CN103421828A - Cloning and application of gene OsAP65 controlling development of paddy rice pollen tube - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering and particularly relates to the cloning, functional verification and application of an aspartic protease gene OsAP65 controlling the development of a paddy rice pollen tube. The nucleotide sequence of the aspartic protease gene OsAP65 is shown as the sequence table SEQ ID NO: 1; the coding region of the aspartic protease gene OsAP65 is positioned from the 129th base to 2024th base of the gene segment; the sequence of the protein of the aspartic protease gene OsAP65 is shown as the sequence table SEQ ID NO: 2. A research result shows that the cloned aspartic protease gene OsAP65 participates in the development process of the paddy rice pollen tube and has a significant value for theoretical research such as the molecular mechanism in the development of the paddy rice pollen tube.

Description

Control clone and the application of paddy pollen pipe development gene OsAP65

Technical field

The present invention relates to the plant gene engineering technology field.Be specifically related to clone, functional verification and an application of controlling the aspartate protease gene OsAP65 of paddy pollen pipe growth.

Background technology

The life cycle of higher plant alternately is comprised of gametophytic generation and sporophyte generation, and gametophytic growth and fertilization process are to realize heterogamous committed step.Microgametophyte is as one of amphigenetic participant of higher plant, and its origin and development is most important for the digenesis process of plant.And the growth of pollen germination and pollen tube is the important step in Male Gametophyte Development.Sprout the pollen tube and present the polar growth pattern, only to an end elongation, organoid in pollen tube in normal growth presents typical compartmentation and distributes, a large amount of secretion vesicas is rich in pollen tube apical growth district, and inferior top zone thereafter contains the various organoids such as a large amount of plastosomes, golgi body, endoplasmic reticulum and vesicle.The top Fast Growth of pollen tube depends on the accumulation on top of a large amount of cell wallss and cell membrane component, many genes participate in this process of regulation and control (Cheung, Deng .2008.Structural and Signaling Networks for the Polar Cell Growth Machinery in Pollen Tubes.Annu Rev PlantBiol, 59:547-572).No matter be that pollen germination is suppressed or the appearance of the polar growth of pollen tube is abnormal, all can cause plant microgamete transfer efficiency to reduce.In the sufficient Arabidopis thaliana of VANGUARD1 (VGD1), first function obtains the pectinesterase enzyme of the pollen tube specifically expressing of evaluation, after the pollen of vgd1 mutant drops on column cap, can sprout and invade column cap, but poor growth in the style conduit, thereby cause in mutant only having angle fruit top section could produce a small amount of seed, it is unstable when the VGD1 transgenation also can cause pollen tube to be sprouted in vitro, phenomenon (the Jiang that explosion occurs, Deng .2005.VANGUARD1encodes a pectin methylesterase that enhances pollen tube growth in theArabidopsis style and transmitting tract.Plant Cell, 17:584-596).

Aspartate protease (Aspartic proteases, APs) be one of four large lytic enzymes, extensively be present in (Davies in animal, plant and microorganism, Deng .1990.The structure and function of the aspartic proteinases.Annual review of biophysics and biophysicalchemistry, 19:189-215; Rawlings, wait .1995.Families of aspartic peptidases, and those of unknown catalyticmechanism.Methods Enzymol, 248:105-120).In animal and microorganism, the function of relevant AP conducts in-depth research, the AP that an obvious example of wherein finding in the mankind is a film grappling has the activity (Yan of the beta-secretase of Alzheimer's disease, Deng .1999.Membrane-anchored aspartyl protease with Alzheimer ' s disease beta-secretase activity.Nature, 402:533-537).In plant, about the functional study of AP roughly can be divided into: syngenesis and reproduction isolation, plant senescence and programmed cell death, the processing that participates in protein is reacted with degradation process and participation environment stress.Have 69 AP family members in Arabidopis thaliana, Ge etc. are screened more than 40 member's mutant wherein, find a mutant called after: PROMOTION OF CELL SURVIVAL1 (PCS1), research find after the PCS1 transgenation to cause female and male gametophyte can not normal delivery and grow in embryo's programmed cell death (Ge waits .2005.An Arabidopsisaspartic protease functions as an anti-cell-death component in reproduction and embryogenesis.EMBO reports.6:282-288).At least there are 96 AP member (Chen in paddy rice, Deng .2009.Aspartic proteases gene family in rice:Gene structureand expression, predicted protein features and phylogenetic relation.Gene, 442:108-118), in relevant paddy rice, the gene functional research of aspartate protease also relatively lags behind, except controlling the S5 gene (Chen of Indica round-grained rice hybrid fertile, Deng .2008.A triallelicsystem of S5 is a m ajor regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice.Proc Natl Acad Sci U S A, 105:11436-11441) and OsCDR1 (Prasad, Deng .2009.Overexpression ofrice (Oryza sativaL.) OsCDR1leads to constitutive activation of defense responses in rice and Arabidopsis.Mol Plant MicrobeInteract, 22:1635-1644) outside the known function, also structure and the expression of oryzasin1 and OsAsp1 are analyzed, not clear (the Asakura of concrete biological function, Deng .1995.Rice aspartic proteinase, oryzasin, expressed during seed ripening and germination, has a gene organization distinct from those of animal and microbial aspartic proteinases.Eur J Biochem, 232:77-83, Bi, Deng .2005.The rice nucellin gene ortholog OsAsp1encodesan active aspartic protease without aplant-specific insert and is strongly expressed in early embryo.Plant Cell Physiol, 46:87-98).

