CN102242110B - Application of a vector with male-sterile gene TCL - Google Patents

Abstract

The invention which belongs to the technical field of gene engineering relates to a male-sterile gene TCL, a coded protein thereof, a promoter thereof, a plasmid thereof and a host cell thereof. The male-sterile gene TCL of the invention is a gene which codes the following protein of (a) or (b): (a) a protein which is composed of an amino acid sequence represented by SEQ ID NO:2; and (b): a protein which has the activity of influencing tapetal programmed cell death, is derived from (a), and is obtained through substituting, deleting or adding one or some amino acids. The invention also relates to the promoter with a base sequence represented by SEQ ID NO:3, the protein with the amino acid sequence represented by the SEQ ID NO:2, and the plasmid and the host cell of the gene with a base sequence represented by SEQ ID NO:1. The TCL gene of the present invention has the function of maintaining tapetal programmed cell death of the paddy rice anther,and the deletion of the gene function can cause tapetal cell necrosis and male sterility of the paddy rice.

Description

Application with carrier of male sterility gene TCL

Patent application of the present invention is Chinese invention patent application number 201010301369.8, February 8 2010 applying date, applicant " Shanghai Communications University ", the dividing an application of denomination of invention " male sterility gene TCL, encoded protein and promotor, plasmid and host cell ".

Technical field

The present invention relates to a kind of gene, encoded protein and promotor, plasmid and host cell of gene engineering technology field, be specifically related to a kind of application with carrier of male sterility gene TCL.

Background technology

Paddy rice is one of topmost food crop of China, and it is staple food with rice that there is 60% above population in China, is maximum in the world rice producing country and country of consumption.3,000 ten thousand hectares of China paddy rice year sown areas account for 20% of the world; 1.85 hundred million tons of output account for the nearly 1/3 of the world, and 6.35 tons/hectare of yield per unit are higher by 65% than 3.85 tons/hectare of global mean yields.Paddy rice remains at about 40% of total amount in China's grain yield, occupied nearly half of the country, and one of them important reasons is exactly that very important effect has been brought into play in the invention of male sterible series of rice and heterotic widespread use.The F1 that parent's combo of the different genetic backgrounds of organic sphere produces is for generation having stronger growth potential and resistance, hybrid vigour that Here it is than its father and mother.The crop heterosis utilization is the important channel of improving crop yield, the applying of hybrid rice, and for huge contribution has been made in the increases in grain production in China and even the world, and the crop male sterile line is effectively to utilize heterotic prerequisite and basis.Utilize male sterible series of rice to produce cenospecies, the hybrid vigour that makes paddy rice is able to widespread use and just becomes possibility producing.Therefore, the research novel male sterible series of rice of seed selection is also furtherd investigate its Regulation Mechanism, is further to excavate paddy rice high-quality and super high-yielding kind prerequisite and basis.

The growth of rice male organ flower pesticide is the process of a complexity, relates to the division differentiation of flower pesticide cell, the reduction division of pollen mother cell and the growth courses such as mitotic division of pollen, the unusual normal development that all can influence pollen of any growth course.The suede adhesion coating is the innermost layer cell in the anther wall cell, directly contacts with sporule with pollen mother cell, and this confluent monolayer cells is the active and the most vigorous confluent monolayer cells of metabolism in each confluent monolayer cells of flower pesticide.After microsporocyte reduction division is finished, tapetal cell begins degraded by the mode of programmed death, and be the further growth of sporule and ripe synthetic and secrete a large amount of nutritive substances and outer wall composition, so the death of this confluent monolayer cells is the basis that forms ripe and fertile flower powder.On the other hand, the normal formation of the degraded of tapetum and extine exists and interdepends, inseparable a kind of relation.Existing gene engineering technique is to be applied on the basis that is based upon the abundant research of plant development process molecular regulation mechanism and understanding, therefore, the further investigation rice tapetum is grown and sporule ripening process, molecular regulation mechanism and regulated and control network and to be applied be basis producing the novel male sterile plant of research and development system.The death and the normal development of sporule outer wall that hinder tapetal cell are the effective ways of cultivating rice male-sterile plants system.But at present the molecular regulation network that rice tapetum is grown and extine composition and molecular regulation mechanism thereof and network are not clear.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, a kind of application with carrier of male sterility gene TCL is provided.TCL gene of the present invention is the anther-specific expression gene, this gene has the function of keeping Rice Anther tapetal cell programmed death, the disappearance of this gene function causes tapetal cell to necrose, and influences the growth of pollen and outer wall thereof then, causes the male sterile of paddy rice.

