CN103571870A

CN103571870A - Method for increasing content of selenium in organism

Info

Publication number: CN103571870A
Application number: CN201310536167.5A
Authority: CN
Inventors: 储成才; 张联合; 胡斌
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2013-11-02
Filing date: 2013-11-02
Publication date: 2014-02-12
Anticipated expiration: 2033-11-02
Also published as: CN103571870B

Abstract

The invention discloses applications of a protein OsPT2 and relevant biological materials to the increment of the selenium content in an organism and particularly discloses an application of a transgenosis method for increasing the content of selenium in a cultivated organism. The method comprises the step of introducing an OsPT2 gene to an acceptor material to obtain a transgenic organism, wherein the selenium content of the transgenic organism is higher than that of the acceptor material, the OsPT2 gene is used for encoding a protein (a) or a protein (b) as follows: a protein (a) composed of an amino acid sequence as shown in a sequence 2 in a sequence table, and a protein (b) which is derived from the protein (a), is relevant to the absorbing ability of plants to selenite and is obtained by substituting and/or deleting and/or adding one or more amino acid residues which are homologous with the sequence 2 in the sequence table or in the amino acid sequence. The OsPT2 gene can be used for increasing and improving the content of selenium in the organism.

Description

A kind of method that improves selenium content in organism

Technical field

The present invention relates to a kind of method that improves selenium content in organism.

Background technology

Selenium is the essential trace element of humans and animals, and it forms the avtive spot of Selenoperoxidase and bring into play its effect with seleno-cysteine form, have anti-oxidant, improve the multiple important physiological functions such as immunizing power and preventing cancer.Selenium also plays an important role at the absorption that regulates vitamin A, C, E and K and consumption, the aspects such as generation that participate in the synthesizing of coenzyme A and ubiquinone, immune stimulatory sphaeroprotein and antibody.In addition, selenium also has the male fertility of maintaining, supports the effects such as anti HIV-1 virus and delaying human body caducity.Some endemic illness are as all low relevant with Selenium in Environment level in Keshan disease, Kaschin-Beck disease, endemia fluorosis, cretinism, region cancer etc.Between selenium and cancer, there is obvious negative correlativing relation, selenium level obviously affects the expression of oncogene and antioncogene, and the generation of oral carcinoma, laryngocarcinoma, nasopharyngeal carcinoma, mammary cancer, colorectal carcinoma, gastrointestinal cancer, liver cancer, lung cancer, esophagus cancer etc. all follows blood selenium to reduce.Long-term edible selenium deficiency food can reduce Abwehrkraft des Koepers because selemium nutrition is not enough, digestive system function is impaired, nervous disorders, affect one's power of vision, renal function, reproductive function, sexual function, bring out VITAMIN, protein and energy deficiency malnutrition, cataract, mazoitis, hepatitis, AIDS, not pregnant disease, Keshan disease, Kaschin-Beck disease, hypertension, hemolytic anemia etc. more than 20 and plant human diseases.

Under normal circumstances, people, mainly from plant food, especially obtain selenium (1) in cereal.Because plant food selenium content is lower, the selenium that human body obtains average every day is difficult to meet health demand (2).Although foliage-spray selenium fertilizer can improve crop kernel selenium content and human body is taken the photograph selenium amount, has also improved agricultural-food production cost simultaneously, even brings potential environmental risk (3,4,5).In addition, foliage-spray is affected by strong wind and rainy weather also, larger to the stabilizing influence of Rice Kernel selenium content.And can not only improve selenium content in cereal kernel by genetic improvement approach, and can also reduce the fertile amount of application of selenium, be the desirable approach (6,7) that improves Rice Kernel selenium content.

Under normal circumstances, selenium in the soil solution mainly with Se ^2-, Se ⁰, Se ₂o ₃ ^2-, SeO ₃ ^2-and SeO ₄ ^2-there are (8) in form.Selenate and selenite are the main selenium forms that can be absorbed by plants.Redox potential and pH can the existence form of remarkably influenced selenium in soil.Under the high condition of oxidation potential, SeO ₄ ^2-it is principal mode; Under oxidation potential moderate condition, SeO ₃ ^2-or HSeO ₃ ^-main existence form (9).Research shows, sulphur translocator has participated in selenate in plant and absorbed (8,10,11,12).High affine sulphur translocator AtSultr1; 2 are identified (13,14) by screening selenate resistant mutants.Different from selenate, plant absorbs selenite mechanism and does not also study clear completely.Past research shows, selenite diffuses into (15) in roots of plants by passive.Respiration inhibitor only can suppress selenite absorption 20% and 30% and support this hypothesis (16,17).Yet azanol and DNP suppress respectively selenite absorption 72% and 86%(18 under pH4.0 condition).(19) that this species diversity is mainly caused the impact of selenite form in absorption liquid by pH.Selenous acid is a kind of weak diacid, and pKa1 and pKa2 are respectively 2.57 and 6.60.Under condition of different pH, selenite is with H ₂seO ₃, SeO ₃ ^2-and HSeO ₃ ^-there are (19,20,21) in form.Different selenium form ratios are with pH alter a great deal (19,20,21).Rice root can absorb H by aquaporin ₂seO ₃the selenite of form (Zhang et al., 2006).

