CN103571869A - Method for improving fertility of indica-japonica rice hybrids by virtue of LRK1 gene transformation - Google Patents

Abstract

The invention belongs to the field of transgenic technologies, and relates to a method for improving the fertility of indica-japonica rice hybrids by virtue of LRK1 gene transformation. According to the method, rice LRK1 genes are transformed into indica calluses by virtue of a gene gun microprojectile bombardment method, and rice plants for expressing the LRK1 genes are obtained through recovery culture. Experimental results show that the LRK1 genes can be used for successfully transforming rice and hybridizing rice with japonica rice varieties such as Nipponbare and Akihikari respectively, and the F1-generation seed setting rates are greatly higher than the control so as to achieve the seed setting rate level of regular varieties. Rice is provided with wide compatibility and can overcome the infertility characteristic of indica-japonica hybrids after being transformed by the rice LRK1 genes provided by the invention and the homologous genes thereof. The invention provides a low-cost and high-benefit novel method for utilization for the advantages of indica-japonica rice intersubspecific hybrids.

Description

A kind of method of utilizing LRK1 gene transformation to improve Indica round-grained rice hybrid fertile

Technical field

The invention belongs to field of transgenic technology, relate to the method that improves Indica round-grained rice hybrid fertile, especially a kind of method of utilizing LRK1 gene transformation to improve Indica round-grained rice hybrid fertile.

Background technology

Recent years, the existing numerous reports of reason of Rice intersubspecific hybrid sterility, also inquire into some extent to the Genetic Mechanisms of its sterility; Research shows, the sterile reason of inter subspecific hybrid is many-sided, wherein, in some combinations, hybrid dysgenesis is caused by pollen abortion, separately have in some combinations and show as again and be with the abortion of a certain specific allelic megagamete part, and be that existing megagamete abortion has microgametophyte abortion in more combination.In prior art, experimental results demonstrate, the sterility of Xian, round-grained rice hybrid is not only controlled by individual gene, but is controlled by a plurality of sterile gene.

The systematic studyes such as Ikehashi 74 kinds of different ecological type rice varieties species hybrid F ₁fertility situation, result shows, some kinds and indica type variety or with the mixing breed of round-grained rice type, its F ₁all can produce very high fertility, thereby propose the concept of wide affine (wide-compatibility) kind, identify subsequently some wide compatibility genes, the most typical wide compatibility gene is to overcome the corresponding S5 of sterile site S-5 ⁿ.Research also shows, is finding all to have found neutral multiple allelomorphos on Shi， corresponding position, megagamete abortion site, i.e. wide compatibility gene; Above-mentioned wide compatibility gene only works to the sterile site of equipotential, inoperative to nonallelic sterile site, or acts on but do not have the effect that can substitute.Some investigators call female sterile gene " wide compatibility gene ", its main points of view is that hybrid sterility main manifestations is that megagamete is sterile, long-grained nonglutinous rice is relative Si and Sj with japonica rice variety Alkie differentiation on certain seat, wide affine kind is Sn at this seat, allelotrope produces fertility when isozygotying, and produces sterility when allelotrope is SiSj heterozygosis, and allelotrope all can be educated while being SnSi or SnSj, therefore, wide compatibility gene concept is with respect to megagamete abortion site.

Zhang Guiquan etc. call " Specific compatible gene " male sterility gene seat, and its main points of view is that hybrid sterility main manifestations is pollen sterility; On single seat, Alkie differentiation is relative Si and Sj, and the Si of differing materials and Sj have different degrees of differentiation; The fertility of hybrid depends on number and the allelic differentiation distance at heterozygosis seat; Allelotrope can produce sterility and can produce fertility when isozygotying when heterozygosis.

Above-mentioned " Specific compatible gene " and " wide compatibility gene " though different in the theoretical foundation of explaining hybrid dysgenesis sex-controlled inheritance, finally all belong in essence that polygene seat controls, all meet single seat position sporinite-gametophyte and make mutually hereditary pattern.From cell levels, say, cause the reason of Hybrid sterility between indica and japonica rice except microgamete abortion and megagamete abortion, also have pollen on column cap, to sprout obstacle, gamete impaired development, endosperm development is obstructed or undesired, flower pesticide does not ftracture, dysontogenesis and dichogamy etc.; In addition, envrionment conditions also has a great impact it, and above-mentioned factor all relates to complicated molecular genetic mechanism.