Summary of the invention

The objective of the invention is to clone a new control paddy pollen pipe development gene OsAP65 and proteins encoded thereof and controlling the developmental application of paddy pollen pipe.Described OsAP65 gene has the nucleotide sequence as shown in sequence table SEQ ID NO:1, also comprise the gene order of showing 90% above homology with the nucleotides sequence shown in SEQ ID NO:1, also comprise because inserting, substitute or lack mutant allele or the derivative that one or more bases produce.The present invention also comprises the aminoacid sequence of the OsAP65 albumen shown in SEQIDNO:2, and there is the above aminoacid sequence of 90% homology with the aminoacid sequence shown in SEQIDNO:2, also comprise because inserting, substitute or lack the functional analogue that one or more amino acid produces.

The T-DNA insertion mutation body offspring of the present invention clone's rice Os AP65 gene has serious inclined to one side separation, be T-DNA insertion mutation body offspring do not have the individual OsAP65-of homozygous mutation/-, only have heterozygous individual OsAP65+/-and the individual OsAP65+ of wild-type /+(see embodiment 1, Fig. 2, table 1), heterozygous plant OsAP65+/-and wild-type plant OsAP65+ /+the reciprocal cross experiment show not have homozygous individual can not normal delivery to cause and (see embodiment 2 because of saltant type pollen, table 2), the OsAP65 gene transformation heterozygous individual OsAP65+ of normal function/-can produce in the offspring the individual OsAP65-of homozygous mutation/-plant (see embodiment 3, Fig. 6, table 3).

The present invention is by the T-DNA insertion mutation body of aspartate protease gene (OsAP) in Screening of Rice, utilize the mutant of OsAP65 to study the effect of this gene in paddy rice microgamete growth course in great detail, offer help for the growth mechanism of understanding Pollen Tubes in depth and the gene regulatory network of plant Devflopment Ofmle Gametophyte, also be expected in addition provide fundamental basis for crop genetic improvement and breeding.

Realize that concrete technical scheme of the present invention is as follows:

1, (creation method of mutant library is shown in paper to utilize existing T-DNA-inserted Mutant Pool in Rice, see Jeong, Deng .2002.T-DNAinsertional mutagenesis for activation tagging in rice.Plant Physiol, 130:1636-1644; Jeong, Deng .2006.Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice.Plant J, 45:123-132) the mutant of screening aspartate protease gene, obtain the lethal family 4A-01549 that isozygotys (purchased from Korea S Pu item University of Science and Technology, see contract), the gene that its T-DNA inserts is LOC_Os07g40260.In order to study conveniently, this research is OsAP65 by this unnamed gene, by this T-DNA insertion mutation body family name osap65 family.The authentication method of this mutant is shown in detailed description in embodiment 1.

2, the present invention analyzed heterozygous OsAP65+/-and wild-type OsAP65+ /+the microgamete growth course, result show isozygoty lethal be due to the pollen tube of OsAP65 saltant type grow exist abnormal, (the seeing embodiment 2) that microgamete can not normal delivery causes.

3, insert and being divided into from checking and function complementation experiment (see embodiment 1, embodiment 3) of mutant character by the T-DNA to mutant OsAP65, successfully cloned and pollen tube development related gene OsAP65.

Detailed technological invention details is listed in " embodiment ".

Advantage of the present invention is:

1. the invention provides new control pollen tube development gene OsAP65 and a proteins encoded thereof, and search out this gene in the developmental effect of control pollen tube.

2. pollen tube has very important effect to offspring's generation of flowering plant, and the OsAP65 gene participates in paddy pollen pipe growth course, and the molecule mechanism scheduling theory research that this gene is grown for the paddy pollen pipe has important value.

The accompanying drawing explanation

Sequence table SEQ ID NO:1 is the nucleotide sequence of the OsAP65 gene of separating clone of the present invention, and sequence length is 2392bp.

Sequence table SEQ IDNO:2 is the aminoacid sequence of protein of the OsAP65 albumen of separating clone of the present invention, 631 amino acid of encoding.

Fig. 1: the phenotype of OsAP65T-DNA insertion mutation family.

A in Fig. 1: heterozygous OsAP65+/-(left side) and wild-type OsAP65+ /+comparison of (right side) ripe plant.

B in Fig. 1: remove the heterozygous OsAP65+ of 3/4 clever shell/-Xiao Hua (left side) wild-type OsAP65+ /+comparison of Xiao Hua (right side).