Inorganic phosphorus translocator is the person of participating in directly that inorganic phosphorus in plant (phosphate) absorbs and transports.In rice genome, have the inorganic phosphorus translocator of 13Ge PHT1 family, OsPT2 is one of these 13 members.Research shows, OsPT2 shows the receptivity to inorganic phosphorus in xenopus leavis oocytes system, and the remarkable reduction that the transgenic paddy rice that OsPT2 interferes simultaneously shows content of inorganic phosphorus, shows that OsPT2 plays a significant role in the phosphoric absorption and transport of paddy rice.（The?Plant?Journal(2009)57,798–809doi:

10.1111/j.1365-313X.2008.03726.xTwo rice phosphate transporters, OsPht1; 2and OsPht1; 6, have different functions and kinetic properties in uptakeand translocationPenghui Ai1, , Shubin Sun1, , Jianning Zhao1, , Xiaorong Fan1, Weijie Xin1, Qiang Guo1, Ling Yu2, Qirong Shen1, Ping Wu3, Anthony J.Miller4and Guohua Xu1, *), the associated sequence information of OsPT2 is shown in LOC_Os03g05640

（ http://rice.plantbiology.msu.edu/）。

Reference

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Summary of the invention

Technical problem to be solved by this invention is to provide the new purposes of the protein OsPT2 or its homologous protein and the associated biomolecule material thereof that derive from paddy rice.

A kind of new purposes provided by the present invention is to cultivate the transgenic plant of organism selenium content raising or the method for other biological.

The method of the genetically modified organism that cultivation selenium content provided by the present invention improves, comprises in receptor biological and imports OsPT2 gene, obtains selenium content higher than the step of the genetically modified organism of described receptor biological; Described OsPT2 genes encoding following a) or b) protein::

A) protein that the aminoacid sequence sequence 2(SEQ ID No.2 in sequence table) forms;

B) in sequence table in the aminoacid sequence shown in sequence 2 through replacement and/or disappearance and/or add that one or several amino-acid residue obtains relevant to plant selenite receptivity by a) derivative protein.

In aforesaid method, described receptor biological can be acceptor spermatophyte, and the genetically modified organism that described cultivation selenium content improves can be cultivates the transgenic plant that seed selenium content improves.

Wherein, in sequence table, sequence 2 is comprised of 528 amino-acid residues.

The present invention also protects the method that improves spermatophyte selenium content.

The method of raising spermatophyte selenium content provided by the present invention, comprises and improves OsPT2 gene expression dose described in object spermatophyte, obtains the transgenic plant that selenium content improves.

In aforesaid method, described raising spermatophyte selenium content can be and improves spermatophyte seed selenium content, and the transgenic plant that described selenium content improves can be the transgenic plant that seed selenium content improves.

In aforesaid method, described in raising object spermatophyte, OsPT2 gene expression dose specifically can be in object spermatophyte and imports described OsPT2 gene.

In aforesaid method, the expression level of the described OsPT2 gene of described transgenic plant is higher than described acceptor spermatophyte.

Described OsPT2 gene specifically can be following 1) or 2) or 3) or 4) shown in gene:

1) its encoding sequence is sequence 1(SEQ ID No.1 in sequence table) the cDNA molecule of 46-1632 position Nucleotide;

2) nucleotide sequence is the cDNA molecule of sequence 1 in sequence table;

3) under stringent condition with 1) or 2) DNA molecule hybridize that limits and cDNA molecule or the genomic dna of code for said proteins;

4) with 1) or 2) the cDNA molecule that limits has more than 90% identity and cDNA molecule or the genomic dna of code for said proteins.

Wherein, in sequence table, sequence 1 is comprised of 1929 Nucleotide.

Above-mentioned stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 2 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.5 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄with in the mixing solutions of 1mM EDTA, hybridize, at 65 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, at 65 ℃, hybridization, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively washes film once.

In aforesaid method, wherein said OsPT2 gene can first be modified as follows, then imports in acceptor material (as acceptor spermatophyte), to reach better expression effect:

1) modify according to actual needs and optimize, so that gene efficient expression; For example, the codon that can have a preference for according to recipient plant changes its codon to meet plant-preference in the aminoacid sequence that keeps OsPT2 gene of the present invention; In optimizing process, preferably can make to keep certain GC content in the encoding sequence after optimizing, to realize best the high level expression of quiding gene in plant, wherein GC content can be 35%, more than 45%, more than 50% or more than approximately 60%;

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

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

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

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

In aforesaid method, described OsPT2 gene imports in described acceptor spermatophyte by the recombinant expression vector that contains described OsPT2 expression casette.

The expression casette of OsPT2 described in the present invention all can contain described OsPT2 gene and start the promotor of described OsPT2 genetic transcription.The expression casette of OsPT2 described in the present invention all refers in host cell, to express the DNA of the OsPT2 shown in SEQ ID No.2, this DNA not only can comprise the promotor that starts described OsPT2 genetic transcription, also can comprise the terminator that stops described OsPT2 genetic transcription.Further, described OsPT2 expression casette also can comprise enhancer sequence.Can be used for promotor of the present invention includes but not limited to: constitutive promoter, the promotor that tissue, organ and growth are special, and inducible promoter.The example of promotor includes but not limited to: the constitutive promoter 35S of cauliflower mosaic virus; From the wound-induced type promotor of tomato, leucine aminopeptidase (" LAP ", the people such as Chao (1999) Plant Physiol120:979-992); From chemical inducible promoter of tobacco, pathogeny 1 (PR1) (being induced by Whitfield's ointment and BTH (diazosulfide-7-carbothioic acid carbothiolic acid S-methyl esters)) that be correlated with; Tomato proteinase inhibitor II promotor (PIN2) or LAP promotor (all available jasmonic acid Yue ester inductions); Heat-shocked promotor (United States Patent (USP) 5,187,267); Tsiklomitsin inducible promoter (United States Patent (USP) 5,057,422); Seed specific promoters, as Millet Seed specificity promoter pF128(CN101063139B (Chinese patent 200710099169.7)), the special promotor of seed storage protein matter (for example, phaseollin, napin, the promotor of oleosin and soybean beta conglycin (people (1985) EMBO such as Beachy is J.4:3047-3053)).They can be used alone or are combined with other plant promoter.All reference cited herein all quote in full.Suitable transcription terminator includes but not limited to: Agrobacterium rouge alkali synthetase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (referring to, such as: the people (I such as Odell ⁹⁸⁵) Nature313:810; The people such as Rosenberg (1987) Gene, 56:125; The people such as Guerineau (1991) Mol.Gen.Genet, 262:141; Proudfoot (1991) Cell, 64:671; The people Genes Dev. such as Sanfacon, 5:141; The people such as Mogen (1990) Plant Cell, 2:1261; The people such as Munroe (1990) Gene, 91:151; The people such as Ballad (1989) Nucleic Acids Res.17:7891; The people such as Joshi (1987) Nucleic Acid Res., 15:9627).

