CN108676812A

CN108676812A - A method of obtaining output increased plant using CRISPR/Cas9 system sudden changes OsHXK1

Info

Publication number: CN108676812A
Application number: CN201810257507.3A
Authority: CN
Inventors: 庄楚雄; 郑少燕
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2018-10-19
Anticipated expiration: 2038-03-27
Also published as: CN108676812B

Abstract

The invention belongs to paddy gene engineering fields, are related to a kind of method using CRISPR/Cas9 system gene editors mutation rice hexokinase OsHXK1 genes to improve yield plant.The present invention builds pU3 gRNA and pU6 the gRNA carriers of the segment containing target sequence according to OsHXK1 gene design target sequences, builds the pCRISPR/Cas9 carriers of the segment containing target sequence, and obtains transgenic seedling.The plant that the method for the present invention has knocked out OsHXK1 is consistent with OsHXK1 RNAi transfer-gen plant phenotypes, realize raising rice yield of the artificial culture without transgene component, simultaneously with utilize the not essential difference of OsHXK1 RNAi transfer-gen plants, it is strong with purpose, rice yield can be significantly improved, is had broad application prospects in rice molecular design and context.

Description

It is a kind of to obtain output increased plant using CRISPR/Cas9 system sudden changes OsHXK1 Method

Technical field

The invention belongs to paddy gene engineering fields, and in particular to a kind of to utilize CRISPR/Cas9 system sudden changes OsHXK1 The method of output increased plant and the gene of this method editor are obtained, the albumen of the gene code is further related to.

Background technology

Rice (Oryza sativa L.) is as important cereal crops and unifacial leaf model plant.Currently, population in the world Quantity increases, and Cultivated Land Area Decrease, environmental pollution is serious, and extreme climate frequently occurs, and rice yield raising faces a severe challenge. Therefore, " experience breeding " can be realized to " orientation efficiently, is accurately educated by carrying out the concept proposition of Molecular design breeding to rice Kind " transformation (Peleman et al., 2003；Wan Jianmin, 2006；Wang Jiankang et al., 2011).In recent years, with CRISPR/ Cas9(Clustered regularly interspaced short palindromic repeats and CRISPR Associated) technology, which is the genome fixed point editing technique of representative, becomes the new hot spot of research of plant breeding teclmiques, is rice Germplasm enhancement provide safe and efficient new way (Xu et al., 2016；Ma et al.,2015).It utilizes The relevant gene of CRISPR/Cas9 technologies fixed point editor control rice yield simultaneously formulates some non-transgenics with important value Rice new germ plasm and mutant for rice basic research, cultivation and basic research to expand new rice variety lay the foundation, It has great theoretical and practical significance.

Bibliography

Peleman J.D and vander Voort J.R.,2003,Breeding by design,Trends Plant Sci,8(7):330-334

Feng Z Y,Zhang B T,Ding W N,et al.Efficient genome editing in plants Using a CRISPR/Cas9system [J] .Cell Research, 2013,23 (10):1229-1232.Ma X L,Zhang Q Y, Zhu Q L, et al.Arobust CRISPR/Cas9system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J].Molecular Plant,2015, 8(8):1274-1284.

Wan Jianmin, 2006, Perspectives of Molecular Design Breeding in Crops, Acta Agronomica Sinica, 32 (3):455-462

Wang Jiankang, Li Huihui, Zhang Xuecai, Yin Changbin, Li Yu, horse is strong-willed, Li Xinhai, Qiu Lijuan, Wan Jianmin, and 2011, in State's Perspectives of Molecular Design Breeding in Crops, Acta Agronomica Sinica, 37 (2):191-201

Invention content

The purpose of the present invention is to provide a kind of methods improving rice yield.

The present invention also aims to provide application of the OsHXK1 genes in terms of providing rice yield.

The present invention also aims to provide that CRISPR/Cas9 systems is utilized to knock out the side that OsHXK1 improves rice yield Method.

The above-mentioned purpose of the present invention is realized by following technological means：

The present invention provides a kind of methods improving rice yield, for by reducing OsHXK1 expressing quantities or resistance The generation of disconnected OsHXK1 albumen.

OsHXK1 is a member in hexokinase family, and in plant, hexose has to pass through Phosphorylation events could be into Enter glycolytic cycle, provides energy for growth and development of plants, the enzyme of catalysis pbosphohexose is referred to as hexokinase (HXK).HXK The perception of Sugar signal is also taken part in plant.It has been acknowledged that rice hexokinase family shares 10 members, is named as OsHXK1 to OsHXK10.

It is related to the growth and development of plant to have been known OsHXK1, energy is provided for growth and development of plants.The present invention passes through Numerous studies are found for the first time, it has unexpectedly been found that, OsHXK1 genes are mutated, OsHXK1 expressing quantities or blocking are reduced The generation of OsHXK1 albumen can improve the yield of rice from multiple characters.

The albumen of the OsHXK1 gene expressions has the amino acid sequence described in SEQ ID No.1.It can as one kind The mode of choosing, OsHXK1 genes have the nucleic acid sequence described in SEQ ID No.2.

