CN103255141A - Paddy rice nitrogen deficiency specific induced expression promoter Y5 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically relates to a paddy rice nitrogen deficiency specific induced expression promoter Y5 (also known as Y5A) and an application thereof. With a chip technology, promoter Y5 (Y5A) downstream gene nitrogen supply induced expression information is obtained. With different paddy rice varieties, Y5A downstream gene nitrogen supply induced expression mode is verified. With a PCR method, the promoter Y5A full length and a 5'-terminal truncated fragment thereof are amplified, and are connected to a promoter vector DX2181b, such that a promoter fusion GUS expression vector is obtained. The constructed vector is used in genetic transformation upon a paddy rice variety Zhonghua11. The regulation of the promoter upon GUS gene is verified in a transformation positive plant. Through Realtime expression level verification and GUS protein activity detection, it is further determined that the promoter is a paddy rice nitrogen deficiency specific induced expression promoter. A functional section is at ATG upstream 546bp.

Description

A kind of promotor Y5 and application of paddy rice nitrogen stress specificity abduction delivering

Technical field

The invention belongs to the plant gene engineering technology field.Be specifically related to a kind of promotor Y5 and application of paddy rice nitrogen stress specificity abduction delivering.

Background technology

Transgenic research is mainly with the genetic expression of constitutive promoter driving purposes at present, and most typical is CaMV35S promotor (Hirt et al., 1990 of separating in the cauliflower mosaic virus; Battraw et al., 1990), and the promotor of some plant origins in recent years, as the ubiquitin gene ubiquitin promotor (Schledzewski et al., 1994) of cloning in the promotor of the actin gene Actinl that from paddy rice, clones and the corn.But the constitutive expression of foreign gene tends to cause the inessential waste of resource, and the accumulation of simultaneously a large amount of heterologous proteins also can be broken the original metabolic balance of plant, hinders normal growth (Nie Lina etc., 2008 of plant; Rai et al., 2009).Kasuga etc. (1999) find under study for action, when using strong composition type expression promoter CaMV35S to start the expression of DREB1A, may play to a certain degree inhibition to the growth of plant, and can alleviate this situation with the promotor of induction type.The promotor of induction type is " open and close " of inducible transcription gene fast and effeciently: can accept inducement signal as required under specific etap of plant, histoorgan or growing environment, inducible gene expression, also can remove at any time and coerce, stop to express (Li Jie etc., 2006).Therefore, obtain the promotor of paddy rice abduction delivering under the nitrogen hunger state, not only can reduce the negative impact that the foreign gene great expression is brought, more can provide regulation and control safely and effectively for genetically engineered improves paddy rice.

In the required various macroelement of plant-growth, nitrogen is limiting plant growth and the primary factor that forms plant biomass.Nitrogen is not only the basis of genetic material, especially the important component part (Lu Jingling, 1994) of protein, nucleic acid, chlorophyll, enzyme, VITAMIN, alkaloid and plant hormone etc.Since nearly half a century, countries in the world are all enriching nitrogenous fertilizer as the important agricultural measures that increases rice yield, particularly after the Green Revolution first time, along with the anti-fertile variety popularization of high yield is used, farmland amount of application of nitrogen fertilizer rapid growth has greatly improved the output (Zhong Daibin etc., 2001) of paddy rice.The excessive of farmland nitrogenous fertilizer used, and the consequent is that nitrogen utilization efficiency reduces and a series of environmental problem.A large amount of losses of nitrogen directly cause the eutrophication (Wang Guanghuo etc., 2003) of groundwater pollution and rivers and lakes.

Along with molecular biology and genetically engineered the developing rapidly of plant field, plant is also more and more clear to the molecular mechanism of nitrogen absorption and transport.A large amount of ammonium salt transhipments is sub, nitrate transport is sub is found, and GS/GOGAT (glu famine synthetic enzyme/NADPH-linked glutamate synthase) circulation assimilation ammonium mechanism is revealed, is the research material that provides a large amount of that absorbs of genetically engineered improvement paddy rice nitrogen.Though verified not oligogenic absorption and transport function by mutant or absorption experiment etc., the overexpression of these genes in the paddy rice body do not reach the purpose that improves the absorption and transport ammonium.Mohammad etc. (2006) transport sub-OsAMT1-1 with the ammonium salt in the corn ubiquitin promotor overexpression paddy rice, improved total ammonium content in the receptivity of ammonium in the paddy rice and the root, but produced the murder by poisoning of ammonium simultaneously, caused the upperground part biomass to reduce in a large number.Drive if change the nitrogen stress evoked promoter into, just might suitably control the absorption of ammonium according to the needs of plant, avoid poisoning.GS participates in NH4 ⁺With the main enzyme that is combined to glutamine (Gln), Cai etc. (2009) spend overexpression paddy rice glutamine synthetase gene GS1 in 11 with 35S promoter in rice varieties; 1, GS1; 2 and intestinal bacteria glutamine synthetase gene glnA, GS activity, total nitrogen content, aminoacids content, soluble proteins etc. all are significantly improved, but individual plant biological yield and grain yield have all significantly descended.Drive if change into for the nitrogen evoked promoter, not only can reduce " waste " that constitutive expression causes, more can guarantee only under the situation that supplies the nitrogen abundance, to promote the assimilation of GS, avoid individual plant to cause biological yield to reduce because nitrogenous source is not enough.

