CN112625101B - Genes, proteins, biomaterials and uses thereof for regulating development of ears - Google Patents

Abstract

The invention discloses a gene, protein, biological material, length polymorphism marker and application. The gene is a major gene for regulating and controlling the traits of the wheat spike and can regulate and control the spike length and the number of small spikes. The amino acid sequence of the protein copied by the gene A, B, D is shown as sequences 4-6 in a sequence table, and the nucleotide sequence is shown as sequences 1-3 in the sequence table. The invention provides clues for researching the development of the wheat ear and provides gene resources for improving the plant ear character by a gene engineering means so as to improve the wheat yield.

Description

Genes, proteins, biomaterials and uses thereof for regulating development of ears

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a gene TaZVHD for regulating development of spike, protein, biological material, molecular marker and application thereof.

Background

Common wheat (Triticum aestivum L.) is one of the most important food crops in the world, and wheat is the main food for about 40% of the population in the world, and the yield of the wheat directly influences the economic development of society and the survival of human beings. Wheat is also the third major grain crop in China, and the annual planting area and the total yield of the wheat account for 25% and 22% of the total grain amount respectively, so that the improvement of the wheat yield plays a very important role in the economic development of China. The three factors determining wheat yield include: ear number, ear grain number and thousand grain weight.

The research finds that the ear part is used as a main functional organ of wheat, and the whole development process directly influences the latter two of three elements of wheat yield. The traits of the spike mainly comprise spike length, small spike number, spike grain number and the like, which are quantitative genetic traits controlled by multiple genes and jointly regulated by major genes and minor genes, and the traits have certain differences under different segregation populations and different environmental conditions. QTL mapping and association analysis are effective means for studying these traits, and some QTL studies on the trait of the wheat spike have been reported (Simmonds et al, 2014; Edae et al, 2014; Sukumuran et al, 2015) at present, wherein individual QTL sites are finely mapped, but it is difficult to isolate and identify related functional genes by conventional forward genetics methods. The important gene for digging and regulating the wheat ear character has important significance for improving the agronomic character and increasing the wheat yield.

The virus-induced gene silencing technology (VIGS) is an emerging technology, is simple to operate and can be used for quickly and effectively analyzing the functions of target genes. VIGS is produced based on the RNA-mediated antiviral mechanism of the plant based on post-transcriptional gene silencing. After the RNA virus infects the plant, a double-stranded RNA structure is formed in the replication process, then the double-stranded RNA structure is cut into 21-25nt siRNA by Dicer enzyme, and then a large amount of siRNA is generated under the action of RNA-dependent RNA polymerase endogenous to the plant, and the siRNA guides an RNA-induced silencing complex (RISC) to degrade a target RNA sequence in a sequence-specific manner. With the progress of research, it has been found that a target gene sequence can be inserted into a viral vector, and a recombinant virus can be engineered to infect plants, and under the action of PTGS, viral RNA is degraded, and the level of endogenous target gene RNA having homology with the inserted sequence is also reduced, so as to achieve a virus-induced gene silencing effect (Benedicto et al, 2004; Burch-Smith et al, 2004). In wheat, VIGS has been applied in many ways, mainly concentrated on seedling stage leaf experiments, and plays an important role in the function research of disease-resistant genes and stress-resistant genes.

In recent years, transcriptomics are researched in different growth and development stages of wheat without tissues, and the process of dynamically regulating and controlling growth and development of gene expression is disclosed. However, transcriptomics often obtain a great number of genes with differential expression, and functional screening of the genes is still needed, so that key regulatory factors are focused, and subsequent research work is facilitated.

Disclosure of Invention

In view of the above, the invention screens and identifies the key positive regulatory factor TaZVHD of ear length and spikelet number by a large amount of researches by using VIGS technology on the basis of the transcriptome sequencing of different stages of young ears of Zheng wheat 366.

According to a first aspect of the present invention there is provided a protein which is a1), a2), a3) or a 4): a1) the amino acid sequence is a protein shown as any one of sequences 1-3 in a sequence table; a2) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of an amino acid sequence shown in any one of sequences 4-6 in a sequence table; a3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in any one of sequences 4-6 in a sequence table; a4) and (b) a protein having at least 95% sequence identity with an amino acid sequence represented by any one of sequences 4 to 6 in the sequence table and having the same function. For example, the protein has an amino acid sequence having at least 99% sequence identity with an amino acid represented by any one of sequences 4 to 6 in a sequence table.