The amino acid sequence of SEQ ID NO.1OsHXK1 albumen

MAAAAVAADQKVVTMTSLREGCACAAPPAAAAPPMPKMAAAQRVVAELREACATPAARLAEVAAAMAGEMEAGLAVE GGSSEMKMIVSYVDSLPTGGEEGSYYALDLGGTNFRVLRVRLAGGGVAERVAREVPIPPGLMSGGGATSECLFGFIA SALAEFVGEEEEEGGLDGGERELGFTFSFPVHQTSIASGTLIRWTKAFAVDDAIGEDVVAALQAAMSERGLDMRVSA LINDTVGTLAAGSYYDEDVVAAVILGTGTNAAYVEDATAIAKLHPSQLPASNTMVINTEWGSFASPCLPLTEFDEAL DQESLNPGEQTYEKLISGMYLGEIVRRVLLKISSRCPSLLGGAGELATPFVLRTPDVSAMHHDETPDLSIVGEKLER TLGIRGTSPEARRMVVEVCDIVATRAARLAAAGIVGILKKIGRVDGGEGRRRRSVVAVDGGLFEHYGKFRRCMESAV RELLGEAAAERVVVKLASDGSGLGAALVAAAHSQRA.

SEQ ID NO.2OsHXK1cDNA nucleotide sequences

ATGGCGGCGGCGGCGGTGGCGGCAGATCAGAAGGTGGTGACGATGACGAGCCTCCGGGAGGGCTGCGCTTGCGCGGC GCCTCCTGCTGCAGCTGCGCCGCCGATGCCGAAGATGGCGGCGGCGCAGAGGGTGGTGGCGGAGCTGAGAGAAGCGT GCGCGACGCCGGCGGCGAGGCTGGCGGAGGTGGCCGCGGCGATGGCCGGCGAGATGGAGGCCGGGCTGGCGGTGGAG GGCGGCAGCAGCGAGATGAAGATGATCGTGTCGTACGTCGACAGCCTCCCCACCGGCGGCGAGGAGGGGTCGTACTA CGCGCTCGACCTCGGCGGCACCAACTTCCGCGTCCTCCGCGTGCGGCTTGCCGGCGGCGGCGTCGCCGAGCGCGTGG CGAGGGAGGTCCCGATCCCTCCCGGCCTCATGTCCGGCGGCGGCGCCACCTCGGAGTGCCTCTTCGGCTTCATCGCC TCCGCGCTAGCCGAGTTCGTCGGCGAGGAGGAAGAAGAAGGCGGCCTCGACGGCGGCGAGAGGGAGCTTGGGTTCAC CTTCTCCTTCCCCGTGCACCAAACCTCCATCGCGTCGGGGACGCTCATCCGGTGGACGAAGGCGTTCGCCGTCGACG ACGCGATCGGCGAGGACGTCGTGGCGGCGCTGCAGGCGGCCATGTCGGAGCGGGGGCTCGACATGCGCGTGTCGGCG CTCATCAACGACACCGTCGGGACGCTCGCCGCGGGCAGCTACTACGACGAGGACGTCGTGGCCGCCGTCATCCTCGG CACCGGCACGAACGCCGCCTACGTCGAGGACGCCACCGCCATCGCCAAGCTACACCCATCGCAGCTGCCAGCATCGA ACACCATGGTGATCAACACCGAGTGGGGCAGCTTCGCCTCGCCGTGCCTCCCATTGACGGAGTTCGACGAAGCACTC GATCAGGAGAGCCTCAACCCCGGCGAGCAGACCTACGAGAAGCTCATCTCCGGGATGTACCTCGGCGAGATCGTCAG GAGGGTCCTCCTCAAGATCTCCTCCCGGTGCCCCTCCCTCCTCGGCGGCGCCGGCGAGCTCGCGACGCCGTTCGTCC TCAGGACACCCGACGTGTCCGCGATGCACCACGACGAGACGCCCGACCTGAGCATCGTCGGCGAGAAGCTGGAACGC ACGCTGGGCATCCGCGGCACGTCGCCGGAGGCGAGGAGGATGGTCGTCGAGGTGTGCGACATCGTCGCCACGAGGGC CGCCCGGCTGGCCGCGGCGGGGATCGTCGGGATCCTGAAGAAGATCGGGAGGGTCGACGGCGGCGAGGGGCGGAGGA GGAGGTCGGTGGTCGCCGTGGACGGCGGGCTGTTCGAGCACTACGGCAAGTTCCGGCGGTGCATGGAGAGCGCGGTG AGGGAGCTGCTCGGAGAGGCGGCGGCGGAGAGGGTGGTCGTCAAGCTCGCCAGCGACGGCTCCGGGCTCGGCGCCGC As an implementation, the present invention is by passing through OsHXK1 genes by CCTGGTTGCAGCTGCTCACTCGCAGAGAGCATAA Mutation, to reduce OsHXK1 expressing quantities or block the generation of OsHXK1 albumen.