The present invention has excavated the promotor of a nitrogen stress inducible gene expression by chip, verify in three different varietiess with real-time quantitative realtime PCR, determined further that by the paddy rice stable conversion this promotor is a promotor that nitrogen stress is induced simultaneously, for genetically engineered improvement paddy rice provides new material to absorbing of nitrogen.

Summary of the invention

The objective of the invention is to overcome existing technological deficiency, a kind of application of nitrogen stress inducible gene expression promotor in paddy rice is provided.The applicant is with this promotor called after promotor Y5.The purposes of Y5 nitrogen stress inducible gene expression in paddy rice is as follows: chip results shows, at the warm and fine precious Shan 97 of rice varieties Japan when nitrogen stress is coerced 3～7d, the downstream gene of promotor Y5 control is about 10 times and 70 times of abduction deliverings in root respectively, abduction delivering not then during portion organizes on the ground.In the 3rd kind, spend in 11, find that by realtime PCR checking the downstream gene of promotor Y5 control is induced more than 20 times when nitrogen stress 3～7d.Therefrom spend amplification promotor Y5 full length sequence fusion gus gene stable conversion paddy rice in 11 (claiming ZH11 again) material, the transgenic positive material is carried out nitrogen stress coerce processing, find that nitrogen stress coerces the remarkable abduction delivering of back gus gene expression amount in root, and the gus protein activity is compared and is obviously risen before inducing.Further verified the characteristic of promotor Y5 nitrogen stress abduction delivering.

The present invention is achieved in that

The present invention is with the fine (English name: Nipponbare of paddy rice Japan, a public rice varieties material, and finished the rice varieties of genome sequencing) and precious Shan 97 (rice varieties that China is public, from Jiangxi Province Academy of Agricultural Sciences) as base mateiral, water planting seedling to the 5 leaf phase, branch different time points nitrogen stress is coerced and is handled sampling (nitrogen stress 1h, nitrogen stress 1d, nitrogen stress 3d, nitrogen stress 7d, recover behind the nitrogen stress 7d for nitrogen 2h, recover for nitrogen 1d), to continue seedling that normal nutritive medium (seeing embodiment) cultivates in contrast, utilize full genome cDNA chip express spectra as technique means (Yang Rong etc., 1999), obtained nitrogen stress gene of high power abduction delivering all in the root of two kinds, having obtained gene upstream sequence according to the information of these genes at NCBI website (www.ncbi.nlm.nih.gov) is its promoter sequence, simultaneously, the applicant by same disposal methods spend 11 (to claim ZH11 again in the 3rd rice varieties, the public kind that comes from Institute of Crop Science, Chinese Academy of Agricultural Science), and by realtime PCR verified the expression pattern that this gene nitrogen stress is induced.Promoter sequence is merged gus reporter gene to be building up to promoter vector DX2181b and (by the crop genetic improvement National Key Laboratory at applicant place commercial carrier pCAMBIA1381 to be transformed GUS closure and restriction enzyme site, see Fig. 2) on, carry out agriculture bacillus mediated genetic transformation again, spend 11 in the rice transformation kind, obtain positive transformed plant.Detect the gus gene expression amount of conversion positive plant and find that the GUS expression amount that nitrogen stress is handled behind the 3d significantly rises, its active obvious enhancing of nitrogen stress processing back is found in the gus protein detection.

The invention has the advantages that:

(1) the invention provides a kind of application of nitrogen stress inducible gene expression promotor in paddy rice.In three different rice varieties, verified the expression pattern that the expression amount nitrogen stress high power of this promotor downstream gene is induced.Promotor merges in the gus gene stable conversion paddy rice positive plant, all significantly rises after the active nitrogen stress of GUS expression amount and GUS is handled.

(2) the present invention has found that first promotor Y5 is the promotor of a nitrogen stress high power inducible gene expression, and the function section is in the upstream 546bp of its regulatory gene initiator codon ATG, for assimilation how effectively to control nitrogen in the genetically engineered improvement paddy rice provides new material.

(3) promotor of using among the present invention and downstream gene thereof can provide support for the Nutrition and Metabolism research of cereal crop such as paddy rice and other crop.

Description of drawings

Sequence table SEQ ID NO:1 is the promotor Y5A nucleotide sequence of (be promotor Y5 of the present invention, A represents this promotor complete sequence), sequence total length 1305bp.

Sequence table SEQ ID NO:2 is the nucleotide sequence of promotor section Y5B of one of them brachymemma of promotor Y5A, and the sequence total length is 1062bp.

Sequence table SEQ ID NO:3 is the nucleotide sequence of promotor section Y5C of one of them brachymemma of promotor Y5A, and the sequence total length is 784bp.