According to a second aspect of the present invention, there is provided a gene which is b1), b2), b3) or b 4): b1) a nucleotide sequence encoding the protein; or b2) has a nucleotide sequence shown in any one of sequences 1-3 in the sequence table; or b3) a nucleotide sequence which is at least 75% identical to the nucleotide sequence defined in b1) or b2) and which encodes the protein; b4) a nucleotide sequence which hybridizes with the nucleotide sequence defined by b1), b2) or b3) under stringent conditions and encodes the protein. The 758 th to 769 th bases of the gene nucleotide sequence can be deleted.

According to a third aspect of the present invention, there is provided a biomaterial that modulates development of ears, the biomaterial being any one of c1) to c9) below: c1) an expression cassette containing the gene; c2) a recombinant vector containing the gene; c3) a recombinant vector comprising the expression cassette of c 1); c4) a recombinant microorganism containing the gene; c5) a recombinant microorganism comprising the expression cassette of c 1); c6) a recombinant microorganism comprising the recombinant vector of c2) or c 3); c7) a transgenic plant cell line comprising the gene of claim 3; c8) a transgenic plant cell line comprising the expression cassette of c 1); c9) a transgenic plant cell line comprising the recombinant vector of c2) or c 3).

According to a fourth aspect of the present invention, there is provided the use of said protein or said gene or said biological material for modulating development of a plant ear, in particular the use of said protein or said gene or said biological material for modulating plant ear length and/or spikelet number and/or ear grain number.

According to a fifth aspect of the present invention there is provided a length polymorphic marker which detects whether a plant variety has said base deletion.

According to the sixth aspect of the invention, a method for regulating the wheat head trait is provided, which comprises constructing a VIGS vector from the gene, transforming a plant with the constructed VIGS vector, and silencing the gene in the plant.

The plant may be a dicot or a monocot, such as a monocot, preferably a gramineae, for example the plant may be selected from wheat, rice, sorghum, maize, barley, oats, rye, etc.

On the basis of transcriptome sequencing of different development periods of young ears of Zheng wheat 366, the positive regulatory factor TaZVHD of ear length and spikelet number is obtained by using the VIGS technology, the expression mode is analyzed, effective molecular markers are developed, and theoretical basis and gene resources are provided for breeding the spike length and the spikelet number of wheat.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an optical micrograph of the 6 developmental stages of ZM366 young ear transcriptome sequencing.

FIG. 2 shows the number of gene expressions at 6 developmental stages.

FIG. 3 is a diagram showing the distribution of differentially expressed genes at different developmental stages.

FIG. 4 is the expression of TaZVHD-2A, TaZFHD-2B and TaZVHD-2D genes in samples of 6 stages of transcriptome sequencing.

FIG. 5 is the analysis of the expression pattern of TaZVHD gene in different wheat tissues and organs and different development periods of ear.

Fig. 6 and 7 are graphs showing the gene expression levels and spike phenotype changes after silencing 2 segments of TaZFHD in Apogee wheat using the VIGS technique. VIGS can effectively silence a target gene TaZVHD, and the wheat spike length and the number of spikelets are observed to be increased.

FIG. 8 is a sequence alignment of 14 extremely long and short panicle material.

Fig. 9 is a developed STR marker. The marker can effectively distinguish whether TaZVHD has 12bp insertion deletion variation in different materials.

FIG. 10 is a correlation analysis of the molecular markers of TaZVHD with ear length and spikelet number in a population of non-natural populations.

Detailed Description

To make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to better research the key genes influencing the development of the panicle, the inventor adopts a high-throughput sequencing technology to analyze the key gene changes of the large panicle mutant and the wild spikelet at the early stage of differentiation, utilizes a virus-induced gene silencing system to research candidate genes, and develops effective molecular markers.

Compared with the prior art, the invention has the following advantages:

1. according to the invention, by sequencing transcriptome of different development periods of young ears of Zheng wheat 366, key candidate genes influencing the development of the ear of wheat are screened and identified, and the specific high-expression gene TaZVHD of the young ears is screened and identified by combining with the analysis of a gene expression mode.