C in Fig. 1: heterozygous OsAP65+/-(left side) and wild-type OsAP65+ /+comparison of (right side) flower pesticide.

Fig. 2: OsAP65 genotype identification.

In Fig. 2, the structure of A:OsAP65 gene and T-DNA insertion point are analyzed.Initiation codon (ATG) and termination codon (TAA) mark, the exon of solid black box indicating OsAP65 gene, and black line means the intron of OsAP65 gene; Hollow dark square means UTR, and trilateral means the position that OsAP65T-DNA inserts.P1, P2 means two genome primers across T-DNA, P3 means to be arranged in the border primer on OsAP65 mutant T-DNA.

B in Fig. 2: be the genotype detection that an OsAP65T-DNA inserts the heterozygous plant offspring.

The mutant plant is genotypic to be determined: can amplify target product when P1 and P3 pairing, because the T-DNA Insert Fragment is too large, the product of P1 and P2 pairing amplification can't obtain; The wild-type plant is due to the insertion that there is no T-DNA, so P1 and P3 pairing amplification do not have product, but can utilize primer P1 and P2 pairing amplification to obtain target product; OsAP65 heterozygosis T-DNA insert transfer-gen plant when P1 and P2 pairing and P1 and P3 pairing section can increase and obtain product.24 T ₃For the 2nd, 6,7,12,13,14,15,17,18,19,22 and 24 strains in individual plant, be heterozygous phenotype (meaning with H), 12 remaining individual plants are wild-type phenotype (meaning with W).

Fig. 3: observe OsAP65 Average pollen number and pollen maturation rate.

A in Fig. 3: wild-type OsAP65+ /+pollen of plant observes after IKI dyeing.

B in Fig. 3: heterozygous OsAP65+/-pollen of plant observes after IKI dyeing.

C in Fig. 3: wild-type OsAP65+ /+plant and heterozygous OsAP65+/-Average pollen number (left side) and pollen maturation rate statistics (right side) in each flower pesticide of plant.

Fig. 4: utilize the cytology method to observe OsAP65 pollen.

A in Fig. 4: wild-type OsAP65+ /+observe after the DAPI of plant pollen dyeing.

B in Fig. 4: heterozygous OsAP65+/-observe after the DAPI of plant pollen dyeing.

V means trophonucleus, and S means Generative cell nuclei.

C and D: wild-type OsAP65+ in Fig. 4 /+the scanning electron microscope result of plant pollen.Bar=50 μ m in Fig. 4 C; Bar=10 μ m in Fig. 4 D.

E in Fig. 4: wild-type OsAP65+ /+transmission electron microscope results of plant pollen.Bar＝5μm。

F and Fig. 4 G: heterozygous OsAP65+ in Fig. 4/-the scanning electron microscope result of plant pollen.Bar=50 μ m in Fig. 4 F; Bar=10 μ m in Fig. 4 G.

H in Fig. 4: heterozygous OsAP65+/-transmission electron microscope results of plant pollen.Bar＝5μm。

I in Fig. 4: wild-type OsAP65+ /+the external sprouting result of pollen of plant.

J in Fig. 4: heterozygous OsAP65+/-the external sprouting result of pollen of plant.

The external sprouting result of pollen of the complementary plant of K:OsAP65 in Fig. 4.

L in Fig. 4: to from wild-type OsAP65+ /+, heterozygous OsAP65+/-and the pollen germination rate of complementary plant carry out statistical.

The experiment of Fig. 5 .OsAP65 functional complementation.

A in Fig. 5: complementary carrier pU2301-FLAG structural representation.

The part 1 in embodiment 3 is shown in the structure explanation of this carrier, increased with primer in the complete coding region that is about to gene OsAP65, after the PCR product is connected to the pGEM-T carrier, select the positive colony that there is no sudden change by order-checking, cut by Kpn I and Bgl II enzyme the pGEM-T carrier that is connected with OsAP65, reclaim the OsAP65 gene fragment, (transforming pCAMBIA2301 by this chamber doctor Sun Qianwen gets to be cloned into carrier pU2301-FLAG, plasmid pCAMBIA2301 is given by Australian CAMBIA laboratory Http:// www.cambia.org/daisy/cambia/materials/overview.html) the upper novel plasmid formed.PU2301-FLAG empty carrier transformed plant is as negative control.

In Fig. 5, the B:OsAP65 functional complementation is tested carrier OsAP65-pU2301-FLAG structural representation used.

The complementary T1 plant genotype detection that transforms of Fig. 6 .OsAP65.

P1, P2 and P3 primer are with the primer of OsAP65T-DNA insertion mutation body genotype identification.P1+P2 detects T-DNA insertion mutation body genome band, and P1+P3 detects T-DNA insertion mutation body belt carrier.

Embodiment

Be described in further detail understanding the present invention below in conjunction with accompanying drawing, but not the present invention be construed as limiting.