In an embodiment of the present invention, in described OsPT2 expression casette, the promotor that starts described OsPT2 genetic transcription is rice actin promotor (Actin), and the terminator that stops described OsPT2 genetic transcription is Agrobacterium octopine synthetic enzyme terminator (Ocs-T).

The recombinant expression vector that available existing plant expression vector construction contains described OsPT2 expression casette.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.As pGWB412, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb(CAMBIA company) etc.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of transcribing as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean stores protein gene) 3 ' end all has similar functions.While using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene (gus gene of luminophor, luciferase genes etc.), antibiotic marker gene (as is given the nptII gene to kantlex and associated antibiotic resistance, give the bar gene to weedicide phosphinothricin resistance, give the hph gene to microbiotic hygromycin resistance, with the dhfr gene of giving methatrexate resistance, give the EPSPS gene to glyphosate resistance) or anti-chemical reagent marker gene etc. (as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.

In a specific embodiment of the present invention, described OsPT2 gene imports in described acceptor spermatophyte by the recombinant expression vector that contains described OsPT2 expression casette.The recombinant expression vector that contains described OsPT2 expression casette is pCambia-OsPT2, and pCambia-OsPT2 is that the fragment between the XbaI of pCambia2300Actin1 and PstI site is replaced with to encoding sequence is that in sequence table, the OsPT2 gene shown in sequence 1 46-1632 position Nucleotide obtains OsPT2 expression vector.

Described OsPT2 expression vector can be by being used Ti-plasmids; plant virus carrying agent; directly delivered DNA; microinjection, the conventional biotechnological means such as electroporation imports vegetable cell (Weissbach, 1998; Method for Plant Molecular Biology VIII; Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).

Above, described transgenic seed plant is interpreted as and not only comprises the first-generation transgenic plant that described gene transformation object plant is obtained, also comprises its filial generation.For transgenic plant, can in these species, breed this gene, also available traditional breeding method enters this transgenosis other kind of same species, in commercial variety.Described transgenic plant comprise seed, callus, whole plant and cell.

Above, described spermatophyte specifically can be angiosperm; Further, described angiosperm can be monocotyledons and dicotyledons, as paddy rice, wheat, corn, soybean, Chinese sorghum, vegetables etc.

The new purposes of another kind provided by the present invention is OsPT2 or the biomaterial relevant to the OsPT2 application in regulating plant selenite receptivity;

Described OsPT2 be following a) or b) protein:

A) protein that the aminoacid sequence shown in sequence 2 forms in sequence table;

B) in sequence table in the aminoacid sequence shown in sequence 2 through replacement and/or disappearance and/or add that one or several amino-acid residue obtains relevant to plant selenite receptivity by a) derivative protein;

The described biomaterial relevant to OsPT2 is following B1) to B7) in any:

B1) the encode nucleic acid molecule of described OsPT2;

B2) contain B1) expression cassette of described nucleic acid molecule;

B3) contain B1) recombinant vectors of described nucleic acid molecule or contain B1) recombinant vectors of described expression cassette;

B4) contain B1) recombinant microorganism of described nucleic acid molecule or contain B2) recombinant microorganism of described expression cassette or contain B3) recombinant microorganism of described recombinant vectors;

B5) contain B1) transgenic plant cells of described nucleic acid molecule system or contain B2) the transgenic plant cells system of described expression cassette or contain B3) the transgenic plant cells system of described recombinant vectors;

B6) reduce the nucleic acid molecule that described OsPT2 expresses;

B7) contain B6) expression cassette of described nucleic acid molecule, recombinant vectors, recombinant microorganism or transgenic plant cells system.

In above-mentioned application, described regulation and control selenate receptivity be presented as following at least any:

1) regulation and control (improve or reduce) root system of plant selenite uptake rate;

2) selenite content in regulation and control (improve or reduce) root system of plant and cauline leaf;

3) selenium content in seed in regulation and control (improve or reduce) spermatophyte.

Above, described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA or hnRNA etc.In above-mentioned application, B1) described nucleic acid molecule is following 1) or 2) or 3) or 4) shown in gene:

1) its encoding sequence is the cDNA molecule of the 46-1632 position Nucleotide of sequence 1 in sequence table;

2) nucleotide sequence is the cDNA molecule of sequence 1 in sequence table;

4) with 1) or 2) the cDNA molecule limiting has cDNA molecule or the genomic dna of more than 90% identity and the described OsPT2 that encodes;

Above, described recombinant microorganism specifically can be bacterium, yeast, algae and fungi.Wherein, bacterium can be from Escherichia (Escherichia), Erwinia (Erwinia), agrobacterium tumefaciens belongs to (Agrobacterium), Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Rhodopseudomonas (Pseudomonas), Bacillus (Bacillus) etc.Described transgenic plant cells is non-plant reproductive material.

Term used herein " identity " refers to the sequence similarity with natural acid sequence." identity " can be with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequences can use per-cent (%) to represent, it can be used for evaluating the identity between correlated series.

