CN110129471A

CN110129471A - It is a kind of for detecting the nucleic acid sequence and its detection method of rice plants ZUPM01

Info

Publication number: CN110129471A
Application number: CN201910325815.XA
Authority: CN
Inventors: 毛传澡; 徐纪明
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-04-15
Filing date: 2019-04-15
Publication date: 2019-08-16

Abstract

The present invention relates to a kind of for detecting the nucleic acid sequence and its detection method of rice plants ZUPM01, and the nucleic acid sequence of the rice plants includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series.Rice plants ZUPM01 of the present invention has a preferable tolerance to glyphosate herbicidal and low-phosphorus stress, and detection method can quickly and accurately identify in biological sample whether include transgenic paddy rice event ZUPM01 DNA molecular.

Description

It is a kind of for detecting the nucleic acid sequence and its detection method of rice plants ZUPM01

Technical field

The present invention relates to plant biotechnology fields.Specifically, being related to a kind of for detecting the core of rice plants ZUPM01 Acid sequence and its detection method, more particularly to the transgenic paddy rice thing of a kind of tolerance glyphosate herbicidal application and low-phosphorus stress Part ZUPM01 and for detect in biological sample whether include specific transgenic paddy rice event ZUPM01 nucleic acid sequence and its inspection Survey method.

Background technique

Weeds in field and crop competition water, fertilizer, light and growing space, directly affect crop yield and quality.Permitted simultaneously More weeds are the vector of crop pathogens and pest again, are one of important biomolecule restriction factors of crop yield.According to joint Food and Agriculture Organization, state statistics, annual up to 95,000,000,000 dollars because of grain-production loss caused by weeds of the whole world, is equivalent to loss 3.8 hundred million tons of wheats are roughly equal to more than half of global wheat yield in 2009.It is poor in 95,000,000,000 dollars of economic loss Developing country bear about 70,000,000,000 dollars (FAO.The lurking menace of weeds [J/OL] (http: // Www.fao.org/news/story/en/item/29402/icode/), 2009-08-11.).Therefore, field is efficiently controlled Weeds are one of the important measures for promoting increases in grain production.In China, the weeds type of hazard rice has more than 40 kinds, wherein endanger compared with Big weeds have more than 10 kinds.It can make rice underproduction 10-20% in general time weeds, more up to 30-50% when serious.In addition, As China's people in the countryside are toward the quickening of urban migration speed, the scale and mechanization of Rice Cropping be one it is foreseeable become Gesture, this makes traditional artificial weeding mode become unrealistic.Currently, widely applied selective herbicide amount of application in the market Greatly, residual life, is long, is easy to influence the normal growth of second stubble crop.The steriland herbicides such as glyphosate have efficient, low toxicity, easily drop The features such as solution, noresidue.But their weedings cannot be used directly in the growth period of crop without selectivity.Pass through transgenic technology The rice for cultivating such resistance to steriland herbicide can overcome this problem.Spraying 1-2 times in rice growing season can effectively solve Certainly weed problem reduces the dosage and input cost of herbicide.Therefore, herbicide-resistant transgenic paddy rice has boundless Application value and market potential.

Phosphorus is the important composition ingredient of plant nucleic acid in vivo, phosphatide and ATP, as plant energy i (in vivo) transfer substance, The entire metabolic process that vivo protein can be activated, regulate and control plant.Among a variety of inorganic nutrients needed for plant, phosphorus conduct One of most important element, it is extremely difficult to be obtained from soil.Although phosphorus content is abundant in the ecosystem, can be plant assimilating The chemical species of the phosphorus of absorption are but mainly Phos-phosphate (Pi).Phosphate distributed pole is unbalanced in soil, and mostly Number phosphate can not all move freely, thus be unfavorable for absorption (the Raghothama K G.Phosphate of root system Acquisition [J] .Annual Review of Plant Physiology and Plant Molecular Biology, 1999,50 (1): 665-693.).Therefore, the supply situation of soil available phosphorus and plant become the absorbability of phosphorus nourishing One of main determining factor for plant growth and development (Schachtman D P, Reid R J, and Ayling S M.Phosphorus Uptake by Plants:From Soil to Cell [J] .Plant Physiol, 1998,116 (2): 447-53.)。

Tellurian phosphorus ore is just being gradually decreased as a kind of non-renewable resources in the case where the mankind constantly exploit utilization.The U.S. The data of geological prospecting show that global phosphate exploitation total amount is 100,016,000 tons within 2008, and the demand of chemical fertilizer is not Come in 5 years to increase with the rate of annual 2.5%-3%.If things go on like this, world phosphate resource can only support human demand again 125 years or so (Gilbert N.Environment:The disappearing nutrient [J] .Nature, 2009,461 (7265): 716-8.).Meanwhile the phosphorus excessively applied can also cause huge harm to environment；Therefore, improve crop to phosphorus element Absorption and using to ecology and agricultural economy be all of great significance.

Known foreign gene is influenced in the intracorporal expression of plant by their chromosome location, it may be possible to due to dyeing Matter structure (such as heterochromatin) or transcription regulatory element (such as enhancer) are close to integration site.Thus, it usually needs screening is a large amount of Event be possible to identify can be with commercialized event (event that the target gene imported obtains optimal expression).Example Such as, have been observed that the expression quantity of quiding gene there may be very big difference between event in plant and other organisms；In table On the space reached or time mode may there is also differences, such as between different plant tissues transgenosis relative expression exist it is poor Different, this species diversity shows that actual expression pattern may be pre- with the transcription regulatory element institute in the gene construct according to importing The expression pattern of phase is inconsistent.It is thus typically necessary to generate hundreds and thousands of different events and filter out from these events Single incident with transgene expression amount and expression pattern desired for the purpose of being commercialized.With expected transgenosis table Event up to amount and expression pattern can be used for that transgenosis is penetrated into other by sexual cutcross using conventional breeding methods In genetic background.The transgenic expression characteristics of original transformant are maintained by the offspring that this Crossing system generates.Using this Kind strategy pattern may insure there is reliable gene expression in many kinds, and these kinds can well adapt to locality Growth conditions.

It will be beneficial that the presence of particular event, which is able to detect, so that whether the offspring for determining sexual hybridization includes target gene. In addition, the method for detection particular event also will be helpful to abide by relevant laws and regulations, such as thrown from the food of recombination crops It needs to obtain official approval before entering market and is marked.Transgenosis is detected by any well known polynucleotides detection method Presence be all it is possible, such as polymerase chain reaction (PCR) or using polynucleotide probes DNA hybridization.These detections Method is usually focused on common genetic elements, such as promoter, terminator, marker gene etc..Therefore, unless with insertion turn The sequence of the adjacent chromosomal DNA of gene DNA (" flanking DNA ") be it is known, above-mentioned this method cannot be used to distinguish Different events, especially those events generated with identical DNA construct.Insertion is spanned so often utilizing at present The pair of primers of the junction of transgenosis and flanking DNA identifies transgenosis particular event by PCR, specifically includes The first primer in flanking sequence and the second primer comprising insetion sequence.

Summary of the invention

The object of the present invention is to provide a kind of for detecting the nucleic acid sequence and its detection method of rice plants ZUPM01, turns Trans-genetic hybrid rice event ZUPM01 has preferable tolerance to glyphosate herbicidal and low-phosphorus stress, and detection method can be accurate Rapidly identify biological sample in whether include specific transgenic paddy rice event ZUPM01 DNA molecular.

To achieve the above object, the present invention provides a kind of nucleic acid sequence, the nucleic acid sequence include SEQ ID NO:1 or Its complementary series, and/or SEQ ID NO:2 or its complementary series, the nucleic acid sequence are originated from transgenic paddy rice event ZUPM01.

Further, the nucleic acid sequence include SEQ ID NO:3 or its complementary series, and/or SEQ ID NO:4 or its Complementary series.

Further, the nucleic acid sequence includes SEQ ID NO:5 or its complementary series.

The SEQ ID NO:1 or its complementary series are in transgenic paddy rice event ZUPM01 in 5 ' ends of insetion sequence A length near insertion junction is the sequence of 22 nucleotide, the SEQ ID NO:1 or its complementary series The DNA sequence dna for spanning the left side flap genomic dna sequence of rice insertion point and the 5 ' end of left margin of insetion sequence includes The SEQ ID NO:1 or its complementary series can be accredited as the presence of transgenic paddy rice event ZUPM01.The SEQ ID NO:2 or its complementary series are attached positioned at insertion junction in 3 ' ends of insetion sequence in transgenic paddy rice event ZUPM01 A close length is the sequence of 22 nucleotide, and the SEQ ID NO:2 or its complementary series span the right side of insetion sequence The DNA sequence dna of 3 ' end of boundary and the right side flap genomic dna sequence of rice insertion point, comprising the SEQ ID NO:2 or Its complementary series can be accredited as the presence of transgenic paddy rice event ZUPM01.

In the present invention, the nucleic acid sequence can be inserted into sequence for the SEQ ID NO:3 or its complementary series transgenic Any portion of at least 11 or more continuous polynucleotides (the first nucleic acid sequence) of column, or be the SEQ ID NO: 3 or its complementary series in 5 ' left side flap oryza sativa genomic dna regions any portion of at least 11 or more continuous multicores Thuja acid (second nucleotide sequence).The nucleic acid sequence may further be derived from or be complementary to comprising the complete SEQ ID to be same A part of the SEQ ID NO:3 of NO:1.When the first nucleic acid sequence is used together with second nucleotide sequence, these nucleic acid Sequence includes DNA primer group in the DNA cloning method for generating amplified production.It is produced using DNA primer in DNA cloning method Raw amplified production is that can diagnose transgenic paddy rice event ZUPM01 or thereafter when including the amplified production of SEQ ID NO:1 The presence in generation.Well known to those skilled in the art, the first and second nucleic acid sequences need not be only made of DNA, may also comprise The mixture or DNA, RNA of RNA, DNA and RNA or it is other not as the nucleotide of one or more polymerase templates or its The combination of analog.In addition, heretofore described probe or primer should be at least about 11,12,13,14,15,16,17, 18, the length of 19,20,21 or 22 continuous nucleotides can be selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID Nucleotide described in NO:3, SEQ ID NO:4 and SEQ ID NO:5.When selected from SEQ ID NO:3, SEQ ID NO:4 and When nucleotide shown in SEQ ID NO:5, the probe and primer can be for length at least about 21 to about 50 or More continuous nucleotides.The SEQ ID NO:3 or its complementary series are in transgenic paddy rice event ZUPM01 in insertion sequence Column 5 ' ends be located at insertion junction near a length be 732 nucleotide sequence, the SEQ ID NO:3 or Its complementary series by 486 nucleotide rice left side flap genomic dna sequence (the nucleotide 1-486 of SEQ ID NO:3), 74 The pPHF1G9A-1300 construct left margin DNA sequence dna (the nucleotide 487-560 of SEQ ID NO:3) of a nucleotide and 172 3 ' the end DNA sequences (the nucleotide 561-732 of SEQ ID NO:3) of the CaMV 35S terminator of nucleotide form, and include institute The presence of transgenic paddy rice event ZUPM01 can be accredited as by stating SEQ ID NO:3 or its complementary series.

The nucleic acid sequence can be any portion of the SEQ ID NO:4 or its complementary series transgenic insetion sequence At least 11 or more the continuous polynucleotides (third nucleic acid sequence) divided, or be the SEQ ID NO:4 or its complementation Any portion of at least 11 or more the continuous polynucleotides the (the 4th in 3 ' right side flap oryza sativa genomic dna regions in sequence Nucleic acid sequence).The nucleic acid sequence may further be derived from or be complementary to the institute comprising the complete SEQ ID NO:2 to be same State a part of SEQ ID NO:4.When third nucleic acid sequence is used together with the 4th nucleic acid sequence, these nucleic acid sequences are being produced It include DNA primer group in the DNA cloning method of raw amplified production.Amplification using DNA primer to being generated in DNA cloning method Product is can to diagnose transgenic paddy rice event ZUPM01 or the presence of its offspring when including the amplified production of SEQ ID NO:2.

