CN102352367A - Clone and application of semi-dominant gene qGL3 capable of controlling grain length and grain weight of rice kernel - Google Patents

Clone and application of semi-dominant gene qGL3 capable of controlling grain length and grain weight of rice kernel Download PDF

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CN102352367A
CN102352367A CN2011103252782A CN201110325278A CN102352367A CN 102352367 A CN102352367 A CN 102352367A CN 2011103252782 A CN2011103252782 A CN 2011103252782A CN 201110325278 A CN201110325278 A CN 201110325278A CN 102352367 A CN102352367 A CN 102352367A
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张红生
张晓军
蓝虹霞
王建飞
王才林
唐海娟
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Nanjing Agricultural University
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Abstract

The invention relates to the technical field of plant genetic engineering and discloses clone and application of semi-dominant gene qGL3 capable of controlling grain length and grain weight of rice kernel. In the invention, a main-effective QTL (quantitative trait loci) SEQ ID NO:1 which is capable of simultaneously controlling the grain length and grain weight of rice kernel and a semi-dominant allelic gene SEQ ID NO:3 having advantageous function are separated and cloned; the two genes have the capacities of modifying the yield and appearance quality of rice; and at the same time, the two homologous genes of the two genes in rice are cloned and a fact that the two homologous genes have regulation effect on the length of rice kernel as well is proved. Thus, the gene qGL3 and the advantageous allelic gene qGL3-D thereof as well as the two homologous genes of the gene qGL3 all can be applied to crop genetic modification.

Description

An a kind of clone and an application of controlling a rice grain grain length and a heavy semi-dominant gene qGL3
Technical field
The present invention relates to the plant gene engineering technology field; Relate to an a kind of clone and an application of controlling the heavy semi-dominant gene qGL3 of rice grain grain length and grain, be specifically related to one and be positioned at control seed grain length with the grain heavy main gene clone and crop improvement application of imitating semi-dominant gene qGL3 of paddy rice trisome on long-armed.
Background technology
Along with the growth of world population, paddy rice is faced with as main food crop improves pressing for of output.Estimate the year two thousand thirty, rice yield need improve could satisfy human needs more than 40%.Simultaneously, along with human living standard's raising, more and more higher requirement has also been proposed for the exterior quality of paddy rice.The people of SOUTHERN CHINA, America and European countries is partial to like elongated grain type, and NORTH CHINA, Japan and Korea S are partial to like short round shaped grain type (Unnevehr et al., 1992).In order to satisfy human needs, rice genetic breeding scholar has carried out more and more deep research to the output of paddy rice with the grain type.
The yield traits of paddy rice is by the quantitative character of controlled by multiple genes, shows as voriability, is subjected to the influence of environment bigger.In the complicated yield factor of paddy rice, directly constitute the factor and comprise that grain is heavy, every fringe grain husk is spent number, number of productive ear and setting percentage.In addition, the output that proterties such as plant height, tiller number, fringe type, blade profile also can the remote effect Rice Population.The formation of output is these numerous proterties coefficient results under certain environmental conditions.
Although grain heavily is the quantitative character that is subjected to controlled by multiple genes, its this proterties of higher heritability explanation can be isolated some major gene loci.Grain is heavy can be decomposed into grain length, grain is wide, grain is thick and a plurality of components such as grouting density.As far back as 1980, Takeda and saito just reported that rice grain length is controlled by single-gene LK-f; Mckenziedeng (1983) report thinks that grain length is by 2-3 or more Gene Handling.Along with computer technology is improvement and the mass development of molecule marker of the quantitative inheritance analytical procedure on basis, what People more and more was many studies paddy rice grain weight and particle shape by means of the QTLs analytical procedure.Numerous investigators adopt different genetic group to navigate to heavy and about 315 (the http://www.gramene.org/ in QTL site particle shape of relevant grain; By in September, 2011); Almost be distributed on all 12 karyomit(e)s of paddy rice, and grain is heavily contributed big QTL to concentrate to be distributed in karyomit(e)s such as the 2nd, 3,5,6,8.This site that wherein has has a large amount of repetitions probably, is between identical chromosomal region with clone's GS3 like QTL LK-4 (t), gl-3, gw3.1, GW3 about grain length.
Closely during the last ten years because the completion of paddy gene group plan, some paddy rice grain weight, grain number per spike, tiller, rice yield genes involveds such as plant height, plant type obtain separation.Have a plurality of re-correlation genes to be cloned, comprise the gene GS3 of the control grain length on the 3rd karyomit(e), SRS3 be positioned at the 5th chromosomal D1; Be positioned at the wide gene qSW5/GW5 of grain that controls on the 2nd karyomit(e) on wide GW2 of grain and the 5th karyomit(e).GS3 is the BC that is equaled to utilize in 2006 length grain rice material bright extensive 63 and short grain material river 7 structures by Fan Chuchuan 3F 2The colony location obtains.It is one and is positioned near the transmembrane protein in paddy rice trisome kinetochore.GS3 exists three kinds of differences to block the allelic variation at position; Come from bright extensive 63 site and (cut all functions territory; The albumen complete deactivation) the long grain of control type seed (9.91 ± 0.10mm; Mean ± SD); The site (albumen of complete function) that comes from precious Shan 97 forms medium grain (8.08 ± 0.07mm); The site (cut C end TNFR and VWFC functional domain, only keep the OSR functional domain of N end) that comes from river 7 forms short grain (6.30 ± 0.09mm) (Mao et al., 2010).GS3 for the adjusting of seed length through the control cell fission regulate clever shell longitudinally cell number regulate (Mao et al., 2010).D1 is Ashikari etc. and Fujisawa etc. (1999) finds that the afunction sudden change d1 of this gene can form abnormal short seed, its proteic a subunit of G of encoding, and this proteinoid is considered to the molecular switch that the hormone regulating and controlling approach is in the acceptor downstream.The SRS3 gene is by discovering through mutant such as Kitagawa (2010).Mutator gene srs3 forms abnormal grain through the length that reduces clever shell cell number reduction longitudinally grain, and it is a member in kinesin-13 family.GW2 (Song et al., 2007) and GW5/qSW5 (Wan et al., 2008; Shomura et al., 2008) be the QTL site of two control rice grain width, these two genes all are that the sudden change of afunction type causes the separation of seed grain husk shell lateral cell to increase, and then cause the seed of the increase formation broad of lateral cell number.Preliminary study proof GW2 is the E3 ubiquitin ligase of a RING type; And qSW5/GW5 is the albumen of a unknown function; Yeast is two assorted finds that its interact proteins also are a kind of poly ubiquitin, and two sites width of having regulated seed through same ubiquitin protein enzyme body molecular pathways is still waiting further research.
Fine Mapping has also been arrived in other control paddy rice grain QTL site heavy or the grain type can be through the degree of molecular marker assisted selection; Gw8.1 (Xie et al. for example; 2006); Gw9.1 (Xie et al.; 2008) and GW6 (Guo et al.; 2009), still also obtain clone and further functional verification.Research heavy about grain or the grain type has also obtained certain progress (Gegas et al., 2010) in other food crop.