B6) nucleic acid molecule that the described OsPT2 of described reduction expresses specifically can be SEQ _forward-X-SEQ _oppositely

（I）

Described SEQ _forwardbe selected from SEQ ID No.1;

Described SEQ _oppositelysequence and described SEQ _forwardsequence reverse complemental;

Described X is described SEQ _forwardwith described SEQ _oppositelybetween intervening sequence, in sequence, described X and described SEQ _forwardand described SEQ _oppositelyall not complementary.In a specific embodiment of the present invention, described SEQ _forwardnucleotides sequence classify the 147-451 position Nucleotide of SEQ ID No.1 as.In an embodiment of the invention, SEQ _forward-X-SEQ _oppositely(809bp altogether), SEQ _forwardsequence be the 147-451 position Nucleotide (altogether 305bp) of SEQ ID No.1, the sequence of X is sequence 4(109bp altogether), be the sequence of potato GA20 oxydase First Intron, SEQ _oppositelysequence (305b altogether _p) and SEQ _forwardsequence reverse complemental.

The nucleic acid molecule of described OsPT2 of encoding specifically can be the cDNA molecule of sequence 1 in sequence table.

In above-mentioned application, described plant can be monocotyledons or dicotyledons, as corn, paddy rice, wheat.

Of the present invention experimental results show that, the root system selenite uptake rate utmost point of the transgenic paddy rice that OsPT2 gene expression amount improves is significantly higher than wild-type paddy rice, in root system and cauline leaf, selenite content is significantly higher than wild-type paddy rice, and in seed, selenium content is significantly higher than wild-type paddy rice; The root system selenite uptake rate of the transgenic paddy rice that OsPT2 gene expression amount reduces is significantly lower than wild-type paddy rice, and in root system and cauline leaf, selenite content is significantly lower than wild-type paddy rice, and in seed, selenium content is significantly lower than wild-type paddy rice.OsPT2 gene can be used for the selenium content in raising and improved seed plant seed.

Below in conjunction with specific embodiment, the present invention is described in further details.

Accompanying drawing explanation

Fig. 1 is the physical map of pCambia2300Actin1.

Embodiment

Following embodiment is convenient to understand better the present invention, but the present invention is not limited in these embodiment.Experimental technique in following embodiment, if no special instructions, is ordinary method.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.

In paddy rice in following embodiment, spend (spending paddy rice in being called again No. 11) (Tong H No. 11, Jin Y, Liu W, Li F, Fang J, Yin Y, Qian Q, Zhu L, Chu C (2009) DWARF AND LOW-TILLERING, a new member of GRAS family, plays positive roles in brassinosteroid signaling in rice.Plant is J.58:803-816) heredity of the public Ke Cong Chinese Academy of Sciences and the acquisition of developmental biology institute, this biomaterial related experiment of the present invention of only attaching most importance to is again used, not can be used as other purposes and uses.

Binary vector pCambia2300Actin1(Lin A^ in following embodiment, Wang Y^, Tang J^, Xue P, Li C, Liu L, Hu B, Yang F, Loake GJ, Chu C (2012) Nitric oxide and protein s-nitrosylation are integral to hydrogen peroxide induced leaf cell death in rice, Plant Physiol.158:451-464) heredity of the public Ke Cong Chinese Academy of Sciences obtains with developmental biology institute, this biomaterial related experiment of the present invention of only attaching most importance to is again used, not can be used as other purposes uses.The physical map of pCambia2300Actin as shown in Figure 1.

Determination of Selenium method in following embodiment is as follows:

Glassware used is immersed in 10%HCl solution in advance in order to avoid selenium contamination.By oven dry with mix sample (100mg) and weigh, then put in 100ml alimentary canal.Add 5ml mixing acid (HNO ₃: HClO ₄=4:1).Under 25 ℃ of conditions, after standing diel, under 150 ℃ of conditions, clear up to clarification.After cooling, digestion solution is settled to 25ml with deionized water, then uses selenium content in ICP-MS working sample.It is pure that all reagent is top grade.Digest tealeaves standard specimen (GBW07605(GSV-4) (0.072mg kg with sample simultaneously ^-1se) and blank.Selenium average recovery rate is between 89 and 93%.

The structure of embodiment 1, OsPT2 gene overexpression carrier and OsPT2 gene interference carrier

1, the structure of OsPT2 gene overexpression carrier pCambia-OsPT2

Extract in paddy rice and spend the total RNA of seedling in tri-leaf period No. 11, adopt the increase 726bp full-length cDNA of coding OsPT2 of RT-PCR method.Wherein,

1) extraction of the total RNA of plant: choosing and spending the seedling of No. 11 in 100mg paddy rice in tri-leaf period is material, grinds in liquid nitrogen, and the lyophilized powder grinding in liquid nitrogen is transferred in the 1.5ml centrifuge tube containing 1ml Trizol reagent (Invitrogen), fully mixes; Place 5 minutes for 25 ℃; In every pipe, add the fresh chloroform of 0.2ml, violent jolting 15 seconds, 25 ℃ of incubation 2-3 minute; 12,000rpm, 4 ℃, centrifugal 15 minutes; The water 0.5ml of supernatant is transferred in a new 1.5ml centrifuge tube, add 0.5ml Virahol, place for 25 ℃ and within 10 minutes, make RNA precipitation; 12,000rpm, 4 ℃, centrifugal 10 minutes; Remove supernatant, RNA precipitation is cleaned 2 times with 1ml75% ethanol, super clean bench blows to half-dried; With 50 μ l DEPC-ddH ₂the resuspended precipitation of O, 60 ℃ of water-baths 10 minutes, obtain RNA solution to dissolve RNA precipitation.By-70 ℃ of preservations after this RNA solution packing, the standby template of doing reverse transcription.