Acquisition and the phenotypic evaluation of embodiment 1:OsAP65 mutant

The present embodiment paddy rice T-DNA insertion mutation body used is utilized carrier pGA2715 to build to form that (construction process of this mutant library is shown in paper: Jeong by Korea S Pu item University of Science and Technology, Deng .2002.T-DNA insertional mutagenesis for activation tagging in rice.PlantPhysiol, 130:1636-1644).The process for screening and identifying of mutant is: the Mutant Rice seed obtained from above-mentioned paddy rice T-DNA insertion mutation side wing sequence library (RiceGE:http: //signal.salk.edu/cgi-bin/RiceGE), be sowed at rice seedling bed after seed soaking, vernalization according to a conventional method, transplant after 20 days to land for growing field crops that (planting density is 5 cun * 8 cun, the experimental plot that the plantation place is Hongshan District, Hubei China province Wuhan City Assessment In Shizishan Region Hua Zhong Agriculture University), paddy rice planting method routinely carries out field management.Through land for growing field crops screening and genotype detection (concrete grammar sees below described), obtain the lethal family of isozygotying, the original number of this family is 4A-01549 (purchased from Korea S Pu item University of Science and Technology).This T-DNA be inserted in second exon of paddy gene LOC_Os07g40260 (download address: Http:// signal.salk.edu/cgi-bin/RiceGE? LOCATION=24132687& CHROMOSOMF=chr07& INTFRVAL=20), the family called after osap65 family (being osap65 gene family of the present invention) that the applicant inserts this T-DNA, (this OsAP65T-DNA inserts the phenotype of family and sees Fig. 1) this family has been indexed in Rice mutant pool (RiceGE:http: //signal.salk.edu/cgi-bin/RiceGE), and all resources of this mutant library all can studied personnel be asked for.

More detailed method is as described below:

Authentication step to this mutation type surface is as follows: for the lethal phenotype that confirms to isozygoty is to cause because the intragenic T-DNA of OsAP65 inserts, the present invention has carried out being divided into from detection of T-DNA insertion point flanking sequence and mutant phenotype to 166 T3 for transfer-gen plant.Be divided into from the concrete steps that detect and be: (1) is at OsAP65 gene T-DNA insertion point both sides design one pair of genes group primer P1 (5 '-ATCGTCGCTCGGTTAGTTT-3 ') and P2 (5 '-GATTACCCCTCGCCTTCTC-3 '), carrier primer P3 of design on T-DNA (5 '-TTGGGGTTTCTACAGGACGTAAC-3 '), as shown in A in Fig. 2.At T ₃In plant, the OsAP65 T-DNA that isozygotys inserts plant and only has when P1 and P3 pairing and just can amplify target product; And in insert in T-DNA the product>10kb make P1 and P2 pairing amplification, can't obtain corresponding PCR product (identifying that so the genotypic method of plant is the universal method in field) at the PCR response procedures of applicant's setting.The wild-type plant is due to the insertion that there is no T-DNA, so P1 and P3 pairing amplification do not have product, but can utilize primer P1 and P2 pairing amplification to obtain target product.OsAP65 heterozygosis T-DNA insert transfer-gen plant when P1 and P2 pairing and P1 and P3 pairing section can increase and obtain product.

(2) application CTAB method (Murray waits .1980.Rapid isolation ofhigh molecular weight plant DNA.Nucleic AcidsResearch, 8:4321-4326) is from 166 strain T ₃As template, with combination of primers P1+P3 in step (1) and P1+P2, carry out pcr amplification for the total DNA of difference extracting in individual plant.Reaction system is 20 μ l, specifically comprise: DNA profiling 2 μ l, the Taq enzyme reaction buffer solution 2 μ l of 10 times of volumes or the GC buffer I reaction buffer of 2 * volume (are acted on behalf of purchased from precious biotechnology Dalian company limited,, Japan Takara company) 10 μ l (during the P1+P3 combination of primers by the Taq enzyme reaction buffer solution of 10x volume, during the P1+P2 combination of primers with the GC buffer I reaction buffer of 2 * volume); 2mM dNTP2 μ l; 10 μ M primer 0.2 μ l; 0.3 the Taq of unit enzyme, add distilled water to 20 μ l.Reaction parameter arranges as follows: 94 ℃ of 5min; 94 ℃ of 40sec, 58 ℃ of 40sec, 72 ℃ of 1min, 30cycles; 72 ℃ of 10min, 25 ℃ of 1min.(3) by reaction product through agarose gel electrophoresis, judge the genotype of each individual plant according to the banding pattern on gel.Experimental result shows, 166 strain T ₃In individual plant, 70 pnca gene types are OsAP65 heterozygous (OsAP65+/-), and other 96 pnca gene types are wild-type (OsAP65+ /+) (as table 1).This mutant phenotype that shows the OsAP65 mutant is lethal for isozygotying.