Summary of the invention
The objective of the invention is from paddy rice one of separating clone and control the complete coding region segment DNA sheet degree of the semi-dominant allele of heavy main effect QTL of rice grain grain length and grain and advantage function thereof simultaneously, utilize the output of this improvement of genes paddy rice and the ability of exterior quality.Cloned simultaneously and proved that two homologous genes of this gene in paddy rice have the regulating effect to rice grain length equally.
A kind of rice grain length semi-dominant gene qGL3 heavy with grain that control, nucleotide sequence is shown in SEQ ID NO:1.
Described gene qGL3 encoded protein, promptly among the SEQ ID NO:1 the 219th～3230 the translation pairing amino acid polypeptide, aminoacid sequence is shown in SEQ ID NO:2.
The advantage allelotrope qGL3-D of described gene qGL3, nucleotide sequence is shown in SEQ ID NO:3.
Described advantage allelotrope qGL3-D encoded protein, by the 219th～3230 pairing amino acid polypeptide of translation among the SEQ ID NO:3, aminoacid sequence is shown in SEQ ID NO:4.
The homologous gene qGL3-L1 of said gene qGL3 in paddy rice, nucleotide sequence shown in SEQ ID NO:5, its encoded protein sequence shown in SEQ ID NO:6, promptly among the SEQ ID NO:5 99～2774 the translation pairing amino acid polypeptides.
The homologous gene qGL3-L2 of said gene qGL3 in paddy rice, nucleotide sequence shown in SEQ ID NO:7, its encoded protein sequence shown in SEQ ID NO:8, promptly among the SEQ ID NO:7 78～3107 the translation pairing amino acid polypeptides
The application of described gene qGL3 in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
The application of described gene qGL3-D in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
The application of described gene qGL3-L1 in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
The application of described gene qGL3-L2 in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
Beneficial effect of the present invention:
(1) applicant found a big grain japonica rice germplasm N411 from paddy rice local variety resource material in 2005, and its thousand seed weight reaches 71.1 ± 0.22 grams.Subsequently respectively with granule long-grained nonglutinous rice family 05-643, the type positive and negative hybridization of kind 93-11 of middle grain and backcross, heavy and correlated character is launched research to grain.Preliminary research shows that grain re-correlation proterties does not have cytoplasmic effect.Through N411/05-643 F 2The qtl analysis of colony; Detect 1 contribution rate in SSR molecule marker RM6266-RM3350 interval and reach 52.5% the main grain length QTL:qGL3 of imitating; This site reaches 31% to the heavy contribution rate of grain simultaneously; This site physical location is positioned at about the downstream 8.5Mb of the grain length controlling gene GS3 that has reported, and sequential analysis shows that N411,05-643,93-11 have identical GS3 mutational site with bright extensive 63.From F 2Individuality and 05-643,93-11 that selection carries the big grain of RM6266-RM3350 parent marking type backcross respectively, further the BC through 93-11 being backcrossed producing 2F 2The qtl analysis of colony has confirmed that this site is for the grain length monofactorial inheritance effect heavy with grain.Utilize seed length isolating four BC that backcross of single-factor to occur on this basis 2F 3Colony is positioned at qGL3 in about 46.6kb between Indel mark xj39 and the xj26 interval (Fig. 4).
Two in this section prediction expressing genes are carried out cloning and sequencing find that relatively the cDNA sequence of one of them gene does not have difference between two parents in two parents.And there is the sudden change of two places in an other gene between big grain parent N411 and granule parent 93-11.Be specially: the site (SEQ ID NO:3) that comes from big grain parent N411 is compared with the site that comes from granule parent 93-11 (SEQ ID No.1): a place is positioned at the disappearance of a single bases G of-72 positions existence in the 5`UTR zone of cDNA sequence; Another place is positioned at mononucleotide replacement of the 10th coding region+1092 positions on the exon: C → A, and then causes amino acid whose replacement of protein: 364 aspartic acid (Asp, D) be transformed into L-glutamic acid (Glu, E), D364E; With this unnamed gene that sudden change of two places takes place is qGL3-D.The expression ratio of two genes between background parent and near isogenic line found that there is not differential expression in two genes between parent and near isogenic line.The candidate gene that the gene that has sequence difference is decided to be qGL3.To the structure of this gene and the prediction and the analysis of proteins encoded product, find that this gene is made up of 21 exons and 20 introns.Coding region length is 3012bp, 1003 the amino acid whose polypeptide of encoding.Prediction finds that its encoded polypeptides is serine/threonine phosphoric acid (ester) enzyme that contains 2 kelch repeating structure territories according to protein functional domain.In paddy rice, this gene is the little family that is made up of three homologous genes, and the applicant is with its difference called after qGL3 (SEQ ID NO:1), qGL3-L1 (SEQ ID NO:5) and qGL3-L2 (SEQ ID NO:7).Allelotrope to from two parents is further analyzed discovery; Its advantage allelotrope qGL3-D be positioned at 4 of protein 36s aspartic acid (Asp, D) be transformed into L-glutamic acid (Glu, E); D364E, this sudden change occurs on the D position of conservative motif AVLDT of structural domain kelch (Fig. 5) just.Real-Time PCR expression analysis to qGL3 and two other homologous gene qGL3-L1 and qGL3-L2 is found; This family gene is expressed in the different development stage tissue of the root of paddy rice, stem, blade, leaf sheath, tassel, and predominant expression is in middle and later periods (Fig. 6) that fringe is grown.Promotor (SEQ ID NO:9) through with this gene connects the transgenosis promotor GUS expression activity analysis that gus gene carries out; Confirmed the result of Real-Time PCR equally; Can find that simultaneously qGL3 is in the periphery of the middle arteries and veins of the vascular tissue of the tip of a root, stem, blade, leaf sheath, summit vegetative point, the base portion of prematurity grain husk shell, sophisticated clever shell; And the base portion predominant expression of gynoecium, and expression (Fig. 7) in the no longer sophisticated flower pesticide.Through finding in the Subcellular Localization in the onion epidermis this gene coded protein: the albumen of wild-type (shown in SEQ ID NO:2) is distributed in the whole tenuigenin; Distribution does not have superiority in nucleus; And the albumen (shown in SEQ ID NO:4) after the sudden change is distributed in the tenuigenin, and advantage is distributed in (Fig. 8) in the nucleus simultaneously.The change of inferring the proteic Subcellular Localization of this genes encoding is the key of its performance different effects.Wild type gene qGL3 (sequence is shown in SEQ ID NO:1) entire reading frame was connected among the expression vector PCAMBIA-1300S, spent in 11 transgenosis T in the rice transformation kind 0Generation and transgenosis T 1All show the grain type that shortens for the positive individual plant of overexpression, and fringe type and plant type are not had remarkably influenced (Figure 10).Functional domain with two predictions of this genes encoding blocks respectively simultaneously, and like Figure 12, two are blocked the back polypeptide 296 of joining regions are amino acid whose in the middle of existing and stride foldedly, make up in the expression vector rice transformation kind and spend 11, OX-Kelch transgenosis T 0Generation is positive to be crossed and expresses plant and show the grain type that shortens and OX-PP2Ac transgenosis T 0Generation is positive to cross the grain type of expressing plant do not change (Figure 12).This explanation mainly comes from the kelch structural domain in the regulatory function to rice grain length, and little with PP2Ac structural domain relation.In rice varieties, spend in 11 RNAi to interfere T to target gene 0For transgenic positive individual plant and T thereof 1Find that for the analysis of transgenic positive individual plant diminish (Figure 10) all taken place the seed of expression inhibiting plant.