2) RT-PCR: get the above-mentioned RNA solution of 1 μ l, use DEPC-ddH ₂100 times of O dilutions, by spectrophotometric determination RNA concentration.With reference to RT-PCR test kit (Promega) specification sheets, according to the quantitative result of RNA, get the above-mentioned RNA solution that contains 2 μ gRNA, add 1.0 μ g Oligo dT primers, use DEPC-ddH ₂o is supplemented to 15 μ l, mixes rear 70 ℃ of sex change 5 minutes, ice bath 5 minutes.Of short duration centrifugal after, add 25 μ l reverse transcription mixtures (5 μ l M-MLV5 * Reaction Buffer, 6 μ l dNTP Mixture (2.5mM), 1 μ l M-MLV Reverse Transcriptase, 0.5 μ l RNase Inhibitor, 12.5 μ l DEPC-ddH ₂o).After mixing, 42 ℃ of water-baths complete transcriptive process,reversed for 1 hour; 75 ℃ of water-baths make ThermoScript II inactivation in 10 minutes, obtain the mixture that contains the first chain cDNA.

Get above-mentioned the first chain cDNA of 1 μ l as the template of PCR, by following system, carry out PCR reaction: 0.2 μ l LA Taq(5U/ μ l), 10 μ l2 * GC buffer, 1.8 μ l dNTPs, 0.5 μ l5 ' end primer (10 μ M), 0.5 μ l3 ' end primer (10 μ M), adds ddH ₂o final volume 20 μ l.

Wherein, 5 ' end primer: 5 '- tCTAGAgCTTATAACTTTGCAGCTTGAGG-3 ' (underscore sequence is XbaI site), 3 ' end primer: 5 '- cTGCAGgGGAAAGTTCACAAAATCTCACA-3 ' (underscore sequence is PstI site) is primer, carries out pcr amplification, obtains PCR product.

Reclaim PCR product and check order, result shows that the nucleotides sequence of this PCR product classifies sequence 3 in sequence table as.This PCR product is OsPT2 gene, and its encoding sequence is that in sequence table, sequence 1 is from 5 ' end 46-1632 position Nucleotide, and encoding amino acid sequence is the protein OsPT2 of sequence 2 in sequence table.

After this PCR product is cut with XbaI and PstI enzyme, this fragment is cloned into plasmid vector

The XbaI of pCambia2300Actin1 and PstI site, obtain recombinant vectors pCambia-OsPT2.

PCambia-OsPT2 replaces with the OsPT2 gene shown in sequence 1 10-1835 position Nucleotide (being sequence 3) in sequence table by the fragment between the XbaI of pCambia2300Actin1 and PstI site to obtain OsPT2 expression vector.

2, the structure of OsPT2 gene interference carrier pCambia-inOsPT2

By OsPT2 gene interference fragment SEQ _forward-X-SEQ _oppositely(be total to 809b _p) insert _pthe SalI of Cambia2300Actin1 and PstI site, obtain recombinant vectors pCambia-inOsPT2.PCambia-inOsPT2 replaces with OsPT2 gene interference fragment by the fragment between the SalI of pCambia2300Actin1 and PstI site to obtain OsPT2 gene interference carrier.Wherein, SEQ _forwardsequence be the 147-451 position Nucleotide (altogether 305bp) of SEQ ID No.1.The sequence of X is sequence 4(109bp altogether), be the sequence of potato GA20 oxydase First Intron.SEQ _oppositelysequence (altogether 305bp) and SEQ _forwardsequence reverse complemental.

Embodiment 2, utilize OsPT2 albumen and associated biomolecule material adjusting and controlling rice thereof to the receptivity of selenite and cultivate the transgenic paddy rice that seed selenium content improves or reduces

1, cultivate transgenic paddy rice

According to document (Liu X, Bai X, Wang X, Chu C.2006.OsWRKY71, a rice transcription factor, is involved in rice defense response.Journal of Plant Physiology 164 (8): 969 – 979) utilize agriculture bacillus mediated method for transformation, the OsPT2 gene overexpression carrier pCambia-OsPT2 that embodiment 1 is built proceeds in paddy rice and spends and obtain turning pCambia-OsPT2 paddy rice No. 11, the OsPT2 gene interference carrier pCambia-inOsPT2 that embodiment 1 is built proceeds in paddy rice and spends and obtain turning pCambia-inOsPT2 paddy rice No. 11, pCambia2300Actin1 is proceeded in paddy rice and spends and obtain turning pCambia2300Actin1 paddy rice No. 11, concrete grammar is as follows:

With reference to electric exciter (EasyJecT Plus electric exciter, Britain EquiBio company) operational guidance, pCambia-OsPT2, pCambia-inOsPT2 and pCambia2300Actin1 are transformed separately to Agrobacterium EHA105(Biovector Co. with electric shocking method respectively, the catalog number (Cat.No.) Biovec-11 of LTD company), through the resistant panel screening containing kantlex, obtain proceeding to the recombinant bacterium of pCambia-OsPT2, called after EHA105/pCambia-OsPT2, proceeds to

The recombinant bacterium of pCambia-inOsPT2, called after EHA105/pCambia-inOsPT2, proceeds to

The recombinant bacterium of pCambia2300Actin1, called after EHA105/pCambia2300Actin1.