The SEQ ID NO:4 or its complementary series are in transgenic paddy rice event ZUPM01 in 3 ' ends of insetion sequence A length near insertion junction is the sequence of 913 nucleotide, the SEQ ID NO:4 or its complementary series By no (rouge alkali synthetase) transcription terminator sequences (the nucleotide 1-254 of SEQ ID NO:4) of 254 nucleotide, 219 The pPHF1G9A-1300 construct right margin DNA sequence dna (the nucleotide 255-473 of SEQ ID NO:4) of a nucleotide and 440 The rice integration site right side flap genomic dna sequence (the nucleotide 474-913 of SEQ ID NO:4) of nucleotide forms, and includes The SEQ ID NO:4 or its complementary series can be accredited as the presence of transgenic paddy rice event ZUPM01.

The SEQ ID NO:5 or its complementary series are that the length of characterization transgenic paddy rice event ZUPM01 is 7306 cores The sequence of thuja acid, the genome and genetic elements for specifically including are as shown in table 1.Include the SEQ ID NO:5 or its complementation Sequence can be accredited as the presence of transgenic paddy rice event ZUPM01.

The genome and genetic elements that 1 SEQ ID NO:5 of table includes

1: unit bp.

The nucleic acid sequence or its complementary series can be used in DNA cloning method to generate amplicon, the inspection of the amplicon Survey diagnosis biological sample transgenic rice event ZUPM01 or the presence of its offspring；The nucleic acid sequence or its complementary series can For in nucleotide detection method, to detect the presence of biological sample transgenic rice event ZUPM01 or its offspring.

To achieve the above object, the present invention also provides the DNA of test sample transgenic rice event ZUPM01 a kind of Existing method, comprising:

Contact sample to be tested in nucleic acid amplification reaction at least two primers；

Carry out nucleic acid amplification reaction；

Detect the presence of amplified production；

The amplified production includes SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or its complementary series；

The amplified production is originated from transgenic paddy rice event ZUPM01.

In the above-mentioned technical solutions, the amplified production further includes SEQ ID NO:6 or its complementary series, and/or SEQ ID NO:7 or its complementary series.

Specifically, the primer includes the first primer and the second primer, the first primer be selected from SEQ ID NO:1 or its Complementary series, SEQ ID NO:8 and SEQ ID NO:10；Second primer be selected from SEQ ID NO:2 or its complementary series, SEQ ID NO:9 and SEQ ID NO:11.

Contact sample to be tested with probe, the probe includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series, the source probe transgenic rice event ZUPM01；

Hybridize the sample to be tested and the probe under stringent hybridization conditions；

Detect the hybridisation events of the sample to be tested and the probe.

The stringent condition can in 6 × SSC (sodium citrate), 0.5%SDS (lauryl sodium sulfate) solution, Hybridize at 65 DEG C, is then respectively washed film 1 time with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS.

Further, the probe further includes SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or it is mutual Complementary series.

Selectively, at least one fluorophor label of at least one described probe.

Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:1 or Its complementary series or SEQ ID NO:2 or its complementary series, the marker nucleic acid molecules are originated from transgenic paddy rice event ZUPM01；

Hybridize the sample to be tested and the marker nucleic acid molecules under stringent hybridization conditions；

The hybridisation events of the sample to be tested and the marker nucleic acid molecules are detected, and then are educated by marker auxiliary Kind analysis is to determine that herbicide tolerant and/or low-phosphorus stress tolerance and marker nucleic acid molecules are chain on science of heredity 's.

Further, the marker nucleic acid molecules further include SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

To achieve the above object, the present invention also provides a kind of DNA detection kit, including at least one DNA molecular, institutes Stating DNA molecular includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series, be can be used as pair There is one of DNA primer of specificity or probe in transgenic paddy rice event ZUPM01 or its offspring；The DNA molecular, which is originated from, to be turned Trans-genetic hybrid rice event ZUPM01.

It further, further include SEQ ID NO:6 or its complementary series or SEQ when the DNA molecular is as probe ID NO:7 or its complementary series.

To achieve the above object, protect rice plants from the damage as caused by herbicide the present invention also provides a kind of Method plants the big Tanaka of at least one transgenic rice plant including that will be applied to containing effective dose glyphosate herbicidal, The transgenic rice plant successively includes 561-6647 SEQ ID NO:1, SEQ ID NO:5 nucleic acid in its genome It include SEQ ID NO:5 in the genome of sequence and SEQ ID NO:2 or the transgenic rice plant；The transgenosis Rice plants have the tolerance to glyphosate herbicidal.

To achieve the above object, protect rice plants from as caused by the low-phosphorous element of soil the present invention also provides a kind of The method of damage is included in low-phosphorous concentration soil and plants at least one transgenic rice plant, the transgenic rice plant In its genome successively include SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2, It or include SEQ ID NO:5 in the genome of the transgenic rice plant；The transgenic rice plant has to low-phosphorous The tolerance of stress.

To achieve the above object, the present invention also provides it is a kind of control rice cultivation plant big Tanaka weeds method, Plant the big Tanaka of at least one transgenic rice plant including that will be applied to containing effective dose glyphosate herbicidal, described turn Trans-genetic hybrid rice plant in its genome successively comprising SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and It include SEQ ID NO:5 in the genome of SEQ ID NO:2 or the transgenic rice plant；The transgenic paddy rice is planted Object has the tolerance to glyphosate herbicidal.

To achieve the above object, the present invention also provides a kind of cultures has the rice plant of tolerance to glyphosate herbicidal The method of object, comprising:

An at least rice paddy seed is planted, includes the nucleic acid sequence of specific region, institute in the genome of the rice paddy seed The nucleic acid sequence for stating specific region successively includes SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ The nucleic acid sequence of ID NO:2 or the specific region includes SEQ ID NO:5；

The rice paddy seed is set to grow up to rice plant；

The rice plant is sprayed with effective dose glyphosate herbicidal, harvest does not have the specific region with other The plant of nucleic acid sequence compares the plant with the plant injury weakened.

To achieve the above object, the present invention also provides a kind of rice plants cultivated and have tolerance to low-phosphorus stress Method characterized by comprising

An at least rice paddy seed is planted in low-phosphorous soil, includes specific region in the genome of the rice paddy seed Nucleic acid sequence, the nucleic acid sequence of the specific region successively include 561-6647 SEQ ID NO:1, SEQ ID NO:5 cores The nucleic acid sequence of acid sequence and SEQ ID NO:2 or the specific region includes SEQ ID NO:5；

The rice paddy seed is set to grow up to rice plant；

Harvest has the plant injury weakened compared with the plant of other nucleic acid sequences for not having the specific region Plant.

To achieve the above object, the present invention also provides a kind of rice plants for generating and having tolerance to glyphosate herbicidal The method of strain, including, 561-6647 nucleic acid sequences of SEQ ID NO:5 are introduced in the genome of Xiang Suoshu rice plant, and So that the genome of the rice plant successively include SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2, or the genome of the rice plant is made to include SEQ ID NO:5, the rice of selection tolerance glyphosate Plant.

Specifically, the method for generating the rice plant that there is tolerance to glyphosate herbicidal, comprising:

By transgenic paddy rice event ZUPM01 the first parent rice plant to glyphosate herbicidal with tolerance and lack Second parent's rice plant sexual hybridization of few glyphosate tolerant, to generate a large amount of progeny plants；

The progeny plant is handled with glyphosate herbicidal；

The progeny plant of selection tolerance glyphosate.

To achieve the above object, the present invention also provides a kind of rice plants for generating and having tolerance to low-phosphorus stress Method, which is characterized in that including introducing 561-6647 nucleic acid sequences of SEQ ID NO:5 into the genome of the rice plant Column, and the genome of the rice plant is made successively to include 561-6647 SEQ ID NO:1, SEQ ID NO:5 nucleic acid Sequence and SEQ ID NO:2, or the genome of the rice plant is made to include SEQ ID NO:5, selection is resistant to the low-phosphorous side of body Urgent rice plant.

Specifically, the method for generating the rice plant that there is tolerance to low-phosphorus stress, comprising:

By to low-phosphorus stress have tolerance transgenic paddy rice event ZUPM01 the first parent rice plant with lack it is low Second parent's rice plant sexual hybridization of P deficiency tolerance, to generate a large amount of progeny plants；

The progeny plant is handled with low-phosphorus stress；

The progeny plant of selection tolerance low-phosphorus stress.

To achieve the above object, the present invention also provides a kind of composition for being produced from transgenic paddy rice event ZUPM01, The composition is rice, straw, rice husk or seed rice.

To achieve the above object, the present invention also provides it is a kind of by transgenic paddy rice event ZUPM01 production agricultural product or Commodity, the agricultural product or commodity are that rice flour, rice bran oil, rice bran, rice embryo, rice gluten, rice starch, Rice Bran oil or rice bran are more Sugar, cosmetics or filler.

In nucleic acid sequence and its detection method of the present invention for detecting rice plants, defined below and method can be more The present invention is defined well and those skilled in the art is instructed to implement the present invention, it is unless otherwise mentioned, general according to this field Lead to the conventional usage of technical staff to understand term.

" rice " refers to rice (Oryza sativa), including all plant varieties that can be bred with rice, including Wild seed rice and those belong to the plant bred between the permission species of Oryza.

The "comprising" refers to " including but not limited to ".

Term " plant " includes that whole plant, plant cell, plant organ, plant protoplast, plant can therefrom again It is complete in raw plant cell tissue cultures, plant callus, vegetation bed (plant clumps) and plant or plant part Whole plant cell, the plant part such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stalk, root, the tip of a root, flower Medicine etc..The part for the genetically modified plants being interpreted as in the scope of the invention includes but is not limited to plant cell, protoplast, group It knits, callus, embryo and flower, stem, fruit, Ye Hegen, the above plant part are originated from advance with DNA molecular conversion of the invention And the genetically modified plants being therefore at least partly made of transgenic cell or its filial generation.

Term " gene " refers to the nucleic acid fragment of expression specific protein, including adjusting sequence (5 ' the non-volumes before coded sequence Code sequence) and coded sequence after adjusting sequence (3 ' non-coding sequence)." natural gene ", which refers to, is naturally found to have its own Adjust the gene of sequence." mosaic gene " refer to be not natural gene any gene, it includes non-natural discovery adjusting and Coded sequence." endogenous gene " refers to natural gene, and the natural gene is located in organism genome its natural place. " foreign gene " is the alien gene being not present in the existing genome for being biology and originally, also refers to and imports through Transgenic procedures The gene of recipient cell.Foreign gene may include the natural gene or mosaic gene of insertion non-native organism." transgenosis " It is the gene that genome is had been incorporated by Transformation Program.The site that recombinant DNA has been inserted into Plant Genome can claim For " insertion point " or " target site ".

" flanking DNA " may include the genome being naturally present in the organism of such as plant or be drawn by conversion process External source (heterologous) DNA entered, such as segment relevant to transformation event.Therefore, flanking DNA may include natural and exogenous DNA Combination.In the present invention, " flanking region " or " flanking sequence " or " genome frontier district " or " genome border sequence " refer to The base-pair of at least 3,5,10,11,15,20,50,100,200,300,400,1000,1500,2000,2500 or 5000 is longer Sequence, be located at initial external source insertion DNA molecular immediately upstream or downstream and with initial external source be inserted into DNA molecular phase It is adjacent.When the flanking region is located at upstream, it is referred to as " left margin flank " or " 5 ' flank " or " 5 ' flanking genomic area " Or " 5 ' flanking sequence of genome " etc..When the flanking region is located at downstream, it is referred to as " right margin flank " or " 3 ' sides The wing " or " 3 ' flanking genomic area " or " 3 ' flanking sequence of genome " etc..