Because the complicacy of Hybrid sterility between indica and japonica rice mechanism, at present only successful separating clone wide compatibility shape gene S5 ⁿ, but S5 ⁿgene can only solve the sterility being caused by sterile site S-5, can not overcome the sterility being caused by other sterile sites, therefore, also need to continue to excavate wide compatibility gene and the Specific compatible gene of cloning other, only have a plurality of megagamete compatibility genes and the polymerization of microgamete compatibility gene in same parent, a plurality of wide spectrum affinity parental breeds at a plurality of compatibility genes of seed selection polymerization seat, make above-mentioned parent and various japonica rice or rice variety hybridization all there is affinity, both can eliminate microgametophyte abortion, can eliminate megagamete abortion again, further improve setting percentage and the adaptability to envrionment conditions, contribute to solve subspecies indica and japonica hybrid sterility problem, real realization directly utilizes subspecies indica and japonica hybrid advantage.

There is report to disclose, with rice-cultivating and the hybridization of Jiangxi Dongxiang Wild Rice, be material with the gene building that backcrosses, by chromosomal localization and genomic sequence analysis separating clone from the gene of the genomic group coding transmembrane receptor protein LRK of Dongxiang Wild Rice, have 8 join end to end bunch family members that collection is arranged, respectively called after LRK1, LRK2, LRK3, LRK4, LRK5, LRK6, LRK7 and LRK8.In some rice variety, only contain 7 LRK genes, lack LRK1 gene; In japonica rice, LRK2, LRK3 and LRK5 gene are not expressed; In long-grained nonglutinous rice, lack LRK1 gene, LRK3 and LRK5 gene are not expressed.

Said gene bunch is positioned at No. 2 chromosomal ends of paddy rice, and its DNA sequence dna has been logined at international genome database NCBI, and clone is numbered AY756174 and DQ195081.Through rice seedling Molecular Detection, confirm, in fine 8 the LRK genes of japonica rice Japan, have 5 genetic expressions, be respectively LRK1, LRK4, LRK6, LRK7 and LRK8.One of long-grained nonglutinous rice 9311(super hybridized rice parent) in, also there are 5 LRK genetic expressions, be respectively LRK2, LRK4, LRK6, LRK7 and LRK8.

Till settled the present, there is not yet the report that utilizes this group LRK gene to improve Indica round-grained rice hybrid fertile aspect.

Summary of the invention

The object of this invention is to provide a kind of method that improves Indica round-grained rice hybrid fertile, be specifically related to a kind of method that the LRK1 of utilization gene transformation improves Indica round-grained rice hybrid fertile; Described method is utilized LRK1 gene and homogenic carrier rice transformation kind thereof, in order to overcome Indica round-grained rice intersterility.In embodiments of the invention, by the over-express vector Transformation of Indica Rice kind 9311 of LRK1 gene, the fertility of the cross-fertilize seed such as long-grained nonglutinous rice 9311 and japonica rice Japan are fine, Qiu Guang is increased substantially.

The Argine Monohydrochloride sequence of LRK1 gene of the present invention is as shown in SEQ ID NO2.

In the present invention, described oryza sativa l. RK1 gene be aminoacid sequence as the albumen of SEQ ID NO2, also comprise and having and variant form oryza sativa l. RK1 albumen identical function, SEQ ID NO.2 sequence; Above-mentioned variant form comprises (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several (being generally in 20 at C-terminal and/or N-terminal, being preferably in 10, is more preferably in 5) amino acid; For example, in the art, while replacing with the close or similar amino acid of performance, conventionally can not change the function of protein; Again such as, at C-terminal and/or N-terminal, add one or several amino acid and conventionally also can not change the function of protein; It also comprises active fragments and the reactive derivative of oryza sativa l. RK1 albumen.

In the present invention, the nucleotide sequence of described oryza sativa l. RK1 gene is as shown in SEQ ID NO1.