The phenotype analytical that the insertion of one of them homologous gene qGL3-L1 is knocked out mutant is found simultaneously: the function of qGL3-L1 lacks fully and causes seed obviously to diminish, simultaneously to the mistake express transgenic T of qGL3-L1 and qGL3-L2 0Represent type analysis to find to express the homologous gene qGL3-L1 of qGL3 and seed that qGL3-L2 can faintly make paddy rice increases Figure 13 D.This explanation qGL3 and homologous gene qGL3-L1 thereof and qGL3-L2 have similar function on adjusting rice grain length.
Through molecule marker selection acquisition is the near isogenic line 93-11NIL-qGL3-D analysis discovery of the allelotrope qGL3-D of the next arrogant grain of background importing parent N411 with 93-11: the advantage allelic variation qGL3-D of gene qGL3 (shown in SEQ ID NO:3) is elongated except making seed for paddy rice; Outside grain heavily increases, there is not change on the statistics (like table 2 for other agronomy phenotype; Figure 13).Utilize this near isogenic line 93-11NIL-qGL3-D and 93-11 hybridization simultaneously and recover system and extensively account for the acquisition F of the cross combination of 63S structure with two-line sterile line as two-line hybrid rice 1The grain type analysis find that the allelic variation that comes from big grain parent can make seed length increase (Figure 14) equally under heterozygous state.The near isogenic line (the short grain equipotential background gs3 of GS3) that rice varieties heat is ground background is analyzed discovery with the black glutinous near isogenic line in rice varieties Longli (the short grain equipotential background d1 of D1) simultaneously, and qGL3 can cover the effect (Figure 15) that gs3 and d1 make the seed contraction in length.
To sum up presentation of results: wild-type grain length controlling gene qGL3 (SEQ ID NO:1) in spend under 11 backgrounds, still downward modulation takes place to raise seed to be shortened diminish.And the advantage allelic variation gene qGL3-D (SEQ ID NO:3) of qGL3 occurs in the locational aspartic acid of the D (Asp of the conservative motif AVLDT in the kelch structural domain; D) be transformed into L-glutamic acid (Glu; E); The albumen that has caused this genes encoding is from a large amount of entering nucleus of kytoplasm; And then brought into play the function that makes rice grain elongated, this is the acquired sudden change of a kind of function.Proved that simultaneously two homologous gene qGL3-L1 and the qGL3-L2 of qGL3 in paddy rice has a regulatory function heavy with grain to the rice grain grain length equally.And the advantage allelotrope qGL3-D of qGL3 (SEQ ID NO:3) is as the genetics downstream gene of a control rice grain length; Can well save upstream gene such as gs3; The negative regulation effect of d1 etc., the semidominance characteristic makes it can be used to recover the improvement that is and then is used for hybrid rice breeding simultaneously.
(2) said gene of cloning among the present invention is that the high yield and high quality breedings of cereal crop such as paddy rice provides new genetic resources; Also for clone's genes involved in other crops provides technological borrowing, also can produce evidence for the molecular evolution research of dicotyledonous crops such as cereal crop such as paddy rice, wheat, corn, jowar and soybean, rape.
Description of drawings
Fig. 1: the general technical route map that the present invention uses.
Fig. 2: the molecule marker genetic map and the localized QTLs that utilize N411 and 05-643 to make up.
The BC of Fine Mapping colony that Fig. 3: N411 and 93-11 make up 2F 3In 206 strain seed phenotype number of times distribution plans at random, the fill style shows the genotype in qGL3 site.
The Fine Mapping procedure chart of Fig. 4: qGL3, the individual number of the reorganization between mark in 2968 individuals shown in the A.; B. the individual number of the reorganization shown between further smaller area; High precision linkage analysis between phenotype C. and genotype; D. shown in according to the physical distance between mark xj39 shown in the fine genome annotation of Japan and xi26.
Fig. 5: Gramineae is represented the kelch structural domain Partial Protein comparison of qGL3 homologous gene family in plant and the Arabidopis thaliana, and arrow is represented the allelic variation of qGL3-D among the big grain parent N411: D364E.
The semi-quantitative expressed analysis of Fig. 6: qGL3 and homologous gene qGL3-L1 and qGL3-L2 and real-time quantitative Real-Time PCR expression analysis.
A. semi-quantitative expressed analysis shown in, the expression analysis of real-time quantitative shown in the B.; Wherein: R: root, C: stem, LB: blade, LS: the elementary fringe of leaf sheath, PP:2cm-3cm, YP:8cm-10cm children fringe, MP: the ripe tassel that just will extract out, YL: unpumped spire.
Fig. 7: qGL3 gene promoter GUS expression activity is analyzed.
Fig. 8: qGL3 gene wild-type and the proteic Subcellular Localization of advantage equipotential.
93-11-qGL3-GFP representes the Subcellular Localization figure of qGL3 gene wild-type protein, and N411-qGL3-D-GFP representes the proteic Subcellular Localization of advantage equipotential after the qGL3 transgenation.
Fig. 9: the structure restriction enzyme site figure that crosses express transgenic carrier pCAMBIA-1304, pCAMBIA-1300S and RNA interference vector pCK303.
Wherein, A is the structure restriction enzyme site figure of pCAMBIA-1304, and B is the structure restriction enzyme site figure of pCAMBIA-1300S, and C is the structure restriction enzyme site figure of pCK303.
Figure 10: cross expression and RNAi and interfere the expression of transgenic line to detect.
Wherein, A is semi-quantitative expressed analytical results figure, and B is a real-time quantitative expression analysis statistical graph as a result.
Figure 11: wild type gene is crossed and is expressed and RNAi interference transgenic line agronomy phenotype and tassel, seed phenotype.
Wherein, A is that wild type gene is crossed expression and RNAi interferes transgenic line agronomy phenotype; B is that wild type gene is crossed expression and RNAi interferes transgenic line tassel phenotype; C is that wild type gene is crossed expression and RNAi interferes transgenic line seed phenotype.
Figure 12: the pairing polypeptide of mistake express transgenic vector construction in break-in facility territory, and the corresponding seed phenotype of transfer-gen plant.A is the pairing polypeptide synoptic diagram of mistake express transgenic vector construction in break-in facility territory; B is the corresponding seed phenotype of transfer-gen plant; Wherein OX-qGL3 representes the pairing polypeptide synoptic diagram of mistake express transgenic vector construction of qGL3; OX-kelch representes that the kelch functional domain among the qGL3 crosses the pairing polypeptide synoptic diagram of express transgenic vector construction, and OX-PP2Ac representes that the PP2Ac functional domain among the qGL3 crosses the pairing polypeptide synoptic diagram of express transgenic vector construction.