The Transformation Program of the random integration of exogenous DNA is caused to will lead to the transformant containing different flanking regions, the difference Flanking region is that each transformant institute specificity contains.When recombinant DNA is introduced into plant by conventional hybridization, flanking region is logical Chang Buhui changes.Transformant also can be containing between heterologous insertion DNA and the section of genomic DNA or between two sections of genomic DNAs Or the unique engagement between two sections of allogeneic dna sequence DNAs." engagement " is the point of two specific DNA fragmentation connections.For example, engagement exists In the position of insert DNA connection flanking DNA.Junction is also present in the organism of conversion, and two of them DNA fragmentation is to repair Adorn linking together for the mode found from native organism." engagement DNA " refers to the DNA comprising junction.

The present invention provides the transgenic paddy rice event of referred to as ZUPM01 and its offspring, the transgenic paddy rice event ZUPM01 is rice plants ZUPM01 comprising the Plants and Seeds and its plant cell of transgenic paddy rice event ZUPM01 or Its renewable part, the plant part of the transgenic paddy rice event ZUPM01, including but not limited to cell, pollen, ovule, Flower, bud, root, stem, fringe, inflorescence, leaf and the product from rice plants ZUPM01, such as rice, straw, rice husk or seed rice and stay Biomass in rice crop field.

Transgenic paddy rice event ZUPM01 of the present invention contains a DNA construct, when it is expressed in plant cell, The transgenic paddy rice event ZUPM01 obtains the tolerance to glyphosate herbicidal and/or low-phosphorus stress.The DNA construct Comprising an expression cassette, expression cassette includes the suitable promoter and suitable Polyadenylation letter for expressing in plant Number sequence, the promoter, which is operably connected, encodes the gene of 5- enol-pyrovyl shikimic acid -3- phosphate synthase (G9A), institute The nucleic acid sequence for stating G9A albumen has tolerance to glyphosate herbicidal.The DNA construct includes another expression cassette, table Up to the suitable promoter and suitable polyadenylation signal sequence that box includes for expressing in plant, the promoter It is operably connected and encodes the rice phosphate transporter assistance transport factor (OsPHF1) gene, the nucleic acid of the OsPHF1 albumen Sequence has tolerance to low-phosphorus stress.Further, the promoter can be the suitable promoter separated from plant, including Composing type, induction type and/or tissue-specific promoter, the suitable promoter includes but is not limited to cauliflower mosaic virus (CaMV) 35S promoter, figwort mosaic virus (FMV) 35S promoter, ubiquitin protein (Ubiquitin) promoter, actin (Actin) promoter, soil Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) promoter, chapter Fish alkali synzyme (OCS) promoter, Cestrum (Cestrum) yellow leaf curl virus promoter, patatin (Patatin) promoter, ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylase/oxygenase (RuBisCO) promoter, glutathione S-transferase Enzyme (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium Rhizogenes) RolD promoter and Arabidopsis (Arabidopsis thaliana) Suc2 promoter.The polyadenosine Polyadenylation signal sequence can be the suitable polyadenylation signal sequence to work in plant, the suitable polyadenylic acid Changing signal sequence includes but is not limited to synthesize from soil Agrobacterium (Agrobacterium tumefaciens) nopaline The polyadenylation signal sequence of enzyme (NOS) gene derives from cauliflower mosaic virus (CaMV) 35S terminator, derives from The polyadenylation signal sequence of protease inhibitors II (PIN II) gene and derive from alpha-tubulin (α-tubulin) The polyadenylation signal sequence of gene.

In addition, the expression cassette can also include other genetic elements, the genetic elements include but is not limited to enhance Son and signal peptide/transit peptides.The expression of gene can be enhanced in the enhancer, and the enhancer includes but is not limited to cigarette Careless etch virus (TEV) translation activity factor, CaMV35S enhancer and FMV35S enhancer.Signal peptide/the transit peptides can be with Guide G9A Protein transport to extracellular or intracellular specific organelle or compartment, for example, utilizing encoding chloroplast transit peptide Sequence targets chloroplaset, or utilizes ' KDEL ' to retain sequence and target endoplasmic reticulum.

" glyphosate " refers to the salt of N- phosphonomethylglycine and it, is handled with " glyphosate herbicidal " and refers to use Any one is handled containing the herbicide formulations of glyphosate.In order to reach ebd and to certain glyphosate system The selection of agent utilization rate is no more than the technical ability of common agronomic technique personnel.Herbicide formulations using any one containing glyphosate Processing contains the field of the vegetable material from transgenic paddy rice event ZUPM01, raw by the weeds in the field are controlled It is long, and the growth or yield of the vegetable material from transgenic paddy rice event ZUPM01 are not influenced.

The DNA construct is introduced in plant using method for transformation, and the method for transformation includes but is not limited to agriculture bar Bacterium (Agrobacterium) mediated transformation method, Gene Knock-out Mice and pollen tube channel conversion method.

The Agrobacterium_mediated method is the common method of Plant Transformation.The exogenous DNA gram that will be introduced into plant Between the grand left and right boundary consensus sequence to carrier, i.e. the area T-DNA.The carrier is transformed into agrobatcerium cell, then, The agrobatcerium cell is organized for infection plant, and the area T-DNA of the carrier comprising exogenous DNA is inserted into plant gene In group.

The Gene Knock-out Mice is with carrier bombardment plant cell (the biological bullet that particle mediates comprising exogenous DNA Hit conversion).

The pollen tube channel conversion method is that natural pollen tube channel (also known as pollen is formed by after pollinating using plant Pipe guides tissue), through megarchidium channel, exogenous DNA is carried into blastular.

After conversion, it is necessary to have from the plant tissue regenerating plants of conversion, and using suitable label selection The offspring of exogenous DNA.

DNA construct is the combination that DNA molecular is interconnected, and this combination provides one or more expression cassettes.DNA Construct preferably can the self-replacation in bacterial cell, and contain different restriction endonuclease sites plasmid, Contained restriction endonuclease sites provide functioning gene element, i.e. promoter, introne, leader sequence, volume for importing The DNA molecular of code sequence, 3 ' terminator regions and other sequences.Expression cassette contained in DNA construct includes providing courier Genetic elements necessary to the transcription of RNA, the expression cassette can be designed as expressing in prokaryotic cell or eukaryocyte.This hair Bright expression cassette is designed to most preferably express in plant cell.

Transgenosis " event " is as obtained from converting plant cell with heterologous DNA construct, that is, includes at least one Expression of nucleic acid box containing target gene is inserted into Plant Genome to generate plant population by transgene method, then The raw plant population, and selection have the specific plant of insertion specific gene group site feature.Term " event " refers to including different The original transformant of source DNA and the offspring of the transformant.Term " event " also refers to transformant and other kinds containing allogeneic dna sequence DNA Offspring obtained from sexual hybridization is carried out between individual, even if after be returned repeatedly with backcross parent, from transformant The insertion DNA and flanking genomic dna of parent exists in the same chromosome location in filial generation.Term " event " also refers to DNA sequence dna from original transformant, the DNA sequence dna include insertion DNA and with the close adjacent flanking genomes sequence of insertion DNA Column, which, which is expected, is transferred in filial generation, the filial generation by containing insertion DNA parental department (such as original transformant and its It is selfed the filial generation generated) sexual hybridization is carried out with the parental department without containing insertion DNA and is generated, and the filial generation is received comprising mesh Mark the insertion DNA of gene.

" recombination " refers to the DNA that generally can not be found and therefore generate by manual intervention in nature in the present invention And/or the form of albumen and/or organism.This manual intervention can produce recombinant DNA molecules and/or recombinant plant." the weight It is that isolated sequence section obtains, such as passes through chemistry in other cases that group DNA molecular ", which is by two kinds of artificial combination, Synthesis operates isolated nucleic acid segment by genetic engineering technology.The technology for carrying out nucleic-acid manipulation is well-known.

Term " transgenosis " includes any cell, cell line, callus, tissue, plant part or plant, above base Because type due to heterologous nucleic acids there are due to change, " transgenosis " includes Transgenics initially changed in this way and by most The offspring individual that first Transgenics are generated by sexual hybridization or vegetative propagation.In the present invention, term " transgenosis " does not wrap (chromosome the or extrachromosomal) change by conventional plant breeding method or the natural genome that event occurs is included, it is described It is natural that for example random allogamy of event, non-recombinant virus infection, non-recombinant Bacterial Transformation, non-recombinant swivel base or spontaneous prominent occurs Become.

" heterologous " refers to that the first molecule is not found usually and the second molecular combinations in nature in the present invention.For example, Molecule can be originated from the first species and be inserted into the genome of the second species.Therefore this molecule for host be it is heterologous and It is artificially introduced in the genome of host cell.

The transgenic paddy rice event ZUPM01 that there is tolerance to glyphosate herbicidal is cultivated, passes through following steps: first Make first parent's rice plants and second parent's rice plants sexual hybridization, so that the first generation progeny plant of multiplicity is produced, The first parent rice plants are made of the rice plants of cultivation transgenic rice event ZUPM01 and its offspring, this turns base Because rice event ZUPM01 and its offspring are by having tolerance to glyphosate herbicidal and/or low-phosphorus stress using of the invention Property expression cassette converted obtained from, second parent's rice plants, which lack, has glyphosate herbicidal and/or low-phosphorus stress There is tolerance；Then the progeny plant that there is tolerance to glyphosate herbicidal and/or low-phosphorus stress is selected, can be cultivated pair Glyphosate herbicidal and/or low-phosphorus stress have the rice plants of tolerance.These steps, which may further include, makes glyphosate And/or the progeny plant of low-phosphorus stress tolerance is returned with second parent's rice plants or third parent's rice plants, so Afterwards by with glyphosate herbicidal apply low-phosphorus stress or by molecular marked compound relevant to character such as comprising transgenosis water 5 ' the ends and 3 ' ends of insetion sequence identify in rice event ZUPM01 the DNA molecular of bond site) identification select filial generation, To generate the rice plants that there is tolerance to glyphosate herbicidal and/or low-phosphorus stress.

It will also be appreciated that two different genetically modified plants can also hybridize to generate containing there are two independent, separation The offspring of the foreign gene of formula addition.It is all homozygous for the available foreign gene added to two of the selfing of appropriate offspring The Progeny plants of son.It is also as previously described to be expected to the backcrossing of parental plant and with the cutcross of non-transgenic plant , vegetative propagation is also same.

Term " probe " is the nucleic acid molecules of one section of separation, is combined with conventional detectable label or report point above Son, for example, radioactive isotope, ligand, chemiluminescent agent or enzyme.This probe is complementary with a chain of target nucleic acid , in the present invention, probe is complementary with a DNA chain from transgenic paddy rice event ZUPM01 genome, no matter the gene Group DNA is plant or the kind that transgenic paddy rice event ZUPM01 is also derived from from transgenic paddy rice event ZUPM01 or seed Son or extract.Probe of the invention not only includes DNA or ribonucleic acid, further include specifically with target dna Sequence combines and can be used for detecting the existing polyamide and other probe materials of the target dna sequence.

Term " primer " is the nucleic acid molecules of one section of separation, is hybridized by nucleic acid, annealed combination to complementary target dna On chain, heterozygote is formed between primer and target dna chain, then under the action of polymerase (such as archaeal dna polymerase), along mesh DNA chain is marked to extend.Primer pair of the invention is related to its application in target nucleic acid sequence amplification, for example, passing through polymerase chain Formula reacts (PCR) or other conventional nucleic acid amplification methods.