The present invention realizes by following technical scheme:

First aspect the present invention relates to a kind of male sterility gene TCL, this genes encoding following (a) or (b) described protein:

(a) protein of being formed by the aminoacid sequence shown in the SEQ ID NO:2,

(b) aminoacid sequence in (a) is through replacing, lack or adding one or several amino acid and have the protein of being derived by (a) that influences tapetal cell programmed death activity.

Preferably, the base sequence of described gene is shown in SEQ ID NO:1.

Second aspect the invention still further relates to a kind of promotor that starts described genetic expression, and its base sequence is shown in SEQ IDNO:3.

The third aspect the invention still further relates to aforesaid male sterility of rice gene TCL encoded protein matter, and the aminoacid sequence of this protein is shown in SEQ ID NO:2.

Fourth aspect the invention still further relates to a kind of plasmid, and this plasmid comprises the gene of base sequence shown in SEQ ID NO:1.

Preferably, described plasmid also comprises the promotor of base sequence shown in SEQ ID NO:3.

The 5th aspect the invention still further relates to a kind of host cell, and this host cell comprises the gene of base sequence shown in SEQ ID NO:1.

Preferably, described host cell also comprises the promotor of base sequence shown in SEQ ID NO:3.

Compared with prior art, the present invention has following beneficial effect: the present invention has vital role at the programmed death of excavating and utilizing the early stage tapetum of paddy rice sporule aspect pollen and outer wall thereof, fertility, can or suppress the TCL gene by gene knockout and cause the new male sterible series of rice of unusual dead acquisition of tapetal cell, be used for producing cenospecies, have very important use in agriculture production.

Description of drawings

The morphological observation synoptic diagram of Fig. 1 tcl mutant plant;

Fig. 2 tcl mutant flower pesticide tapetal cell programmed death signal detection synoptic diagram;

Location, Fig. 3 TCL seat synoptic diagram;

The morphological observation figure of the complementary plant of Fig. 4 tcl;

Fig. 5 tcl mutant and wild-type 9522 anther tissues section synoptic diagram;

Fig. 6 TCL promoter activity analysis chart.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Term " coding influences the gene of the albumen of tapetal cell programmed death activity " refers to encode and has the nucleotide sequence of the synthetic monooxygenase active polypeptide of regulation and control extine cutin monomer, as nucleotide sequence and the degenerate sequence thereof of base sequence shown among the SEQ ID NO:1 the 1st～2040.Degenerate sequence refers to, is arranged in the 1st～2040 Nucleotide of encoder block of SEQ ID NO:1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO:1 in the 1st～2040 nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO:2 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO:1 in from the nucleotide sequence of the nucleotide sequence hybridization of the 1st～2040 in Nucleotide.This term also comprise with SEQ ID NO:1 in from the homology of nucleotide sequence at least 70% of the 1st～2040 in Nucleotide, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.This term also comprises encoding to have the variant form of open reading frame sequence among the SEQ ID NO:1 with the albumen of the identical function of natural adjusting and controlling rice flower pesticide tapetal cell programmed death.These variant forms comprise (but being not limited to): several (are generally 1～90, preferably 1～60, more preferably 1～20,1～10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

Term " influences the albumen of tapetal cell programmed death activity " and refers to have the SEQ ID NO:2 polypeptide of sequence of adjusting and controlling rice flower pesticide tapetal cell death protein activity.This term also comprises the variant form that has with the PHD finger Protein S EQ ID NO:2 sequence of natural adjusting and controlling rice flower pesticide tapetal cell programmed death.These variant forms comprise (but being not limited to): several (are generally 1～50, preferably 1～30, more preferably 1～20,1～10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.In the art, when replacing with the close or similar amino acid of performance, can not change the function of protein usually; Add one or the common function that also can not change protein of several amino acid at C-terminal and/or N-terminal.