Wherein, it is described sport nucleic acid sequence shown in SEQ ID No.2 by addition, substitution or missing one or Several bases.

The mode of gene editing in the prior art may be used in above-mentioned mutation, as ZFN, TALEN or CRISPR gene are compiled The technology of collecting can also use also undeveloped new gene editing technology, in short, the one or more of nucleic acid sequence can be realized Base addition, substitution or missing can be used.

As a preferred embodiment, improving rice yield using CRISPR/Cas9 system sudden changes OsHXK1.Specifically Ground designs the sgRNA sequences based on CRISPR/Cas9 for OsHXK1 genes, will contain the DNA for encoding the sgRNA sequences Segment is connected in the carrier for carrying CRISPR/Cas, rice transformation, realizes the rite-directed mutagenesis to rice Os HXK1 genes.

OsHXK1 genes are numbered in different databases：In the GenBank (http of NCBI:// Www.ncbi.nlm.nih.gov/) its number is Os07g0446800；In Rice Genome Annotation Project (RGAP) network address：http:Its number of //rice.plantbiology.msu.edu/ is LOC_Os07g26540.

As a preferred embodiment, the target sequence (sgRNA) has 5 '-N_XIt is tied shown in-NGG-3 ' Structure,；Can also be 5 '-N_X- NAG-3 ' or 5 '-N_X-NGA-3’；

Wherein N indicates A, T, any one in C and G；

Wherein, X represents base sequence.

Feature after mutation show as between NGG or the base of the upstreams NAG or NGA the 3rd and the 4th base be inserted into base or In its 5 ' and/or 3 ' loss base.

As exemplary embodiment in the present invention, the target sequence has SEQ ID NO.5 or SEQ ID Nucleic acid sequence shown in NO.15.

More specifically, being specifically included using the CRISPR/Cas9 system sudden changes OsHXK1 methods for improving rice yield following Step：

(1) the sgRNA carriers of the segment containing target sequence are built；

Specifically, in step (1), the target primer pair of anamorphic zone cohesive end first moves to after being denaturalized adapter-primer Annealing is completed in room temperature cooling；Primer after annealing is linked on the sgRNA carriers after digestion, is tested through PCR amplification and sequencing Demonstrate,prove positive plasmid；

As preferred embodiment, in step (1), the carrier of the sgRNA is pU3-gRNA and/or pU6- gRNA；

As preferred embodiment, in step (1), the target primer pair with cohesive end has such as SEQ ID NO.6 With nucleic acid sequence shown in SEQ ID NO.7；Or with nucleic acid sequence shown in SEQ ID NO.16 and SEQ ID NO.17；

(2) the pCRISPR/Cas9 carriers of the segment containing target sequence are built；

Specifically, step (2) is to cut the sgRNA expression cassettes for containing target site sequence segment from sgRNA carriers, then It is connected on the pCRISPR/Cas9 carriers of the expression cassette containing Cas9；

Optionally, one or more target sequence can be connected to the pCRISPR/Cas9 carriers of the expression cassette containing Cas9 On.

Such as a target sequence is connected on the pCRISPR/Cas9 carriers of the expression cassette containing Cas9, then it only need to be in step (2) a sgRNA carrier is built when；Multiple target sequences are such as connected to the pCRISPR/Cas9 carriers of the expression cassette containing Cas9 On, then corresponding target quantity sgRNA carriers need to be only built at step (1), in step, in step (2), then by multiple targets Sequence is scaled off from sgRNA carriers, is commonly connected on the pCRISPR/Cas9 carriers of a Cas9 expression cassette.

As preferred embodiment, the target sequence has shown in SEQ ID NO.5 and/or SEQ ID NO.15 Nucleic acid sequence.

Embodiment more preferably connects target sequence shown in SEQ ID NO.5 and SEQ ID NO.15 simultaneously It is connected on the pCRISPR/Cas9 carriers of the expression cassette containing Cas9.

(3) it converts；

Specifically, step (3) is that will contain the pCRISPR/Cas9 carrier rice transformation callus of target, through screening, is divided Change and seedling of taking root, transfer-gen plant is planted in solarium；Positive transgenic plant is identified by hygromycin；

As preferred embodiment, in step (3), pass through Agrobacterium-mediated genetic transformation or Bombardment-Mediated Transformation PCRISPR/Cas9 carriers are to Rice Callus；

As preferred embodiment, the method further includes：

(4) to the identification in mutational site；

Specifically, step (4) is to extract the DNA of positive plant, and the design identification above-mentioned DNA of primer amplification after purified, is surveyed Sequence analyzes catastrophe.

As preferred embodiment, in step (4), using the primer pair described in SEQ ID No.3 and SEQ ID No.4 It is identified.

Compared with prior art, the present invention has the advantage that is with advantageous effect：

1, present invention firstly discovers that, OsHXK1 genes are related with rice yield, are mutated to it, reduce OsHXK1 eggs White expression quantity or the generation for blocking OsHXK1 albumen can improve the yield of rice from multiple characters, can be used as rice One effective target spot of genetic breeding.

