATCTCGGGGG GCACCGCGCT 60ACAAATGGCG TCAAGCGCCA TCCTATCCTC ATGAAGCACG ACGCCGCCTG GATCGATGGC 120GCCCCACAGG CACCGGGCTT GCAGTTTGAT CTTCATCAGC AAAGACCAAT CGTTGTAGTT 180AGTTCTTGTC AACAGCGGGT AGTTTGCCGC ACCTCCTGCT TCCTTGACCA CGCGGTGGAT 240CACCAGCCCA CGGTCGCTCC CGCCGCTGCC CGAGGCGCGG CCGCCGGAAC CTTGAAACAT 300GCACGGACGG TGACGCCGAC CGCCGGCGCC GGGGAAGTTC CGTGCGTCCT CGAGGCATGA 360AGACACCCTC AAGAGCGCGA TCTCGCAGCT ACTACTAGAT ACATCGTGTA AGGTTTATTT 420CCAACACGGC CGGCCGTGGC TTGTGGTCCT ACGCCTTACG CGCCCACCAT TTTTTATTAT 480TTATTTGTGA AACTGACATG TGGGTCCCAC GAGGTTTATT ATTTTTCAGA TCGAATTGCC 540ACATCAGTGC CACGTCAGAT CGAGACCAAG TCAAATTAGC CACGTAGGCG CACACGTCAG 600CCAAAACCGC CGTCAAAACT GCCGAGGGAT CTAATCTGCA CCGGTTTTAA TAGTTGTAGG 660GACCCGTTGT ATATGGTTTT GCGGTTGAAG GACGGTAAAT TGGATTCGTT GACAAGTTAA 720GGGACCTTAG ATGAACTTAT TCCTTTTATA TTTGCACAGG CCTAATTTCA AGTCCAGCCC 780AGCTTTCTTC AGCCTGTTTG ATAATTCTCT CTAGCTTATT ACAGCCGTGG GAGAGGAGAT 840ATACAGCTAC AAGATTACAA GTCGATGTAT ACAGCAAACC CATGAGCTGA TTGCCTGATT 900AGACGCGGTA AGAATGCATC CCTGAGAAGC AAATGCATCA CCAAATTTGT AGCTTAGATA 960AATGCTGTGA CCTGCAAGAA AACAAAATTA AAATCAAAAG AAAAGAAAAG CGCAGGTAAT 1020TGACACCCCA CGCATACAAG TGTAGATGCA TAACACGTTC ATCTAATCAT CTTAATTAGA 1080CTTAGGTAAA ACTACAATGA GGTTTATGTC CTACGGAATG ACGATAAGCT AGCAGCACAG 1140AGGCACAGAT CATATCGTCT CCAGACTCAA GTGCACGTTG ATCATTCGCT CACTGCTTCA 1200TCGATCATCC CTTTGTCGAG GCGTTAGTTG GCAGGCACTA ATAGCTACAG TAAAGTAAAG 1260AGCAACGTGC CAACGTACGC ACGCTAACGT GAGTCATGTA GCGTAATTCC AAGTTCTTTT 1320TTTTTTGTCA GCACGTACAA GCAGCCGCTA GCCTCGCCCT GCATGAGAAG CTCGCGGCGC 1380GCCACCAAAC TGGCAGGCAC TCAGCTCGCT GCTGGTCCCG CACGTCGCCA CACGATCGAC 1440GTACGCACGC GAGCGAGATC CACCGATGGT TTACGCGTAC GCGACGCTCA CACATCCCCC 1500GGTGCCCAAC AGAAACCACA CACCACCCGC ACGAAAAAAA CCGAACCGCA CGTGCGCGCG 1560CGCTCCACGC ACACCCCAAA CAGACGGCAC GGCGGGAGCG CGCGCGCGCA CGCGAGCCGA 1620GGAGAAAACA AACGGGGGAA ACAAGCTGGA AAAGCAAAAG GGGAAAAGAA CGGAGCGGAG 1680GCTTCACCCA CGGCCACCGC GACGGCCACC AGCGTGCGGT GCAATGCAAC GTACGCCAAG 1740CCGAAACGGC AGGCAGCATC GCGCACGCAC GCACACACAG GCCACAGCAC ACGCGAGCGA 1800CGTACGCGAG TGCATGCAGA TGCATGCGCG GGGCTCGCGC GAGACCGGCC GATGGGTTCG 1860CTTCTCTTCT CTCTCCCGTC CCGTTGCGTC GTCATGGACA AAAGTCGGTT TTGCTTTTGG 1920TTTTTTGGTT CTGAGACTGA CGTGCGGGCC AGCGTACGCC TGCGTGCCCC GCATGTCATC 1980GTCGACACCG GCCGGGGACC GCGTAAAATG TGTTGCGGGA GGGAGAGGGG GAGAGAGAGA 2040TCGCGCGGGC TTCACGCAAC GGCGCTACAA ATAGCCACCC ACACCACCAC CCCCTCTCTC 2100ACCATTCCTT CAGTTCTTTG TCTATCTCAA GACACAAATA ACTGCAGTCT CTCTCTCTCT 2160CTCTCTCTCT GCTTCACTTC TCTGCTTGTG TTGTTCTGTT GTTCATCAGG AAGAACATCT 2220GCAAGGTATA CATATATGTT TATAATTCTT TGTTTCCCCT CTTATTCAGA TCGATCACAT 2280GCATCTTTCA TTGCTCGTTT TTCCTTACAA ATAGTCTCAT ACATGCTAAT TTCTGTAAGG 2340TGTTGGGCTG GAAATTAATT AATTAATTAA TTAATTGACT TGCCAAGATC CATATATATG 2400TCCTGATATT AAATCTTCGT TCGTTATGTT TGGTTAGGCT GATCGATGTT ATTCTAGAGT 2460CTAGAGAAAC ATACCCAGGG GTTTTCCAGC TAGCTCCACA AGATGGTGGG CTAGCTGACC 2520TAGATTTAAG TCTCACTCTT TCTAATTATT TGATATTAGA TCATTTTCTA ATATTTGCGT 2580CTTTTTTTAT TCTAGAGTCT AGATCTTGTG TTCAACTCTC GTTAAATCAT GTCTCTCGCC 2640ACTGGAGAAA CAGATCAGGA GGGTTTATTT TGGGTATAGG TCAAAGCTAA GATTGAAATT 2700CACAAATAGT AAAATCAGAA TCCAACCAAT TTTAGTAGCC GAGTTGGTCA AAGGAAAATG 2760TATATAGCTA GATTTATTGT TTTGGCAAAA AAAAATCTGA ATATGCAAAA TACTTGTATA 2820TCTTTGTATT AAGAAGATGA AAATAAGTAG CAGAAAATTA AAAAATGGAT TATATTTCCT 2880GGGCTAAAAG AATTGTTGAT TTGGCACAAT TAAATTCAGT GTCAAGGCTT TGTGCAAGAA 2940TTCAGTGTGA AGGAATAGAT TCTCTTCAAA ACAATTTAAT CATTCATCTG ATCTGCTCAA 3000AGCTCTGTGC ATCTCCGGGT GCAACGGCCA GGATATTTAT TGTGCAGTAA AAAAATGTCA 3060TATCCCCTAG CCACCCAAGA AACTGCTCCT TAAGTCCTTA TAAGCACATA TGGCATTGTA 3120ATATATATGT TTGAGTTTTA GCGACAATTT TTTTAAAAAC TTTTGGTCCT TTTTATGAAC 3180GTTTTAAGTT TCACTGTCTT TTTTTTTCGA ATTTTAAATG TAGCTTCAAA TTCTAATCCC 3240CAATCCAAAT TGTAATAAAC TTCAATTCTC CTAATTAACA TCTTAATTCA TTTATTTGAA 3300AACCAGTTCA AATTCTTTTA GGCTCACCAA ACCTTAAACA ATTCAATTCA GTGCAGAGAT 3340CTTCCACAGC AACAGCTAGA CAACCACCAT GTCGGCTCTC 3400