Among the embodiment, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing the Rice Anther tapetal cell programmed death related polypeptide of present embodiment, adjusting and controlling rice flower pesticide tapetal cell death protein encoding sequence operationally can be connected in expression regulation sequence, thereby form adjusting and controlling rice flower pesticide tapetal cell programmed death correlative protein expression carrier.

Term " operationally is connected in " and refers to a kind of like this situation, and namely some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promoter regulation sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

Term " host cell " is eukaryotic cell, and eukaryotic host cell commonly used comprises yeast cell, rice cell and other vegetable cell.

Associated nucleotide full length sequence or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to the disclosed relevant nucleotide sequence of present embodiment, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

Except producing with recombination method, the fragment of albumen available solid phase technique is also produced by direct peptide synthesis among the embodiment.Can carry out by hand or automatically at external synthetic protein.Can distinguish each fragment of chemosynthesis present embodiment albumen, be connected to produce the molecule of total length then with chemical process.

Term " promotor " refers to the section of DNA sequence for RNA polymerase identification, combination and transcriptional start.Synthetic being called of RNA, transcribe under DNA instructs, the initial promotor control by DNA of transcribing.

Embodiment utilizes gamma-rays that japonica rice 9522 strains are handled, and for selecting the unusual mutant of anther development, selecting the separation ratio is research object for the recessive single-gene mutant of 3:1 at F2 in the plantation back.Obtain the new recessive male sterile mutant of a paddy rice tcl.By hereditary localization method, at first the mutator gene seat is positioned between paddy rice the 9th karyomit(e) InDel mark Ch901 and the Lh901.Further Fine Mapping, we have developed 5 pairs between these two marks have polymorphism InDel molecule marker, and this locus is positioned between Lh903-8 and the Lh903-5, and physical distance is 63.6kb.This zone comprises 7 encoding genes, gene and the Arabidopis thaliana male sterility gene MS1 homology of one of them coding PHD finger albumen, by twice order-checking to this gene, there is the insertion of a T base at second intron place of this gene of discovery mutant tcl, determines that by the function complementation experiment analysis dysfunction of male sterility gene TCL has caused the sterile of paddy pollen.By sterility of anthers mutant tcl development of floral organs being carried out tissue slice, scanning electron microscope, chromosome observation each period, after the sudden change of finding this gene can cause the paddy rice sporule to discharge from tetrad, tapetal cell does not have the PCD signal, sporule tapetal cell in mid-term is expanded gradually, and the tapetal cell content is infiltrated in the flower pesticide chamber, sporule begins degraded, WUHUAFEN grain in the mature anther; Adding molecule mark GUS analysis revealed TCL gene by promotor expresses in the early stage tapetum of sporule and sporule; RT-PCR shows that also the TCL gene is specific expressed in flower pesticide, does not all express in root, stem, leaf and flower glume; Biochemical measurement analysis revealed mutant flower pesticide surface wax composition increases; Complementation test shows that the TCL gene can recover the phenotype of mutant.Illustrate that this gene function is the pollen development that has influence on paddy rice really, this influence may be to realize by the programmed death of regulating the flower pesticide tapetal cell.

Embodiment 1

The acquisition of tcl mutant plant and morphologic observation

With ⁶⁰Co gamma-ray and mutagenesis japonica rice 9522 seeds, treatment dosage is 280Gy, gets mutant.To the F2 of mutagenesis in generation a male sterile mutant three generations backcross, obtain the mutant tcl of the genetic stability of recessive single-gene regulation and control.All vegetable materials are planted in the test base, Academy of Agricultural Sciences, Shanghai City.Tcl mutant and japonica rice 9522 are backcrossed, and F1 is for being to educate all, and separating appears in generation in selfing F2, and wherein normal plant is 189, and mutant strain is 55(χ ²=0.65), shows that this male sterile mutation type surface is caused by a monokaryon transgenation.Morphological observation to tcl mutant plant.As Fig. 1, the phenotype contrast of wild-type and mutant tcl shows: the solid back of wild-type spike of rice sagging (left side), and the shaky vertical (right side) that is of tcl mutant small ear, wild-type flower pesticide comprises mature pollen and is rendered as yellow (left side) (B, C), mutant tcl flower pesticide is faint yellow and WUHUAFEN grain (right side) (B, C).