2, the present invention is rice yield using the CRISPR/Cas9 system sudden changes OsHXK1 methods for obtaining output increased plant It improves and a kind of effective way is provided；The not essential difference with the mutant that is obtained using chemistry, physical mutagenesis, only purpose By force, small to the damage of genome, evade the possibility risk that transgenosis is brought；

3, the regulating effect of yield controlling gene of the present invention is good, and compared with wild type, plant grain length increases, number of grain per ear Increase, rice yield can be significantly improved, while non-transgenic material can be obtained.This

4, technology of the invention can improve yield from multiple yield traits, and main grain number per spike increases；Evil number is divided to increase；Grain length Increase obviously, and it is existing generally from 1-3 yield traits raising yield.As only from grain length, or only wide etc. from grain, or from optimization Fertilization mode such as improves nitrogen utilization efficiency and improves rice yield.

The present invention is described in further details with reference to the accompanying drawings and detailed description.

Description of the drawings

Fig. 1 are the phenotype of the small ear of the CHXK1-1 obtained in the present invention, and wherein ZH11 indicates to spend 11 in wild rice, CHXK1-1 indicates mutation OsHXK1 transgenic lines；

The phenotype for the effectively point evil number comparison that Fig. 2 are the CHXK1-1 obtained in the present invention, wherein ZH11 indicate wild type 11, ZH11-CHK1-1, ZH11-CHK1-2 is spent to indicate the Target-HXK1-U3 and Target- of mutation OsHXK1 respectively in rice The transgenic line of HXK1-U6；

The phenotype for the number of grain per ear statistics that Fig. 3 are the CHXK1-1 obtained in the present invention, wherein ZH11 indicate wild type water 11, ZH11-CHK1-1, ZH11-CHK1-2 is spent to indicate the Target-HXK1-U3 and Target- of mutation OsHXK1 respectively in rice The transgenic line of HXK1-U6；

Fig. 4 are the phenotype of the grain length of the CHXK1-1 obtained in the present invention, and wherein ZH11 is indicated in wild rice 11, ZH11-CHK1-1, ZH11-CHK1-2 is spent to indicate the Target-HXK1-U3 and Target-HXK1- of mutation OsHXK1 respectively The transgenic line of U6.

The single plant yield statistics that Fig. 5 is the CHXK1-1 obtained in the present invention, wherein wherein ZH11 is indicated in wild rice 11, ZH11-CHK1-1 is spent to indicate the transgenic line of the Target-HXK1-U3 and Target-HXK1-U6 of mutation OsHXK1.

Specific implementation mode

The embodiment tested below is that explanation is expanded on further to the present invention, is not limitation of the present invention.Following reality It applies and specific experiment condition and method is not specified in example, used technological means is usually well-known to those skilled in the art normal Rule means.

Embodiment 1 is spent in 11 in japonica rice variety and obtains output increased using CRISPR/Cas9 system sudden changes OsHXK1 Plant is as follows：

(1) target sequence designs：

Target-HXK1-U3(SEQ ID NO.5)：GTGACGATGACGAGCCTCC

(2) the pU3-gRNA vector constructions containing Target-HXK1-U3：

The target primer pair Target-HXK1-U3F (SEQ ID NO.6) of anamorphic zone cohesive end first： GTTGGTGACGATGACGAGCCTCC, Target-HXK1-U3R (SEQ ID NO.7)：AAACGGAGGCTCGTCATCGTCAC； Room temperature cooling is moved to after adapter-primer is denaturalized and completes annealing, and the primer after annealing, which is linked to the pU3-gRNA after digestion, to be carried On body；Through PCR amplification and sequence verification positive plasmid；

(3) the pCRISPR/Cas9 vector constructions of the segment containing Target-HXK1：

Expression cassette containing Target-HXK1-U3 segments is cut from pU3-gRNA carriers, is then attached to containing Cas9 On the pCRISPR/Cas9 carriers of expression cassette；

(4) acquisition of transfer-gen plant：

PCRISPR/Cas9 carriers containing target are passed through into flower in Agrobacterium-mediated genetic transformation method rice transformation kind 11 (ZH11) callus；Through screening, breaks up and seedling of taking root is identified by transfer-gen plant plantation in solarium by hygromycin Positive transgenic plant；

(5) identification in transfer-gen plant mutational site：

The genomic DNA for extracting positive plant, with primer C1F (SEQ ID NO.3)：ATGGCGGCGGCGGCGGTGGC and C1R(SEQ ID NO.4)：GAACCCAAGCTCCCTCTCGC expands said gene group DNA, and the purified Hou Song companies of product survey Sequence, sequencing result is compared with the WT lines sequence before transgenosis, referring specifically to SEQ ID NO.8-SEQ ID NO.14；Point Analyse catastrophe；