Sequence table SEQ ID NO:4 is the nucleotide sequence of promotor section Y5D of one of them brachymemma of promotor Y5A, and the sequence total length is 546bp.

Sequence table SEQ ID NO:5 is the encoding sequence of the downstream gene of Y5 regulation and control, sequence total length 477bp, 158 amino acid of encoding.

Sequence table SEQ ID NO:6 is the sequence of encoded protein matter of the downstream gene of Y5 regulation and control.

Fig. 1. be general technical route map of the present invention.

Fig. 2. the structural representation of carrier DX2181b.Fig. 2 a is carrier pCAMBIA1381 carrier structure figure before DX2181b transforms; Fig. 2 b is the structural representation of carrier DX2181b; Fig. 2 c is the multiple clone site structure of carrier DX2181b.

Fig. 3. promotor merges the structural representation of GUS expression vector stable conversion paddy rice fragment.

Fig. 4. the expression pattern of promotor Y5 downstream gene in chip material.Fig. 4 a is chip data, is presented at this gene nitrogen stress induced strong in the chip data of Japan's fine (nip) and two kinds of precious Shan 97 (ZS); Fig. 4 b is realtime checking expression amount result with chip with batch precious Shan 97 materials extracting RNA again, shows the nitrogen stress induced strong equally.

Fig. 5. during verifying with realtime, spends promotor Y5 downstream gene the expression pattern in 11.Fig. 5 a is over-ground part result (shoot); Fig. 5 b is root knot fruit (root).

Fig. 6. after promotor Y5 merged GUS stable conversion paddy rice, the gus gene expression amount was with the variation before and after the nitrogen stress in the different positive individual plant of each brachymemma pattern.Figure 61,62,63,64 is followed successively by the result of promotor total length Y5A and promotor section Y5B, Y5C, Y5D, the different transgenic positive strain system of different digital numbering representative of back.

Fig. 7. after promotor Y5 merged GUS stable conversion paddy rice, the gus protein activity was with the variation before and after the nitrogen stress in the different positive individual plant of each brachymemma pattern.Figure 71,72,73,74 is followed successively by the result of promotor total length Y5A and promotor section Y5B, Y5C, Y5D, the different transgenic positive strain system of different digital numbering representative of back.

Embodiment

Following examples further define the present invention, and have described pattern, genetic transformation and expression level and the measuring method of GUS activity and the pattern of the regulating and expressing of this promotor in paddy rice of promotor Y5 and the expression of brachymemma promotor section regulatory gene thereof.Implement example according to following description and these, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification to the present invention, so that its suitable various uses and condition.

Below in conjunction with accompanying drawing the present invention is further described in detail:

The acquisition of the definite and sequence of the regulation and control model of embodiment 1 promotor Y5

For excavating the promotor of nitrogen stress inducible gene expression in the paddy rice, for genetically engineered improvement paddy rice Nitrogen Absorption provides new promotor material, we have designed the technological line figure in the accompanying drawing 1, and have selected for use two kinds of public conventional rice kinds as experiment material: the warm and fine precious Shan 97 of Japan.The application chip technology is selected the new gene of nitrogen being coerced significant reaction, and this technology can be used for a large amount of genes of detection by quantitative in different time expression level (Yang Rong etc., 1999).