Table 1OsAP65T ₃For plant genotype identification result

Embodiment 2: mutation type surface is observed

1. reciprocal cross experiment

The ratio that separates that inserts heterozygous plant due to wild-type in the self-pollination offspring of OsAP65 heterozygous plant and T-DNA is approximately 1: 1, depart from the Mendelian inheritance ratio within 1: 2: 1, (T-DNA inserts and isozygotys: T-DNA inserts heterozygosis: wild-type).So we tentatively think that this mutant is the Development of Gametophytes deficient mutants.In order to determine that this mutant is affect microgamete to grow also sufficient megagamete growth, or female, microgamete growth all is affected, and we have carried out the reciprocal cross experiment.Do female parent with the OsAP65 heterozygous plant, the pollen of wild-type plant (bright extensive 63, or claim MH63, the rice varieties of Chinese establishing in large scale) is done male parent and is hybridized, and its offspring separates than being respectively 1.43: 1 (67: 47).When the OsAP65 heterozygous plant is cooked male parent, do female parent with wild-type plant (Zhenshan 97a, or title ZS97A, the rice varieties of Chinese establishing in large scale) and hybridized, its offspring is all the wild-type plant.By above the analysis showed that, T-DNA inserts the megagamete of mutant is grown and do not impacted, and the transfer efficiency of the microgamete of mutant obviously reduces (table 2).In sum, OsAP65 is a microgamete developmental defect type mutant.

The reciprocal cross experimental result genetic analysis of table 2OsAP65

2. the growth course of cytological observation pollen

Result based on to the OsAP65 genetic analysis, we know that OsAP65 is the microgamete deficient mutants.The whole growth course of Rice Anther is from tetrad discharges sporule, arrives at ovule to pollen tube and completes the double fertilization end.Wherein any one stage is affected, the growth of microgamete is all likely interrupted, and (Rice Anther is grown and is divided into 8 periods, details, with reference to Feng, wait .2001.Pollen development andits stages in rice (Oryza sativa L.) .Chinese J.Rice Sci15:21-28).At first by IKI, the mature pollen of heterozygous plant and wild-type plant being dyeed, (document Zhou is shown in concrete operations, Deng .2011.Pollen semi-sterilityl encodes a kinesin-1-likeprotein important for male meiosis, anther dehiscence, and fertility in rice.Plant Cell, 23:111-129), the starch of finding OsAP65 pollen enriches as broad as long (A in Fig. 3, B image).Simultaneously we are added up to the Average pollen number in each flower pesticide and mature pollen rate that (document Hirose is shown in concrete operations, Deng .2010.Disruption of a gene for rice sucrose transporter, OsSUT1, impairspollen function but pollen maturation is unaffected.J Exp Bot, 61:3639-46), purpose is in order to compare the Average pollen number in heterozygous plant and each flower pesticide of wild-type plant, to prove the formation whether the saltant type pollen granule is arranged.Result shows, the Average pollen number in the Average pollen number in heterozygous plant flower pesticide and wild-type plant flower pesticide is obviously difference not, the formation that the saltant type pollen granule is arranged also just has been described and can reach maturity (Fig. 3 C).

(document Han is shown in concrete operations to the method that we then utilize DAPI to dye, Deng .2011.Rice Importin β 1gene affects pollentube elongation.Mol Cells, 31:523-530), observed the nuclear developmental state of mature pollen of OsAP65 heterozygous plant and wild-type plant, there is 2 spermatid nucleuses and 1 trophonucleus (A in Fig. 4, B image) by relatively finding that OsAP65 saltant type pollen is the same with wild-type pollen.Then (document Zhou is shown in concrete operations to utilize the method for scanning electron microscope (SEM), Deng .2011.Pollen semi-sterilityl encodesa kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice.Plant Cell, 23:111-129), observed the formalness of heterozygosis and wild-type mature pollen, result shows relatively there is no obviously to distinguish (the C in Fig. 4 from the POLLEN MORPHOLOGY of heterozygous plant and wild-type, D, F, the G image).(document Li is shown in concrete operations further to utilize the method for transmission electron microscope (TEM), Deng .2011.Rice APOPTOSIS INHIBITOR5coupled with two DEAD-Box adenosine5 '-triphosphate-dependent RNAhelicases regulates tapetum degeneration.Plant Cell, 23:1416-34), observed the internal structure of OsAP65 heterozygous and wild-type mature pollen, find that fovilla and pollen wall do not have too big-difference (Fig. 4 E, H).These results show, the sudden change of OsAP65 does not affect the maturation of paddy pollen.

Because OsAP65 pollen there is no too big-difference in stage of maturity and wild-type pollen, so whether we further detect them and there are differences at germination process.(document Han is shown in concrete operations will to carry out external sprouting experiment from the mature flower powder of wild-type, OsAP65 heterozygous and complementary plant under identical condition, Deng .2011.Rice Importin β 1gene affects pollen tube elongation.Mol Cells, 31:523-530), result shows that the germination rate of wild-type pollen reaches 79.64%, germination rate from the pollen of heterozygous plant is 56.78%, and the germination rate 72.23% of the pollen of complementary plant (Fig. 4 I-L).Therefore we think that the OsAP65 mutant is because pollen tube is grown existence extremely, causes not having T-DNA to insert homozygous individual.