WT(SEQ ID NO.8)：

GGTGGCGGCAGATCAGAAGGTGGTGACGATGACGAGCCTCCGGGAGGGCTGCGCTTGCGCGGCGCCTCC

CHXK1-1(SEQ ID NO.9)：

GGTGGCGGCAGATCAGAAGGTGGTGACGATGACGAG*CTCCGGGAGGGCTGCGCTTGCGCGGCGCCTCC

CHXK1-2(SEQ ID NO.10)：

GGTGGCGGCAGATCAGAAGGTGGTGACGATGACGAG* CTTCCGGGAGGGCTGCGCTTGCGCGGCGCCTCC

CHXK1-3(SEQ ID NO.11)：

GGTGGCGGCAGATCAGAAGGTGGCGA* GAGGGAGCTTGGGTTCGGAGGGCTGCGCTTGCGCGGCGCCTCC

CHXK1-4(SEQ ID NO.12)：

**********GATCAGAAGGTGGTGACGATGACGAGGCTCCGGGAGAGCTGCGCTTGCGCGGCGCCTCC

CHXK1-5(SEQ ID NO.13)：

GGTGGCGGCAGATCAGAAGGTGGTGACGATGACGAGCCA***************************CC

CHXK1-6(SEQ ID NO.14)：

GGTGGCGGCAGATCAGAAGGTGGTGACGATGACGAGCC****************************CC

Wherein, CHXK1-1, CHXK1-2, CHXK1-3, CHXK1-4, CHXK1-5 and CHXK1-6 indicate different transgenosis Strain；WT indicates wild type；In sequence "A”、“G" indicate that the base being mutated, " * " indicate base deletion；The missing of base and prominent Change illustrates rite-directed mutagenesis success.By the sequence of SEQ ID NO.9-SEQ ID NO.14 it is found that the genetically modified plants of the present invention are equal Rite-directed mutagenesis success.

Embodiment 2 is spent in 11 in japonica rice variety and obtains output increased using CRISPR/Cas9 system sudden changes OsHXK1 Plant is as follows：

It is as follows：

(1) target sequence designs：

Target-HXK1-U6(SEQ ID NO.15)：ATCCCTCCCGGCCTCATGTC

(2) the pU6-gRNA vector constructions containing Target-HXK1-U6 segments,

The target primer pair Target-HXK1-U6F (SEQ ID NO.16) of anamorphic zone cohesive end first： GGCATCCCTCCCGGCCTCATGTC, Target-HXK1-U6R (SEQ ID NO.17)： AAACGACATGAGGCCGGGAGGGA；Room temperature cooling is moved to after adapter-primer is denaturalized and completes annealing, by the primer strand after annealing It is connected on the pU6-gRNA carriers after digestion；Through PCR amplification and sequence verification positive plasmid；

Expression cassette containing Target-HXK1-U6 segments is cut from pU6-gRNA carriers, is then attached to containing Cas9 On the pCRISPR/Cas9 carriers of expression cassette；

(4) acquisition of transfer-gen plant：

(5) identification in transfer-gen plant mutational site：

The genomic DNA for extracting positive plant, with primer C1F (SEQ ID NO.3)：ATGGCGGCGGCGGCGGTGGC and C1R(SEQ ID NO.4)：GAACCCAAGCTCCCTCTCGC expands said gene group DNA, and the purified Hou Song companies of product survey Sequence, sequencing result is compared with the WT lines sequence before transgenosis, referring specifically to SEQ ID NO.18-SEQ ID NO.23； Analyze catastrophe；

WT(SEQ ID NO.18)：

GTGGCGAGGGAGGTCCCGATCCCTCCCGGCCTCATGTCCGGCGGCGGCGCCACCTCGGAG

CHXK2-1(SEQ ID NO.19)：

GTGGCGAGGGAGGTCCCGATCCCTCCCGGC*****GTCCGGCGGCGGCGCCACCTCGGAG

CHXK2-2(SEQ ID NO.20)：

GTGGCGAGGGAGGTCCCGATACCTCCCGGCCTCGTGTGTCGCGATCC*GCGCCACCTCGGAG

CHXK2-3(SEQ ID NO.21)：

GTGGCGAGGGAGGTCCCGATCCCTCC*GGCCTC**GAC*GGCGGCGGCGCCACCTCGGAG

CHXK2-4(SEQ ID NO.22)：

GTGGCGAGGGAGGTCCCGATACCTCCCGGCCTCGTGTCCGGCGGCGGCGCCACCTCGGAG

CHXK2-5(SEQ ID NO.23)：

GTGGCGAGGGAGGTCCCGAAGCCTCC*GGCCTCATGTCCGGCGGCGGCGCCACCTCGGAG

Wherein, CHXK2-1, CHXK2-2, CHXK2-3, CHXK2-4, CHXK2-5 indicate different transgenic lines；WT tables Show wild type；In sequence "A”、“G" indicate that the base being mutated, " * " indicate base deletion；The missing of base and mutation illustrate to pinpoint It is mutated successfully.By the sequence of SEQ ID NO.19-SEQ ID NO.23 it is found that the present invention the equal rite-directed mutagenesis of genetically modified plants at Work(.

By embodiment 1 and embodiment 2 it is found that no matter using pU3-gRNA pU6-gRNA carrier constructions, in the present invention The target sequence being related to can successfully be mutated OsHXK1 genes.