2. The invention silences TaZVHD in USU-Apogee spring wheat (Chinese academy of sciences) by utilizing a virus-induced gene silencing system, and the reduction of spike length and spikelet number is observed, which indicates that the gene is a positive regulatory factor influencing spike development.

3. The invention develops molecular markers by analyzing the sequence of TaZVHD in the wheat varieties with extreme phenotype of big ear and small ear, and verifies in natural population containing 130 parts of materials.

The invention uses the young ears of Zheng wheat 366 (plant institute of Chinese academy of sciences) which are widely planted in Huang-Huai-wheat area and are in different development stages for transcriptomics research. In order to find out key genes for regulating development of panicle, the inventor discovers 11221 genes which are differentially expressed at different stages in total by analyzing transcriptome of young panicle at different stages of early development. Screening some differential expression genes, and screening the tasfhd candidate gene by combining gene expression pattern analysis. A virus-induced gene silencing system is utilized to construct a BSMV-VIGS vector of three copy conservative sections of the gene, TaZVHD is silenced in USU-Apogee spring wheat, and the reduction of spike length and spikelet number is observed, which indicates that the gene is a positive regulatory factor influencing spike development. By analyzing the sequences of TaZFHD in 12 extreme phenotype wheat varieties of large and small ears, an analytical marker was developed and its effectiveness was verified in a natural population containing 130 material.

First, transcriptome sequencing analysis

The sequencing samples were 3666 young ears of zheng wheat at different developmental stages, 3 biological replicates each, for a total of 18 samples (figure 1 shows the morphology of ZM366 young ears at different stages of development).

1. Sequencing and transcript Assembly and Gene differential expression analysis

Transcriptome sequencing can efficiently and quickly obtain all transcripts of biological tissues. After removing the low quality readings, approximately 15Gb readings were obtained for each sample. Aligned to the wheat reference genome (TGACCv 1, Ensembl Plants), 84.66% to 88.24% of the reads are aligned to the genome, with 70.84% to 74.08% of the reads aligned to a single site in the genome. Splicing of transcripts was performed using software and compared to database known wheat transcripts (Trapnell et al, 2012; Pertea et al, 2015), and for newly assembled transcripts, their coding capacity was predicted using software (Kong et al, 2007; Finn et al, 2016). And calculating the expression amount of the gene by using the FPKM value, wherein if the average value of three repetitions of the FPKM of a certain gene in at least one period is more than 1, the gene is considered to be expressed, and otherwise, the gene is considered not to be expressed. Based on the 6 stages of 18 samples, a total of 47574 expressed genes were obtained, and the number of expressed genes significantly increased as the ear development process varied from S1 to S6 (FIG. 2). Gene differential expression analysis was performed using DEGseq (Wang et al, 2010), comparing the expression levels of FDR ≦ 0.05 and | log2Ratio | ≧ 1, resulting in a total of 11221 differentially expressed genes at different stages of development of the young ear of ZM366 (as shown in FIG. 3).

2. Transcription factor analysis

In ZM366 young ear transcriptome data, 1008 genes encoding transcription factors are differentially expressed at different stages of ear development, including 128 AP2 genes and 65 MADS-box genes, which are reported to play an important role in inflorescence meristem development. Zhd (zinc finger homeodomain) is a zinc finger protein homeodomain, is a plant-specific transcription factor family, and has few functional studies, and transcriptome data indicates that the expression level of TaZFHD is significantly changed in the tassel development process (as shown in fig. 4).

Second, TaZVHD expression pattern analysis

1. Preparation of Total RNA and cDNA

Various tissues and organs of Zheng wheat 366, including 5 stages of root, stem, leaf and ear development (lengths of 0.5mm, 1mm, 3mm and 5mm, respectively, and flowering period) and 3 stages of kernel development (5 days, 10 days and 15 days after flowering), were sampled, total RNA was extracted using Trizol, and cDNA was obtained from the total RNA using Thermo-revertAid First Strand cDNA kit (Thermo-Fisher Scientific, Shanghai, China).