The length of probe and primer is usually 11 polynucleotides or more, preferably 18 polynucleotides or more, More preferably 24 polynucleotides or more, most preferably 30 polynucleotides or more.This probe and primer are in height Specifically hybridize under degree stringent hybridization condition with target sequence.Although being different from target dna sequence and being protected to target dna sequence The probe for holding hybridization ability can design by conventional method, however, it is preferred to, probe and primer in the present invention There is complete DNA sequence dna identity with the continuous nucleic acid of target sequence.

It can be determined by conventional method based on the primer and probe of flanking genomic dna and insetion sequence of the invention, For example, by separating corresponding DNA molecular from from the vegetable material of transgenic paddy rice event ZUPM01, and determining should The nucleic acid sequence of DNA molecular.The DNA molecular includes transgene insert sequence and rice genome flank region, and the DNA divides The segment of son may be used as primer or probe.

Nucleic acid probe and primer of the invention hybridizes with target dna sequence under strict conditions.Any conventional nucleic acid is miscellaneous It hands over or amplification method may be used to identify in sample from the presence of the DNA of transgenic paddy rice event ZUPM01.Nucleic acid point Son or its segment can carry out specific hybrid with other nucleic acid molecules in any case.As the present invention uses, if two A nucleic acid molecules can form antiparallel double-strandednucleic acid structure, so that it may say that the two nucleic acid molecules are able to carry out specifically to each other Property hybridization.If two nucleic acid molecules show complete complementarity, claiming one of nucleic acid molecules is another nucleic acid point " complement " of son.As the present invention uses, when each nucleotide and another nucleic acid molecules of a nucleic acid molecules The corresponding nucleotide mutual added time, then the two nucleic acid molecules is claimed to show " complete complementarity ".If two nucleic acid molecules can be with Enough stability phase mutual crosses then claim to make them anneal and be bonded to each other under the conditions of at least conventional " low stringent " The two nucleic acid molecules are " minimum level is complementary ".Similarly, if two nucleic acid molecules can be mutual with enough stability Hybridization then claims the two nucleic acid molecules to have to make them anneal and be bonded to each other under the conditions of conventional " height is stringent " " complementarity ".Deviateing from complete complementarity can permit, as long as not exclusively to prevent two molecules from being formed double for this deviation Chain structure.In order to enable a nucleic acid molecules as primer or probe, it is only necessary to it is adequately complementary to guarantee that it has in sequence Property, so that stable duplex structure can be formed under used specific solvent and salinity.

As the present invention uses, substantially homologous sequence is one section of nucleic acid molecules, and the nucleic acid molecules are in high stringency Specific hybrid can occur with the complementary strand of another section of nucleic acid molecules to match down.Promote the suitable stringent of DNA hybridization Condition is then used under the conditions of 50 DEG C for example, about being handled under the conditions of 45 DEG C with 6.0 × sodium chloride/sodium citrate (SSC) 2.0 × SSC washing, these conditions are well known to those skilled in the art.For example, the salinity in washing step can be selected About 2.0 × SSC, 50 DEG C to high stringency of about 0.2 × SSC, 50 DEG C from Low stringency conditions.In addition, washing step In temperature condition can be increased to about 65 DEG C of high stringency from about 22 DEG C of room temperature of Low stringency conditions.Temperature strip Part and salinity can all change, can also one of them remain unchanged and another variable changes.Preferably, originally Invention a nucleic acid molecules can under moderate stringency, such as at about 2.0 × SSC and about 65 DEG C with SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, in SEQ ID NO:6 and SEQ ID NO:7 Specific hybrid occurs for any segment of one or more nucleic acid molecules or its complementary series or above-mentioned sequence.It is highly preferred that A nucleic acid molecules of the invention under high stringency with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, one or more nucleic acid molecules or its complementary series in SEQ ID NO:6 and SEQ ID NO:7, Or specific hybrid occurs for any segment of above-mentioned sequence.In the present invention, preferred marker nucleic acid molecules have SEQ ID Any segment of NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7 or its complementary series or above-mentioned sequence.

Another preferred marker nucleic acid molecules of the present invention and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or Any segment of SEQ ID NO:7 or its complementary series or above-mentioned sequence is with 80% to 100% or 90% to 100% Sequence identity.SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7 may be used as plant breeding side Marker in method is to identify the offspring of genetic cross.Probe can be this by any one with hybridizing for target dna molecule Method known to the technical staff of field detects, these methods include but is not limited to that fluorescent marker, resists radioactive label Body class label and chemiluminescent labeling.

About the amplification (for example, passing through PCR) for using specific amplimer to carry out target nucleic acid sequence, " stringent item Part " refers to the condition for only allowing primer pair target nucleic acid sequence to hybridize in the hot amplified reaction of DNA, has and target core The primer of the corresponding wild-type sequence of acid sequence (or its complementary series), can be and excellent in conjunction with the target nucleic acid sequence Choosing generates unique amplified production, amplified production, that is, amplicon.

Term " specific binding (target sequence) " refers to probe under stringent hybridization conditions or primer only and comprising target Target sequence in the sample of sequence hybridizes.

As the present invention uses, " by the DNA of amplification " or " amplicon " refer to the target as nucleic acid-templated a part The nucleic acid amplification product of nucleic acid sequence.For example, in order to determine rice plants whether by containing transgenic paddy rice event of the present invention ZUPM01 is generated by sexual hybridization mode, or whether the rice sample acquired from field includes transgenic paddy rice event Whether ZUPM01 or extracts from rice, such as coarse powder, powder or oil include transgenic paddy rice event ZUPM01, from rice plants group The DNA that tissue samples or extract extract can be generated by using the nucleic acid amplification method of primer pair for transgenic paddy rice thing The presence of the DNA of part ZUPM01 is diagnostic amplicon.The primer pair includes one in Plant Genome and inserts The first primer of the adjacent flanking sequence of the exogenous DNA insertion point entered, and the second primer of the exogenous DNA from insertion. Amplicon has certain length and sequence, and the sequence is also diagnostic to the transgenic paddy rice event ZUPM01.

The length range of amplicon can be the combination length of primer pair plus a nucleotide base pair, preferably plus about 50 nucleotide bases pair more preferably add about 250 nucleotide bases pair, most preferably add about 450 Nucleotide base to or more.

Optionally, primer pair can include entire insertion to generate from the flanking genomic sequence of the insertion two sides DNA The amplicon of nucleotide sequence.One in the primer pair of plant genome sequences can be located at away from insertion DNA sequence dna At a certain distance from, which may range from a nucleotide base to about 20,000 nucleotide bases pair.Term " amplification The use of son " has been particularly intended to exclude the primer dimer formed in the hot amplified reaction of DNA.

Nucleic acid amplification reaction can be realized by any nucleic acid amplification reaction method known in the art, including polymerization Enzyme chain reaction (PCR).Various nucleic acid amplification methods have been well-known to those skilled in the art.PCR amplification method has been sent out Open up the genomic DNA of amplifiable 22kb and the phage DNA of 42kb.Other DNA cloning sides of these methods and this field Method can be used for the present invention.The exogenous DNA array of insertion and flanking DNA sequence from transgenic paddy rice event ZUPM01 can be with By being expanded using genome of the provided primer sequence to transgenic paddy rice event ZUPM01, PCR is expanded after amplification Increase the DNA sequencing of son or the DNA progress standard of clone.

DNA detection kit based on DNA cloning method contains DNA primer molecule, they are under reaction condition appropriate On specific hybrid to target dna and expand diagnostic amplicon.Kit can provide the detection method based on Ago-Gel Or many methods of checkout and diagnosis amplicon known in the art.Containing with SEQ ID NO:3 or SEQ ID NO:4's Any part in rice genome area is homologous or complementary and any part with the transgenosis insert district of SEQ ID NO:5 The kit of homologous or complementary DNA primer is provided by the present invention.Particularly identify useful in DNA cloning method draw Object expands 5 ' transgenosis/genome with transgenic paddy rice event ZUPM01 to being SEQ ID NO:8 and SEQ ID NO:9 A part of homologous diagnostic amplicon in area, wherein amplicon includes SEQ ID NO:1.Identification has in DNA cloning method Primer pair further includes SEQ ID NO:10 and SEQ ID NO:11, and amplification turns with the 3 ' of transgenic paddy rice event ZUPM01 A part of homologous diagnostic amplicon of gene/genomic region, wherein amplicon includes SEQ ID NO:2.As DNA primer Other DNA moleculars can be selected from SEQ ID NO:5.

Amplicon caused by these methods can be detected by multiple technologies.One of method is Genetic Bit Analysis, this method devise one across the DNA of insertion DNA sequence dna and adjacent flanking genomic DNA sequence widow Nucleotide chain.The oligonucleotide chain is fixed in the micropore of a microwell plate, after carrying out PCR amplification to target area ( A primer is respectively used in insetion sequence and in adjacent flanking genomic sequence), single stranded PCR products can be with fixed few nucleosides Sour chain is hybridized, and the template as single base extension, which has used archaeal dna polymerase and be next The ddNTPs of expected base specific markers.Result can be obtained by fluorescence or ELISA class method.Signal represent insertion/ The presence of flanking sequence illustrates that amplification, hybridization and single base extension are successful.

Another method is Pyrosequencing (pyrosequencing) technology.This method devises one across insertion The oligonucleotide chain of DNA sequence dna and adjacent genomic DNA binding site.By the single-stranded of the oligonucleotide chain and target area Then and DNA PCR product (primer is respectively used in insetion sequence and in adjacent flanking genomic sequence) is hybridized, Polymerase, ATP, sulfonyl enzyme, luciferase, apyrase, adenosine -5 '-phosphorus sulfate and luciferin are together It is incubated.It is separately added into dNTPs, measures the optical signal of generation.Optical signal represents the presence of insertion/flanking sequence, says Bright amplification, hybridization and single base or polybase base extension are successful.

Fluorescence polarization also can be used for detecting amplicon of the present invention a kind of method (Chen X, Levine L, and Kwok P Y.Fluorescence polarization in homogeneous nucleic acid analysis [J] .Genome Res, 1999,9 (5): 492-8.).Need to design one in this way across insertion DNA sequence dna and phase The oligonucleotide chain of adjacent genomic DNA binding site.The single stranded PCR products of the oligonucleotide chain and target area (are being inserted Enter in sequence and respectively use in adjacent flanking genomic sequence a primer) hybridized, then with archaeal dna polymerase and one The ddNTP of kind fluorescent marker is incubated together.Single base extension will lead to insertion ddNTP.This insertion can use fluorescence Instrument measures the change of its polarization.The change of polarization represents the presence of insertion/flanking sequence, illustrates amplification, hybridization and single alkali Base extension is successful.

Taqman is described as a kind of detect and is mentioned with method existing for quantitative analysis DNA sequence dna, this method in manufacturer It is discussed in detail in the operation instruction of confession.It is now briefly illustrated below, designs one across insertion DNA sequence dna and adjacent base Because of the FRET oligonucleotide probe of group flank binding site.The FRET probe and PCR primer are (in insetion sequence and adjacent side A primer is respectively used in wing genome sequence) circular response is carried out in the presence of heat-stabilised poly synthase and dNTPs.FRET probe Hybridization lead to the division of fluorescence part and quencher moieties and the release of fluorescence part on FRET probe.The generation of fluorescence signal The presence of insertion/flanking sequence is represented, illustrates amplification and hybridization is successful.

Based on Hybridization principle, for detecting the suitable technology of the vegetable material from transgenic paddy rice event ZUPM01 also It may include Southern blot hybridization, Northern blot hybridization and in situ hybridization.Particularly, the suitable technology includes temperature Probe and sample are educated, is washed to remove whether unbonded probe and detection probe have hybridized.The detection method depends on The type of the label appended by probe for example, can detecte radiolabeled probe by X-ray exposure and imaging, or passes through Substrate conversion realizes that color change can detecte the probe of enzyme label.