By EHA105/pCambia-OsPT2, EHA105/pCambia-inOsPT2 and

EHA105/pCambia2300Actin1 spends in the callus of No. 11 (hereinafter to be referred as wild-type paddy rice) in Introduced into Rice separately respectively, then with the sterilized water washing 4-5 time containing 300mg/L cephamycin, after aseptic filter paper blots, goes to N ₆d ₂s ₁on substratum, a screening generation; After two weeks, be transferred to N ₆d ₂s ₂on substratum, screened for two generations (2 weeks/generation); Taking-up is screened eugonic resistant calli through 3 generations, is transferred to division culture medium (1), upper, in differentiation culture case (illumination 12 hours every days, 12 hours dark, 28 ℃ of daytimes, 25 ℃ of nights), cultivates 7 days; Then be transferred to division culture medium (2) upper, in differentiation culture case, be cultured to generation regrowth.Plant strong plantlets and rootage on Rooting and hardening-off culture base of regeneration; When seedling grows to 10 centimetres of left and right, open container closure film, hardening 2-3 days, then seedling is moved into phytotron cultivation, obtain 15 strain T0 generations turn pCambia-OsPT2 paddy rice (strain title be respectively Ox-1, Ox-2, Ox-3 ..., Ox15), 12 strain T0 generations turn pCambia-inOsPT2 paddy rice (strain title be respectively Ri-1, Ri-2, Ri-3, Ri-4, Ri-5 ..., Ri-12) and 10 strain T0 generation turn pCambia2300Actin1 paddy rice (that strain title is respectively is empty 1, empty 2 ..., empty 10).

In aforesaid method, used medium is as table 1.

Table 1, substratum

2, the evaluation of transgenic paddy rice

1) PCR identifies

In 2 strain T0 generations of getting at random that step 1 obtains, turn pCambia-OsPT2 paddy rice (strain title is respectively Ox-1 and Ox-3), 2 strain T0 generations and turn pCambia-inOsPT2 paddy rice (strain title is respectively Ri-2 and Ri-5) and 2 strain T0 generation and turn pCambia2300Actin1 paddy rice (strain title be respectively empty 1 and sky 2).Carry out as follows PCR evaluation: get rice leaf and extract genomic dna, use relevant primer to carry out transgenic paddy rice positive identification.Positive identification the primer is the contained NPTII gene primer of pCambia2300Actin1, and its sequence is

5'-CTCGTCAAGAAGGCGATAGA-3' and 5'-TGTCATCTCACCTTGCTCCT-3', target sequence is the partial sequence of NPT II gene, prediction object product sheet segment length 479bp.

2) RT-PCR identifies

In 2 strain T0 generations that strain title is respectively to Ox-1 and Ox-3, turn 2 strain T0 generations that pCambia-OsPT2 paddy rice, strain title be respectively Ri-2 and Ri-5 and turn pCambia-inOsPT2 paddy rice, strain title and be respectively 2 strain T0 generations of empty 1 and empty 2 and turn in pCambia2300Actin paddy rice and 2 strains and spend No. 11 paddy rice to carry out as follows quantitative PCR:

Get and in rice seedling, extract mRNA, and transcribe respectively and obtain cDNA.Utilize fluorescence real-time quantitative PCR method, take cDNA as template, with 1 μ l5 ' end primer 1 (10 μ M) (5 '-ctcttctcacggcagttcat-3 '), 1 μ l3 ' end primer 1 (10 μ M) (5 '-gctgaagatgtccttctgga-3 ') is primer, and the OsPT2 gene expression abundance of above-mentioned paddy rice is detected.Using Actin gene as internal reference, take 5'-ACCATTGGTGCTGAGCGTTT-3' and 5'-CGCAGCTTCCATTCCTATGAA-3' as primer amplification Actin simultaneously.Reagent for quantitative analysis is SYBR Green Realtime PCR Master Mix(TOYOBO).Instrument is the real-time fluorescence quantitative PCR instrument Mx3000P of U.S. Stratagene company.Draw 1 μ l the first chain cDNA solution, dilute 50 times as template, by following system, carry out PCR reaction: 10 μ l SYBR Green Realtime PCR Master Mix, 4 μ l templates, 1 μ l5 ' end primer 1 (10 μ M), 1 μ l3 ' end primer 1 (10 μ M), adds ddH2O final volume 20 μ l.Experiment in triplicate, is carried out significance of difference analysis with t-Test.Result shows, at Actin gene as internal reference in the situation that, compare with wild-type paddy rice (OsPT2 gene expression amount is designated as 1), the OsPT2 gene expression amount utmost point of the T0Dai Zhuan pCambia-OsPT2 Ox-1 of rice strain and Ox-3 is significantly higher than wild-type paddy rice (p < 0.01), strain title is respectively the 2 strain T0 of Ri-2 and Ri-5 for turning pCambia-inOsPT2 paddy rice extremely significantly lower than wild-type paddy rice (p < 0.01), strain title is respectively empty 1 and empty 22 strain T0 for turning pCambia2300Actin1 paddy rice and wild-type paddy rice without significant difference (p > 0.05).The OsPT2 gene expression amount of wild-type paddy rice is designated as to 1, the OsPT2 gene expression amount of the T0Dai Zhuan pCambia-OsPT2 Ox-1 of rice strain and Ox-3 is respectively 33.45 ± 0.93 and 62.53 ± 10.04, the 2 strain T0 that strain title is respectively Ri-2 and Ri-5 are respectively 0.042 ± 0.003 and 0.046 ± 0.005 for the OsPT2 gene expression amount that turns pCambia-inOsPT2 paddy rice, and strain title is respectively empty 1 and for the OsPT2 gene expression amount that turns pCambia2300Actin1 paddy rice, is respectively 0.97 ± 0.14 and 1.12 ± 0.089 with empty 22 strain T0.

3, the expression of OsPT2 changes and can significantly change selenite absorption and selenium accumulation

In 2 strain T0 generations that the strain title of results in step 2 is respectively Ox-1 and Ox-3, turn 2 strain T0 generations that pCambia-OsPT2 paddy rice, strain title be respectively Ri-2 and Ri-5 and turn pCambia-inOsPT2 paddy rice, strain title and be respectively 2 strain T0 generations of empty 1 and empty 2 and turn the seed (T1 is for seed) that pCambia2300Actin1 rice plant is tied, and T1 is implanted in and under equivalent environment, gathers in the crops the seed (T2 is for seed) that T1 ties for plant for seed.