Embodiment 3 is identified using the transfer-gen plant yield traits that CRISPR/Cas9 system sudden changes OsHXK1 is obtained

Positive transfer-gen plant culture will be accredited as to maturation by PCR, observe the plant forms of different times, as a result As shown in Figure 1, obtained homozygous lines transfer-gen plant all shows the phenotype that number of grain per ear obviously increases.Transformed plant with Adjoining tree spends 11 to compare in japonica rice variety, has significant change in following four yield traits：Main grain number per spike increases (see Fig. 3)； Point evil number increases (see Fig. 2)；Grain length increases (see Fig. 4)；Single plant yield increases (see Fig. 5).

The rice that the present invention has carried out OsHXK1 mutation comprehensively improves the yield of rice from multiple yield traits.With The wild rice for not carrying out mutation OsHXK1 is compared, and main grain number per spike increases 10%-20%, and evil number is divided to increase 10%-20%；Grain It is long to increase by 8%；Single plant yield increases by 5%.

Rice yield depends on three factors, i.e., spike number, number of grain per ear and grain weight on unit area.Wherein rice grain shape is long Short is the factor of determination of grain weight, and usual grain length is longer, and seed can be heavier.The increase of number of grain per ear can also dramatically increase rice Yield.

Embodiment described above is only the preferred embodiment of the present invention, it is noted that for the general of the art For logical technical staff, without departing from the technical principles of the invention, several improvements and modifications can also be made, these change Protection scope of the present invention is also should be regarded as into retouching.

Sequence table

<110>Agricultural University Of South China

<120>A method of obtaining output increased plant using CRISPR/Cas9 system sudden changes OsHXK1

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 498

<212> PRT

<213>Rice (Oryza sativa L.)