(Tyagi S and Kramer F R.Molecular can also be detected to sequence using molecular labeling Beacons:probes that fluoresce upon hybridization [J] .Nat Biotechnol, 1996,14 (3): 303-8.).One is designed to visit across the FRET oligonucleotides of insertion DNA sequence dna and adjacent flanking genomic binding site Needle.The unique texture of the FRET probe causes it to contain secondary structure, which can keep fluorescence portion in short distance Point and quencher moieties.The FRET probe and PCR primer (respectively use one in insetion sequence and in adjacent flanking genomic sequence A primer) in the presence of heat-stabilised poly synthase and dNTPs carry out circular response.By successful PCR amplification, FRET probe and mesh The hybridization of mark sequence leads to the forfeiture of probe secondary structure, thus separate fluorescence part and quencher moieties spatially, Generate fluorescence signal.The generation of fluorescence signal represents the presence of insertion/flanking sequence, illustrates amplification and hybridization is success 's.

The method of other descriptions, such as the method that microfluid (microfluidics) provides separation and DNA amplification sample And equipment.Photoinitiator dye is for detecting and measuring specific DNA molecular.Include the electronic sensor or knot for detecting DNA molecular Close specific DNA molecular receive pearl and thus can be detected receive test tube (nanotube) equipment for detecting DNA of the invention points Son is useful.

Method that composition and DNA detection field of the present invention describes or known can be used to develop DNA inspection Test agent box.The kit is conducive to identify the DNA that whether there is transgenic paddy rice event ZUPM01 in sample, can also use In the rice plants for cultivating the DNA containing transgenic paddy rice event ZUPM01.The kit can contain DNA primer or spy Needle at least part for being derived from or being complementary to SEQ ID NO:1,2,3,4 or 5, or contains other DNA primers or probe, , with being derived from or being complementary to DNA contained in the genetically modified element of DNA, it is anti-that these DNA sequence dnas can be used for DNA cloning for it It answers, or as the probe in DNA hybridization method.Transgenosis that is containing in rice genome and illustrating in Fig. 1 and table 1 The DNA structure of insetion sequence and rice genome binding site includes: being located at the rice of 5 ' end of transgene insert sequence ZUPM01 left side flap genome area, a part of insetion sequence of the left boundary area (LB) from Agrobacterium, first table Up to box by maize ubiquitin protein gene promoter (ubiquitin promoter), it is operably connected to the conjunction of maize acetyl lactic acid At on the signal peptide sequence (AHAS) of enzyme gene, it is operably connected to the 5- alkene of the glyphosate tolerant in radioresistant cocci On alcohol-pyruvoyl shikimic acid -3- phosphate synthase gene (G9A), and the 35S for being operably connected to cauliflower mosaic virus is terminated It is formed on sub- rouge alkali synthetase gene terminator (CaMV 35S terminator), second expression cassette is spent by cauliflower The 35S promoter maize ubiquitin protein gene promoter (355 promoter of CaMV) of mosaic virus, is operably connected to rice Phosphate cotransporter body is assisted in transport factor sequence (OsPHF1), and is operably connected to rouge alkali synthetase gene terminator It is formed, a part of insetion sequence in the right side boundary region (RB) from Agrobacterium, Yi Jiwei on (no terminator) Rice plants ZUPM01 right side flap genome area (SEQ ID NO:5) in 3 ' end of transgene insert sequence.In DNA cloning In method, the DNA molecular as primer can be appointing from transgenic paddy rice event ZUPM01 transgenic insetion sequence What part is also possible to any part from the region of DNA domain of flank rice genome in transgenic paddy rice event ZUPM01.

Transgenic paddy rice event ZUPM01 can be combined with other Transgenic Rices, such as herbicide (such as glufosinate-ammonium, Dicamba etc.) tolerance rice, or carry anti insect gene Transgenic Rice.All these difference transgenic events Various combinations, the breeding together with transgenic paddy rice event ZUPM01 of the invention, can provide pest-resistant and resist a variety of herbicides simultaneously It is resistant to the improvement hybrid transgenic rice varieties of low-phosphorus stress.These kinds turn base compared to non-transgenic kind and unisexuality shape Because kind can show the superior features such as yield promotion.

The present invention provides a kind of for detecting the nucleic acid sequence and its detection method of rice plants, transgenic paddy rice event ZUPM01 has the function of being resistant to agriculture herbicide and/or low-phosphorus stress containing glyphosate.The rice plant expression of the character is resistance to Radioresistant cocci 5- enol-pyrovyl shikimic acid -3- phosphate synthase (G9A) albumen and rice phosphate of glyphosate resistance are transported Body assists transport factor protein (OsPHF1), assigns plant to the tolerance of glyphosate and low-phosphorus stress.Present invention inspection simultaneously Sequence in survey method comprising SEQ ID NO:1 or its complementary series, the sequence comprising SEQ ID NO:2 or its complementary series, SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series can be used as DNA primer or probe to produce It is raw to be diagnosed as transgenic paddy rice event ZUPM01 or the amplified production of its offspring, and can be quick, accurate, stable identify Derived from the presence of the vegetable material of transgenic paddy rice event ZUPM01.

BRIEF DESCRIPTION OF THE SEQUENCES:

5 ' transgenic insert locus left side flap oryza sativa genomic dnas in SEQ ID NO:1 transgenic paddy rice event ZUPM01 With each 11 nucleotide sequences of transgenic fragment left margin；

3 ' transgenic insert locus transgenic fragment right margins and the right side in SEQ ID NO:2 transgenic paddy rice event ZUPM01 Each 11 nucleotide sequences of flank oryza sativa genomic dna；

It is attached positioned at insertion junction in 5 ' ends of insetion sequence in SEQ ID NO:3 transgenic paddy rice event ZUPM01 A close length is the sequence of 732 nucleotide；

It is attached positioned at insertion junction in 3 ' ends of insetion sequence in SEQ ID NO:4 transgenic paddy rice event ZUPM01 A close length is the sequence of 913 nucleotide；

5 ' left side flap Rice Genome Sequence of SEQ ID NO:5, entire T-DNA sequence and 3 ' right side flap rice genomes Sequence；

SEQ ID NO:6 is located at the sequence inside SEQ ID NO:3, span 5 ' left side flap Rice Genome Sequences, PPHF1G9A-1300 construct left margin DNA sequence dna and CaMV 35S terminator sequence；

SEQ ID NO:7 is located at the sequence inside SEQ ID NO:4, spans no transcription terminator, pPHF1G9A- 1300 construct right margin DNA sequence dnas and 3 ' right side flap Rice Genome Sequences；

The first primer of SEQ ID NO:8 amplification SEQ ID NO:6；

The second primer of SEQ ID NO:9 amplification SEQ ID NO:6；

The first primer of SEQ ID NO:10 amplification SEQ ID NO:7；

The second primer of SEQ ID NO:11 amplification SEQ ID NO:7；

The first primer of SEQ ID NO:12 PCR detection G9A；

The second primer of SEQ ID NO:13 PCR detection G9A；

The first primer of SEQ ID NO:14 PCR detection OsPHF1；

The second primer of SEQ ID NO:15 PCR detection OsPHF1；

The primer of SEQ ID NO:16 acquisition right margin flanking sequence；

The primer of SEQ ID NO:17 acquisition right margin flanking sequence；

The primer of SEQ ID NO:18 acquisition right margin flanking sequence；

The primer of SEQ ID NO:19 acquisition right margin flanking sequence；

The primer of SEQ ID NO:20 acquisition right margin flanking sequence；

The primer of SEQ ID NO:21 acquisition right margin flanking sequence；

The primer of SEQ ID NO:22 acquisition right margin flanking sequence；

The primer of SEQ ID NO:23 acquisition right margin flanking sequence；

The primer of SEQ ID NO:24 acquisition right margin flanking sequence；

The primer of SEQ ID NO:25 acquisition right margin flanking sequence；

The primer of SEQ ID NO:26 acquisition right margin flanking sequence；

The primer of SEQ ID NO:27 acquisition right margin flanking sequence；

The primer of SEQ ID NO:28 acquisition right margin flanking sequence；

The primer of SEQ ID NO:29 acquisition right margin flanking sequence；

Probe in SEQ ID NO:30 Southern hybridization check；

Probe in SEQ ID NO:31 Southern hybridization check；

Below by drawings and examples, technical scheme of the present invention will be described in further detail.

Detailed description of the invention

The structural schematic diagram of Fig. 1 transgene insert sequence and rice genome binding site.

The physical map of Fig. 2 recombinant expression carrier pPHF1G9A-1300.Each element English and abbreviation meaning are listed below:

The 35S promoter of CaMV 35S promoter cauliflower mosaic virus (CaMV).

OsPHF1 encodes phosphate transporter and assists the transport factor, promote rice to the absorption of phosphorus and Transhipment.

The terminator of no terminator rouge alkali synthetase gene.

The T-DNA right border sequence of T-Border (right) Agrobacterium C58, needed for T-DNA transfer.

The plasmid stabilisation site of pVS1 sta pVS1 plasmid.

The replication origin of pVS1 rep pVS1 plasmid.

The site bom of pBR322 bom pBR322 plasmid migrates the action site of protein mop.

The replication origin of pBR322 ori pBR322 plasmid.

Kanamycin (R) encodes aminoglycoside phosphotransferase albumen, assigns bacterium kalamycin resistance.

The T-DNA left margin sequence of T-Border (left) Agrobacterium C58, needed for T-DNA transfer.

The 35S terminator of CaMV 35S terminator cauliflower mosaic virus (CaMV).

G9A is originated from radioresistant cocci, encodes EPSPS albumen, generates glyphosate resistance.

The signal peptide sequence of AHAS maize acetyl lactic acid synthase gene.

The promoter of ubiquitin promoter Maize Ubiquitin gene.

The insertion copy number Southern marking of Fig. 3 ZUPM01 target gene OsPHF1 hybridizes figure.A:OsPHF1 probe is miscellaneous Intersection graph；The area B:T-DNA restriction enzyme site and probe location schematic diagram, the stripe size after digital representation digestion, unit kb are used Probe location is labeled in lower part with line segment.Swimming lane 1-25 respectively indicates different DNA samples.1,7,13,19,20:DNA Marker, stripe size are labeled in the leftmost side, unit kb；2,8,14,21: blank；3,9,22: Plasmid DNA；4,10,15,16, 23: 9311 genomic DNA of non-transgenic；5,11,17,24:ZUPM01 T₅Genomic DNA；6,12,18,25:ZUPM01 T₆Base Because of a group DNA；3-6:EcoRI digestion；9-12:PvuII digestion；15-18:NheI digestion；22-25:HindIII digestion.

The insertion copy number Southern marking of Fig. 4 ZUPM01 target gene G9A hybridizes figure.A:G9A probe hybridization figure； The area B:T-DNA restriction enzyme site and probe location schematic diagram, the stripe size after digital representation digestion, unit kb, probe used Position is labeled in lower part with line segment.Swimming lane 1-18 respectively indicates different DNA samples.1,7,13:DNA Marker, stripe size It is labeled in the leftmost side, unit kb；2,8,14: blank；3,9,15: Plasmid DNA；4,10,16: 9311 genomic DNA of non-transgenic； 5,11,17:ZUPM01 T₅Genomic DNA；6,12,18:ZUPM01 T₆Genomic DNA；3-6:NheI digestion；9-12: HindIII digestion；15-18:SacI digestion.