Embodiment 3: complementation test

1. the structure of complementary carrier

The construction process of complementary carrier OsAP65-pU2301-FLAG is as follows: the design primer carries out the amplification of (from ATG, not comprising TAA) of OsAP65 coding region sequence.By primer 65CDS-KpnI-F2 (5 '-GGGGTACCATGGCGAGTCGCCGGTCGCGTCGGC-3 ') and 65OE-R2 (5 '-GAAGATCT TAGCGGTTGGAGCTCTTGCTCAG-3 ') amplification OsAP65CDS sequence, the PCR product carries out agarose electrophoresis, reclaim the purpose fragment from sepharose, purpose fragment and pGEM-T carrier (are acted on behalf of purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, be U.S. Promega company) connect, (electric conversion instrument is Eppendorf company product to the method that the connection product transforms by electricity, applied voltage of the present invention is 1800V, concrete operations are with reference to the working instructions of this instrument) import in intestinal bacteria DH10B (purchased from Invitrogen company), add 300 μ l LB substratum recovery 40min, getting 200 μ l is applied to containing penbritin, the LA flat board of the chloro-3-indoles-β of the bromo-4-of 5--D-semi-lactosi (X-gal) and different Nei Ji-β-D-thiogalactoside (IPTG), cultivate 8-10h (LA and the reference of LB formula: Pehanorm Brooker for 37 ℃, " molecular cloning experiment guide " third edition, Science Press, 2002).Choose white mono-clonal, enlarged culturing extracting plasmid, enzyme cut and check order after select do not have the sudden change the clone, carry out enlarged culturing, cut with Bgl II enzyme the purpose fragment that is connected the pGEM-T carrier with Kpn I and obtain external source, reclaim the OsAP65 gene fragment, (transforming pCAMBIA2301 with this chamber doctor Sun Qianwen gets to be cloned into carrier pU2301-FLAG, see Sun, Deng .2008.Rice jmjC domain-containing gene JMJ706encodes H3K9demethylase required forfloral organ development.Proc NatlAcad Sci U SA, 105:13679-13684, carrier information is shown in A in Fig. 5, pCAMBIA2301 is given by Australian CAMBIA laboratory, http://www.cambia.org/daisy/cambia/materials/overview.html) the upper novel vector OsAP65-pU2301-FLAG (B in Fig. 5) formed.The complementary carrier OsAP65-pU2301-FLAG electricity built is proceeded in Agrobacterium (Agrobacterium tumefaciens) EHA105 (purchased from CAMBIA company).Bacterial strain called after EHA105-OsAP65-pU2301-FLAG after conversion.

2. genetic transformation

Adopt agriculture bacillus mediated genetic transforming method bacterial strain EHA105-OsAP65-pU2301-FLAG to be imported in the heterozygous genes type callus of OsAP65 mutant, through preculture, infect, cultivate altogether, screening has G418 (for screening a kind of microbiotic of positive transgenosis callus, purchased from the white Bioisystech Co., Ltd in Yuanping City, Beijing) callus of resistance, differentiation, take root and the acclimatization and transplants land for growing field crops obtains the transfer-gen plant (patent documentation that the method for agriculture bacillus mediated genetic transformation is previous referring to the applicant, patent No. ZL200710053552.9, the title of invention: the separating clone of rice wide compatibility gene S 5 and application, patent publication No.: CN101200725, license day: on 04 21st, 2010), according to identical method, empty carrier pU2301-FLAG is imported to the heterozygous genes type callus of OsAP65 mutant, using the transfer-gen plant that obtains as negative control.

Result shows, complementary carrier OsAP65-pU2301-FLAG is transformed to the heterozygous genes type that imports the OsAP65 mutant (OsAP65+/-) callus by agriculture bacillus mediated method, obtains altogether independently complementary transformation seedlings 18 strains, and wherein 8 strains are positive.Field planting derives from 3 parts of T ₀The plant of future generation of family, result shows, 3 T ₁Insert homozygous individual (in Table 3, Fig. 6) for all isolating T-DNA in the family plant.Detected equally the T that proceeds to the pU2301-FLAG empty carrier ₁For family, result shows, transforms the T of pU2301-FLAG empty carrier ₁For not having T-DNA to insert homozygous individual in plant, there is no to recover the phenotype (table 3) of isozygotying lethal.These results of study further show that OsAP65T-DNA inserts the family T-DNA that do not isozygoty and inserts and individually determine that the sudden change that is due to the OsAP65 gene causes.

The complementary T that transforms of table 3OsAP65 ₁For the genotype detection result

The agriculture bacillus mediated genetic transformation reagent arrived involved in the present invention and formula are as follows:

(1) reagent and solution abbreviation

6-benzyladenine (6-BA); Kinetin (KT); Naphthylacetic acid (NAA); Indolylacetic acid (IAA); 2,4 dichlorophenoxyacetic acid (2,4-D); Syringylethanone (AS); Caseinhydrolysate (CH); Totomycin (HN); Dimethyl sulfoxide (DMSO) (DMSO); The a large amount of composition solution of N6 (N6max); N6 is composition solution (N6min) in a small amount; The a large amount of composition solution of MS (MSmax); MS trace ingredients solution (Msmin).