1) expression of OsPT2 changes and can significantly change selenite absorption

Strain title be respectively Ox-1 and Ox-3 turn pCambia-OsPT2 paddy rice T2 for seed, strain title be respectively Ri-2 and Ri-5 turn pCambia-inOsPT2 paddy rice T2 for seed, strain title be respectively empty 1 and empty 2 turn pCambia2300Actin1 paddy rice T2 for seed and in spend No. 11 rice paddy seeds to be handled as follows respectively:

Rice paddy seed, after surface sterilization and washed with de-ionized water, is placed on culture dish filter paper, under 35 ℃ of conditions, in dark place, sprouts.When radicle 2cm is long, rice seedling is transferred to middle cultivation of Kimura's nutritive medium (each solute concentration of full dose Kimura's nutritive medium of table 5 is reduced by half) of 1/2 concentration, artificial growth chamber intensity of illumination is 300 μ mol m ^{– 2}s ^{– 1}, illumination/dark is 16/8h(24/18 ℃), humidity is 67%.After 3 days, seedling is transferred in full dose Kimura nutritive medium (solvent is water, and solute is as table 5) and continues to cultivate 3 days, select the consistent seedling of growth and cultivate in 18L container.Be adjusted to 5.5 with 1mM HCl solution and 1mM NaOH solution by pH every day.Within every 4 days, change one time of nutrition liquid.After 8 days, get seedling for testing:

The expression of a, OsPT2 changes and changes the uptake rate of plant to selenite

Experiment in triplicate, repeats 3 strain rice seedling roots without selenium solution [100 μ M KH at every turn ₂pO ₄, 100 μ M Ca(NO ₃) ₂with 100 μ M MgCl ₂, pH5.5, solvent is water] in be transferred to 2 μ M Na after pre-treatment 0.5h ₂seO ₃after absorbing 3 hours in the aqueous solution (pH5.5) with ice-cold solution [5mM MES, 0.5mM Ca(NO ₃) ₂with 0.5mM K ₂sO ₄(pH5.5), solvent is water] clean, then in scavenging solution, soak 15min to remove the selenium of surface adsorption.After root being blotted with filter paper, get root for Determination of Selenium.According to following formula, calculate the uptake rate of selenite: selenite content=selenium concentration * constant volume/root dry weight/soak time.With t-Test, carry out significance of difference analysis.

B, experiment in triplicate, repeat 3 strain rice seedling roots without selenium solution [100 μ M KH at every turn ₂pO ₄, 100 μ M Ca(NO ₃) ₂with 100 μ M MgCl ₂, pH5.5, solvent is water] in be transferred to 2 μ M Na after pre-treatment 0.5h ₂seO ₃after absorbing 4 days in the aqueous solution (pH5.5), use ice-cold solution [5mM MES, 0.5mM Ca(NO ₃) ₂with 0.5mM K ₂sO ₄(pH5.5), solvent is water] clean, then in scavenging solution, soak 15min to remove the selenium of surface adsorption.After root being blotted with filter paper, get root and cauline leaf (whole plant is removed the remainder after root) for Determination of Selenium.According to following formula, calculate in root selenite content in selenite content and cauline leaf: selenite content=selenium concentration * constant volume/sample (root/cauline leaf) dry weight.With t-Test, carry out significance of difference analysis.

Result is as shown in table 2, shows at Na ₂seO ₃in the aqueous solution, process after 3 hours, the uptake rate utmost point of the selenite of the T2Dai Zhuan pCambia-OsPT2 Ox-1 of rice strain and Ox-3 is significantly higher than wild-type paddy rice (p < 0.01), strain title is respectively the T2 of Ri-2 and Ri-5 for turning pCambia-inOsPT2 paddy rice extremely significantly lower than wild-type paddy rice (p < 0.01), and strain title is respectively empty 1 and empty 2 T2 for turning pCambia2300Actin1 paddy rice and wild-type paddy rice without significant difference (p > 0.05).

The uptake rate of the selenite of each rice strain of table 2.

Strain	Uptake rate (the mgkg of selenite ^-1Dry weight h ^-1）
		Ox-1	1.12±0.08**
Ox-3	1.92±0.14**
		Ri-2	0.49±0.07*
Ri-5	0.51±0.06*
		Empty 1	0.68±0.07
Empty 2	0.67±0.08
		Wild-type paddy rice	0.71±0.1

Note: * * represent strain and in spend No. 11 (wild-type paddy rice) to have utmost point significant difference (P<0.01 level), * represent strain and in spend No. 11 (wild-type paddy rice) there were significant differences (P<0.05 level).

Result is as shown in table 3, shows at Na ₂seO ₃in the aqueous solution, process after 4 days, the selenite content of the T2Dai Zhuan pCambia-OsPT2 Ox-1 of rice strain and Ox-3 root and cauline leaf is significantly higher than wild-type paddy rice (p < 0.05), strain title is respectively the T2 of Ri-2 and Ri-5 for turning pCambia-inOsPT2 paddy rice significantly lower than wild-type paddy rice (p < 0.05), and strain title is respectively empty 1 and empty 2 T2 for turning pCambia2300Actin1 paddy rice and wild-type paddy rice without significant difference (p > 0.05).

Selenite content (μ gg in each rice strain's root of table 3. and cauline leaf ^-1dry weight)

Strain

Root

Cauline leaf

Ox-1	120.85±3.64*	12.4±1.36*
			Ox-3	134.46±13.48*	13.62±0.48*
Ri-2	72.08±10.9*	7.42±0.93*
			Ri-5	75.59±9.5*	6.44±0.45*
Empty 1	96.12±8.15	8.96±0.97
			Empty 2	93.21±9.54	9.15±1.2
Wild-type paddy rice	94.36±4.60	9.05±1.56

Note: * represent strain and in spend No. 11 (wild-type paddy rice) there were significant differences (P<0.05 level).