The seed of the warm and fine precious Shan 97 of Japan was soaked seed 3 days for 37 ℃, vernalization 2 days, husky training was emerged in one week, transplanted seedlings in paddy rice pancebrin water planting (mill water culture nutrient solution composition: 1.44mM NH ₄NO ₃, 0.3mM NaH ₂PO4,0.5mM K ₂SO ₄, 1.0mM CaCl ₂, 1.6mM MgSO ₄, 0.17mM NaSiO ₃, 50 μ M Fe-EDTA, 0.06 μ M (NH ₄) ₆Mo ₇O ₂₄, 15 μ M H ₃BO ₃, 8 μ M MnCl ₂, 0.12 μ M CuSO ₄, 0.12 μ M ZnSO ₄, 29 μ M FeCl ₃, 40.5 μ M Citric acid, pH value 5.5 is specifically referring to Yoshida et al., 1976), being cultured to for 5 leaf phases, the nutritive medium that part paddy rice seedling is moved to nitrogen stress (does not add NH in the above-mentioned nutritive medium ₄NO ₃Get final product) in carry out nitrogen and coerce processing, with the seedling that continues to cultivate with pancebrin as contrast.According to the experiment needs, treatment time point design is as follows: recover for nitrogen 2h behind nitrogen stress 1h, nitrogen stress 1d, nitrogen stress 3d, nitrogen stress 7d, the nitrogen stress 7d, recover for nitrogen 1d.Top and root dividually during sampling are wrapped with masking foil respectively, place liquid nitrogen to preserve.Extracting sample RNA and reverse transcription become cDNA, and (concrete steps are referring to the SSIII reverse transcription test kit specification sheets of invitrogen company, article No.: Cat.No.18080-093), the sample of each time point is delivered to Beijing Boao Biological Co., Ltd make full genome cDNA chip (Yang Rong etc., 1999), chip data to feedback is analyzed, from chip results, find, downstream gene nitrogen stress 3～7d back root part in these two rice varieties by promotor Y5 regulation and control has the high power abduction delivering, overground part reactionless (Fig. 4 a, Fig. 4 b).In order to be sure of whether this promotor has same regulation and control model in different varieties, the applicant with a collection of material of coercing of having spent 11 kinds in another rice varieties, verifies that this promotor downstream gene is to the reaction of nitrogen again.Realtime checking result (method of Realtime PCR referring to the working instructions of TAKARA commercial reagents box, article No. code:DRR041A): in spend that nitrogen stress 3～7d root also has 30 times induce (Fig. 5 a, Fig. 5 b) in 11.The regulation and control model that promotor Y5 is described conforms to chip results really.The applicant with chip probe sequence input RGAP website (//rice.plantbiology.msu.edu/) carry out Blast to obtain by the complete genome sequence of regulatory gene and accession number LOC_Os12g36840.According to gene order information, checked in gene upstream sequence at NCBI (www.ncbi.nlm.nih.gov), the applicant as promotor total length and called after Y5A, passes through PCR method with this Gene A TG upstream 1305bp, be template to spend total DNA of 11 in the rice varieties, amplification has obtained this fragment.For further verifying and dwindling this promotor to the scope of nitrogen response regulatory element, the applicant is punctured into three sections with this promotor full length sequence by 5` end brachymemma pattern again simultaneously, difference called after: Y5B (ATG upstream 1062bp), Y5C (ATG upstream 784bp), Y5D (ATG upstream 546bp), obtain the fragment of brachymemma with the method for PCR, again fragment is connected on the carrier DX2181b of promotor fusion gus reporter gene, carry out agriculture bacillus mediated genetic transformation again, spend 11 in the rice transformation kind, obtain positive transformed plant.Detect the gus gene expression amount of conversion positive plant and find that the GUS expression amount that nitrogen stress is handled behind 3～7d significantly rises, its active obvious enhancing of nitrogen stress processing back is found in the gus protein detection.

Embodiment 2: the structure of promoter expression conversion carrier

Known total length promoter sequence (seeing that sequence table SEQ ID NO:1 is described) and truncated sequence Y5B (seeing that sequence table SEQ IDNO:2 is described) thereof according to gene Y5, Y5C (seeing that sequence table SEQ ID NO:3 is described), Y5D (seeing that sequence table SEQ ID NO:4 is described) designs primer (primer sequence sees Table 1), be template to spend total DNA of 11 in the rice varieties, amplification obtains Y2B (1062bp), Y2C (784bp) and the Y5D (546bp) of promotor Y5A full length sequence (1305bp) and brachymemma respectively.When increasing these four fragments, all added identical restriction endonuclease sites HindIII (shown in table 1-1) at the 5` of four pairs of primers end, therefore the fragment that obtains of amplification can be carried out enzyme with restriction endonuclease HindIII and cut, be connected to then promoter vector DX2181b go up (carrier DX2181b be the researchist of applicant place crop genetic National Key Laboratory at commercial carrier pCAMBIA1381, transform on the basis of a plasmid of openly reporting from Australia and using and to obtain.This carrier includes the screening-gene of Totomycin: hygromycin (R), the structure of carrier DX2181b is described referring to Fig. 2), and then with the primer of table shown in the 1-2 clone who obtains is checked order, determine that gene is connected on the DX2181b carrier by correct direction and (see shown in Figure 2).

Utilize classical agriculture bacillus mediated method for transformation (Agrobacterium EHA105, from Australian CAMBIA laboratory, the concrete operation method that transforms is referring to Elizabeth et.al., 1993), in rice varieties, spend the expression that starts gus gene in 11 with Y5A and brachymemma promotor thereof.Investigate the transgenic positive plant, find can cause the GUS expression amount significantly to rise behind the nitrogen stress 3d, gus protein is active obviously to be increased.

The PCR primer of table 1-1 the present invention design is right

The sequencing primer of table 1-2 the present invention design

Embodiment 3:Y5 promotor total length and brachymemma promoter fragment thereof merge the rice conversion experiment behind the GUS

After Y5 promotor total length and brachymemma promoter fragment thereof be connected respectively to carrier DX2181b and go up, adopts agriculture bacillus mediated transgenic method, the paddy rice positive plant that obtains transforming, specifically step of converting is as described below:

The correct clone's that obtains plasmid (DX2181b-Y5A, DX2181b-Y5B, DX2181b-Y5C and DX2181b-Y5D) the rice genetic transformation system by Agrobacterium (EHA105 is provided by Australian CAMBIA laboratory) mediation imported to spend in 11 in the rice varieties, through the callus of cultivating in advance, infecting, cultivating altogether, screening having hygromycin resistance, break up, take root, practice transplantation of seedlings, obtain transformed plant.Agriculture bacillus mediated paddy rice (japonica rice subspecies) genetic conversion system mainly adopts the method for the basic enterprising one-step optimizations of people (1994) report such as Hiei.