2. Design of primers for gene isolation and expression detection

According to Chinese spring sequencing information, full-length primers TaZVHDcdsF (5'-TCCTCTCGTGCGGACATG-3') and TaZVHDcdsR (5'-GCTAGACGCTGATGGGGG-3') are designed by using Primer5 software (Premier Biosoft, Palo alto, Calif.) to clone genes and isolate TaZVHD transcripts. PCR amplification was performed using GeneAmp PCRSystem 9700(Thermo-Fisher Scientific) using high fidelity Phushion DNA polymerase (Thermo-Fisher Scientific). PCR amplification procedure: denaturation at 98 ℃ for 2 min; 35 cycles: 15 seconds at 98 ℃, 30 seconds at 58 ℃ and 1 minute at 72 ℃; followed by final extension at 72 ℃ for 10 min. The PCR product was purified from 1.0% agarose gel using the Tiangen TIANgel Midi purification kit (Tiangen Co.). The PCR product was cloned into pGEM-T Easy plasmid vector (Promega Corporation, Madison, Wis., USA). The recombinant plasmid was then transformed into E.coli DH5 alpha cells (TaKaRa, China). Randomly selecting 24 single colonies, and identifying by using primers TaZVHDcdsF and TaZVHDcdsR to obtain fragments with consistent sizes. The positive clone was sequenced by commercial company (Huada gene, Shenzhen, China), and the three copies of TaZVHD were compared to determine the three transcript sequences (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) of TaZVHD.

Primers TaZFHDqF (5'-ATGATGGATCACCTGAGCCTG-3') and TaZFHDqR (5'-CGGTGGAAGCTGCGGTGGCAG-3') are designed in three copies of conserved segments according to a transcript sequence obtained from Zheng wheat 366, and gene expression detection is carried out by taking an Actin gene (F: 5'-ATGGAAGCTGCTGGAATCCAT-3', R: 5'-CCTTGCTCATACGGTCAGCAATAC-3') as an internal reference gene. PCR amplification was performed using Light cycler 96(Roche) with high fidelity Phushion DNA polymerase (Thermo-Fisher Scientific). PCR amplification procedure: denaturation at 98 ℃ for 2 min; 35 cycles: 15 seconds at 98 ℃, 30 seconds at 58 ℃ and 1 minute at 72 ℃; followed by final extension at 72 ℃ for 10 min. In 10 samples including different tissues and ear development stages, the gene was found to be expressed in a higher amount in the early stage of ear development (0.5 cm and 1 cm young ears), indicating that the gene may exert biological functions in the early stage of ear development (FIG. 5).

Third, silencing of TaZVHD affects USU-Apogee ear traits

Firstly constructing a VIGS vector of TaZVHD, infecting the native tobacco, then using tobacco leaves to frictionally inoculate Apogee wheat, detecting the silencing level of the TaZVHD gene, and observing the spike phenotype.

Construction of the VIGS vector

Designing VIGS conservative primers according to ABD conservative sections of the TaZVHD genes of wheat, designing primers in base conservative sections of 300 th to 502 th positions and 527 th to 723 th positions of three copy coding regions (V1F: 5'-CGTGCTGCAGGGCTTCCTCC-3', V1R: 5'-GGGTGAACTTGGTCCGGTGC-3'; V2F: 5'-CCTTCGCCGAGAAGCAGG-3', V2R: 5'-TCCTGGGTTGATGCCGATGC-3'), adding an LIC fragment, F-LIC: 5'-AAGGAAGTTTAA-3', R-LIC: 5'-AACCACCACCACCGT-3' (Ma et al, 2012), and the lengths of the target silencing fragments obtained by using Apogee wheat young ear cDNA as a template through amplification are 203bp and 197bp respectively. And simultaneously carrying out enzyme digestion on the pCa-gamma b empty vector for 3-4 hours by using ApaI, recovering an amplified fragment and an enzyme digestion fragment after the enzyme digestion, carrying out end reaction on a target fragment and the Apa I enzyme-digested vector by using T4 polymerase, treating for 30 minutes at 22-25 ℃, terminating the reaction at 75 ℃ for 10 minutes, adding 2 mu L of the treated vector fragment into 20 mu L of the treated target fragment, standing for half an hour at room temperature after 2 minutes at 65 ℃, and then transforming into a Escherichia coli DH5 alpha cell. The constructed vector was expressed using BS 10: and BS 32: and (4) performing bidirectional sequencing, sequencing the correctly extracted plasmid, transforming the plasmid into EHA105 competent cells, and storing for later use.