Figure 13: qGL3-L1 inserts plant and the seed phenotype knock out mutant, and homologous gene qGL3-L1 and qGL3-L2 cross the seed phenotype of express transgenic plant.
Wherein, A is the plant phenotype of wild-type east Tianjin DJ and mutant qgl3-l1; B is the tassel phenotype of wild-type east Tianjin DJ and mutant qgl3-l1; C is the seed phenotype of wild-type east Tianjin DJ and mutant qgl3-l1, and D is the homologous gene qGL3-L1 of qGL3 and the seed phenotype that qGL3-L2 crosses the express transgenic plant; In spend 11 for receptor parent.
Figure 14: qGL3 is in rice varieties heat and grinds near isogenic line (the short grain equipotential background gs3 of GS3) and the black glutinous near isogenic line in rice varieties Longli (the short grain equipotential background d1 of D1) seed under the B background.
Figure 15: the plant type of background parent 93-11 and near isogenic line 93-11NIL-qGL3-D, tassel, and seed phenotype map.A is the plant type map of background parent 93-11 and near isogenic line 93-11NIL-qGL3-D; B is the tassel map of background parent 93-11 and near isogenic line 93-11NIL-qGL3-D, and C is the seed phenotype map of background parent 93-11 and near isogenic line 93-11NIL-qGL3-D.Figure 16: 93-11 and near isogenic line 93-11NIL-qGL3-D and they and two-line sterile line extensively account for the hybridisation rice tassel of 63S configuration and the comparison of seed.
Wherein A is the comparison diagram that 93-11 and near isogenic line 93-11NIL-qGL3-D and they and two-line sterile line extensively account for the hybridisation rice tassel of 63S configuration;
B is the comparison diagram that 93-11 and near isogenic line 93-11NIL-qGL3-D and they and two-line sterile line extensively account for the hybridisation rice seed of 63S configuration.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Be interpreted as, these concrete embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.Do not indicate the experimental technique of actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of being advised according to each company's sell goods.The SSR molecule marker RM series of wherein using is except that specified otherwise, and all the sequence of announcing according to gramene (http://www.gramene.org/) website is synthesized.
Separation and the acquisition of embodiment 1 paddy rice grain length gene qGL3
(1) materials and methods:
1. large seed matter N411 and 05-643,93-11 are hybridized the structure (idiographic flow is referring to Fig. 1) of colony:
(1) utilize rice big grain germplasm N411 (to preserve by paddy rice institute of Agricultural University Of Nanjing germplasm storehouse; Its advantage allelotrope can obtain from No. 5, the agricultural Xian in south; The kind application number of accepting 20110730.0) as the donor material of beneficial gene; With 05-643 (forming), 93-11 hybridization by rich B in rice varieties Guangdong and imperial Mortopl B selection cross; The plantation cross-fertilize seed, selfing obtains F 1With F 1The seed that selfing obtains is planted into individual plant and is constituted F 2Colony is used to locate grain length, the heavy QTL of grain.
(2) F 2Select the long individual plant continuous backcross of seed to give 05-643 or 93-11 in the colony, it is high for BCF to backcross 2Colony is used for the Fine Mapping master and imitates grain length, the heavy QTL of grain.
2. main grain length QTL molecule location and the marker development method of imitating
(1) extracting each strain of colony with the SDS method is that (concrete grammar is referring to molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989)) for DNA.
(2) at first use 976 pairs of SSR primers (according to gramene:http: //www.gramene.org/, the sequence that announce the website is synthetic) large seed matter N411 and kind 05-643,93-11 parent's dna polymorphism is carried out initial analysis.Obtaining has polymorphic mark to be used to make up genetic linkage maps between the parent.
(3) the PCR reaction volume is 10 microlitres, and wherein 10 * buffer1 microlitre has 2.5mM MgCl 2The buffer1 microlitre, 2.5mMdNTPs 0.1 microlitre, Taq enzyme (5 units/microlitre) 0.1 microlitre, template DNA 1 microlitre adds water to 10 microlitres.After the SSR response procedures is 95 ℃ of preparatory sex change 5min, 95 ℃ of sex change 30s, 50-58 ℃ of annealing 30s, 72 ℃ are extended exhibition 30s, circulate 35 times, and last 72 ℃ are extended 10min, and 4 degree are preserved.In the enterprising performing PCR amplification of pcr amplification appearance, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel (containing 7.6 gram acrylamides and 0.4 gram methylene diacrylamide in the 100ml polyacrylamide solution), and silver dyes then, takes a picture statistics.Utilize polymorphism mark to pass through F 2Segregating population is set up linkage map, analyzes the grain type and the heavy QTLs of grain of grain.
(4) marker development: just designing new InDel mark in the localizing objects zone: the rice genome of downloading from the rice genome database website between the positioning area of target gene place is announced sequence; According to the genome sequence comparison difference of the warm and fine 93-11 of Japan, design I nDel mark (seeing table 1).
Table 1. just designs new InDel mark in the localizing objects zone
Figure BDA0000101468560000061
1. the IRGSP Build04 of position shown in
(5) according to chain exchange rule, utilize colony's genotype data to make up the genetic map of paddy rice, used software is Mapmaker3.0, the LOD threshold value is made as 3.0, obtains linkage map.
(6) utilize WinQTLcart2.5 software and IciMapping software to F 2The genotype data of the molecule marker of each individual plant of colony is carried out software analysis, Mapping of QTL s with the grain characters of its corresponding each individual plant.
(2) result and analysis:
Paddy rice japonica rice large seed matter N411 (thousand seed weight 71.1 grams) obtains F with 05-643 (thousand seed weight 17.9 grams) 93-11 (thousand seed weight 28.3 grams) hybridization 1F 1Grain length, thousand seed weight all are in mid-parent or middle parent parent on the low side value.F 2Seed length and thousand seed weight make the number of times distribution plan, show successive quantitative character characteristic, illustrate grain length with the grain heavily be proterties by controlled by multiple genes.Utilization is distributed in 12 chromosomal 107 SSR polymorphism marks, utilizes the F of N411/05-643 2182 individuals in generation have been set up the molecule marker collection of illustrative plates, like Fig. 2.Utilize WinQTLcart2.5 software that grain is weighed and component: grain length, the wide and thick QTLs of carrying out of a grain location, adopt to meet interval graphing method, 1000 iteration of sampling are confirmed the LOD value.The QTLs analysis revealed, the QTLs of control grain length is distributed in the 3rd karyomit(e), and wherein the grain length QTL of contribution rate maximum is between molecule marker RM5864-RM3199, like Fig. 2.
The further employing progressively localized method of progressively backcrossing, Fine Mapping grain length QTL.Utilize the BC of N411/05-643 1F 2Colony, QTL is positioned between the RM6266-RM3350 with purpose.BC from N411/93-11 2F 2In the colony, select through molecule marker, it is less to select the target area, and background is single, and whole strain phenotype is backcrossed to 93-11 near the individuality of 93-11, at BC 3F 1In the colony, select through molecule marker, select contain the purpose zone again background collect single-strain seed near the individuality of 93-11, plantation forms BC 3F 2About 3000 strains of colony.