Embodiment 2

The programmed death signal detection of tcl mutant flower pesticide tapetal cell

Step 1, the paraffin section preparation of each etap wild-type and mutant tcl flower pesticide

(1) material requested an amount of (be no more than stationary liquid volume 1/20) is got in the penicillin bottle that fills the FAA stationary liquid.Slowly bleed (pumping process 10 minutes) with vacuum pump, vacuum state (＞20atm) place 15 minutes after, slowly venting (deflation course 10 minutes).Can be observed the air vesicle and emerged, at the bottom of sample sinks to bottle, 4 ℃ of preservations;

(2) dehydration; Gradient is respectively: 50% ethanol (plant tissue sink to bottle at the bottom of explanation to bleed be sufficient), 50% ethanol, 60% ethanol, 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, 1/4 dimethylbenzene, 3/4 ethanol, 1/2 dimethylbenzene, 1/2 ethanol, 3/4 dimethylbenzene, 1/4 ethanol, under the room temperature every grade 30 minutes.90% dimethylbenzene, 10% chloroform, 90% dimethylbenzene, 10% chloroform, 90% dimethylbenzene, 10% chloroform, under the room temperature every grade 60 minutes, at last 90% dimethylbenzene, 10% chloroform is filled half of penicillin bottle volume;

(3) with the careful sample flasket that contains 1/2 volume, 90% dimethylbenzene, 10% chloroform that drops into of 3～5 wax disk(-sc)s, and bottle is positioned in the baking oven that is tuned to 42 ℃, bottle is shaken in the wax dissolving gently after about 15 minutes, the paraffin of dissolving is evenly distributed, add 3～5 wax disk(-sc)s again.The back bottle is gradually full several times, keeps after 30 minutes, material is changed over to rapidly with tweezers be placed with in the new penicillium bottle that melts good wax disk(-sc) (on 60 ℃ baking oven), spends the night.Sooner or later respectively change wax 1 time every day, oozes best 2～3 day time of wax;

(4) embedding; Solvent paraffin in the canoe of going into to roll well, is clipped to material in the canoe with the tweezers that burn heat, and sets the position.The wax for the treatment of the surface coagulates slightly, canoe is dipped in rapidly in the cold water of having got ready, places at least 2 hours.Available clean container installed and places 4 ℃ to deposit after embedded wax stone dried;

(5) open 45～50 ℃ in baking oven in advance, the slide glass shelf is wrapped with disposable glove, put in the baking oven.Open 42 ℃ in exhibition sheet machine, (every adds water 2～3ml) to the water on the preheating slide glass.Roughly repair earlier piece, the meticulous piece of repairing again, 8～10 microns of slice thicknesses.42 ℃ of exhibition sheets use the dustless paper of wiping away when blotting.42 ℃ of exhibitions got final product in 1 minute.The slice, thin piece of Zhan Haohou is put on 45～50 ℃ the slide shelf of baking oven, wraps with disposable glove, in order to avoid pollute, roasting sheet is more than 24 hours.

Step 2, the section pre-treatment

The method of section pre-treatment and PCD signal detection derives from the apoptosis testing product reagent of Promega company

The experimental procedure content that box (017959) provides is undertaken by the method that company provides, and experimental procedure is as follows

The section dewaxing will be cut into slices and be immersed fresh 100% dimethylbenzene 5min, repeat once;

100% washing with alcohol 5min;

The dehydration of ethanol gradient, 100%, 95%, 85%, 70%, 50% 3min at different levels;

Wash 5min among the 0.85%NaCl;

PBS washs 5min;

37% formaldehyde solution is diluted to 3.7% with PBS, the immersion of will cutting into slices, immobilization material 15min;