In the present invention, the nucleotide sequence of described oryza sativa l. RK1 gene comprises that coding has the nucleotide sequence of the polypeptide of oryza sativa l. RK1 protein-active, as 32-3181 position nucleotide sequence and degenerate sequence thereof in SEQ ID NO.1; This degenerate sequence refers to, is arranged in the encoder block 32-3181 position Nucleotide of SEQ IDNO.1 sequence, the sequence that has one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces; Due to the degeneracy of codon, therefore with SEQ ID NO.1 in 32-3181 position nucleotide sequence homology be low to moderate the sequence that approximately 70% degenerate sequence also can be encoded out described in SEQ ID NO.2; It also comprises can be under moderate stringent condition, more preferably under height stringent condition, with in SEQ ID NO.1 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 32-3181 position; Or comprise with SEQ ID NO.1 in from the homology at least 70% of the nucleotide sequence of Nucleotide 32-3181 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

The invention provides the method for utilizing LRK1 gene transformation to improve Indica round-grained rice hybrid fertile, it is characterized in that, it comprises step:

(1) LRK1 gene coded sequence is inserted to plant expression vector construction recombinant expression vector;

(2) Rice Callus is processed in induction;

(3) use the recombinant expression vector rice transformation callus of step (1) gained;

(4) Rice Callus of renewal cultivation step (3) gained.

In the inventive method, in step (1), plant expression vector can be conventional paddy rice expression vector; Build the method for recombination sequence with reference to the standard method of < < molecular cloning > >, for example, LRK1 gene coded sequence is cloned into the multiple clone site of plant expression vector;

Described plant expression vector can be the plant expression vector that commercially available or bibliographical information is crossed, such as pCAMBIA1304 etc.

In the inventive method, in step (3), adopt particle gun micropellet bombardment method to realize recombinant expression vector rice transformation callus, in addition, also can join with other conventional gene transformation methods;

In the inventive method, renewal cultivation Rice Callus is first containing on the inducing culture of selective agent, not grow in step (4), and then screens containing on the substratum of selective agent, and by the callus illumination cultivation after screening; Described selective agent is Totomycin.

In the inventive method, in described step (4) when screening, the method that can adopt selective agent concentration progressively to improve, also can coordinate illumination condition to carry out (adopt for example in one embodiment of the invention, the screening culture medium of the following 30mg/ of containing and 50mg/L Totomycin, coordinate dark cultivation to screen for approximately two weeks simultaneously); After having screened, the callus that gained contains LRK1 gene, can after differentiating plantlet, then proceed to Rooting and hardening-off culture base by callus illumination cultivation on division culture medium of surviving after screening, after growing up, moves into greenhouse.

The method that improves paddy rice indica-japonica hybrid fertility with LRK1 gene transformation of the present invention, adopts particle gun micropellet bombardment method, by oryza sativa l. RK1 gene transformation Rice Callus, obtains the rice plant of expressing LRK1 gene through renewal cultivation; Experimental result shows, the paddy rice that success transforms is with japonica rice variety Japan is fine, autumn light philosophy is hybridized, its F ₁for setting percentage, respectively significantly higher than contrast, reach the setting percentage level of frequent kind.

On the other hand, the invention provides and turn the new application of oryza sativa l. RK1 gene aspect raising Indica round-grained rice hybrid fertile.

In the present invention, oryza sativa l. RK1 gene and homologous gene rice transformation thereof by sequence as shown in SEQ ID NO2, make it have wide compatibility, can overcome indica-japonica hybrid sterile gene.The present invention provides the novel method of a kind of low cost, high benefit for the utilization of rice indica subspecies Heterosis.

Accompanying drawing explanation

Fig. 1 is the expression vector structure iron containing LRK1 gene, and wherein, the position that LRK1 gene inserts expression vector has arrow to indicate.

Fig. 2 is the comparison that turns LRK1 gene 9311, contrasts 9311 plant types.

Fig. 3 for turning LRK1 gene 9311, contrast 9311 respectively with the fine hybrid F of Japan ₁fertility comparison.

Fig. 4 for turning LRK1 gene 9311, contrast 9311 respectively with the fine hybrid F of Japan ₁setting percentage comparison.