To BC 3F 2The preceding at random 212 strain individual plants of colony are received seed, and the length and width of investigating seed are thick, analyze the molecular marker data of colony target area simultaneously, like Fig. 3.To remaining BC 3F 2The both sides label screening exchange strain of colony is through investigating the phenotypic data of exchange strain, the inner marker of combining encryption, the position (Fig. 4) of affirmation QTL.
Examination to 2968 strain separating individual; 91 exchange strains between molecule marker SSR mark RM15561 and RM15630 have been obtained; Encrypt the molecule marker between two marks, and the sequence of the sparse zone contrast Japan warm and fine 9311 of molecule marker is developed new InDel mark.Utilize the molecular marker analysis reorganization of encrypting individual, confirm the particular location that reorganization takes place.Binding analysis seed length data is carried out the high precision linkage analysis, and qGL3 is confirmed within the 46.6kb between InDel mark xj39 and the xj26, is divided into from (Fig. 4) with mark xj24, xj18.
Individual according to the reorganization that obtains simultaneously: the allelotrope qGL3-D that under the 93-11 background, imports N411; Interval span from InDel mark xj39 strain system of about 700kb to RM3601 pass through selfing pure and mild after, confirm as the near isogenic line 93-11NIL-qGL3-D of grain length gene qGL3.
2, the clone of gene
According to the fine genome sequence of Japan; The rice genome annotation system (MSU Rice Genome Annotation (Osa1) Release6.1:http: //rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/) in this interval prediction 5 genes; Wherein three is transposon or retrotransponsons, the EST information that has no.The gene of two other supposition: LOC_Os03g44484 and LOC_Os03g44500 have part EST information, but do not have total length eDNA sequence.According to projections sequences, we designed LOC_Os03g44500 gene primers qGL3-F (SEQ ID NO: 10), qGL3-R (SEQ ID NO: 11) and LOC_Os03g44484 Gene Primer G12-F (SEQ ID NO: 12), G12R (SEQ ID NO: 13); each of the primer pairs and two parents N411 93-11 panicle cDNA templates were amplified from the two parents N411 and 93-11 spike Department of cDNA templates were successfully amplified from these two genes (conventional PCR procedure, the annealing temperature appropriate adjustments) by TA cloning, the amplified sequences connected to pMD18-Tsimple vector (purchased from Takara), transforming competent cells of E. coli DH5a, coated ampicillin resistance on LB plates, pick monoclonal positive by PCR detection (points to another using their respective gene primers), the number of individual clones sequenced delivery GenScript biotech companies.The eDNA sequence of relatively finding LOC_Os03g44484 does not have difference in two parent N411 and 93-11.And there are two place's differences in the cDNA sequence of LOC_Os03g44500 in big grain parent N411 and 93-11: compare with the allelotrope (SEQ ID NO:1) from 93-11, come the disappearance of a single bases G of-72 positions existence in 5`UTR zone of the allelotrope (SEQ ID NO:3) of arrogant grain parent N411; Be positioned at mononucleotide replacement of the 10th coding region+1092 positions on the exon, C → A, and then cause amino acid whose replacement of protein: 364 aspartic acid (Asp, D) be transformed into L-glutamic acid (Glu, E), D364E.We are decided to be the candidate gene of qGL3 with the LOC_Os03g44500 among the 93-11 parent thus, and sequence is SEQ ID NO:1; And with its allelic variation called after qGL3-D from next arrogant grain parent N411, sequence is SEQ ID NO:3.
The information biology prediction finds that qGL3 is made up of 21 exons and 20 introns.Coding region length is 3012bp, 1003 the amino acid whose polypeptide of encoding.Prediction finds that its encoded polypeptides is one and contains 2 kelch multiple serine/threonine phosphoric acid (ester) enzyme (kelch repeat-containing serine/threonine phosphatase) according to protein functional domain.In paddy rice, three members are contained in this subgene family, are respectively LOC_Os03g44500; LOC_Os05g05240 and LOC_Os12g42310, we are with its difference called after qGL3, qGL3-L1, qGL3-L2.In Arabidopis thaliana, its homologous gene belongs to one four member's subfamily, is respectively AtBSU1, AtBSL1, and AtBSL2, AtBSL3, they are positive regulating factors of BRs signal pathway.
Embodiment 2 qGL3 and homologous gene family expression analysis thereof
On NCBI, the EST profiles database analysis of paddy rice is found: three members of the qGL3 family of paddy rice have expression in multiple tissue, especially in dividing vigorous tender tissue.In order better to understand the expression pattern of this gene family; MRNA sequence according to gene annotation and est database splicing; We have designed three gene qGL3, qGL3-L1, qGL3-L2 specific expressed primer separately to qGL3Seq-F (SEQ ID NO:14), qGL3Seq-R (SEQ ID NO:15); QGL3-L1Seq-F (SEQ ID NO:16), qGL3-L1Seq-R (SEQ ID NO:17) and qGL3-L2Seq-F (SEQ ID NO:18), qGL3-L2Seq-R (SEQ ID NO:19).Utilize the method for semi-quantitative analysis that the expression of this gene family is analyzed.
Were extracted from 93-11 and 93-11NIL-qGL3 root (R), stems (C), just out of the blade (LB), leaf sheath (LB), 2cm-3cm primary spike (PP), 8cm-10cm panicle (YP), just to draw the mature ears (MP) of the RNA (using the day as the company's RNA extraction kit) and reverses the cDNA, reverse reagent system is used:
Figure BDA0000101468560000081
RT Master Mix Perfect Real Time, purchased from Takara.Utilize OsRac1 as confidential reference items (primer to OsRacl-F (SEQ ID NO:20), OsRacl-R (SEQ ID NO:21)) mode transfer plate concentration to consistent.Utilize the primer expression primer of three gene specifics right then: qGL3Seq-F/qGL3Seq-R; QGL3-L1Seq-F/qGL3-L1Seq-R and qGL3-L2Seq-F/qGL3-L2Seq-R analyze three homologous genes expression in each tissue through reacting with a PCR.As can be seen from the figure, qGL3 gene family member has expression in each tissue, and especially the expression amount in blade is the highest.Simultaneously can find out that the expression amount of each tissue among background parent 93-11 and the near isogenic line 93-11NIL-qGL3 does not have difference (Fig. 6 A).Thereby in the real time fluorescent quantitative analysis of back, near isogenic line is not analyzed.
Expression for further careful relatively qGL3 and homologous gene qGL3-L1 thereof, qGL3-L2; It is right that qPCR design of primers instrument that we provide through qPCR primer design tool website (http://www.quantprime.de/main.php page=home) has designed the special primer of qGL3, qGL3-L1, three genes of qGL3-L2; Be followed successively by: qP1-F (SEQ ID NO:22), qP1-R (SEQID NO:23); QP2-F (SEQ ID NO:24), qP2-R (SEQ ID NO:25); QP3-F (SEQ ID NO:26), qP3-R (SEQ IDNO:27) further analyzes the expression of three genes in each tissue through the method for Quantitative PCR.What concrete reagent adopted is:
Figure BDA0000101468560000082
Premix Ex Taq TM// (Perfect Real Time), available from Takara company, that the fluorescent PCR appearance uses is the 7500fast of ABI company.Unpumped spire (YL) has been added in this analysis on semiquantitative basis; Be employed on same 96 orifice plate and carry out three repetitions simultaneously; Internal control gene adopts is 18S (primer to 18S-F (SEQ ID NO:28), 18S-R (SEQ ID NO:29)) result such as Fig. 6 B.