2. Agrobacterium injection ben-zeng tobacco

Taking out the agrobacterium EHA105 strain from a refrigerator at the temperature of minus 80 ℃ for unfreezing, inoculating the agrobacterium EHA105 strain to 10mL of LB liquid culture medium with corresponding resistance, carrying out shaking culture at 220rpm for 12-16 hours, and sucking 500 mu L of bacterial liquid culture to inoculate to 50mL of LB liquid culture medium with corresponding resistance. Shaking culture at 220rpm for 12-16 hours. Centrifuging at 5000rpm for 10min, discarding the supernatant, adding 10mL resuspension (200. mu.M acetosyringone, 10Mm MES, 10mM MgCl)₂) Resuspending and OD adjustment₆₀₀To 0.7. And mixing the agrobacterium tumefaciens heavy suspension carrying pCaBS alpha, pCaBS beta and pCa gamma bLIC in equal volume, standing for 3 hours, and injecting the benthic tobacco which grows well in 6-8 leaf stages and has no stress. And slightly poking a pinhole on the flat leaf by using a syringe needle, pressing the pinhole on the back of the tobacco leaf by using a finger, and sucking the bacterial liquid mixture by using a 1mL syringe without the needle to inject from the front of the leaf so as to ensure that the bacterial liquid infiltrates the whole leaf as much as possible. Transferring the tobacco after injection to an illumination incubator for culture, collecting the ill injection leaf blades on 7-10 days, wrapping with tinfoil paper, weighing, and storing in a refrigerator at-80 deg.C for later use.

3. Tobacco leaf juice friction inoculated wheat

The collected tobacco leaves were removed from the freezer, transferred to a mortar pre-cooled with liquid nitrogen, ground thoroughly, added with 20mM sodium phosphate buffer pH 7.0 at a ratio of 1: 1(w/v), ground to a homogenate, and gauze filtered to remove plant debris. Adding a small amount of diatomite, stirring uniformly, and performing friction inoculation on two leaves of the Apogee wheat for a new period by using the obtained juice. The junction of the stem and the leaf is pressed to be fixed, and the proper amount of juice is dipped and rubbed on the wheat leaves for 3-5 times from bottom to top, the force is not too large, and the noise can be generated during the rubbing. And (3) observing the virus phenotype 10-12 days after inoculation, respectively stripping young ears of plants with BSMV:00 and BSMV: TaZVHD, and collecting the young ears into a 1.5mL centrifuge tube for extracting RNA. After filling, investigating the properties of the tested wheat, including the properties of spike length, small spike number, spike grain number, grain length, grain width, grain weight and the like.

Multiple VIGS infection experiments are carried out in wheat by utilizing 2 silencing target segments of TaZVHD, and the results show that when TaZfHD2 is silenced, compared with BSMV control, the spike length change and the change trend of the gene silencing level are consistent and positively correlated, while the change of the spike number, the spike grain number, the thousand grain weight, the grain length and the grain width and the change trend of the gene silencing level are inconsistent and have no correlation with the gene silencing level (figure 6 and figure 7), and similar experiment results are obtained by silencing two different segments of the gene. Therefore, TaZfHD2 may be involved in regulating ear length and ear grain number.

Four, the two haplotypes of TaVP15 have significant difference

To understand the variation of TaZVHD genome level, we selected 14 varieties with extreme panicle length traits (the phenotype data are shown in Table 1), including 7 long panicle materials and 7 short panicle materials, and connected the gene coding sequence to pEASY-Blunt vector for sequencing. The sequence alignment showed that there was a 12bp insertion of the coding region 758-769 of TaZfHD2-B in both materials (FIG. 8), resulting in four amino acids increase. A length polymorphism marker is designed, whether the detected variety has 12bp base deletion is determined according to the STR typing result, and two haplotypes of the gene can be clearly distinguished (figure 9).