Fig. 5 for turning LRK1 gene 9311, contrast 9311 respectively with autumn light hybrid F ₁fertility comparison.

Fig. 6 for turning LRK1 gene 9311, contrast 9311 respectively with autumn light hybrid F ₁setting percentage comparison.

Embodiment

The functional verification of embodiment 1LRK1 gene

(1) build LRK1 expression vector

Employing pCAMBIA1304 is expression vector, and Dongxiang Wild Rice LRK1 gene is inserted into pCAMBIA1304 carrier 35S group type promotor downstream; Contain the expression vector structure of LRK1 gene as shown in Figure 1;

(2) LRK1 gene transformation experiment and result

One of the main parent 9311 who adopts current domestic super hybridized rice is transgene receptor; 9311 mature seeds are shelled and are seeded in dedifferentiation plant tissue after sterilization and cultivate evoked callus on base; After callus induction 2-3 week, adopt particle gun micropellet bombardment method, the pCAMBIA1304 plasmid DNA of the purifying containing LRK1 gene is imported to 9311 callus cells; On the MS substratum that adds 30ppm Totomycin, cultured continuously 3-4 week screening resistant cell line, then transfers to the clone of screening division culture medium induction and sprouts and take root;

(3) transfer of conversion processing test-tube plantlet and transformed plant offspring's Molecular Detection

The 9311 test-tube plantlet transfer fields that LRK1 gene transformation is processed, and in tillering phase, get blade extraction DNA employing PCR expansion discharge technique and carry out Molecular Detection, in the autumn in 2005, obtain positive T ₀generation; Winter in 2005, at Hainan Island adding generation, obtain transgenosis T ₁in generation, amount to 7 strains;

(4) LRK1 transgenosis T ₄for strain and japonica rice hybridization F ₁fertility investigation and statistics

By LRK1 transgenosis T ₄for strain, contrast 9311, Japan is fine and autumn light in kind in 2009 in Shanghai, between heading stage, utilization turns LRK19311, contrasts 9311 and autumn light hybridization fine with Japan respectively, cross-fertilize seed was planted respectively in Shanghai and Jining summer in 2010, and the ripening stage is examined or check the setting percentage of each cross combination; The setting percentage (species test result is as shown in table 1) of each combination investigation 10 strain stem fringe:

Table 1

Combination title	Average setting percentage is tested in Jining	Average setting percentage is tested in Shanghai
			Turn LRK19311/ Japan fine	49.6%	67.8%
Contrast 9311/ Japan is fine	1.7%	19.5%
			Turn LRK19311/ autumn light	80.7%	84.8%
Contrast 9311/ autumn light	12.1%	42.1%

Result is as shown in table 1, turns the hybrid F of the fine and autumn light of LRK19311 and japonica rice Japan ₁setting percentage increase substantially, surpass contrast combination more than 40%; Turn LRK1 gene and make 9311 setting percentages that improved indica-japonica hybrid, there is wide compatibility feature.

The method of embodiment 2 oryza sativa l. RK1 gene transformation long-grained nonglutinous rices 9311

(1) select LRK1 total length ORF section (seeing sequence table), adopt PCR method to be increased from Dongxiang Wild Rice, and the synthetic joint that inserts is cloned into plant expression vector pCAMBIA1304, acquisition LRK1 expression vector pCAMBIA1304/LRK1;

(2) inducing paddy rice callus culture base

1) induction and subculture medium: MS+2mg/L2,4-D;

2) height oozes substratum: MS+2mg/L2,4-D+46.67g/L sorbyl alcohol+46.67g/L N.F,USP MANNITOL;

3) first round screening culture medium: MS+2mg/L2,4-D+30mg/L Totomycin;

4) second take turns screening culture medium: MS+2mg/L2,4-D+50mg/L Totomycin;

5) division culture medium: MS+3mg/L6-BA+0.5mg/LNAA+50mg/L Totomycin;

6) Rooting and hardening-off culture base: 1/2MS+0.1mg/LNAA;