From Fig. 6 B, can find out; Whole gene family all has expression in each tissue; And qGL3 and qGL3-L2 are higher than qGL3-L1 expression amount; Has more similar expression pattern simultaneously; In fringe along with the trend that expression amount rises all appears in the whole family of developmental process; QGL3-L2 keeps higher expression at the tender blade of children always in sophisticated blade and the leaf sheath, be different from the ascendant trend gradually of qGL3.
Promoter (SEQIDNO:9) to gene qGL3; We have designed primer qGL3Pro-F(SEQIDNO:30; Restriction enzyme site PstI) and qGL3Pro-R(SEQIDNO:31; Restriction enzyme site NcoI); The genomic DNA that utilizes big grain parent N411 is as template; Obtained the promoter sequence of gene qGL3 by pcr amplification; To obtain the PCR product clones by T-A; Be connected on the pMD-18TsimpleT-simple carrier and (buy) from Takara; By heat shock method transformed into escherichia coli DH5a competent cell; LB is dull and stereotyped for coating ammonia benzyl resistance; The picking monoclonal is by PCR positive detection (utilizing primer to qGL3Pro-F and qGL3Pro-R); Positive monoclonal is delivered biotech firm's order-checking, and the bacterial strain that sequence is correct shakes bacterium and extracts the plasmid that has target fragment.On the vector plasmid pCAMBIA-1304 (restriction enzyme site structure iron such as Fig. 9 A) that the plasmid that obtains is utilized restriction enzyme Pst I and Nco I enzyme to cut to be connected to same enzyme and to cut, formation recombinant plasmid pCAMBIA-qGL3Pro-GUS.With this plasmid through spending 11 in the agrobacterium-mediated transformation rice transformation kind.To positive T 0(concrete grammar is with reference to molecular cloning: lab guide) to carry out the GUS staining analysis for transfer-gen plant.Connect the transgenosis promotor GUS expression activity analysis that gus gene carries out; Confirmed the result of Real-Time PCR equally; Can find that simultaneously qGL3 is in the periphery of the middle arteries and veins of the vascular tissue of the tip of a root, stem, blade, leaf sheath, summit vegetative point, the base portion of prematurity grain husk shell, sophisticated clever shell; And the base portion predominant expression of gynoecium, and expression (Fig. 7) in the no longer sophisticated flower pesticide.
The Subcellular Localization of embodiment 3 qGL3 wild-type proteins and advantage equipotential albumen qGL3-D
Utilize primer to qGL3GFP-F(SEQIDNO:32); QGL3GFP-R(SEQIDNO:33) qGL3(that will be cloned into respectively on the T carrier sees specific embodiment one) and the full length coding region of allelic variation gene qGL3-D increase out; Be connected to (purchase is from Takara) on the pMD-18Tsimple carrier by the T-A clone; By heat shock method transformed into escherichia coli DH5a competent cell; LB is dull and stereotyped for coating ammonia benzyl resistance; The picking monoclonal is by PCR positive detection (utilizing primer to qGL3Seq-F/qGL3Seq-R); Positive monoclonal is delivered biotech firm's order-checking, and the bacterial strain that sequence is correct shakes bacterium and extracts the plasmid that has target fragment.The plasmid that obtains is utilized Smal I enzyme to cut to be connected on the vector plasmid pBI121 that same enzyme cuts, constitute recombinant plasmid pBI121-qGL3 and pBI121-qGL3-D.To connect product transformed into escherichia coli Top10 competent cell; LB is dull and stereotyped for coating ammonia benzyl resistance; The picking mono-clonal is through PCR positive detection (utilizing primer to qGL3Seq-F/R); And through enzyme cut the checking its connection directivity; Positive monoclonal is delivered biotech firm's order-checking, the thalline that correctly connects is expanded the pBI121 plasmid after numerous extraction is integrated.Plasmid is transformed the epidermic cell of onion through particle bombardment.After the incubated overnight, fluorescent microscope is sought down and is transformed successful cell.
As can be seen from Figure 8: the albumen 93-11-qGL3-GFP that does not undergo mutation (SEQ ID NO:2) has distribution in whole cell, the tenuigenin enrichment region after plasmolysis has the gathering of fluorescent signal.And the albumen N411-qGL3-D-GFP (SEQ ID NO:4) after the sudden change also is covered with whole cell, but in nucleus, has more accumulation, and this possibly obtain directly related with proteic sudden change back function.
The paddy rice transgenic research of embodiment 4 qGL3 genes
(1) the qGL3 wild-type is crossed and is expressed and RNAi expression interference research
Utilize qGL3 (the seeing specific embodiment one) coding region that primer will be cloned on the T carrier 0XqGL3-F (SEQ ID NO:34) 0XqGL3-R (SEQ ID NO:35) to increase out; Be connected on the pMD18-Tsimple carrier through the T-A clone; Through heat shock method transformed into escherichia coli DH5a competent cell; LB is dull and stereotyped for coating ammonia benzyl resistance; The picking mono-clonal is through PCR positive detection (utilizing primer to qGL3Seq-F/R); Positive monoclonal is delivered biotech firm's order-checking, and the bacterial strain that sequence is correct shakes bacterium and extracts the plasmid that has target fragment.Utilize restriction enzyme KpnI and Sal I while enzyme to cut the plasmid that has target fragment; Run glue and reclaim small segment; With the pCAMBIA-1300S vector plasmid that utilizes Kpn I and Sal I enzyme to cut equally (through carrier pCAMBIA-1300 transforming (Zhou et al.; 2009), the plasmid structure iron is shown in Fig. 9 B) run the big fragment that glue reclaims and utilize the T4 ligase enzyme to connect.To connect product heat shock method transformed into escherichia coli DH5a competent cell; LB is dull and stereotyped for the coating kalamycin resistance; The picking mono-clonal (utilizes primer to qGL3Seq-F through the PCR positive detection; QGL3Seq-R); Positive monoclonal is delivered biotech firm's order-checking; The bacterial strain that sequence is correct shakes bacterium and extracts the plasmid that has target fragment, is the qGL3 wild-type and crosses expression plasmid: pCAMBIA-1300S-qGL3.The qGL3 wild-type is crossed expression plasmid pCAMBIA-1300S-qGL3 transform the Agrobacterium competent cell; LB is dull and stereotyped for the coating kalamycin resistance; Detecting the positive through PCR behind the picking mono-clonal (utilizes primer to qGL3Seq-F; QGL3Seq-R); With positive strain with in spend 11 callus to cultivate altogether, through repeatedly having obtained independently transgenic line of four strains after the differentiation.With T 0Seed kind for the individual plant results becomes plant, utilizes the qGL3Seq-F (SEQ ID NO:14) that strides intron, and qGL3Seq-R (SEQ ID NO:15) primer is to carrying out positive verification.Each different strains system all has positive individual plant to occur.To positive individual plant OX-1, OX-2 and OX-3 carry out expression analysis and phenotype investigation.