<400> 1

Met Ala Ala Ala Ala Val Ala Ala Asp Gln Lys Val Val Thr Met Thr

1 5 10 15

Ser Leu Arg Glu Gly Cys Ala Cys Ala Ala Pro Pro Ala Ala Ala Ala

20 25 30

Pro Pro Met Pro Lys Met Ala Ala Ala Gln Arg Val Val Ala Glu Leu

35 40 45

Arg Glu Ala Cys Ala Thr Pro Ala Ala Arg Leu Ala Glu Val Ala Ala

50 55 60

Ala Met Ala Gly Glu Met Glu Ala Gly Leu Ala Val Glu Gly Gly Ser

65 70 75 80

Ser Glu Met Lys Met Ile Val Ser Tyr Val Asp Ser Leu Pro Thr Gly

85 90 95

Gly Glu Glu Gly Ser Tyr Tyr Ala Leu Asp Leu Gly Gly Thr Asn Phe

100 105 110

Arg Val Leu Arg Val Arg Leu Ala Gly Gly Gly Val Ala Glu Arg Val

115 120 125

Ala Arg Glu Val Pro Ile Pro Pro Gly Leu Met Ser Gly Gly Gly Ala

130 135 140

Thr Ser Glu Cys Leu Phe Gly Phe Ile Ala Ser Ala Leu Ala Glu Phe

145 150 155 160

Val Gly Glu Glu Glu Glu Glu Gly Gly Leu Asp Gly Gly Glu Arg Glu

165 170 175

Leu Gly Phe Thr Phe Ser Phe Pro Val His Gln Thr Ser Ile Ala Ser

180 185 190

Gly Thr Leu Ile Arg Trp Thr Lys Ala Phe Ala Val Asp Asp Ala Ile

195 200 205

Gly Glu Asp Val Val Ala Ala Leu Gln Ala Ala Met Ser Glu Arg Gly

210 215 220

Leu Asp Met Arg Val Ser Ala Leu Ile Asn Asp Thr Val Gly Thr Leu

225 230 235 240

Ala Ala Gly Ser Tyr Tyr Asp Glu Asp Val Val Ala Ala Val Ile Leu

245 250 255

Gly Thr Gly Thr Asn Ala Ala Tyr Val Glu Asp Ala Thr Ala Ile Ala

260 265 270

Lys Leu His Pro Ser Gln Leu Pro Ala Ser Asn Thr Met Val Ile Asn

275 280 285

Thr Glu Trp Gly Ser Phe Ala Ser Pro Cys Leu Pro Leu Thr Glu Phe

290 295 300

Asp Glu Ala Leu Asp Gln Glu Ser Leu Asn Pro Gly Glu Gln Thr Tyr

305 310 315 320

Glu Lys Leu Ile Ser Gly Met Tyr Leu Gly Glu Ile Val Arg Arg Val

325 330 335

Leu Leu Lys Ile Ser Ser Arg Cys Pro Ser Leu Leu Gly Gly Ala Gly

340 345 350

Glu Leu Ala Thr Pro Phe Val Leu Arg Thr Pro Asp Val Ser Ala Met

355 360 365

His His Asp Glu Thr Pro Asp Leu Ser Ile Val Gly Glu Lys Leu Glu

370 375 380

Arg Thr Leu Gly Ile Arg Gly Thr Ser Pro Glu Ala Arg Arg Met Val

385 390 395 400

Val Glu Val Cys Asp Ile Val Ala Thr Arg Ala Ala Arg Leu Ala Ala

405 410 415

Ala Gly Ile Val Gly Ile Leu Lys Lys Ile Gly Arg Val Asp Gly Gly

420 425 430

Glu Gly Arg Arg Arg Arg Ser Val Val Ala Val Asp Gly Gly Leu Phe

435 440 445

Glu His Tyr Gly Lys Phe Arg Arg Cys Met Glu Ser Ala Val Arg Glu

450 455 460

Leu Leu Gly Glu Ala Ala Ala Glu Arg Val Val Val Lys Leu Ala Ser

465 470 475 480

Asp Gly Ser Gly Leu Gly Ala Ala Leu Val Ala Ala Ala His Ser Gln

485 490 495

Arg Ala

<210> 2

<211> 1497

<212> DNA

<213>Rice (Oryza sativa L.)

<400> 2

atggcggcgg cggcggtggc ggcagatcag aaggtggtga cgatgacgag cctccgggag 60

ggctgcgctt gcgcggcgcc tcctgctgca gctgcgccgc cgatgccgaa gatggcggcg 120

gcgcagaggg tggtggcgga gctgagagaa gcgtgcgcga cgccggcggc gaggctggcg 180

gaggtggccg cggcgatggc cggcgagatg gaggccgggc tggcggtgga gggcggcagc 240

agcgagatga agatgatcgt gtcgtacgtc gacagcctcc ccaccggcgg cgaggagggg 300

tcgtactacg cgctcgacct cggcggcacc aacttccgcg tcctccgcgt gcggcttgcc 360

ggcggcggcg tcgccgagcg cgtggcgagg gaggtcccga tccctcccgg cctcatgtcc 420

ggcggcggcg ccacctcgga gtgcctcttc ggcttcatcg cctccgcgct agccgagttc 480

gtcggcgagg aggaagaaga aggcggcctc gacggcggcg agagggagct tgggttcacc 540

ttctccttcc ccgtgcacca aacctccatc gcgtcgggga cgctcatccg gtggacgaag 600

gcgttcgccg tcgacgacgc gatcggcgag gacgtcgtgg cggcgctgca ggcggccatg 660

tcggagcggg ggctcgacat gcgcgtgtcg gcgctcatca acgacaccgt cgggacgctc 720

gccgcgggca gctactacga cgaggacgtc gtggccgccg tcatcctcgg caccggcacg 780

aacgccgcct acgtcgagga cgccaccgcc atcgccaagc tacacccatc gcagctgcca 840

gcatcgaaca ccatggtgat caacaccgag tggggcagct tcgcctcgcc gtgcctccca 900

ttgacggagt tcgacgaagc actcgatcag gagagcctca accccggcga gcagacctac 960

gagaagctca tctccgggat gtacctcggc gagatcgtca ggagggtcct cctcaagatc 1020

tcctcccggt gcccctccct cctcggcggc gccggcgagc tcgcgacgcc gttcgtcctc 1080

aggacacccg acgtgtccgc gatgcaccac gacgagacgc ccgacctgag catcgtcggc 1140

gagaagctgg aacgcacgct gggcatccgc ggcacgtcgc cggaggcgag gaggatggtc 1200

gtcgaggtgt gcgacatcgt cgccacgagg gccgcccggc tggccgcggc ggggatcgtc 1260

gggatcctga agaagatcgg gagggtcgac ggcggcgagg ggcggaggag gaggtcggtg 1320

gtcgccgtgg acggcgggct gttcgagcac tacggcaagt tccggcggtg catggagagc 1380

gcggtgaggg agctgctcgg agaggcggcg gcggagaggg tggtcgtcaa gctcgccagc 1440

gacggctccg ggctcggcgc cgccctggtt gcagctgctc actcgcagag agcataa 1497

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

atggcggcgg cggcggtggc 20

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

gaacccaagc tccctctcgc 20

<210> 5

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

gtgacgatga cgagcctcc 19

<210> 6

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

gttggtgacg atgacgagcc tcc 23

<210> 7

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

gttggtgacg atgacgagcc tcc 23

<210> 8

<211> 69

<212> DNA

<213>Rice (Oryza sativa L.)

<400> 8

ggtggcggca gatcagaagg tggtgacgat gacgagcctc cgggagggct gcgcttgcgc 60

ggcgcctcc 69

<210> 9

<211> 68

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

ggtggcggca gatcagaagg tggtgacgat gacgagctcc gggagggctg cgcttgcgcg 60

gcgcctcc 68

<210> 10

<211> 69

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

ggtggcggca gatcagaagg tggtgacgat gacgagcttc cgggagggct gcgcttgcgc 60

ggcgcctcc 69

<210> 11

<211> 69

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

ggtggcggca gatcagaagg tggcgagagg gagcttgggt tcggagggct gcgcttgcgc 60

ggcgcctcc 69

<210> 12

<211> 59

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

gatcagaagg tggtgacgat gacgaggctc cgggagagct gcgcttgcgc ggcgcctcc 59

<210> 13

<211> 41

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

ggtggcggca gatcagaagg tggtgacgat gacgagccac c 41

<210> 14

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

ggtggcggca gatcagaagg tggtgacgat gacgagcccc 40

<210> 15

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 15

atccctcccg gcctcatgtc 20

<210> 16

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 16

ggcatccctc ccggcctcat gtc 23

<210> 17

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 17

aaacgacatg aggccgggag gga 23

<210> 18

<211> 60

<212> DNA

<213>Rice (Oryza sativa L.)