The testing result of Fig. 5 transformation event ZUPM01.A: left margin detection, the expected size 682bp of target stripe；B: the right Boundary's detection, the expected size 835bp of target stripe；M: molecular weight standard, be followed successively by from top to bottom 2kb, 1kb, 750bp, 500bp, 250bp,100bp；N: negative control wild type 9311；P: negative control pPHF1G9A-1300 plasmid；W: water；T₅、T₆: table respectively Show T₅And T₆For transformation event；

Specific embodiment

This application involves transformation event ZUPM01 refer to rice self-mating system 9311 be receptor obtained by genetic transformation The rice plant of foreign gene insert (T-DNA insert) is inserted between specific gene group sequence.In a particular embodiment, Transgenosis expression carrier used thereof has physical map shown in FIG. 1, and obtained T-DNA insert is with SEQ ID NO:5's Sequence shown in 487-6866 nucleotide.Transformation event ZUPM01 can refer to this transgenic protocol, can also refer to by this The combination of T-DNA insert or T-DNA insert and flanking sequence in the obtained genome of process, or can refer to by this The rice plant that one transgenic protocol obtains.In specific example, which is also applied for same expression vector and converts other Receptor kind, thus the plant that T-DNA insert is inserted into same genomic locations and is obtained.Transformation event ZUPM01 may be used also With refer to as above-mentioned plant carry out vegetative propagation, sexual propagation, it is double-diminished or double breeding or above combination obtained from offspring plant Object.

The building of 1 conversion carrier of embodiment

Skeleton carrier used in the present invention is the binary Ti vectors p1300-UBQAHAS- after pCAMBIA1300 vector modification 1174s, carrier have included G9A expression casette.OsPHF1 expression casette is got on by the building of In-Fusion technology.According to The rice phosphate transporter that the website TIGR provides (http://www.tigr.org/) transports helper factor 1 (OsPHF1) gene CDNA sequence, design primer, using the root cDNA of japonica rice OryzasativaLcv.Nipponbare (Nipponbare) as template, amplification obtain OsPHF1 base Behind the code area of cause, it is cloned into pMD-19 carrier (35S:PHF1:NOS/CPB), using the carrier as template, includes using both ends The primer amplification of HindIII restriction enzyme site obtains 35S:PHF1:NOS expression cassette product of the both ends with HindIII restriction enzyme site.So P1300-UBQAHAS-1174s carrier and 35S:PHF1:NOS product are used into HindIII digestion afterwards, and product is put in In- Escherichia coli are converted after 42 DEG C of incubation 30min in Fusion reaction solution, the monoclonal crossed by PCR and digestion verification is selected, obtains To the expression vector for being named as pPHF1G9A-1300.

Resulting vehicle size is 12.684kb, and carrier physical map is shown in Fig. 1.

2 rice transformation of embodiment

Destination gene expression carrier is transferred to rice using agrobacterium-mediated transformation by the present invention, and agrobacterium strains used are EHA105.Then callus induction after rice paddy seed disinfection co-cultures to infect callus with Agrobacterium, cultivates screening quilt by selection The callus of conversion carries out plant regeneration on Selective agar medium later.Detailed process is as follows:

1) inducing paddy rice callus: taking Mature seed of rice, and full bright and clean no bacterial plaque is selected in artificial or mechanical dejacketing Seed, be put into 100ml sterile beaker, pour into 70% alcohol (15ml) disinfection 2min；Alcohol is removed, 100ml 30% is added Sodium hypochlorite (NaClO) solution impregnates 30min；Liquor natrii hypochloritis is gone, cleans seed 4-5 times with sterile distilled water, finally A time immersion 30min.Seed is placed on aseptic filter paper and is blotted, is placed in mature embryo induced medium, 20-30, every ware；Behaviour It finishes with sealed membrane (Micropore^TMSurgical Tape) culture dish is sealed, in 28 DEG C of illumination box cultures.Secretly training Evoked callus under the conditions of supporting, needs 7~10 days；Culture dish is opened on superclean bench, is divided naturally with tweezers picking Embryo callus (faint yellow, densification in spherical), be placed in subculture medium, in 28 DEG C of dark cultures, 1 week subculture.

2) Agrobacterium is cultivated: picking conversion has the Agrobacterium monoclonal of purpose expression vector in 15ml YEP culture solution (containing corresponding antibiotic), 28 DEG C, 12~16h of 250rpm shaken cultivation to bacterium solution OD₆₀₀For 0.8-1.0.

3) co-culture the selection with resistant calli: at room temperature by cultured bacterium solution, 4000rpm is centrifuged 10min, Remove supernatant.The Rice Callus for growing to a certain size is chosen, agrobacterium suspension is put into, 80rpm is trained altogether on horizontal shaker Support 30min；Callus is taken out, is placed on sterile filter paper and drains 30-40min；By callus be placed in one it is sterile On the co-cultivation base of filter paper, 25 DEG C dark culture 3 days.

4) selection culture: callus take out, with sterile water wash 5-6 times, need therebetween not failure of oscillation swing.Again with containing 300mg/ The sterile water wash of L carbapen (Carb) 2 times, rocks 30min on horizontal shaker every time, is finally placed in aseptic filter paper On drain 2 hours.The callus dried is transferred to the selection training of carbapen containing 300mg/L (Carb) and corresponding screening pressure Support base on, 28 DEG C dark culture 14 days, altogether carry out two-wheeled selection, until the resistant calli of graininess is grown.

5) induction of resistant calli is broken up and takes root: the kanamycin-resistant callus tissue 3- for the color cadmium yellow that picking comes from different callus It 5, moves into the plastic jar equipped with differential medium, seals with sealing film, be put into (16h/ in constant temperature (25 DEG C) culturing room 8h), seedling differentiation (about 40 days) are waited.To seedling length to 3cm or so, old root and callus are cut off from seedling base portion with scissors, is put Enter strong sprout in root media (about 1 week).

6) acclimatization and transplants: breaking up intact test tube for seedling root and cauline leaf and choose, and opens sealed membrane, appropriate distilled water is added Or sterile water (preventing the long bacterium of culture medium), hardening 2~3 days, agar is then washed away, for transplanted seedling into greenhouse soil alms bowl, detection turns base Because of the plant positive.

The screening of 3 transformant of embodiment

(1) target gene Molecular Detection is carried out for transformation seedlings to 110 T0 that conversion obtains.It is set according to two gene orders Two PCR primers pair are counted, first primer pair primer sequence is respectively SEQ ID NO:12 and SEQ ID NO:13, and second is drawn Object is respectively SEQ ID NO:14 and SEQ ID NO:15 to primer sequence.Take conversion seedling leaf extract genomic DNA, according to Lower PCR parameter is expanded:

Reaction system:

Response procedures:

Two target gene amplified fragments sizes of positive transformant compare the amplified fragments size one of PCR with positive plasmid It causes, respectively 600bp and 296bp, harvests the T of 77 positive single plants₁Seed.

(2) to T₁-T₃For plant through herbicide screening, the screenings such as segregation ratio is investigated obtain number be OsPHF1-9311-1, 4 strains of OsPHF1-9311-2, OsPHF1-9311-3, OsPHF1-9311-4.

(3) in field to T₄-T₅For OsPHF1-9311-1, OsPHF1-9311-2, OsPHF1-9311-3, OsPHF1- The preliminary economical character of 9311-4 transformant is identified, determines that OsPHF1-9311-1 (i.e. ZUPM01) Comprehensive Traits are prominent.

In terms of yield traits, ZUPM01 and 9311 is in plant height, effective fringe, spike length, total grain number, bear fruit grains, setting percentage, single plant Without significant difference (p > 0.05) (table 2) in terms of yield, dry matter, mass of 1000 kernel.

2 ZUPM01 yield of table and species test character investigation result

Numerical value is indicated with the duplicate mean+SD of 3 secondary pollutants, using each character between LSD method analysis different materials The significance of difference (α=0.05).

The flanking sequence of 4 transformation event ZUPM01 exogenous array of embodiment and rice genome insertion position

The primer (SEQ ID NO:16-SEQ ID NO:29) for designing Flanking sequence isolation, simultaneously using FPNI-PCR amplification The method of sequencing obtains the right side flap sequence (SEQ ID NO:5 6867-7306) of transformant ZUPM01, and according to gene Group sequence is sequenced to obtain left side flap sequence (SEQ ID NO:5 1-486) by PCR amplification.In PlantGDB database In (http://www.plantgdb.org/OsGDB/cgi-bin/blastGDB.pl) with BLASTN tool by flanking sequence with Rice Genome Sequence carries out homologous comparison analysis, is reference sequences with MSU 7.Transformant ZUPM01 is inserted into rice known to analysis At the position genome C hr06:2736689-2736704.

The operating procedure of FPNI-PCR is as follows:

1) oryza sativa genomic dna is extracted.

2) template reacted using the genomic DNA in step 1) as first round PCR, reaction system are as follows:

Response procedures are as follows:

95 DEG C, 2.5min；(94 DEG C, 10sec；62 DEG C, 30sec；72 DEG C, 2min) × 2 circulations；

94 DEG C, 10sec；

25 DEG C, 2min；

72 DEG C (5.1%ramp), 2min；

[94 DEG C, 10sec；62 DEG C, 30sec；72 DEG C, 2min；94 DEG C, 10sec；62 DEG C, 30sec；72 DEG C, 2min；94 DEG C, 10sec；44 DEG C, 30sec；72 DEG C, 2min；]

× 5 circulations；

72 DEG C, 5min；

25 DEG C, 10min.

3) the second wheel PCR amplification is carried out by template of first round PCR product corresponding in step 2).

Reaction system is as follows:

Response procedures are as follows:

95 DEG C, 1min 30sec；

[94 DEG C, 10sec；62 DEG C, 30sec；72 DEG C, 2min] × 30 circulations；

72 DEG C, 7min；

25 DEG C, 10min.

It 4) is that template carries out third round PCR amplification with the second wheel PCR product (50 times of dilution) corresponding in step 3).

Reaction system is as follows:

Response procedures are as follows:

95 DEG C, 1min 30sec；

[94 DEG C, 10sec；62 DEG C, 30sec；72 DEG C, 2min] × 30 circulations；

72 DEG C, 7min；

25 DEG C, 10min.

5) product of third round PCR electrophoresis detection in 1% (w/v) 1 × TAE Ago-Gel is taken, 250bp or more is recycled DNA fragmentation.

6) segment of recycling is connected into carrier T, 16 DEG C of connections overnight.

7) connection product in conversion 6).

8) picking positive colony shakes bacterium upgrading grain, and plasmid is sent to be sequenced.

9) sequencing result uses BLASTN tool in PlantGDB database, using MSU database as reference gene group, with water Rice genome sequence carries out homologous comparison, using best matching result as insertion point.

The overall length insetion sequence for expanding and being sequenced ZUPM01 is further segmented by over-lap PCR.Transformant ZUPM01 is practical Insetion sequence and left and right flank Rice Genome Sequence are as shown in SEQ ID NO:5.

3 Flanking sequence isolation the primer information of table

1: unit bp.

The copy number of 5 transformation event ZUPM01 of embodiment detects

The copy number of foreign gene insertion is determined using the method that the Southern marking hybridizes.

The insertion copy number hybridization check of OsPHF1 genetic fragment chooses tetra- kinds of EcoRI, PvuII, NheI and HindIII limits Property restriction endonuclease difference digestion positive control pPHF1G9A-1300 plasmid, 9311 genomic DNA of negative control wild type and difference processed (T from generation to generation₅And T₆) ZUPM01 transformant genomic DNA.It is marked after running glue transferring film with OsPHF1 gene probe (SEQ ID NO:30) Note.Results of hybridization is as shown in Fig. 3 A.The probe location and restriction enzyme EcoRI, PvuII, NheI of target gene OsPHF1 It is as shown in Figure 3B with the restriction enzyme site of HindIII.