(1260～1743bp) design primers are to RNAi-qGL3F2 (SEQ ID NO:36)/RNAi-qGL3R2 (SEQ ID NO:37) and RNAi-qGL3F1 (SEQ ID NO:38)/RNAi-qGL3R1 (SEQ ID NO:39) among the SEQ ID NO:1 to the special zone of qGL3.T carrier to insert qGL3 is a template; With RNAi-qGL3F2 and RNAi-qGL3R2 is primer, and amplification obtains forward and inserts fragment, inserts carrier pCK303 through restriction enzyme site Spe I and Sac I; The restriction enzyme site structure iron is (ZHEN WANG shown in Fig. 9 C; CHANGBIN CHEN, YUNYUAN XU, RONGXI JIANG; YE HAN; ZHIHONG XU and KANG CHONG, Plant Molecular BiologyReporter 22:409-417, December 2004).T carrier to insert qGL3 is a template; With RNAi-qGL3F1 and RNAi-qGL3R1 is primer; Fragment is oppositely inserted in amplification; This reverse fragment of inserting is inserted into the successful insertion forward of sequence verification through restriction enzyme site Kpn I and BamH I and inserts between the restriction enzyme site of Kpn I and BamH I of segmental pCK303 carrier; Deliver make up successful carrier to the order-checking of Takara biotech firm; Proving correctness obtains qGL3 RNAi interference vector: pCK303-qGL3.The pCK303-RNAi interference vector that correctly makes up through spending 11 (concrete grammar such as the above-mentioned express transgenic of crossing) in the agrobacterium-mediated transformation rice transformation kind, has been obtained independently transgenosis T of 23 strains altogether 0For strain be.With growing way 18 strains preferably is to plant into plant; Utilization is positioned at LAZ-F (SEQ IDNO:50) and 303P1 (the SEQ ID NO:51) primer on the carrier; PCR detects the positive of each strain system: whether the integral body of confirming plant had after positive the appearance; Choosing representative individual plant verifies once more; Final selected three positive strains are Ri-4, and Ri-5 and Ri-6 carry out further expression analysis.
With three independently positive individual plant OX-1 that express in the strain system that cross; OX-2 and OX-3; With three individual plant Ri-4 in the positive strain system of RNAi independently; Spend 11 expression of carrying out qGL3 simultaneously to identify in Ri-5 and Ri-6 and the wild-type, analyzed the expression of expressing and interfering qGL3-L1 and qGL3-L2 in the strain system (the primer is identical with expression analysis primer among the embodiment 2) simultaneously.Get each individual plant each expression of gene situation of unfolded arrow leaf analysis just.
Like Figure 10 through semi-quantitative expressed analysis revealed (utilizing primer) to qGL3Seq-F/R; Tangible raising has taken place in the expression amount of crossing the target gene qGL3 that expresses strain system; And interfere plant that the obvious expression reduction has taken place, and do not make a difference for homogenic expression.The real-time quantitative expression analysis is found, crosses the expression amount of expressing plant tangible raising has all taken place.The expression amount of target gene qGL3 has received obvious suppression in the RNAi plant; Have only in the wild-type plant about 10%; Two other homogenic expression amounts have also received interference to a certain degree simultaneously, but the degree that suppresses is not clearly, have approximately interfered to fall 10%～50%.
The phenotype investigation is found: no matter be the expression positive plant, or obvious variation does not all take place the whole plant type of RNAi interference plant, has only the length of seed and weight to take place significantly to diminish, (Figure 11).
(2) qGL3 wild-type functional domain blocked expression study
To the prediction of gene qGL3 proteins encoded functional domain, the qGL3 encoded protein has two tangible structural domain kelch and PP2Ac structural domain, like Figure 12.In the rice grain length adjustment, bring into play keying action in order further to detect two which positions of functional domain; We pass through the section Auele Specific Primer to OXqGL3-F (SEQ ID NO:34; Restriction enzyme site Kpn I) and OXkelch-R (SEQ ID NO:40; Restriction enzyme site Sal I); Amplify fragment from the T carrier that has qGL3, this fragment coding is removed the polypeptide OX-kelch of PP2Ac functional domain.Utilize Auele Specific Primer to OXPP2A-F (SEQ ID NO:41; Restriction enzyme site Kpn I) and OXqGL3-R (SEQ ID NO:35; Restriction enzyme site Sal I), amplify fragment from the T carrier that has qGL3, this fragment coding is removed the polypeptide OX-PP2Ac (as shown in figure 12) of kelch functional domain.Be building up to respectively on the expression vector pCAMBIA-1300s, and formed two and cross expression vector pCAMBIA-1300S-OX-kelch and pCAMBIA-1300S-OX-PP2Ac.Through spending 11 in the agrobacterium-mediated transformation rice transformation kind.Investigate discovery OX-kelch transgenosis T through phenotype 0Generation is positive to be crossed and expresses plant and show the grain type that shortens and OX-PP2Ac transgenosis T 0In generation,, the variation (Figure 12) on the statistical significance did not take place in the positive grain type of expressing plant of crossing.This explanation comes from the kelch structural domain in the regulatory function to rice grain length, and with the PP2Ac structural domain, and about 290 the amino acid whose link zones relation between kelch and the PP2Ac is little, like Figure 12.
(4) homologous gene qGL3-L1 and qGL3-L2 cross expression study and insertion knocks out the mutation type surface analysis in the qGL3 paddy rice
Compare through information biology; According to information of forecasting; We have designed the specificity clone primer of homologous gene qGL3-L1 and qGL3-L2 in the qGL3 paddy rice to qGL3-L1-F (SEQ ID NO:42)/qGL3-L1-R (SEQ ID NO:43) and qGL3-L2-F (SEQ ID NO:44)/qGL3-L2-R (SEQ ID NO:45); Two the pairing complete CDs of homologous gene zones from the cDNA of rice varieties 93-11, have been amplified; Clone through T-A; Be connected on the pMD18-Tsimple carrier, obtain recombinant plasmid T-qGL3-L1 and T-qGL3-L2.Through heat shock method transformed into escherichia coli DH5a competent cell; LB is dull and stereotyped for coating ammonia benzyl resistance; The picking mono-clonal is through PCR positive detection (utilizing clone's primer to qGL3-L1-F/R and qGL3-L2-F/R); Positive monoclonal is delivered biotech firm's order-checking; The qGL3-L1 sequence is SEQ ID NO:5, and the qGL3-L2 sequence is SEQ ID NO:7; The bacterial strain that sequence is correct shakes bacterium and extracts the plasmid have target fragment and preserve subsequent use.Utilize the primer that has restriction enzyme site to qGL3-L1-KpnF (SEQ ID NO:46, restriction enzyme site KpnF) and qGL3-L1-SalR (SEQ ID NO:47, restriction enzyme site SalR) afterwards respectively; QGL3-L2-KpnF (SEQ ID NO:48; Restriction enzyme site KpnF) and qGL3-L2-SalR (SEQ ID NO:49; Restriction enzyme site SalR) from the plasmid that has target gene, amplifies two homologous gene qGL3-L1 and qGL3-L2 respectively; Then through enzyme cut be connected to expression vector pCAMBIA-1300S on 35S promoter after, obtained two homogenic expression vector pCAMBIA-1300S-qGL3-L1 and pCAMBIA-1300S-qGL3-L2 of crossing.Through spending 11 (concrete grammar such as the above-mentioned express transgenic of crossing) in the agrobacterium-mediated transformation rice transformation kind.Some transgenic positive T have been obtained respectively 0For strain be.It is very faint with the seed growth that qGL3-L2 can make paddy rice to analyze the homologous gene qGL3-L1 that found to express qGL3 after the seed of positive individual plant, Figure 13 D.