2, the expression of OsPT2 changes and changes selenium content in rice grain

Strain title be respectively Ox-1 and Ox-3 turn pCambia-OsPT2 paddy rice T2 for seed, strain title be respectively Ri-2 and Ri-5 turn pCambia-inOsPT2 paddy rice T2 for seed, strain title be respectively empty 1 and empty 2 turn pCambia2300Actin1 paddy rice T2 for seed and in spend No. 11 rice paddy seeds in testing station, Lingshui, Inst. of Genetics and Development Biology, CAS Hainan, to plant respectively.Each strain cultivated area is respectively 10m ², repeat for 3 times.Soil is clay, and pH is 5.2.Organic matter, available nitrogen, rapid available phosphorus and quick-acting potassium content are respectively 19.62g kg ^-1, 123.67mg kg ^-1, 32.64mg kg ^-1with 137.23mg kg ^-1.Full selenium and effectively selenium content are respectively 0.18mg kg ^-1with 5.23 μ g kg ^-1.Each strain way to manage is identical.After paddy rice maturation, results seed, artificial shelling shell, then measures (the μ gg of selenium content in brown rice ^-1dry weight).

The selenium content of the result ，T2Dai Zhuan pCambia-OsPT2 Ox-1 of rice strain as shown in table 4 and Ox-3 seed is significantly higher than wild-type paddy rice (p < 0.05), and strain title is respectively the T2 of Ri-2 and Ri-5 for turning

PCambia-inOsPT2 paddy rice is significantly lower than wild-type paddy rice (p < 0.05), and strain title is respectively empty 1 and empty 2 T2 for turning pCambia2300Actin1 paddy rice and wild-type paddy rice without significant difference (p > 0.05).

The selenium content of each rice strain's seed of table 4.

The solute of table 5, full dose Kimura's nutritive medium

?	mg/L	?	mg/L
				(NH ₄) ₂SO ₄	48.20	H ₂BO ₃	2.86
KH ₂PO ₄	24.80	CuSO ₄·5H ₂O	0.08
				KNO ₃	18.50	ZnSO ₄·7H ₂O	0.22
K ₂SO ₄	15.90	MnCl ₂·4H ₂O	1.81
				Ca(NO ₃) ₂.4H2O	86.17	Na2MoO4.2H2O	0.128
MgSO ₄7H2O	65.90	?	?
				Fe-EDTA solution *	1ml	?	?
Na ₂SiO ₃	200	?	?

* 20mM Fe-EDTA solution: dissolve 5.57g FeSO ₄7H ₂o, in 200ml matrass, dissolves 7.45g Na ₂eDTA is in 200ml distilled water.Heating FeSO ₄7H ₂o solution, adds Na ₂eDTA solution constantly stirs.After cooling, quantitatively in the volumetric flask of 1L.

Claims

1. the method for cultivating the genetically modified organism of selenium content raising, comprises in receptor biological and imports OsPT2 gene, obtains selenium content higher than the step of the genetically modified organism of described receptor biological; Described OsPT2 genes encoding following a) or b) protein:

B) in sequence table in the amino acid sequence homologous shown in sequence 2 or aminoacid sequence through replacement and/or disappearance and/or add that one or several amino-acid residue obtains relevant to plant selenite receptivity by a) derivative protein.

2. method according to claim 1, is characterized in that: described OsPT2 gene is following 1) or 2) or 3) or 4) shown in gene:

2) nucleotide sequence is the cDNA molecule of sequence 1 in sequence table;

3. method according to claim 1 and 2, it is characterized in that: described OsPT2 gene imports in described receptor biological by the recombinant expression vector that contains described OsPT2 expression casette, in described OsPT2 expression casette, the promotor that starts described OsPT2 genetic transcription is rice actin promotor, and the terminator that stops described OsPT2 genetic transcription is Agrobacterium octopine synthetic enzyme terminator.

4. improve the method for spermatophyte seed selenium content, comprise and improve OsPT2 gene expression dose in object spermatophyte, obtain the transgenic plant that seed selenium content improves; Described OsPT2 genes encoding following a) or b) protein:

5. according to arbitrary described method in claim 1-4, it is characterized in that: described acceptor spermatophyte is paddy rice, wheat, corn, soybean, Chinese sorghum or vegetables, described object spermatophyte is paddy rice, wheat, corn, soybean, Chinese sorghum or vegetables.

6.OsPT2 or the application of the biomaterial relevant to OsPT2 in regulating plant selenite receptivity;

Described OsPT2 be following a) or b) protein:

The described biomaterial relevant to OsPT2 is following B1) to B7) in any:

B1) the encode nucleic acid molecule of described OsPT2;

B2) contain B1) expression cassette of described nucleic acid molecule;

B6) reduce the nucleic acid molecule that described OsPT2 expresses;

7. application according to claim 6, is characterized in that: B1) described nucleic acid molecule is following 1) or 2) or 3) or 4) shown in gene:

2) nucleotide sequence is the cDNA molecule of sequence 1 in sequence table;

3) under stringent condition with 1) or 2) DNA molecule hybridize that limits and cDNA molecule or the genomic dna of the described OsPT2 that encodes;

4) with 1) or 2) the cDNA molecule limiting has cDNA molecule or the genomic dna of more than 90% identity and the described OsPT2 that encodes.

8. application according to claim 7, is characterized in that: the nucleic acid molecule of the described OsPT2 that encodes is the cDNA molecule of sequence 1 in sequence table.

9. according to arbitrary described application in claim 6-8, it is characterized in that: described regulation and control selenate receptivity be presented as following at least any:

1) regulating plant root system selenite uptake rate;

2) selenite content in regulating plant root system and cauline leaf;

3) selenium content in seed in regulation and control spermatophyte.

10. according to arbitrary described application in claim 6-9, it is characterized in that: described plant is paddy rice.