The PBS washed twice, each 5min;

Excessive moisture is removed in suction, and section is placed on the smooth desktop; Every Proteinase K that adds 100 μ L20 μ g/mL covers anther tissue; Hatch 8～10min; (annotate: Proteinase K helps staining agent infiltration histocyte in the subsequent step; For best effect, should select the suitableeest incubation time; 4～6 μ m slabs can the proper extension time; But time expand, may cause in the subsequent wash step material being split away off from slide; )

PBS washs 5min;

Fixing 5min in the PBS solution of 3.7% formaldehyde;

PBS washs 5min; (should prepare this moment DNase1 handles over against according to)

Step 3, the detection of tapetum PCD signal

Pat slide to remove excessive moisture, coated with 100 μ L level pads, balance 5～10min under the room temperature;

In equilibrium process, according to every section 45 μ L level pads, 5 μ L Nucleotide Mix and 1 μ LrTdT enzyme are prepared the rTdT incubation buffer; Annotate: the rTdT incubation buffer should be placed on ice, and lucifuge;

Negative contrast: prepare the incubation buffer of a no rTdT enzyme, comprise 45 μ L level pads, 5 μ L NucleotideMix and 1 μ L deionized water; Handle negative contrast section according to step 3～12;

Over against photograph: add under the 100 μ L DNase I damping fluid room temperatures and hatch 5min; Pat the branch that anhydrates, add the enzyme buffer liquid that 100 μ L contain the DNase I of 5.5～10units/mL, hatch 10min under the room temperature; Remove excessive moisture, in the deionized water of abundance, wash 3～4 times; Handle over against shining section according to step 1～12;

Annotate: the DNase I is handled cell can make chromosomal dna fragmentization, and exposes 3 ' of a large amount of Gong mark-OH; Should use the another one staining jar to handle over against shining section, because the activity of residual DNase I can be introduced strong background to experimental group;

At 5cm ²Equilibrium region adds 50 μ L rTdT incubation buffer, does not allow the cell drying; Plastic film coatedly can cut half use, the edge be turned up be convenient to remove;

Make reaction reagent evenly distribute with plastic film coated cell is covered; Put into capacity thieving paper in wet box bottom, add water to supersaturation; To cut into slices in wet box 37 ℃ and hatch 60min, end reaction is carried out; With aluminium foil wet box is wrapped up and to avoid light direct beam;

With deionized water 20 * SSC being diluted by 1: 10 is 2 * SSC, pours staining jar into; Throw off plastic film coatedly, will cut into slices and immerse among the SSC 15min with termination reaction; (annotate: SSC must guarantee that before use all salt all dissolves)

PBS washing three times, each 5min is with unconjugated fluorescence 12-dUTP flush away;

Propidium iodide (PI) is diluted to final concentration 1 μ g/mL in PBS, pours in the staining jar; Section is put into, and 10min dyes in the dark;

Deionized water wash three times, each 5min;

Get rid of excessive moisture, with thieving paper cell periphery moisture is inhaled and gone;

Drip anti-fluorescent quenching mountant, cover glass mounting at slide glass;

Seal around the cover glass with the nail varnish of cleaning, dry 5～10min;

In case of necessity, slide is placed in the wet box preservation of spending the night of 4 ℃ of dark; Observe green fluorescence under 520 ± 20nm, (annotate: PI all dyes redness with apoptotic cell and non-apoptotic cell to observe the red fluorescence of PI under＞620nm; Fluorescence 12-dUTP then only is combined in the nuclear of apoptotic cell).

Among Fig. 2, A to D is the PCD signal detection of wild-type 9522 flower pesticide cells; A, no any PCD signal in the flower pesticide cell of the 6th etap; Can both detect the signal of PCD in the flower pesticide (Fig. 2 C) of the flower pesticide of the 8th etap (Fig. 2 B), the 9th etap and the flower pesticide of the 10th etap (Fig. 2 D).E to H is the PCD signal detection of mutant tcl flower pesticide cell; No any PCD signal is detected in the flower pesticide (Fig. 2 F) of the flower pesticide cell of the 6th etap (Fig. 2 E), the 8th etap, the flower pesticide of the 9th etap (Fig. 2 G) and the flower pesticide of the 10th etap (Fig. 2 H).