TABLE 1 phenotypic data for 14 varieties with extreme panicle length traits

The base deletion conditions of more than 130 wheat varieties of a natural population are detected, the distribution frequency and mean value difference of corresponding characters of two haplotypes are analyzed, and the result shows that 35 parts of materials belong to a deletion type, 103 parts of materials belong to a non-deletion type, and the plant height, tillering number and under-spike stem mean value difference of the deletion type and the non-deletion type are not obvious. For the ear traits, the average value of the deleted ear length is higher than that of the non-deleted ear length, and extremely obvious difference level is achieved. The average value of the number of the deletion spikelets is higher than that of the number of the non-deletion spikelets, and the obvious difference level is achieved. The mean number of spikelets with deletion was higher than the mean number of spikelets without deletion, with no significant difference (figure 10, indicates P <0.05, indicates P < 0.01). Therefore, the deletion of 758 to 769 bases of TaZfHD2-B may be an excellent mutation in ear traits.

Finally, it should be noted that: the above-mentioned embodiments are only specific embodiments of the present invention, which are used for illustrating the technical solutions of the present invention and not for limiting the same, and the protection scope of the present invention is not limited thereto, although the present invention is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Reference to the literature

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Brenchley R, Spannagl M, Pfeifer M, et al, 2012, Analysis of the broken coal genome using a hold-genome shotgun sequence Nature 491, 705-.

Burch-Smith TM,Anderson JC,Martin GB,Dinesh-Kumar SP,2004.Applications and advantages of virus-induced gene silencing for gene function studies in plants.Plant J 39,734-46.

Edae EA,Byrne PF,Haley SD,Lopes MS,Reynolds MP,2014.Genome-wide association mapping of yield and yield components of spring wheat under contrasting moisture regimes.Theor Appl Genet 127,791-807.

Finn RD,Coggill P,Eberhardt RY,et al.2016.The Pfam protein families database:towards a more sustainable future.Nucleic Acids Research 44,D279-285.

Kong L,Zhang Y,Ye ZQ,Liu XQ,Zhao SQ,Wei L,Gao G.2007.CPC:assess the protein-coding potential of transcripts using sequence features and support vector machine.Nucleic Acids Research 35,W345-349.

Pertea M,Pertea GM,Antonescu CM,Chang TC,Mendell JT,Salzberg SL.2015.StringTie enables improved reconstruction of a transcriptome from RNA-seq reads.Nature Biotechnology 33,290-295.

Simmonds J, Scott P, et al, 2014.Identification and independency evaluation of a stable yield and yield grain weight QTL on chromosome 6A of hexagonal reel (Triticum aestivum L.). BMC Plant Biol 14,191.

Sukumaran S,Dreisigacker S,Lopes M,Chavez P,Reynolds MP,2015.Genome-wide association study for grain yield and related traits in an elite spring wheat population grown in temperate irrigated environments.Theor Appl Genet 128,353-63.

Trapnell C,Roberts A,Goff L,Pertea G,Kim D,Kelley DR,Pimentel H,Salzberg SL,Rinn JL,Pachter L.2012.Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.Nature Protocols 7,562-578.

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cggcaccgga ccaagttcac cccggagcag aaggagagga tgcgcgcctt cgccgagaag 540

caggggtggc gcatcaaccg cgacgacggc ggcgccctcg agcgcttctg cctcgagatc 600

ggcgtcaagc ggaacgtcct caaggtctgg atgcacaacc acaagcacca gctcgcctcg 660

cccacctcca ccgcggccgg tatgggcatg ggcatgggga tgggcatcgg catcaaccca 720

ggagtcctcg gcactggcac tgggaccggc gctggcgctg gcgctggcgt tggcgttgga 780

gcgggcaccg gcgacggaga cggagacgac gacgacacgg acgacgactc gcctccacgc 840

gccgccgtct catcgccctc cccctccccc atcagcgtct ag 882

<210> 4

<211> 295

<212> PRT

<213> wheat (Triticum aestivum)