(2) main solution formula

1) N6max mother liquor [10 times of concentrated solutions (10X)]

Dissolve one by one, then under room temperature, be settled to 1000ml.

2) N6min mother liquor [100 times of concentrated solutions (100X)]

Dissolve under room temperature and be settled to 1000ml.

3) Fe ₂EDTA stock solution (100X)

Add 300ml distilled water and ferric sulfate (FeSO in a large triangular flask ₄7H ₂O) 2.78g

Add 300ml distilled water and be heated to 70 ℃ in another large triangular flask, then adding b diammonium disodium edta (Na ₂EDTA2H ₂O) 3.73g

Mix after they all dissolve, keep 2 hours in 70 ℃ of water-baths, be settled to 1000ml, 4 ℃ save backup.

4) VITAMIN stock solution (100X)

Add water and be settled to 1000ml, 4 ℃ save backup.

5) Msmax mother liquor (10X)

Dissolve under room temperature and be settled to 1000ml.

6) Msmin mother liquor (100X)

Dissolve under room temperature and be settled to 1000ml.

7) 2,4-D stock solution (1mg/ml)

2，4-D 100?mg.

1ml 1 N potassium hydroxide dissolves 5 minutes, is settled to 100ml, room temperature preservation after then adding 10ml distilled water dissolve complete.

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

6-BA 100mg.

1ml1 N potassium hydroxide dissolves 5 minutes, is settled to 100ml, room temperature preservation after then adding 10ml distilled water dissolve complete.

9) NAA stock solution (1mg/ml)

NAA 100mg.

1ml 1 N potassium hydroxide dissolves 5 minutes, is settled to 100ml after then adding 10ml distilled water dissolve complete, and 4 ℃ save backup.

10) IAA stock solution (1mg/ml)

IAA 100mg.

1ml 1 N potassium hydroxide dissolves 5 minutes, is settled to 100ml after then adding 10ml distilled water dissolve complete, and 4 ℃ save backup.

11) glucose stock solution (0.5g/ml)

Glucose 125g

Distilled water dissolves and is settled to 250ml, and after sterilizing, 4 ℃ save backup.

12) AS stock solution

AS?0.392g

DMSO 10ml

Divide and be filled in the 1.5ml centrifuge tube, 4 ℃ save backup.

13) 1N potassium hydroxide stock solution

Potassium hydroxide 5.6g

Distilled water dissolves and is settled to 100ml, and room temperature preservation is standby.

14) KT stock solution (1mg/ml)

KT 100mg.

1ml 1 N potassium hydroxide dissolves 5 minutes, is settled to 100ml, room temperature preservation after then adding 10ml distilled water dissolve complete.

(3) culture medium prescription

1) inducing culture

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides and installs to 50ml triangular flask (25ml/ bottle), the sealing sterilizing.

2) subculture medium

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides and installs to 50ml triangular flask (25ml/ bottle), the sealing sterilizing.

3) pre-culture medium

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, the sealing sterilizing.

Use front heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in culture dish poured in packing into.

4) be total to substratum

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, the sealing sterilizing.

Use front heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in culture dish poured in packing into.5) suspension medium

Adding distil water, to 100ml, is regulated pH value to 5.4, divides and installs in the triangular flask of two 100ml, the sealing sterilizing.

Add 1ml glucose stock solution and 100 μ l AS stock solutions before use.

6) select substratum

Adding distil water, to 250ml, is regulated pH value to 6.0, the sealing sterilizing.

Dissolve substratum before using, add 250 microlitre G-418 (50 mg/ml) and 400 microlitre CN (250 mg/ml) packing to pour (25 milliliters/ware) in culture dish into.(annotate: selecting for the first time substratum Pyocianil concentration is 400 mg/litre, and after reaching for the second time, selecting to cultivate Pyocianil concentration is 250 mg/litre).

7) division culture medium

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

Boil and be settled to 1000ml, dividing and install to 100ml triangular flask (50ml/ bottle), the sealing sterilizing.

8) root media

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.8.

Boil and be settled to 1000ml, dividing and install to (25ml/ pipe) in the pipe of taking root, the sealing sterilizing.

(4) agriculture bacillus mediated genetic transformation step

Callus of induce

(1) ripe rice paddy seed is shelled, then use successively 70% Ethanol Treatment 1 minute, 0.15% mercury chloride (HgCl ₂) 15 minutes;

(2) sterilizing washing seed is 4-5 time;

(3) seed is placed on inducing culture;

(4) be placed in dark place and cultivate 5 weeks, 26 ± 1 ℃ of temperature.

The callus subculture

Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark lower the cultivation 2 weeks on subculture medium, 26 ± 1 ℃ of temperature.

Preculture

Select the embryo callus subculture of consolidation and relatively dry, be put in dark lower the cultivation 4 days on pre-culture medium, 26 ± 1 ℃ of temperature.