The method of the key step of genetic transformation of the present invention, substratum and preparation thereof is as described below:

(1) reagent and solution abbreviation

The abbreviation of the used plant hormone of substratum is expressed as follows among the present invention: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (Casein Enzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (N6 macroelement composition solution); N6mix (N6 trace element composition solution); MSmax (MS macroelement composition solution); MSmix (MS trace element composition solution)

(2) solution formula

1) N6 substratum macroelement mother liquor (according to 10 times of concentrated solutions (10X) preparation):

Mentioned reagent is dissolved one by one, be settled to 1000 milliliters with distilled water then.

2) N6 substratum trace element mother liquor (is prepared according to 100 times of concentrated solutions (100X)

Mentioned reagent is settled to 1000 milliliters 2025 degrees centigrade of following dissolvings and with distilled water.

3) molysite (Fe ₂EDTA) stock solution (according to the preparation of 100X concentrated solution)

With 3.73 gram b diammonium disodium edta (Na ₂EDTA2H ₂O) and 2.78 the gram FeSO ₄7H ₂O dissolves respectively, mixes and is settled to 1000 milliliters with distilled water, bathes 2 hours to 70 ℃ of temperature, and 4 ℃ of preservations are standby.

4) VITAMIN stock solution (according to the preparation of 100X concentrated solution)

Adding distil water is settled to 1000 milliliters, and 4 ℃ of preservations are standby.

5) MS substratum macroelement mother liquor (MSmax mother liquor) (according to the preparation of 10X concentrated solution)

Mentioned reagent is dissolved under 20-25 ℃ of temperature, and be settled to 1000 milliliters with distilled water.

6) MS substratum trace element mother liquor (MSmin mother liquor) (according to the preparation of 100X concentrated solution)

Mentioned reagent is dissolved under 20-25 ℃ of temperature, and be settled to 1000 milliliters with distilled water.

7) 2, the preparation of 4-D stock solution (1 mg/ml):

Weigh 100 milligrams of 2,4-D, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, under 20-25 ℃ of temperature, preserve.

8) preparation of 6-BA stock solution (1 mg/ml):

Weigh 100 milligrams of 6-BA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 20-25 ℃ of temperature preserved.

9) preparation of naphthylacetic acid (NAA) stock solution (1 mg/ml):

Weigh 100 milligrams of NAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 4 ℃ of preservations are standby.

10) preparation of indolylacetic acid (IAA) stock solution (1 mg/ml):

Weigh 100 milligrams of IAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 4 ℃ of preservations are standby.

11) preparation of glucose stock solution (0.5 grams per milliliter):

Weigh glucose 125 grams, be settled to 250 milliliters with dissolved in distilled water then, the back 4 ℃ of preservations of sterilizing are standby.

12) preparation of AS stock solution:

Weigh AS 0.392 gram, add 10 milliliters of dissolvings of DMSO, divide to be filled in 1.5 milliliters of centrifuge tubes, 4 ℃ of preservations are standby.

13) 1N potassium hydroxide stock solution

Weigh potassium hydroxide 5.6 grams, be settled to 100 milliliters with dissolved in distilled water, 20-25 ℃ of temperature preserved standby.

(3) be used for the culture medium prescription that rice genetic transforms

1) inducing culture

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boil and be settled to 1000 milliliters, divide and install to 50 milliliters of triangular flasks (25 milliliters/bottle), sterilization according to a conventional method after sealing (sterilized 25 minutes down for 121 ℃, following medium sterilization method is identical with the sterilising method of basal culture medium).

2) subculture medium

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000 milliliters, divides to install to 50 milliliters of triangular flasks (25 milliliters/bottle), seals, as stated above sterilization.

3) pre-culture medium

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, seals, as stated above sterilization.

Use preceding heating for dissolving substratum and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/ware) in the culture dish are poured in packing into.

4) be total to substratum

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, seals, as stated above sterilization.

Use preceding heating for dissolving substratum and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/every ware) in the culture dish are poured in packing into.

5) suspension culture base

Adding distil water to 100 milliliter is regulated pH value to 5.4, divides in the triangular flask that installs to two 100 milliliters, seals, as stated above sterilization.Add 1 milliliter of aseptic glucose stock solution and 100 microlitre AS stock solutions before using.

6) select substratum

Adding distil water to 250 milliliter is regulated pH value to 6.0, seals, as stated above sterilization.

Dissolving substratum before using adds 250 microlitre HN (50 mg/ml) and (25 milliliters/ware) in the culture dish are poured in 400 microlitre CN (250 mg/ml) packing into.(annotate: selecting substratum Pyocianil concentration for the first time is 400 mg/litre, and selecting substratum Pyocianil concentration for the second time and later on is 250 mg/litre).

7) break up substratum in advance

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, seals, as stated above sterilization.

Dissolving substratum before using, 250 microlitre HN (50 mg/ml), 250 microlitre CN (250 mg/ml), (25 milliliters/ware) in the culture dish are poured in packing into.

8) division culture medium

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 6.0.

Boil and be settled to 1000 milliliters with distilled water, divide to install to 50 milliliters of triangular flasks (50 milliliters/bottle), seal, as stated above sterilization.