Simultaneously, after the public mutant database of paddy rice (http://signal.salk.edu/cgi-bin/RiceGE) inquiry, the insertion of having buied the qGL3-L1 gene from the PFGT-DNA mutant library of Korea S knocks out mutant qgl3-l1.Can find that through the phenotype observation disappearance of qGL3-L1 gene is that plant height obviously reduces, like Figure 13 A, tassel shortens like Figure 13 B, and seed obviously shortens little, shown in Figure 13 C.
Embodiment 5 qGL3 gene advantage allelic variations are for GS3, and the functionality advantage of D1 is studied
Grind the cross combination of the black glutinous structure of B and Longli through large seed matter N411 and rice varieties heat, and after the continuous backcross selfing repeatedly, reach the background parent more than 90%, simultaneously segregating generation BC through the molecular marker analysis overall labeling 4F 2The separation of seed grain length simple three peaks occur and distribute; And the single-gene that meets 1: 2: 1 through the individual number in statistical study three peaks separates, we obtained equally with heat grind B and Longli black glutinous be the near isogenic line of background: heat grinds NIL-qGL3 and glutinous NIL-qGL3 is deceived in the Longli.Make discovery from observation, grind the advantage allelic variation of (GS3 is the allelotrope of complete function, can make seed shorter, is shorter than 93-11) qGL3 under the B background in heat and still can bring into play the effect (like Figure 14) that makes seed elongated.The advantage allelic variation of (the D1 gene loses function, makes seed ultrashort, is shorter than heat and grinds B) qGL3 also still can be brought into play the effect (like Figure 14) that makes seed elongated under the black glutinous background in Longli.This illustrates that gene of the present invention is in the position than downstream of hereditary path, can well suppress the restraining effect in middle and upper reaches negative regulation site, seed length control path.
The molecular marker assisted selection breeding experiment of embodiment 6 qGL3
Is repeatedly backcrossing of recurrent parent through big grain parent N411 with breeding backbone parent 93-11 hybridization and with 93-11, the selfing BC that backcrosses afterwards 3F 2In the colony, select to continue to backcross to 93-11, at BC near the 93-11 individual plant that seed length is long simultaneously according to whole plant forms 4F 1In through with the gene line of each individual plant of molecular marker analysis around the qGL3; Select the target area and import minimum individual plant, the context marker type obtains the near isogenic line 93-11NIL-qGL3-D (concrete like embodiment one) that pure and mild small segment inserts near the individual plant selfing of 93-11 simultaneously.Whole plant type as shown in figure 15.Analyzing near isogenic line 93-11NIL-qGL3-D and the recurrent parent 93-11 difference on economical character finds; Through the advantage allelic variation gene qGL3-D of hybridizing the qGL3 that imports the seed length of recurrent parent 93-11 is increased more than the 2mm, be that the grain of paddy rice heavily increases more than 37%.And for the not tangible influence of other agronomy phenotype, what pay special attention to is that grain number per spike is reduced.Concrete phenotypic difference is as shown in table 2.Utilize this near isogenic line 93-11NIL-qGL3-D and two to recover system extensively to account for 63S hybridization structure hybridisation rice F simultaneously 1-2, with common 93-11 and the hybridisation rice F that extensively accounts for the 63S structure 1-1 comparative analysis of carrying out each agronomy phenotype and output is found: contain the allelic near isogenic line 93-11NIL-qGL3-D of qGL3 advantage and can make the grain length of hybrid rice F 1 increase by 17.8% (being increased to 11.26mm from 9.56mm); Grain heavily increases by 30.7% (100-grain weight is increased to 3.498g from 2.676g), like Figure 16.
Table 2: each agronomy phenotype of background parent 93-11 and near isogenic line 93-11NIL-qGL3 is (Nanjing, 2010 positive seasons) relatively
Figure BDA0000101468560000121
QGL3 of the present invention is the title of a gene, it comprises two different allelic variations, and one is big grain length grain, our called after qGL3-D, and one is the short grain of granule, we are called after qGL3.
Figure IDA0000101468660000011
Figure IDA0000101468660000021
Figure IDA0000101468660000041
Figure IDA0000101468660000051
Figure IDA0000101468660000061
Figure IDA0000101468660000071
Figure IDA0000101468660000081
Figure IDA0000101468660000111
Figure IDA0000101468660000121
Figure IDA0000101468660000131
Figure IDA0000101468660000141
Figure IDA0000101468660000151
Figure IDA0000101468660000161
Figure IDA0000101468660000171
Figure IDA0000101468660000191
Figure IDA0000101468660000201
Figure IDA0000101468660000211
Figure IDA0000101468660000221
Figure IDA0000101468660000231
Figure IDA0000101468660000241
Figure IDA0000101468660000251
Figure IDA0000101468660000261
Figure IDA0000101468660000271
Figure IDA0000101468660000291
Figure IDA0000101468660000301
Figure IDA0000101468660000311

Claims (10)

1. control the rice grain length semi-dominant gene qGL3 heavy with grain for one kind, nucleotide sequence is shown in SEQ ID NO:1.
2. the described gene qGL3 of claim 1 encoded protein, aminoacid sequence is shown in SEQ ID NO:2.
3. the advantage allelotrope qGL3-D of the described gene qGL3 of claim 1, nucleotide sequence such as SEQ ID NO:3 institute are not.
4. the described advantage allelotrope of claim 3 encoded protein, sequence is shown in SEQ ID NO:4.
5. the homologous gene qGL3-L1 of the said gene qGL3 of claim 1 in paddy rice, sequence is shown in SEQ ID NO:5, and its encoded protein sequence is shown in SEQ ID NO:6.
6. the homologous gene qGL3-L2 of the said gene qGL3 of claim 1 in paddy rice, sequence is shown in SEQ ID NO:7, and its encoded protein sequence is shown in SEQ ID NO:8.
7. the application of the described gene qGL3 of claim 1 in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
8. the application of the described gene qGL3-D of claim 3 in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
9. the application of the described gene qGL3-L1 of claim 5 in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
10. the application of the described gene qGL3-L2 of claim 6 in crop genetic improvement, the preferably application in improvement crop grain length and/or grain weight.
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