OsPHF1 gene is rice endogenous gene, EcoRI, PvuII, NheI and HindIII digestion negative control 9311 Postgenome hybridizes with label probe and obtains 1 band, and wherein EcoRI digestion can mark 1.5kb band (swimming lane 4), PvuII digestion can mark 2.3kb band (swimming lane 10), and NheI digestion can mark 9.4kb band (swimming lane 15,16), HindIII digestion can mark 15kb band (swimming lane 23).It can be seen that OsPHF gene has a copy in non-transgenic rice. Meanwhile after the tri- kinds of digestion with restriction enzyme labels of EcoRI, PvuII, NheI and HindIII of negative control wild type 9311 The band in addition to endogenous gene is not seen, (swimming lane 2,8,14,21) is shown also without hybridization shaping band in blank control The specificity of hybridization probe.

EcoRI the area T-DNA restriction enzyme site only one, positioned at the left side of OsPHF1 gene probe, by digestion The slug band obtained after ZUPM01 transformant genomic DNA and specific probe hybridization should include the T-DNA sequence of 4.0kb And its unknown sequence of size on the genome of right side, whole fragment length are greater than 4.0kb.Two hybridization items are marked in experiment Band, wherein a 1.5kb band gone out for endogenous OsPHF1 genetic marker, the stripe size that external source OsPHF1 genetic marker goes out is about For 9.4kb (swimming lane 5,6), meet expection.Meanwhile EcoRI does not have restriction enzyme site, therefore positive control marker in carrier framework area Stripe size out is identical as plasmid size, is 12.7kb (swimming lane 3).

PvuII the area T-DNA restriction enzyme site also only one, positioned at the right side of OsPHF1 gene probe, by digestion ZUPM01 transformant genomic DNA and specific probe hybridization after the slug band that obtains should include 6.4kb T-DNA sequence The unknown sequence of size on column and its left side genome, whole fragment length are greater than 6.4kb.Two hybridization items are marked in experiment Band, wherein a 2.3kb band gone out for endogenous OsPHF1 genetic marker, the stripe size that external source OsPHF1 genetic marker goes out is about For 9.3kb (swimming lane 11,12), meet expection.Meanwhile PvuII does not have restriction enzyme site, therefore positive control mark in carrier framework area Remember that stripe size out is identical as plasmid size, is 12.7kb (swimming lane 9).

NheI the area T-DNA restriction enzyme site only one, positioned at the left side of OsPHF1 gene probe, by digestion The slug band obtained after ZUPM01 genomic DNA and specific probe hybridization should include T-DNA sequence and its right side of 3.7kb The unknown sequence of size on the genome of side, whole fragment length are greater than 3.7kb.Two hybridising bands are marked in experiment, wherein One 9.4kb band gone out for endogenous OsPHF1 genetic marker, the stripe size that external source OsPHF1 genetic marker goes out is about 4.3kb (swimming lane 17,18), meets expection.

HindIII is located at OsPHF1 gene probe two sides, can cut the area T-DNA there are two the restriction enzyme sites in the area T-DNA The segment of 2.3kb fixed size out.Two hybridising bands are marked in experiment, wherein one goes out for endogenous OsPHF1 genetic marker 15kb band, the stripe size that external source OsPHF1 genetic marker goes out is 2.3kb (swimming lane 24,25), and with positive control plasmid The stripe size of digestion label is identical (swimming lane 22), show the band of probe label really in the area T-DNA, and OsPHF1 gene There is no recombinations for inside.

The insertion copy number hybridization check of G9A genetic fragment chooses tri- kinds of restriction enzymes of NheI, HindIII and SacI Digestion blank control, positive control pPHF1G9A-1300 plasmid, 9311 genomic DNA of negative control wild type and difference respectively Generation ZUPM01 transformant genomic DNA.It is marked after running glue transferring film with G9A gene probe (SEQ ID NO:31).Results of hybridization As shown in Fig. 4 A.The restriction enzyme site of the probe location of target gene G9A and restriction enzyme NheI, HindIII and SacI are such as Shown in Fig. 4 B.

Restriction enzyme site of the NheI in the area T-DNA has one, positioned at the right side of G9A gene probe, by the ZUPM01 of digestion The slug band obtained after transformant genomic DNA and specific probe hybridization should include T-DNA sequence and its a left side of 2.7kb The unknown sequence of size on the genome of side, whole fragment length are greater than 2.7kb.Single hybridising band is marked in experiment, greatly It is small about 4.3kb (swimming lane 5,6), meet expection.Meanwhile NheI has a restriction enzyme site in carrier framework area, From Left At the position boundary treaty 3.2kb, therefore the stripe size that positive control marker goes out is 6.0kb (swimming lane 3).

HindIII is all located at the right side of G9A gene probe, by digestion there are two the restriction enzyme sites in the area T-DNA The slug band obtained after ZUPM01 transformant genomic DNA and specific probe hybridization should include the T-DNA sequence of 3.8kb And its unknown sequence of size on the genome of left side, whole fragment length are greater than 3.8kb.Single hybridization item is marked in experiment Band, size are about 7.0kb (swimming lane 11,12), meet expection.Meanwhile HindIII does not have restriction enzyme site in carrier framework area, because The stripe size that this positive control marker goes out is 10.4kb (swimming lane 9).

SacI is there are four the restriction enzyme sites in the area T-DNA, wherein the site for being located at G9A gene probe two sides can be T-DNA Area cuts out the segment of 4.6kb fixed size.It is about 4.6kb (swimming lane 17,18) that single hybridising band is marked in experiment, with Positive control plasmid digestion label stripe size it is identical (swimming lane 15), show probe label band really in the area T-DNA, And there is no recombinations for G9A gene internal.Meanwhile tri- kinds of NheI, the HindIII and SacI limitations of negative control wild type 9311 Property endonuclease digestion label after all do not see band (swimming lane 4,10,16), blank control do not see yet band (swimming lane 2,8, 14) specificity of hybridization probe, is shown.

The experimental results showed that, ZUPM01 transformant contains external source OsPHF1 and the G9A genetic fragment singly copied above.

Tolerance of the 6 transformation event ZUPM01 of embodiment to glyphosate herbicidal

Under the field conditions for meeting insulation request, transformant is measured to herbicide by the method for artificial spraying's herbicide The tolerance of glyphosate specifies the validity of transformant herbicide-resistant objective trait.Test setting repeats three times, every cell 1m²(1m × 1m), minizone is every 0.5m.Transgenic paddy rice is arranged in test process, and herbicide spraying, transgenic paddy rice do not spray mesh Mark herbicide, herbicide spraying, corresponding non-transgenic rice do not spray target herbicide to corresponding non-transgenic rice.Weeding Agent spraying concentration is respectively to spray 1 times (1 ×, 200mL/ mus or 82g/ mus) measuring in clear water (0 ×) and field recommended dose, 2 Times (2 ×, 400mL/ mus or 164g/ mus) and 4 times (4 ×, 800mL/ mus or 328g/ mus).

Target herbicide glyphosate spray after respectively 1 week after medication, 2 weeks investigation field run plant numbers, investigate within 4 weeks after medication Field run plant and phytotoxicity seedling number (2~5 grades).After medication at 1 week, 9311 and the transformant plant for spraying clear water control can be normal Survival, but it is weedy；The most of plant for spraying the receptor control 9311 of glyphosate is dead, even if there is small part incomplete Chlorosis blade, lobus cardiacus also dead；And the transformant ZUPM01 for spraying glyphosate various dose can be with normal growth.2 after medication Week and the weeds of 9311 and the transformant plant that after 4 weeks, spray clear water control are more luxuriant, have seriously affected the normal life of rice It is long；Spray all withered death of receptor control 9311 of glyphosate；And spray the transformant ZUPM01 of glyphosate various dose It still can be with normal growth.

1 week after medication, the phytotoxicity investigation result of transformant ZUPM01 was shown in 2 weeks, 4 weeks, at each dosage herbicide Under reason, do not occur phytotoxicity, phytotoxicity rate is 0%, and the seedling (table 4) without there are 4 grades and 5 grades phytotoxicity.According to rice to weeding The criterion of agent tolerance grade can determine that transformant ZUPM01 is excellent to the tolerance grade of target herbicide glyphosate It is elegant.

Tolerance sex expression of 4 ZUPM01 of table to glyphosate

Dosage is indicated with the multiple measured in the recommended dose of herbicide field.Numerical value with the duplicate average value of 3 secondary pollutants ± Standard deviation indicates that statistical analysis is examined using LSD method.

The result shows that receptor control 9311 is not resistant to the glyphosate measured in recommended dose, and transformant ZUPM01 is resistant to By the glyphosate measured at least 4 times of recommended doses, " outstanding " rank is reached to the tolerance of target herbicide, has and preferably pushes away Wide value.

Tolerance of the 7 transformation event ZUPM01 of embodiment to soil low-phosphorus stress

Under the natural conditions of field, if 4 processing, are divided into without phosphorus, low-phosphorous, middle phosphorus and high phosphorus, at many years of this laboratory The field for being practically free of P elements of reason is tested, and the fertilizer applications respectively handled are as follows:

Without phosphorus processing (NP): 0kg/ mus of calcium superphosphate, 30kg/ mus of urea, 20kg/ mus of potassium chloride；

Low-phosphorous processing (LP): 7.5kg/ mus of calcium superphosphate, 30kg/ mus of urea, 20kg/ mus of potassium chloride；

Middle phosphorus handles (MP): 15kg/ mus of calcium superphosphate, 30kg/ mus of urea, 20kg/ mus of potassium chloride；

High phosphorus handles (HP): 30kg/ mus of calcium superphosphate, 30kg/ mus of urea, 20kg/ mus of potassium chloride.

The dry matter weight and phosphorus content for measuring cauline leaf in tillering regularity, boot stage and maturity period (wax ripeness latter stage) respectively, Maturity period measures the dry matter weight and phosphorus content of sword-like leave, fringe stalk, husk and brown rice, and is calculated according to dry matter weight and phosphorus content The cumulant of phosphorus.

Phosphorus nutrient efficiency is made of absorption efficiency, utilization efficiency and transfer efficiency three parts.This project is with plant above ground portion The cumulant of phosphorus measures plant phosphorus absorption efficiency；Unit phosphorus uptake amount of dry matter produced is defined as phosphorus in plant body Utilization efficiency；The percentage that genital area phosphorus accounts for its overground part total phosphorus content after harvest is defined as the transfer efficiency of phosphorus.

Maturity period is converted to per mu yield to processing cell harvest sunning weighing.

After carrying out phosphate fertilizer processing, the full phosphorus in experimental plot, organic matter, available phosphorus and pH value etc. are had detected, the results showed that, nothing Full phosphorus and available phosphorus content are in significant ascendant trend (table 5) in phosphorus, low-phosphorous, middle phosphorus and high phosphorus processing, show that test process is effective, Meet expection.

5 soil physico-chemical property of table

1: unit " mg/kg " as a result derives from 3 duplicate mean values.

The dry matter weight and phosphorus content of sword-like leave, fringe stalk, husk and brown rice are measured in the maturity period, and according to dry matter weight and Phosphorus content calculates the cumulant of phosphorus, and phosphorus absorption, utilization and transfer efficiency has been calculated, the results are shown in Table 6.

The absorption of 6 ZUPM01 P elements of table, utilization and transfer efficiency

Numerical value indicates with the duplicate mean+SD of 3 secondary pollutants, statistical analysis examined using LSD method (α= 0.05).Letter indicates the significance of difference of data between different disposal before "/", and letter indicates the difference of data between different materials after "/" Different conspicuousness." NP, LP, MP, HP " respectively represents without phosphorus processing, low-phosphorous processing, the processing of middle phosphorus and high phosphorus processing.