Embodiment 3

The location of TCL gene and clone

Step 1, target group, with tcl and long-grained nonglutinous rice strain Long Tefu B hybridization, selfing obtains F2 generation, and selecting is target group for male sterile plants wherein;

Step 2, paddy DNA extracts.Adopt improved CTAB method to extract, comprising the steps: to get blade 0.1～0.2 gram (about half sheet) is put in the little mortar, add an amount of liquid nitrogen, be ground to powdery at once, the 2ml centrifuge tube of packing into, add the 1.5xCTAB solution of 700ul100 ℃ of preheating in centrifuge tube, put into 56 ℃ of water-baths behind the careful mixing, take out centrifuge tube after 20 minutes, add equal-volume chloroform/primary isoamyl alcohol, fierce mixing, centrifugal 10 minutes of 13000rpm, get supernatant in new pipe, put more than half an hour for-20 ℃ behind the adding 900ul dehydrated alcohol mixing.The DNA that separates out is centrifugal, centrifugal 10 minutes of 14000rpm.Remove supernatant, will precipitate with 1ml70% ethanol and clean once, centrifugal drying is dissolved in 200ul1/10TE or the water, and 4 ℃ of refrigerators are preserved;

Step 3, the InDel molecular marker analysis.Designed 132 pairs of polymorphism marks altogether, the SSR primer according to reported sequence synthetic (specifically can referring to Http:// www.gramene.org/microsat/ssr.html), other InDel molecule marker designs are according to the nucleotide sequence of having announced that compares 9311 liang of strains of the japonica rice warm and fine long-grained nonglutinous rice of Japan, partial design primer to difference, verify 2 parent japonica rice 9522 and the wide land of the long-grained nonglutinous rice polymorphism between short, the pcr amplification program is: in the 10ul system, and 1ul template, 1ul10pmol/ul Primer1,1ul10pmol/ul Primer2,1ul10 * Buffer (Mg ²⁺), 1ul2mM dNTP, 0.1ul Taq, 3.9ul water; Pass through 6% PAGE gel electrophoresis afterwards, silver staining method detects.

Step 4, colony's compartment analysis is location and Fine Mapping just

Use these polymorphism marks and carry out the pcr amplification analysis, find that in 194 target groups mutator gene is chain with Marker Ch901 and Lh901 performance on No. 9 karyomit(e), further enlarge target group (1162) and design other polymorphism mark location and find that this gene is in apart from between the Lh903-8 of 63.6kb and two Marker of Lh903-5 (Fig. 3 A).This zone comprises 7 encoding genes, the gene Os09g27620 of one of them coding PHD finger albumen is the homologous gene of Arabidopis thaliana male sterility gene MS1, by twice order-checking to this gene, there is the insertion of a T base at second intron place of this gene of discovery mutant tcl.With molecule marker the male sterile individual plant in the target group is carried out gene type assay, the sequence of used mark is as shown in table 1.

Table 1InDel molecule marker and nucleotide sequence thereof

Embodiment 4

The functional analysis of TCL gene

From paddy rice 9522 genomes, use primer

TCL-lpF5:5’-cacc gtcgacGAGTTAACAAGTGGATGGTG-3’，

And TCL-3R1:5 '-TT TggatccACAGTTGAAGGACGGGAACGACAC-3 ';