9) root media

Adding distil water to 900 milliliter is regulated pH value to 5.8 with 1N potassium hydroxide.

Boil and be settled to 1000 milliliters with distilled water, divide to install to (25 milliliters/pipe) in the pipe of taking root, seal, as stated above sterilization.

(4) agriculture bacillus mediated genetic transformation step (EHA105 is provided by Australian CAMBIA laboratory)

3.1 callus of induce

1) will spend 11 rice paddy seeds to shell in the maturation, used 70% Ethanol Treatment then successively 1 minute, 0.15% mercury chloride (HgCl ₂) seed-coat sterilization 15 minutes;

2) wash seed 4-5 time with sterilization;

3) seed is placed on the inducing culture;

4) postvaccinal substratum is placed dark place cultivate 4 weeks, 25 ± 1 ℃ of temperature.

3.2 callus subculture

Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down on the subculture medium.

3.3 pre-the cultivation

Select the embryo callus subculture of consolidation and relatively dry, be put in dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down on the pre-culture medium.

3.4 Agrobacterium is cultivated

1) went up the pre-Agrobacterium EHA105 of cultivation (this bacterial strain is from the agrobacterium strains of CAMBIA company public use) two days, 28 ℃ of temperature having the LA substratum that corresponding resistance selects (LA culture medium preparation with reference to J. Sa nurse Brooker etc., 1998);

2) Agrobacterium is transferred in the suspension culture base, cultivated 2-3 hour on 28 ℃ of shaking tables.

3.5 Agrobacterium is infected

1) pre-incubated callus is transferred in the bottle of the bacterium of having gone out;

2) regulate the suspension of Agrobacterium to OD ₆₀₀0.8-1.0;

3) callus was soaked in agrobacterium suspension 30 minutes;

4) shifting callus blots to the good filter paper of sterilization; Be placed on then on the common substratum and cultivated temperature 19-20 ℃ 3 days.

3.6 callus washing and selection are cultivated

1) aqua sterilisa washing callus is to cannot see Agrobacterium;

2) be immersed in the aqua sterilisa that contains 400 milligrams/L Pyocianil (CN) 30 minutes;

3) shifting callus blots to the good filter paper of sterilization;

4) shift callus to selecting to select on the substratum cultivation 2-3 time, each 2 weeks.

3.7 differentiation

1) kanamycin-resistant callus tissue is transferred on the pre-differentiation substratum in dark place cultivation 5-7 days;

2) callus that shifts pre-differentiation cultivation is cultivated 26 ℃ of temperature under the illumination to division culture medium.

3.8 take root

1) cuts the root that differentiation phase produces;

Then it is transferred to and cultivates 2-3 week, 26 ℃ of temperature in the root media under the illumination.

3.9 transplant

Wash the residual substratum on the root off, the seedling that will have good root system changes the greenhouse over to, keeps moisture moistening at initial several days simultaneously.

Transform in the japonica rice variety and spend 11, obtain transgenosis individual plant T ₀For plant (it is 19 strains that Y5A obtains positive strain, and it is 27 strains that Y5B obtains positive strain, and it is 14 strains that Y5C obtains positive strain, and it is 22 strains that Y5D obtains positive strain).With T ₀Positive strain of generation system receives and plants, and is T ₁For transgenic line.

The checking of embodiment 4:Y5 promotor and brachymemma promoter fragment transfer-gen plant regulation and control model thereof

Measure T respectively ₀For the activity that the promotor of the Y5 promotor total length of transfer-gen plant root and brachymemma is regulated the gus protein of the expression amount of gus gene and coding, find T ₀For the transfer-gen plant of each fragment, all show the pattern of inducing that conforms to chip, i.e. expression amount and the GUS activity of root nitrogen stress processing in 3 days are all obviously induced compared with the control.Measured T simultaneously ₁For transfer-gen plant, also measured phase isogeneous induction result.This has proved also that simultaneously this promotor can regulate and control goal gene to the reaction of nitrogen by genetic transformation.

1.Realtime PCR verification method

The method of Realtime PCR is the working instructions (article No.: DRR041A) of TAKARA commercial reagents box referring to precious biotechnology (Dalian) company limited.Adopt the reaction system of 10 μ l in the experiment, comprise: cDNA template 1.5 μ l (the rice material sample of expression amount to be detected), (GUS detects used left and right sides primer and is respectively qGUS-F and qGUS-R each 0.2 μ M of gene left and right sides primer in this experiment, the left and right sides primer of internal control gene Ubi is respectively qUbi-F and qUbi-R, specifically referring to table 2) and 1U Taq archaeal dna polymerase.The condition of pcr amplification is: 95 ℃ of pre-sex change 30s; 95 ℃ of 5s, 60 ℃ of 34s, 45 circulations; Signal collection is at 60 ℃.

Table 2realtime expression amount detects the primer table

In the detected result (as Fig. 6), after nitrogen stress is coerced 3 days, 4 transgenic positive strain systems of each of each promoter fragment of picked at random have shown behind the nitrogen stress 3d that tangible induced reaction is arranged, and prove that this promotor series is the promotor of regulatory gene nitrogen stress abduction delivering really.