Agrobacterium is cultivated

(1) on the LA substratum with kantlex preculture containing the Agrobacterium EHA105 two days that builds carrier, 28 ℃ of temperature;

(2) Agrobacterium is transferred in suspension medium, cultivates 2-3 hour on 28 ℃ of shaking tables.

Agrobacterium is infected

(1) pre-incubated callus is transferred in the bottle that sterilizing is good;

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

(3) callus is soaked 30 minutes in agrobacterium suspension;

(4) shift callus blots to the good filter paper of sterilizing; Then be placed on common substratum and cultivate 2 days, temperature 19-20 ℃.

Callus washing and selection are cultivated

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

(2) be immersed in containing in the aqua sterilisa of 400 mg/litre Totomycin 30 minutes;

(3) shift callus blots to the good filter paper of sterilizing;

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

Differentiation

(1) kanamycin-resistant callus tissue is transferred to dark place on pre-division culture medium and cultivates 5-7 days;

(2) callus that shifts pre-differentiation culture, to division culture medium, is cultivated under illumination, 26 ℃ of temperature, 5-7 week.

Take root

(1) extract the young plant broken up, cut the root that differentiation phase produces;

(2) then transfer them in root media and cultivate 2-3 week, 26 ℃ of temperature under illumination.

Transplant

Wash the residual substratum on root off, the seedling that will have good root system proceeds to greenhouse, at initial several days, keeps moisture moistening simultaneously.In the greenhouse hardening approximately after about 2 weeks, then the transfer land for growing field crops.

Separation and the order-checking of embodiment 4:OsAP65 coding region

Use this full length gene of method cDNA of cDNA end rapid amplifying (RACE) and PCR primer extension.(anther development is divided into 8 periods to the 8 phase children fringes that the use material is japonica rice Dongjin kind (Korea S Pu item University of Science and Technology is so kind as to give), details are with reference to Feng, Deng .2001.Pollendevelopment and its stages in rice (Oryza sativa L.) .Chinese J.Rice Sci 15:21-28), stand-by in-70 ℃ of refrigerators after liquid nitrogen freezing.Extract RNA according to the described step of TRIZOL reagent (purchased from Invitrogen company) specification sheets, weaker concn to 1 μ g/ μ l ,-70 ℃ of preservations are stand-by.

RACE concrete steps: use SMART ^TMRACE cDNA Amplification Kit test kit (purchased from Clontech company), operation steps is with reference to the specification sheets of this test kit.

The DNA sequence dna of the primer related to is as follows:

The sequence of separating 5 ' end is as follows:

65-5’GSP：5’-ATGTCCTCGCCAAGCACACCACTG-3’

65-5’NGSP：5’-ACTCCTGCGAAGGCGTCCCGATGT-3’

The sequence of separating 3 ' end is as follows:

65-3’GSP：5’-GAAGGGGCTTATTGCTTGGGTGTGT-3’

65-3’NGSP：5’-GCACCTTCAGATTCAGAGGGAGATA-3’

The universal primer that this test kit provides is:

Long?UPM：5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’

Short?UPM：5’-CTAATACGACTCACTATAGGGC-3’

NUPM：AAGCAGTGGTATCAACGCAGAGT

First round PCR reaction conditions:

94 ℃ of denaturations 5 minutes,

94 ℃ of sex change 30 seconds, 72 ℃ 3 minutes, 7 circulations;

94 ℃ of sex change 30 seconds, 67 ℃ are extended 3 minutes, 32 circulations;

67 ℃ of final extensions 7 minutes.

5 ' RACE is used primer UPM+65-5 ' GSP

3 ' RACE is used primer UPM+65-3 ' GSP

Second takes turns the PCR reaction conditions:

94 ℃ of denaturations 5 minutes;

94 ℃ of sex change 30 seconds, 72 ℃ 3 minutes, 5 circulations;

94 ℃ of sex change 30 seconds, 67 ℃ are extended 3 minutes, 25 circulations;

67 ℃ of final extensions 10 minutes.

5 ' RACE is used primer NUPM+65-5 ' NGSP

3 ' RACE is used primer NUPM+65-3 ' NGSP

The last gene model in conjunction with prediction increases out by the coding region of OsAP65 with primer 65CDS-KpnI-F2 and 65OE-R2.

65CDS-KpnI-F2：5’-GGGGTACCATGGCGAGTCGCCGGTCGCGTCGGC-3’

65OE-R2：5’-GAAGATCTTAGCGGTTGGAGCTCTTGCTCAG-3’

Respectively RACE product and PCR product are checked order, analytical sequence finds that coding region (Coding sequence, the CDS) length of gene OsAP65 is 1896bp, and 5 ' UTR length is 128bp, and 3 ' UTR length is 368bp.

Claims (1)

1.OsAP65 gene, controlling the developmental application of paddy pollen pipe, is characterized in that, the nucleotide sequence of this gene is as shown in sequence table SEQ ID NO:1.

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