Note: 1. above-mentioned substratum is all containing 30g/L sucrose+2.5g/L agar, pH5.8; 2. callus of induce, subculture, screening and culturing condition are 26-28 ℃ of dark cultivation, and differentiation, strong plantlets and rootage are 26-28 ℃ and 16 hour photoperiod;

(3) callus induction and processing

1) get the pollination immature seed of latter 12-15 days, under aseptic condition, first with 70% ethanol, embathe 10min, proceed to 0.1% mercuric chloride and soak 20min, sterile water wash 3 times;

2) under aseptic condition, peel off rataria, be inoculated on calli induction media, 26-28 ℃ of dark cultivation after approximately 20 days cut bud, and subculture once;

3) in subculture medium, select growth vigorous, flaxen callus about 30-50 piece (every 3mm left and right), be placed in height and ooze central authorities on substratum, line up in the circle of diameter 2.5cm, cultivate after about 4-5h for transforming;

(4) via Particle Bombardment Transformation

1) particle gun: the high pneumatic gene gun of purchasing from Ningbo Xin Zhi Science and Technology Ltd., model: GJ-1000;

2) prepare particulate bullet;

3) take 60mg tungsten powder (diameter 1um), join in 1.5ml sterilizing centrifuge tube, then add 1ml dehydrated alcohol, vibration 1min, in the centrifugal 10s of 10000rpm, abandons supernatant, after repeating to wash once, bronze is suspended in to existing using or-20 ℃ of preservations in 1ml sterilized water;

4) draw 50ul tungsten powder suspension in 1.5ml centrifuge tube, add successively 5ug DNA, 50ul2.5MCaCl ₂, 20ul0.1M spermidine, vibrate 5 minutes, the centrifugal 20s of 10000rpm, abandons supernatant, with 140ul dehydrated alcohol rinsing twice, adds 60ul dehydrated alcohol, suspends stand-by;

(5) bombardment receptor material

1) particle gun is put on Bechtop, with 70% alcohol, clean vacuum chamber, and can split film, carry granulosa, metal baffle screen (by the supply of material of Ningbo Xin Zhi Science and Technology Ltd.) sterilizes 30 minutes in 70% alcohol, then with aseptic filter paper, blots or blow off residual alcohol;

2) turn on the power switch vacuum pump and helium cylinder valve;

3) can split film pack into fixing, screw;

4) get the tungsten powder dehydrated alcohol suspension of the coated DNA of 10ul, evenly coat and carry granulosa center, be placed on super clean bench and dry up;

5) pack year granulosa and the backstop that are loaded with micro-bullet into micro-bullet launching device, one of particle is faced down;

6) culture dish is placed on pallet, makes callus concentrate on culture dish central authorities;

7) open gas cylinder, regulate pressure 1100Psi;

8) vacuumize, when vacuum tightness reaches desired value, forward VAC key to Hold position;

9) bombard, every ware bombardment 2 times (bombarding for the second time after culture dish 90-degree rotation after bombardment for the first time), presses venting key and makes vacuum meter reading back to zero, takes out sample, oozes continuation on substratum cultivate 12-16h after bombardment in height;

(6) transformed calli screening

1) high callus of oozing on substratum after shooting is proceeded to not containing recovering growth 5-7 days on the inducing culture of selective agent;

2) callus is forwarded in the screening culture medium containing 30mg/L Totomycin, evenly put, secretly cultivate and within 14-17 days, carry out resistance screening for the first time;

3) kanamycin-resistant callus tissue is proceeded in the screening culture medium containing 50mg/L Totomycin, secretly cultivate and within 8-12 days, carry out resistance screening for the second time;

(7) screening of oryza sativa l. RK1 gene transformation plant and detection

1) transform test-tube plantlet Molecular Detection

A, by the illumination cultivation 30 days on division culture medium of the callus of surviving after screening;

B, after differentiating plantlet, plantlet is proceeded to Rooting and hardening-off culture base, after growing up, move into greenhouse;

2) Molecular Detection of transformed plant

The transformed plant of choosing after adopting respectively the assorted method of pcr amplification and genome Southern hybridization to detect, obtains the positive plant of the LRK1 that 7 strains contain conversion altogether.