Annotate: shown in Fig. 6-1 (the GUS expression amount of the different transgenic lines of Y5A detects), 6-2 (the GUS expression amount of the different transgenic lines of Y5B detects), 6-3 (the GUS expression amount of the different transgenic lines of Y5C detects), the 6-4 (the GUS expression amount of the different transgenic lines of Y5D detects).Wherein the different digital numbering represents different transgenic lines, and CK represents normal nutritive medium cultivation, and-N3d represents nitrogen stress processing 3d.All data are the result of root sample.

2, gus protein determination of activity

In order to prove that further this gene promoter also is this expression pattern on translation skill, the applicant has carried out the GUS determination of activity to transgenic seedling, the result shows that the Y5 promotor is consistent with chip results and Realtime result, and the Y5B of brachymemma, Y5C also show the same pattern of inducing, so can determine this gene promoter is the nitrogen stress evoked promoter, and the function section is at ATG upstream 546bp.

Concrete grammar and reagent are as follows:

The fluoroscopic examination of GUS activity

The extraction of 1 albumen and concentration determination

(1) get 600mg paddy rice sample grind into powder in liquid nitrogen, with the powder 1.5ml centrifuge tube of packing into, the GUS that adds 1ml extracts damping fluid, shakes up, leave standstill a few hours after, in 13,000rpm, 4 ℃ of centrifugal 10min, getting supernatant, to put on ice and put 4 ℃ of preservations standby.

(2) making of protein standard curve

Bovine serum albumin (BSA) mother liquor of getting 100 μ g/ml of preparation (accurately takes by weighing 0.100g BSA powder and adds an amount of ddH ₂The O dissolving, constant volume is 100 μ g/ml to 1L again), carry out the BSA gradient dilution by table 3.

The preparation of BSA concentration gradient in table 3 typical curve

From the gradient solution for preparing, get 250 μ L and add 750 μ L Xylene Brilliant Cyanine G dye liquors (according to the difference of reaction system, the preparation in 1: 3 by volume of BSA gradient solution and Xylene Brilliant Cyanine G dye liquor), mixing, room temperature is placed 5min, measure the absorbance value of 595nm, the production standard curve.(3) the sample protein assay is got albumen supernatant 250 μ L, add 750 μ L Xylene Brilliant Cyanine G dye liquors according to 1: 3 ratio of cleer and peaceful Xylene Brilliant Cyanine G dye liquor volume ratio on the albumen, mixing, room temperature is placed 5min, measure the absorbance value of 595nm, according to protein standard curve calculation sample protein content.

The 2GUS fluoroscopic examination

(1) 4-methyl umbellate form ketone (4-MU) solution with 100 μ mol is standard: preparation 4-MU gradient concentration liquid (by the reaction terminating liquid preparation) 0 μ mol/L, 5 μ mol/L, 10 μ mol/L, 20 μ mol/L, 40 μ mol/L, 60 μ mol/L (also can adjust by real signal value per sample by this gradient, be suitable for and get final product) at exciting light 365nm, emission light 455nm, measure the fluorescent value of each sample, the drawing standard curve.

(2) enzyme reaction: in the reaction system of per 200 μ L: get 30 μ L albumen supernatants, add 10 μ L 40mM 4-MUG, place 37 ℃ of reaction 1h, add in the 160ul reaction buffer (37 ℃ of preheatings), this reaction can directly be carried out at 96 hole enzyme plates.

(3) preheating microplate reader, at exciting light 365nm, emission light 455nm measures fluorescent value.According to typical curve, calculate each sample enzyme and live.

(4) the GUS activity is represented with pmol 4-MU/ μ g/min.

GUS extracts damping fluid: 50mM sodium phosphate buffer (pH7.0),

10mM Na ₂-EDTA，

0.1％ Triton X-100，

0.1％ SDS

10mM β one dredges basic ethanol, 4 ℃ of preservations.

Reaction terminating liquid: 0.2M Na ₂CO ₃, (requiring now with the current)

Coomassie brilliant blue G250 solution: take by weighing Coomassie brilliant blue G250 50mg, be dissolved in 95% ethanol 25ml, H ₃PO ₄50ml adds distilled water and is settled to 500ml, filters back 4 ℃ and keeps in Dark Place.

100 μ g/ml BSA graticule solution: 10mg BSA adds distilled water and is settled to 100mL.