<400> 4

Met Met Asp His Leu Ser Leu Val Pro Tyr Glu Gly Gly Ser Gly Ala

1 5 10 15

Gly Gly Asp Ala Gly Ala Lys Tyr Lys Glu Cys Met Arg Asn His Ala

20 25 30

Ala Ala Met Gly Gly Gln Ala Phe Asp Gly Cys Gly Glu Tyr Met Pro

35 40 45

Ala Ser Pro Asp Ser Leu Lys Cys Ala Ala Cys Gly Cys His Arg Ser

50 55 60

Phe His Arg Arg Ala Gly Ser Leu Ala Gly Gly Ala Cys Pro Ala Pro

65 70 75 80

Phe Phe Phe Ser Pro Pro Pro Pro Pro Pro Pro His His His Pro Pro

85 90 95

Pro His His Pro Val Leu Gln Gly Phe Leu Pro Ser Ala Pro Pro Arg

100 105 110

Pro Pro Gln Leu Ala Leu Pro Tyr His Ala Val Pro Ala Ala Trp His

115 120 125

His Ala Leu Leu Asp Pro Ala Arg Ala Gly Ser Glu Thr Pro Pro Arg

130 135 140

Ala Asp Asp Cys Ser Pro Gly Cys Gly Ser Gly Ser Phe Ser Arg Lys

145 150 155 160

Arg His Arg Thr Lys Phe Thr Pro Glu Gln Lys Glu Arg Met Arg Ala

165 170 175

Phe Ala Glu Lys Gln Gly Trp Arg Ile Asn Arg Asp Asp Gly Gly Ala

180 185 190

Leu Glu Arg Phe Cys Leu Glu Ile Gly Val Lys Arg Asn Val Leu Lys

195 200 205

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

210 215 220

Ala Ala Gly Met Gly Met Gly Met Gly Met Gly Ile Gly Ile Asn Pro

225 230 235 240

Gly Ala Leu Gly Thr Gly Thr Gly Thr Gly Ala Asp Ala Gly Val Gly

245 250 255

Val Gly Gly Gly Val Gly Ala Gly Thr Gly Asp Gly Asp Gly Asp Asp

260 265 270

Asp Asp Thr Asp Asp Asp Ser Pro Pro Arg Ala Ala Val Ser Ser Pro

275 280 285

Ser Pro Ser Pro Ile Ser Val

290 295

<210> 5

<211> 293

<212> PRT

<213> wheat (Triticum aestivum)

<400> 5

Met Met Asp His Leu Ser Leu Val Pro Tyr Glu Gly Gly Ser Gly Ala

1 5 10 15

Gly Gly Asp Ala Gly Ala Lys Tyr Lys Glu Cys Met Arg Asn His Ala

20 25 30

Ala Ala Met Gly Gly Gln Ala Phe Asp Gly Cys Gly Glu Tyr Met Pro

35 40 45

Ala Ser Pro Asp Ser Leu Lys Cys Ala Ala Cys Gly Cys His Arg Ser

50 55 60

Phe His Arg Arg Ala Gly Ser Leu Thr Gly Gly Ala Cys Pro Ala Pro

65 70 75 80

Phe Phe Phe Ser Pro Pro Pro Pro Pro Pro Pro His His His Pro Pro

85 90 95

Pro His His Pro Val Leu Gln Gly Phe Leu Pro Ser Ala Pro Pro Arg

100 105 110

Pro Pro Gln Leu Ala Leu Pro Tyr His Ala Val Pro Ala Ala Trp His

115 120 125

His Ala Leu Leu Asp Pro Ala Arg Ala Gly Ser Glu Thr Pro Pro Arg

130 135 140

Ala Asp Asp Cys Ser Pro Gly Cys Gly Ser Gly Ser Phe Gly Arg Lys

145 150 155 160

Arg His Arg Thr Lys Phe Thr Pro Glu Gln Lys Glu Arg Met Arg Ala

165 170 175

Phe Ala Glu Lys Gln Gly Trp Arg Ile Asn Arg Asp Asp Gly Gly Ala

180 185 190

Leu Glu Arg Phe Cys Leu Glu Ile Gly Val Lys Arg Asn Val Leu Lys

195 200 205

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

210 215 220

Ala Ala Gly Ile Gly Met Gly Met Gly Met Gly Ile Gly Ile Asn Pro

225 230 235 240

Gly Val Leu Gly Thr Gly Thr Gly Thr Gly Ala Gly Ala Gly Val Gly

245 250 255

Val Gly Val Gly Ala Gly Thr Gly Asp Gly Asp Gly Asp Asp Asp Asp

260 265 270

Thr Asp Asp Asp Ser Pro Pro Arg Ala Ala Val Ser Ser Pro Ser Pro

275 280 285

Ser Pro Ile Ser Val

290

<210> 6

<211> 293

<212> PRT

<213> wheat (Triticum aestivum)