SEQUENCE LISTING

<110> Fudan University

<120> method of utilizing LRK1 gene transformation to improve Indica round-grained rice hybrid fertile

<160> 2

<170> PatentIn version 3.3

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Leu Gln Ile Leu Arg Ser Ser Ser Lys Leu Thr Thr Leu Leu Ile Ser

420 425 430

Asn Asn Phe Met Asn Glu Ser Ile Pro Asp Asp Asp Arg Ile Asp Gly

435 440 445

Phe Glu Asn Leu Gln Val Leu Asp Leu Ser Gly Cys Ser Phe Ser Gly

450 455 460

Lys Ile Pro Gln Trp Leu Ser Lys Leu Ser Arg Leu Glu Met Leu Val

465 470 475 480

Leu Asp Asn Asn Gln Leu Thr Gly Pro Ile Pro Asp Trp Ile Ser Ser

485 490 495

Leu Asn Phe Leu Phe Tyr Leu Asp Val Ser Asn Asn Asn Leu Thr Gly

500 505 510

Glu Ile Pro Met Ala Leu Leu Gln Met Pro Met Leu Arg Ser Asp Arg

515 520 525

Ala Ala Ala Gln Leu Asp Thr Arg Ala Phe Glu Leu Pro Ile Tyr Ile

530 535 540

Asp Ala Thr Leu Leu Gln Tyr Arg Lys Ala Ser Ala Phe Pro Lys Val

545 550 555 560

Leu Asn Leu Gly Asn Asn Glu Phe Thr Gly Leu Ile Pro Gln Glu Ile

565 570 575

Gly Gln Leu Lys Ala Leu Leu Leu Leu Asn Leu Ser Phe Asn Lys Leu

580 585 590

Tyr Gly Asp Ile Pro Gln Ser Ile Cys Asn Leu Arg Asp Leu Leu Met

595 600 605

Leu Asp Leu Ser Ser Asn Asn Leu Thr Gly Thr Ile Pro Ala Ala Leu

610 615 620

Asn Asn Leu Thr Phe Leu Ile Glu Phe Asn Val Ser Tyr Asn Asp Leu

625 630 635 640

Glu Gly Pro Ile Pro Thr Gly Gly Gln Phe Ser Thr Phe Thr Asn Ser

645 650 655

Ser Phe Tyr Gly Asn Pro Lys Leu Cys Gly Pro Met Leu Thr His His

660 665 670

Cys Ser Ser Phe Asp Arg His Leu Val Ser Lys Gln Gln Gln Asn Lys

675 680 685

Lys Val Ile Leu Val Ile Val Phe Cys Val Leu Phe Gly Ala Ile Val

690 695 700

Ile Leu Leu Leu Leu Gly Tyr Leu Leu Leu Ser Ile Arg Gly Met Ser

705 710 715 720

Phe Thr Thr Lys Ser Arg Cys Asn Asn Asp Tyr Ile Glu Ala Leu Ser

725 730 735

Pro Asn Thr Asn Ser Asp His Leu Leu Val Met Leu Gln Gln Gly Lys

740 745 750

Glu Ala Glu Asn Lys Leu Thr Phe Thr Gly Ile Val Glu Ala Thr Asn

755 760 765

Asn Phe Asn Gln Glu His Ile Ile Gly Cys Gly Gly Tyr Gly Leu Val

770 775 780

Tyr Lys Ala Gln Leu Pro Asp Gly Ser Met Ile Ala Ile Lys Lys Leu

785 790 795 800

Asn Gly Glu Met Cys Leu Met Glu Arg Glu Phe Ser Ala Glu Val Glu

805 810 815

Thr Leu Ser Met Ala Arg His Asp Asn Leu Val Pro Leu Trp Gly Tyr

820 825 830

Cys Ile Gln Gly Asn Ser Arg Leu Leu Ile Tyr Ser Tyr Met Glu Asn

835 840 845

Gly Ser Leu Asp Asp Trp Leu His Asn Lys Asp Asp Asp Thr Ser Thr

850 855 860

Ile Leu Asp Trp Pro Arg Arg Leu Lys Ile Ala Lys Gly Ala Ser His

865 870 875 880

Gly Leu Ser Tyr Ile His Asn Ile Cys Lys Pro Arg Ile Val His Arg

885 890 895

Asp Ile Lys Ser Ser Asn Ile Leu Leu Asp Lys Glu Phe Lys Ala Tyr

900 905 910

Ile Ala Asp Phe Gly Leu Ser Arg Leu Ile Leu Pro Asn Lys Thr His

915 920 925

Val Pro Thr Glu Leu Val Gly Thr Leu Gly Tyr Ile Pro Pro Glu Tyr

930 935 940

Ala Gln Ala Trp Val Ala Thr Leu Lys Gly Asp Val Tyr Ser Phe Gly

945 950 955 960

Val Val Leu Leu Glu Leu Leu Thr Gly Arg Arg Pro Val Pro Ile Leu

965 970 975

Ser Thr Ser Lys Glu Leu Val Pro Trp Val Gln Glu Met Val Ser Asn

980 985 990

Gly Lys Gln Ile Glu Val Leu Asp Leu Thr Phe Gln Gly Thr Gly Cys

995 1000 1005

Glu Glu Gln Met Leu Lys Val Leu Glu Ile Ala Cys Lys Cys Val