<400> 18

gtggcgaggg aggtcccgat ccctcccggc ctcatgtccg gcggcggcgc cacctcggag 60

<210> 19

<211> 55

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 19

gtggcgaggg aggtcccgat ccctcccggc gtccggcggc ggcgccacct cggag 55

<210> 20

<211> 61

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 20

gtggcgaggg aggtcccgat acctcccggc ctcgtgtgtc gcgatccgcg ccacctcgga 60

g 61

<210> 21

<211> 56

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 21

gtggcgaggg aggtcccgat ccctccggcc tcgacggcgg cggcgccacc tcggag 56

<210> 22

<211> 60

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 22

gtggcgaggg aggtcccgat acctcccggc ctcgtgtccg gcggcggcgc cacctcggag 60

<210> 23

<211> 59

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 23

gtggcgaggg aggtcccgaa gcctccggcc tcatgtccgg cggcggcgcc acctcggag 59

Claims

1. a kind of method improving rice yield, which is characterized in that reduce OsHXK1 expressing quantities or block OsHXK1 eggs White generation.

2. according to the method described in claim 1, it is characterized in that, the OsHXK1 albumen has described in SEQ ID No.1 Amino acid sequence；

Preferably, OsHXK1 genes have the nucleic acid sequence described in SEQ ID No.2.

3. according to the method described in claim 2, it is characterized in that, by OsHXK1 genes through mutation,

To reduce OsHXK1 expressing quantities or block the generation of OsHXK1 albumen；

Preferably, described to sport nucleic acid sequence shown in SEQ ID No.2 by addition, substitution or missing one or several A base.

4. according to the method described in claim 3, it is characterized in that, being carried using CRISPR/Cas9 system sudden change OsHXK1 genes High rice yield.

5. according to the method described in claim 4, it is characterized in that, being designed based on CRISPR/Cas9's for OsHXK1 genes DNA fragmentation containing coding sgRNA sequences is connected in the carrier for carrying CRISPR/Cas, turns by target sequence sgRNA sequences Change rice, realizes the knockout to rice Os HXK1 genes.

6. method according to claim 5, which is characterized in that the target sequence has 5 '-N_XIt is tied shown in-NGG-3 Structure；

Alternatively, the target sequence has 5 '-N_X- NAG-3 ' or 5 '-N_XStructure described in-NGA-3 '；

Wherein, N indicates A, T, any one in C and G.

7. according to the method described in claim 5, it is characterized in that, the target sequence have SEQ ID NO.5 and/or Nucleic acid sequence shown in SEQ ID NO.15.

8. according to the method described in claim 5, it is characterized in that, the feature after mutation shows as NGG or the upstreams NAG or NGA Base is inserted between 3rd base and the 4th base or in its 5 ' and/or 3 ' loss base.

9. according to the method described in claim 5, it is characterized in that, the method specifically includes following steps：

(1) the sgRNA carriers of the segment containing target sequence are built；

Preferably, in step (1), the target primer pair of anamorphic zone cohesive end first moves to room temperature after being denaturalized adapter-primer It is cooling to complete annealing；Primer after annealing is linked on the sgRNA carriers after digestion, through PCR amplification and sequence verification sun Property grain；

Preferably, in step (1), the carrier of the sgRNA is pU3-gRNA and/or pU6-gRNA；Preferably, step (1) In, the target primer pair with cohesive end has the nucleic acid sequence as shown in SEQ ID NO.6 and SEQ ID NO.7；Or tool There is nucleic acid sequence shown in SEQ ID NO.16 and SEQ ID NO.17；

Preferably, step (2) is to cut the sgRNA expression cassettes for containing target site sequence segment from sgRNA carriers, is then connected Onto the pCRISPR/Cas9 carriers of the expression cassette containing Cas9；

(3) it converts；

Preferably, step (3) is that will contain the pCRISPR/Cas9 carrier rice transformation callus of target, through screening, differentiation and It takes root seedling, transfer-gen plant is planted in solarium；Positive transgenic plant is identified by hygromycin；

Preferably, it in step (3), is arrived by Agrobacterium-mediated genetic transformation or Bombardment-Mediated Transformation pCRISPR/Cas9 carriers Rice Callus；

Preferably, the method further includes：

(4) to the identification in mutational site；

More preferably；In step (4), identified using the primer pair described in SEQ ID No.3 and SEQ ID No.4.