<400> 6

Met Met Asp His Leu Ser Leu Val Pro Tyr Glu Gly Gly Ser Gly Ala

1 5 10 15

Gly Gly Asp Ala Gly Ala Lys Tyr Lys Glu Cys Met Arg Asn His Ala

20 25 30

Ala Ala Met Gly Gly Gln Ala Phe Asp Gly Cys Gly Glu Tyr Met Pro

35 40 45

Ala Ser Pro Asp Ser Leu Lys Cys Ala Ala Cys Gly Cys His Arg Ser

50 55 60

Phe His Arg Arg Ala Gly Ser Leu Ala Gly Gly Ala Cys Pro Ala Pro

65 70 75 80

Phe Phe Phe Ser Pro Pro Pro Pro Pro Pro Pro His His His Pro Pro

85 90 95

Pro His His Pro Val Leu Gln Gly Phe Leu Pro Ser Ala Pro Pro Arg

100 105 110

Pro Pro Gln Leu Ala Leu Pro Tyr His Ala Val Pro Ala Ala Trp His

115 120 125

His Ala Leu Leu Asp Pro Ala Arg Ala Gly Ser Glu Thr Pro Pro Arg

130 135 140

Ala Asp Asp Cys Ser Pro Gly Cys Gly Ser Gly Ser Phe Ser Arg Lys

145 150 155 160

Arg His Arg Thr Lys Phe Thr Pro Glu Gln Lys Glu Arg Met Arg Ala

165 170 175

Phe Ala Glu Lys Gln Gly Trp Arg Ile Asn Arg Asp Asp Gly Gly Ala

180 185 190

Leu Glu Arg Phe Cys Leu Glu Ile Gly Val Lys Arg Asn Val Leu Lys

195 200 205

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

210 215 220

Ala Ala Gly Met Gly Met Gly Met Gly Met Gly Ile Gly Ile Asn Pro

225 230 235 240

Gly Val Leu Gly Thr Gly Thr Gly Thr Gly Ala Gly Ala Gly Ala Gly

245 250 255

Val Gly Val Gly Ala Gly Thr Gly Asp Gly Asp Gly Asp Asp Asp Asp

260 265 270

Thr Asp Asp Asp Ser Pro Pro Arg Ala Ala Val Ser Ser Pro Ser Pro

275 280 285

Ser Pro Ile Ser Val

290

Claims (7)

1. Use of a protein for modulating development of a ear of a plant, wherein the protein is a1) or a 2): a1) the amino acid sequence is a protein shown in any one of sequences 4-6 in a sequence table; a2) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of an amino acid sequence shown in any one of sequences 4-6 in a sequence table; the plant is wheat.

2. Use of a gene for regulating development of a spike in a plant, wherein the gene is b1) or b 2): b1) a nucleotide sequence encoding the protein of claim 1; or b2) has a nucleotide sequence shown in any one of sequences 1-3 in the sequence table; the plant is wheat.

3. Use of a biomaterial for modulating development of a plant ear, wherein the biomaterial is any one of c1) to c9) as follows: c1) an expression cassette comprising the gene of claim 2; c2) a recombinant vector containing the gene of claim 2; c3) a recombinant vector comprising the expression cassette of c 1); c4) a recombinant microorganism containing the gene of claim 2; c5) a recombinant microorganism comprising the expression cassette of c 1); c6) a recombinant microorganism comprising the recombinant vector of c2) or c 3); c7) a transgenic plant cell line comprising the gene of claim 2; c8) a transgenic plant cell line comprising the expression cassette of c 1); c9) a transgenic plant cell line comprising the recombinant vector of c2) or c 3); the plant is wheat.

4. Use of the protein of claim 1 or the gene of claim 2 or the biomaterial of claim 3 for modulating the ear length of a plant; the plant is wheat.

5. A method for screening for excellent variation in traits in the ear of a plant, which comprises detecting a length polymorphism marker, wherein the length polymorphism marker is an insertion or deletion of 758 to 769 th bases of a nucleotide sequence of the gene according to claim 2, and the plant is wheat.

6. The method of claim 5, wherein the ear trait is ear length.

7. A method for regulating traits of panicle parts of a plant, comprising constructing a VIGS vector from the gene of claim 2, transforming a plant with the constructed VIGS vector, and silencing the gene in the plant; the plant is wheat.

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