The required solution of GUS detection by quantitative

One, phosphoric acid buffer (Na ₂HPO ₄And NaH ₂PO ₄Solution)

Na ₂HPO ₄.12H ₂The molecular weight of O: 358.14

NaH ₂PO ₄.2H ₂The molecular weight of O: 156.01

1,200ml 1mol/L Na ₂HPO ₄Solution

Na ₂HPO ₄.12H ₂O 71.628g

H ₂O is supplemented to 200ml

2, preparation 200ml 1mol/L NaH ₂PO ₄Solution

NaH ₂PO ₄.2H ₂O 31.202g

H ₂O is supplemented to 200ml

Two, 0.1M phosphoric acid buffer (pH7.0)

1mol/L Na ₂HPO ₄ 28.85ml

1mol/L NaH ₂PO ₄ 21.15ml

H ₂O is supplemented to 500ml

Three, 10% sodium lauryl sulphate (SDS) solution

Dissolving 10g SDS is heated to 68 ℃ of hydrotropies in 90ml water, adds several concentrated hydrochloric acids and regulates pH to 7.2, adds water then and is settled to 100ml.

Four, 0.5M EDTA (pH8.0)

Na ₂EDTA.2H ₂The molecular weight of O: 372.24

In 80ml water, add 18.61g Na ₂EDTA.2H ₂O transfers pH to 8.0 (needing the solid NaOH about 2g approximately) with NaOH, is settled to 100ml after the dissolving.

Five, GUS zyme extract

Six, the active liquid (MUG solution) that detects of GUS

MUG (C ₁₆H ₁₆O ₉4-methyl umbellate form ketone-beta-glucuronidase) molecular weight: 352.3

50mg MUG is dissolved in the GUS zyme extract of 3.548ml, being mixed with concentration is the MUG solution of 40mmol/L.

Seven, reaction terminating liquid (0.2mol/L Na ₂CO ₃)

Na ₂CO ₃Molecular weight: 105.99

The Na of preparation 500ml 0.2mol/L ₂CO ₃Solution:

Na ₂CO ₃ 10.6g

H ₂O is supplemented to 500ml

Eight, 4-MU solution preparation

4-MU (4-methyl umbellate form ketone) molecular weight: 176.2

Claim 0.14096g 4-MU solid to be dissolved in 40ml reaction terminating liquid (0.2M Na ₂CO ₃, preparation is as previously mentioned) in, be mixed with the 4-MU solution of 20mM.And then be diluted to 1mM concentration, and be stored in-20 degree, wait until preparation MU typical curve.

GUS detection by quantitative operation steps

One, the preparation of typical curve

With reaction terminating liquid MU mother liquor (1mM, preparation as previously mentioned) is diluted to concentration range at the series standard liquid of 0-100 μ M, makes a typical curve by the fluorescence intensity of measuring them.

Two, GUS enzyme extraction, measuring method:

1, get paddy rice sample to be determined in the mortar of liquid nitrogen precooling, add liquid nitrogen, grinding powder is got the 0.2g left and right sides powder 2ml centrifuge tube of packing into, adds the 1ml zyme extract and puts upside down mixing, puts lixiviate on ice about 4 hours.

2, in 13000rpm, 4 ℃ of centrifugal 10min carefully draw supernatant in another centrifuge tube.Repeated centrifugation is once drawn supernatant.This supernatant is liquid of protease just.

3, get the above-mentioned supernatant of 40 μ l and be diluted to 1ml, measure the content of albumen with foregoing Xylene Brilliant Cyanine G method.

4, get the above-mentioned supernatant of 30 μ l (can carry out suitable adjustment volume according to the protein content of measuring) and join 10 μ l in the detection liquid (40mmol/L 4-MUG solution, preparation are as previously mentioned) of 37 ℃ of preheatings.Rapidly abundant mixing, 37 ℃ of reaction 60min also take out at once and join 160 μ L reaction terminating liquid (0.2mol/L Na ₂CO ₃, preparation is as previously mentioned).

5, use spectrophotofluorometer under excitation wavelength 365nm, emission wavelength 455nm, the fluorescent value of working sample.

6, the GUS activity is represented with pmol 4-MU/ μ g/min.

Interpretation of result:

The result is shown in Fig. 7-1 (gus protein of the different transgenic lines of Y5A is active to be detected), 7-2 (gus protein of the different transgenic lines of Y5B is active to be detected), 7-3 (gus protein of the different transgenic lines of Y5C is active to be detected), 7-4 (gus protein of the different transgenic lines of Y5D is active to be detected).Wherein the different digital numbering represents different transgenic lines, and CK represents normal nutritive medium cultivation, and-N3d represents nitrogen stress processing 3d.All data are the result of root sample.Show among the result, all transform positive strain and tie up to and all show tangible nitrogen stress on the gus protein activity and induce trend in total length promotor Y5A and the brachymemma pattern thereof, further verified the expression pattern that the Y5 nitrogen stress is induced, and its controlling element is in Y5D is ATG upstream 546bp scope.

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Claims (3)

1. the promotor Y5A of a paddy rice nitrogen stress specificity abduction delivering, its nucleotide sequence is shown in sequence table SEQ ID NO:1.

2. the promotor Y5A of a paddy rice nitrogen stress specificity abduction delivering, it also comprises the promotor section Y5B of brachymemma, Y5C and Y5D, their nucleotide sequence are respectively as sequence table SEQ IDNO:2, shown in SEQ IDNO:3 and the SEQ IDNO:4.

3. claim 1 or the 2 described promotor application that induced gene is expressed in paddy rice under nitrogen stress.

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