Amplify the 5255bp fragment that contains TCL full-length gene group sequence, this fragment covers Os09g27620 upstream from start codon 3043bp and gene terminator codon place (not comprising terminator codon).This fragment is inserted among the Gateway carrier PGWB7 by SalI and BamH1, this makes up the correct back of sequence verification and imports Agrobacterium EHA105 by thermal shock, use hereditary means to transform mutant children fringe callus, whether can cause that to observe plant recovers the wild-type proterties.T ₀In generation, obtain complementary plant, and Fig. 5 shows T ₀Generation, complementary plant tcl mutant pollen development recovered, and contained the mature pollen yellowing in the flower pesticide, and T1 can educate for occurring: the separation of sterile plant is than being 3:1 (23 strains can be educated: 7 strains are sterile).This shows that the Os09g27620 gene of cloning is the gene that causes tcl mutant male sterile phenotype; Among Fig. 5, E: epidermal area, En: endodermis, Mi: middle level, T: tapetum, Tds: tetrad, Ms: sporule, DMs: the sporule of degraded, ST: the tapetum that expands.Observe by sterility of anthers mutant tcl development of floral organs being carried out tissue slice each period, the sudden change of finding the TCL gene can cause paddy rice can not degrade at the early stage tapetum of sporule, sporule can not develop into normal mature pollen grain, is thoroughly disintegrated in mutant flower pesticide at last (Fig. 5).Illustrate that the TCL gene is the male sterile gene of adjusting and controlling rice, the degraded of flower pesticide tapetum is had regulating and controlling effect.

Embodiment 5

The TCL promoter activity is analyzed

From paddy rice 9522 genomes, use primer TCL-lpF5:

5 '-cacc GtcgacGAGTTAACAAGTGGATGGTG-3 ' and TCL-pR:

5 '-aaa GgatccCACCATCTTAGGCGCCAT-3 ' amplifies the 3043bp fragment that contains the TCL promoter sequence.The TCL promotor that amplification obtains is inserted among the Gateway carrier pGWB3 by SalI and BamH1.Make up pGWB3:TCL _Pro: GUS is converted in the wild-type rice callus tissue by agrobacterium mediation method.The expression pattern of the activation analysis TCL promotor by analyzing beta-galactosidase, root genetically modified heterozygote plant, stem, leaf, dye by the GUS staining fluid among small ear and the Xiao Hua, tissue after the dyeing is handled with 75% ethanol decolorization, finds that TCL mainly expresses in the flower pesticide in the 8th stage that Rice Anther is grown and the 9th stage.It is fixing that the material of decolouring processing is put into the FAA stationary liquid, dehydration, processing such as infiltration and resin embedding, by section (10 μ m are thick) and ammoniated ruthenium oxychloride dyeing (0.05%, w/v, pH9) 20min is by observing (Leica DM2500) under the opticmicroscope, we find that the TCL gene mainly expresses in tapetum, and in sporule expression are arranged also.Among Fig. 6 A, the 6th etap of 1-; The 8th etap of 2-; The 9th etap of 3-; The 10th stage of 4-; The 10th late period in stage of 5-.Among Fig. 6 B, the St-stamen; Among Fig. 6 C, T, tapetum; Ms, sporule.Arrow among Fig. 6 represents to present the flower pesticide of GUS dyeing.

Claims (3)

1. the application of a male sterility gene TCL, it is characterized in that: the base sequence of described male sterility gene TCL makes the unusual dead of tapetal cell be used for producing cenospecies to obtain new male sterible series of rice by gene knockout or inhibition TCL gene shown in SEQ ID NO:1.

2. application with carrier of male sterility gene TCL; this carrier is plasmid or host cell; it is characterized in that: described application refers to: for the phenotype of the sterile line that causes after the gene Os09g27620 sudden change that recovers a coding of paddy rice PHD finger albumen; the base sequence of described male sterility gene TCL is shown in SEQ ID NO:1; described plasmid is Gateway carrier PGWB7, and described host cell is Agrobacterium EHA105.

3. method of recovering the male sterility of rice that TCL genetically deficient causes, it is characterized in that, from paddy rice 9522 genomes, amplify the 5255bp fragment that contains TCL full-length gene group sequence by primer sequence, and this fragment imported Agrobacterium EHA105 by thermal shock, use hereditary means to transform the mutant callus and realize the recovery of wild-type proterties;

Described primer sequence is:

TCL-lpF5:5’-caccgtcgacGAGTTAACAAGTGGATGGTG-3’，

TCL-3R1:5’-TTTggatcc?ACAGTTGAAGGACGGGAACGACAC-3’；

The base sequence of described TCL gene is shown in SEQ ID NO:1.

Images