Claims (8)

1. a separated DNA sequence is characterized in that, it is meant the waxy gene 5 ' regulating and controlling sequence of water guide rice waxy gene the endosperm tissue specificity expression, and this sequence comprises the nucleotide sequence shown in the SEQ ID NO:1.

2. dna sequence dna according to claim 1 is characterized in that, it comprises the nucleotide sequence shown in the SEQ ID NO:2.

3. an expression vector is characterized in that, this expression vector comprises the described dna sequence dna of claim 1 and heterologous gene encoding sequence that links to each other with this series of operations or natural paddy rice gene antisense sequence.

4. expression vector according to claim 3 is characterized in that, described natural paddy rice gene antisense sequence is a paddy rice waxy gene antisense sequences.

5. the method for production rice plant of efficiently expressing exogenous gene in paddy endosperm, it is characterized in that, with the described expression vector rice transformation of claim 3, the foreign gene encoding sequence that wherein said expression vector contains the described dna sequence dna of claim 1 and links to each other with this series of operations.

6. method according to claim 5 is characterized in that, described foreign gene is the rice protein gene.

7. method of producing the rice plant of paddy rice natural gene expression by inhibitation system, it is characterized in that, with the described expression vector rice transformation of claim 3, the natural paddy rice gene antisense sequence that wherein said expression vector contains the described dna sequence dna of claim 1 and links to each other with this nucleotide sequence operability.

8. method according to claim 7 is characterized in that, the antisense encoding sequence that described natural paddy rice gene antisense sequence is the paddy rice waxy gene.

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