The phosphorus of receptor 9 311 absorbs, utilization and transfer efficiency enhance as soil phosphorus levels increase, and transformant Phosphorus of the ZUPM01 under different phosphorus concentration treatment conditions absorbs, utilization and transfer efficiency are without significant difference.At without phosphorus and low-phosphorous place When reason, the phosphorus of ZUMP01 absorbs, utilization and transfer efficiency are significantly higher than receptor 9 311, and when middle phosphorus and high phosphorus are handled, The phosphorus of ZUPM01 and receptor 9 311 absorbs, there was no significant difference for utilization and transfer efficiency.

The yield under the processing of 4 phosphorus concentrations is investigated, the results are shown in Table 7, compares 9311 yield as soil phophorus is dense The raising of degree is continuously increased, and shows its sensibility to phosphorus processing；And transformant volume variance under the processing of different phosphorus concentrations Not significant, the tolerance for lacking stress to phosphorus is stronger.ZUPM01 transformant yield under without phosphorus and low-phosphorous processing is respectively 597.5 ± 31.8 and 609.7 ± 35.6kg/ mus, it is significantly higher than 9311 (485.4 ± 47.71 and 497.6 ± 56.2kg/ mus), turns Changing body yield under middle phosphorus and high phosphorus processing is respectively 719.1 ± 189.1kg/ mus and 633.1 ± 125.5kg/ mus, and compares nothing Significant difference.

The yield that the different phosphorus concentrations of table 7 handle lower transformant shows (kg/ mus)

Numerical value indicates with the duplicate mean+SD of 3 secondary pollutants, statistical analysis examined using LSD method (α= 0.05).Letter indicates the significance of difference of data under different disposal before "/", and letter indicates the difference of data between different materials after "/" Different conspicuousness." NP, LP, MP, HP " respectively represents without phosphorus, low-phosphorous, middle phosphorus and high phosphorus processing.

The above result shows that the expression of target gene OsPHF1 can enhance rice without phosphorus and low in transformant ZUPM01 Phosphorus nutrient efficiency under the conditions of P deficiency increases rice yield so as to improve the physiological character of rice.

The detection method of 8 transformation event ZUPM01 of embodiment

Such as agricultural product or commodity can be produced by transgenic paddy rice event ZUPM01.If in the agricultural product or commodity Detect enough expression quantity, agricultural product or commodity are expected exists containing can diagnose transgenic paddy rice event ZUPM01 material Nucleotide sequence present in the agricultural product or commodity.The agricultural product or commodity include but is not limited to rice flour, rice bran oil, rice Chaff, rice embryo, rice gluten, rice starch, Rice Bran oil or rice bran polysaccharide and will be as food source for any of animal consumption Other food or additionally as the ingredient in swelling agent or make-up composition for cosmetic use etc..Based on probe or primer Pair nucleic acid detection method and/or kit can be developed to detect such as SEQ ID NO:1 or SEQ ID in biological sample Transgenic paddy rice event ZUPM01 nucleotide sequence shown in NO:2, wherein probe sequence or primer sequence are selected from such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 Shown in sequence, to diagnose the presence of transgenic paddy rice event ZUPM01.

One of detection method are as follows: using PCR method to ZUPM01 two generations (T₅And T₆) specific boundary sequence in plant Column are detected, and PCR primer used is to respectively SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11, PCR reaction system:

Response procedures are as follows:

94 DEG C, 5min；(94 DEG C, 30sec；55 DEG C, 30sec；72 DEG C, 1.0min) × 35 circulations；72 DEG C, 7min；4 DEG C, 5min。

PCR product electrophoresis detection in 1% (w/v) 1 × TAE Ago-Gel is taken, as a result sees Fig. 5.It can in transformation event The set goal band is obtained with amplification, but other non-sourcing cannot then amplify target stripe in the sample of transformation event.

In conclusion transgenic paddy rice event ZUPM01 of the present invention is with higher to glyphosate herbicidal and low-phosphorus stress Tolerance, and whether it includes transgenic paddy rice event ZUPM01's that detection method can be identified quickly and accurately in biological sample DNA molecular.

It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although ginseng It is described the invention in detail according to preferred embodiment, those skilled in the art should understand that, it can be to the present invention Technical solution be modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. a kind of nucleic acid sequence, which is characterized in that the nucleic acid sequence include SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or its complementary series, the nucleic acid sequence are originated from transgenic paddy rice event ZUPM01.

2. nucleic acid sequence according to claim 1, which is characterized in that the nucleic acid sequence include SEQ ID NO:3 or its Complementary series, and/or SEQ ID NO:4 or its complementary series.

3. nucleic acid sequence according to claim 2, which is characterized in that the nucleic acid sequence include SEQ ID NO:5 or its Complementary series.

4. a kind of method existing for DNA of test sample transgenic rice event ZUPM01 characterized by comprising

Carry out nucleic acid amplification reaction；

Detect the presence of amplified production；

The amplified production is originated from transgenic paddy rice event ZUPM01.

5. method existing for the DNA of test sample transgenic rice event ZUPM01, feature exist according to claim 4 In the amplified production further includes SEQ ID NO:6 or its complementary series, and/or SEQ ID NO:7 or its complementary series.

6. method existing for the DNA of test sample transgenic rice event ZUPM01 according to claim 4 or 5, special Sign is that the primer includes the first primer and the second primer, and the first primer is selected from SEQ ID NO:1 or its complementary sequence Column, SEQ ID NO:8 and SEQ ID NO:10；Second primer is selected from SEQ ID NO:2 or its complementary series, SEQ ID NO:9 and SEQ ID NO:11.

7. a kind of method existing for DNA of test sample transgenic rice event ZUPM01 characterized by comprising

Contact sample to be tested with probe, the probe includes SEQ ID NO:1 or its complementary series or SEQ ID NO: 2 or its complementary series, the source probe transgenic rice event ZUPM01；

Detect the hybridisation events of the sample to be tested and the probe.

8. method existing for the DNA of test sample transgenic rice event ZUPM01, feature exist according to claim 7 In the probe further includes SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

9. special according to method existing for the DNA of the test sample transgenic rice event ZUPM01 of claim 7 or 8 Sign is that at least one described probe is marked at least one fluorophor.

10. a kind of method existing for DNA of test sample transgenic rice event ZUPM01 characterized by comprising

Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:1 or it is mutual Complementary series or SEQ ID NO:2 or its complementary series, the marker nucleic acid molecules are originated from transgenic paddy rice event ZUPM01；

The hybridisation events of the sample to be tested and the marker nucleic acid molecules are detected, and then pass through marker assistant breeding point Analysis is to determine that herbicide tolerant and/or low-phosphorus stress tolerance and marker nucleic acid molecules are chain on science of heredity.

11. method existing for the DNA of test sample transgenic rice event ZUPM01 according to claim 10, feature It is, the marker nucleic acid molecules further include SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or it is complementary Sequence.

12. a kind of DNA detection kit, which is characterized in that including at least one DNA molecular, the DNA molecular includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series, can be used as transgenic paddy rice event ZUPM01 or its offspring have one of DNA primer of specificity or probe；The DNA molecular is originated from transgenic paddy rice event ZUPM01。

13. DNA detection kit according to claim 12, which is characterized in that further include when the DNA molecular is as probe SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

14. a kind of method for protecting rice plants from the damage as caused by herbicide, which is characterized in that including that will contain effectively Dosage glyphosate herbicidal is applied to the big Tanaka for planting at least one transgenic rice plant, and the transgenic rice plant exists Successively comprising SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 in its genome, or It include SEQ ID NO:5 in the genome of transgenic rice plant described in person；The transgenic rice plant has to glyphosate The tolerance of herbicide.

15. a kind of method for protecting rice plants from the damage as caused by soil low-phosphorous element, which is characterized in that be included in low At least one transgenic rice plant is planted in phosphorus concentration soil, the transgenic rice plant successively includes in its genome SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 or the transgenic paddy rice are planted It include SEQ ID NO:5 in the genome of object；The transgenic rice plant has the tolerance to low-phosphorus stress.

16. a kind of method for the big Tanaka weeds for controlling rice cultivation plant, which is characterized in that including effective dose grass will be contained Sweet phosphine herbicide is applied to the big Tanaka for planting at least one transgenic rice plant, and the transgenic rice plant is in its gene It successively include SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 or described in group It include SEQ ID NO:5 in the genome of transgenic rice plant；The transgenic rice plant has to glyphosate herbicidal Tolerance.

17. a kind of method that culture has the rice plants of tolerance to glyphosate herbicidal characterized by comprising

An at least rice paddy seed is planted, includes the nucleic acid sequence of specific region, the spy in the genome of the rice paddy seed The nucleic acid sequence for determining region successively includes SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID The nucleic acid sequence of NO:2 or the specific region includes SEQ ID NO:5；

The rice paddy seed is set to grow up to rice plant；

The rice plant is sprayed with effective dose glyphosate herbicidal, harvest does not have the nucleic acid of the specific region with other The plant of sequence compares the plant with the plant injury weakened.

18. a kind of method that culture has the rice plants of tolerance to low-phosphorus stress characterized by comprising

An at least rice paddy seed is planted in low-phosphorous soil, includes the nucleic acid of specific region in the genome of the rice paddy seed Sequence, the nucleic acid sequence of the specific region successively include 561-6647 SEQ ID NO:1, SEQ ID NO:5 nucleic acid sequences The nucleic acid sequence of column and SEQ ID NO:2 or the specific region includes SEQ ID NO:5；

The rice paddy seed is set to grow up to rice plant；

Harvest the plant compared with the plant of other nucleic acid sequences for not having the specific region with the plant injury weakened.

19. a kind of method for generating the rice plant that there is tolerance to glyphosate herbicidal, which is characterized in that including to described 561-6647 nucleic acid sequences of SEQ ID NO:5 are introduced in the genome of rice plant, and make the base of the rice plant Include successively SEQ ID NO:1,56I-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 because organizing, or makes The genome of the rice plant includes SEQ ID NO:5, the rice plant of selection tolerance glyphosate.

20. 9 method for generating the rice plant that there is tolerance to glyphosate herbicidal according to claim 1, feature It is, comprising:

By transgenic paddy rice event ZUPM01 the first parent rice plant to glyphosate herbicidal with tolerance and lack grass Second parent's rice plant sexual hybridization of sweet phosphine tolerance, to generate a large amount of progeny plants；

The progeny plant is handled with glyphosate herbicidal；

The progeny plant of selection tolerance glyphosate.

21. a kind of method for generating the rice plant that there is tolerance to low-phosphorus stress, which is characterized in that including to the rice 561-6647 nucleic acid sequences of SEQ ID NO:5 are introduced in the genome of plant, and make the genome of the rice plant Successively include SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2, or makes described The genome of rice plant includes SEQ ID NO:5, the rice plant of selection tolerance low-phosphorus stress.

22. the generation according to claim 21 has the method for the rice plant of tolerance to low-phosphorus stress, which is characterized in that Include:

By transgenic paddy rice event ZUPM01 the first parent rice plant to low-phosphorus stress with tolerance and lack the low-phosphorous side of body The second parent's rice plant sexual hybridization for compeling tolerance, to generate a large amount of progeny plants；

The progeny plant is handled with low-phosphorus stress；

The progeny plant of selection tolerance low-phosphorus stress.

23. a kind of composition for being produced from transgenic paddy rice event ZUPM01, which is characterized in that the composition is rice, rice Grass, rice husk or seed rice.

24. a kind of agricultural product or commodity produced by transgenic paddy rice event ZUPM01, which is characterized in that the agricultural product or quotient Product are rice flour, rice bran oil, rice bran, rice embryo, rice gluten, rice starch, Rice Bran oil or rice bran polysaccharide, cosmetics or filler.