1010 1015 1020

Lys Gly Asp Pro Leu Arg Arg Pro Thr Met Ile Glu Val Val Ala

1025 1030 1035

Ser Leu His Ser Ile Asp Pro Asp Gly Leu Thr

1040 1045

Claims (10)

1. utilize LRK1 gene transformation to improve a method for Indica round-grained rice hybrid fertile, it is characterized in that, utilize particle gun micropellet bombardment method, by oryza sativa l. RK1 gene transformation Rice Callus, through renewal cultivation, obtain the rice plant of expressing LRK1 gene; It comprises step:

(1) LRK1 gene coded sequence is inserted to plant expression vector construction recombinant expression vector;

(2) Rice Callus is processed in induction;

(3) use the recombinant expression vector rice transformation callus of step (1) gained;

(4) Rice Callus of renewal cultivation step (3) gained.

2. by method claimed in claim 1, it is characterized in that, the Argine Monohydrochloride sequence of described LRK1 gene is as shown in SEQ ID NO2.

3. by method claimed in claim 1, it is characterized in that, described described oryza sativa l. RK1 gene also comprises having and variant form oryza sativa l. RK1 albumen identical function, SEQ ID NO.2 sequence; Described variant form comprises 1-50 amino acid whose disappearance, insertion and/or replacement, and adds one or 20 with interior amino acid at C-terminal and/or N-terminal; And active fragments and the reactive derivative of oryza sativa l. RK1 albumen.

4. by method claimed in claim 1, it is characterized in that, the nucleotide sequence of described LRK1 gene is as shown in SEQ ID NO1.

5. by method claimed in claim 4, it is characterized in that, the nucleotide sequence of described LRK1 gene also comprises that coding has the nucleotide sequence of the polypeptide of oryza sativa l. RK1 protein-active, as 32-3181 position nucleotide sequence and degenerate sequence thereof in SEQ ID NO.1.

6. by method claimed in claim 1, it is characterized in that, in described step (3), adopt particle gun micropellet bombardment method to carry out recombinant expression vector rice transformation callus.

7. by method claimed in claim 1, it is characterized in that, the step of renewal cultivation Rice Callus comprises in described step (4): first containing on the inducing culture of selective agent, do not growing, and then screening containing on the substratum of selective agent, and by the callus illumination cultivation after screening.

8. by method claimed in claim 7, it is characterized in that, described selective agent is Totomycin.

9. by method claimed in claim 7, it is characterized in that, while screening on containing the substratum of selective agent, described selective agent concentration progressively improves and coordinates illumination condition, by survival Rice Callus in division culture medium illumination cultivation, differentiate after plantlet, proceed to Rooting and hardening-off culture base, until move into greenhouse.

10. turn the application of oryza sativa l. RK1 gene in improving Indica round-grained rice hybrid fertile, wherein, the Argine Monohydrochloride sequence of described LRK1 gene is as shown in SEQ ID NO2.

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