CN112266412B

CN112266412B - Two-line hybrid wheat yield heterosis related protein TaCCA1-7D and coding gene and application thereof

Info

Publication number: CN112266412B
Application number: CN202011200730.8A
Authority: CN
Inventors: 公杰; 高世庆; 赵昌平; 刘永杰; 李丹
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-11-02
Filing date: 2020-11-02
Publication date: 2022-03-25
Anticipated expiration: 2040-11-02
Also published as: CN112266412A

Abstract

The invention relates to the field of genetic engineering, in particular to a plant drought-resistant related protein TaCCA1-7D, and a coding gene and application thereof. The amino acid sequence of the protein is shown as SEQ ID NO:1, and the gene sequence is shown as SEQ ID NO:2, respectively. The yield heterosis related protein and the coding gene thereof have important theoretical and practical significance for improving and enhancing the stress resistance of wheat, improving the yield, accelerating the breeding process of stress-resistant molecules and effectively saving water resources.

Description

Two-line hybrid wheat yield heterosis related protein TaCCA1-7D and coding gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a two-line hybrid wheat yield heterosis related protein TaCCA1-7D and a coding gene and application thereof.

Background

Heterosis is an important biological phenomenon in which hybrid progeny excels in parental average in a number of traits such as growth vigor, seed yield, and adaptability. Although researches on heterosis action mechanisms are frequently reported at home and abroad, researchers put forward new theories on physiological, genetic and molecular biological mechanisms of heterosis formation of different crops from different scientific perspectives, and continuous improvement and perfection are still needed.

It is believed that all genes in the progeny of the cross are derived from the independently present alleles of the parental genome and have not been recombined or mutated to form new genes, but F1 shows a significantly superior phenotype. The hypothesis of dominant complementation, super-dominant and epistatic effects is proposed by the predecessors to describe the interaction of parental related genes in the context of heterosis inheritance to explain the genetic basis of heterosis phenomena. The rice is one of the most important grain crops and is a good molecular biological research model plant; the utilization of heterosis in rice greatly promotes the increase of rice yield, so that rice becomes an ideal mode crop for researching the heterosis mechanism. In recent years, with the development of sequencing technology and the progress of experimental technology, many key genes related to heterosis have been cloned and deep functional studies have been carried out.

Recent researches show that the biological clock has a key regulation and control effect on the heterosis of the model plants, namely the arabidopsis thaliana and the crop maize, and shows that the biological clock research has important theoretical value and wide application prospect on agricultural production. Biological clocks are ubiquitous in a wide variety of organisms, from lower bacteria to higher fungi, plants, animals and humans. Circadian rhythm disorders of various levels, such as molecules, biochemistry, cells, physiology, behaviors and the like regulated by a biological clock, cause serious damage to the health and survival of organisms.

The plant biological clock participates in regulating and controlling almost all metabolism and growth and development processes of the plant body, so that the time and space synchronization of the plant biological clock and external environmental conditions (light and temperature cycles) is achieved. The biological clock regulating mechanism makes the plant benefit in the process of participating in environment response, and from gene expression to physiological and biochemical level, the rhythmic oscillation of the biological clock in the near day can reduce unnecessary energy and material consumption, enhance the accumulation of biomass and life vitality, and greatly improve the survival and competitive capacity of the plant. Plant biological clocks are a highly complex, ordered physiological regulatory system: firstly, a sensing system-input channel, red light receptors PHYs, blue light receptors CRYs and other unknown factors which may exist sense the change of environmental signals of natural illumination, temperature, nutritional factors and the like, and then transmit the information of the change to a core oscillator (core oscillator) of a biological clock. The classical model plant Arabidopsis thaliana biological clock core regulatory system now comprises a variety of elements that form a complex, multiple feedback regulatory network. Finally, the response system-output pathway refers to the biological rhythm of the oscillation of a plurality of near-day biological clocks in which the biological clock participates in regulation and control, and relates to various key physiological and biochemical reaction processes in the growth and development of plants, such as: the plant grows periodically, the stomata of the leaves open and close, the leaves move, the petals open and close, the flowering time, the hypocotyl elongation, the synthesis and signal transduction of hormone, abiotic and biotic stress response and the like. The three parts of systems of the biological clock respond to each other to form a complex and ordered whole to regulate and control the growth and development of plants and the adaptation to the external environment.

The dicotyledonous plant Arabidopsis thaliana is a model organism for researching plant biological clock, and the research of Arabidopsis thaliana biological clock makes the clear understanding of the function of plant biological clock system in the aspects of plant growth, development, adaptation to external environment and the like. Calcium ion is one of the most important second messengers in plant cells, and plays an important role in the growth and development of plants and in stress signal transduction. Research shows that the concentration of plant cytoplasm free calcium ions is 24h rhythm oscillation, the oscillation is regulated by blue light and red light, components of biological clock input channel, namely blue light receptor and red light receptor play an important role, and blue light plays an important role in cytoplasm [ Ca²⁺]The level-up regulation requires the joint participation of PHYB, CRY1 and CRY2, in which the biological clock core oscillator component ELF3 acts like a valve to regulate the rhythm of free calcium ions in cells directly by controlling the light input pathway of the biological clock.

The biological clock core oscillator element and the target gene regulated by the element play an important role in both biotic stress and abiotic stress reactions, and the CCA1 and the stomata opening and closing regulated by the LHY and the rhythmic expression of the downstream target gene are very important for the disease resistance of plants. Many target genes regulated by the CCA1 directly or indirectly participate in basic defense reaction and R-gene mediated resistance reaction, so that the plants can predict pathogen infection and pertinently mobilize corresponding defense genes to start expression. The ABA content of the three deletion mutants of the arabidopsis PRR5 PRR7 PRR9 is obviously higher than that of the wild type, and the three deletion mutants are further found to be obviously related to the expression of key genes of an ABA synthetic pathway in the mutants in an up-regulation way, which suggests that the PRR5/PRR7/PRR9 may play an important role in the response of plants to abiotic stresses such as drought, low temperature, high salt and the like. Studies by Legnaioli et al show that ABA can induce expression of TOC 1; in turn, TOC1 may also regulate the periodic expression of ABAR by binding directly to the ABAR promoter region, and thus TOC1 is thought to act as a molecular switch between the drought stress signaling pathway and the biological clock.

Through research on different ecotype parents of arabidopsis thaliana and a hybrid F1, it is found that the biological clock plays an important role in plant heterosis. The research of Ni and the like shows that in the daytime, the expression of a biological clock core oscillator coding gene CCA1(LHY) in an Arabidopsis F1 plant is regulated and controlled by an apparent factor to inhibit the expression, and the expression level of target genes (participating in chlorophyll and starch synthesis and the like) is increased, so that the content of chlorophyll and starch in a hybrid F1 is higher than that of the parent, the photosynthesis is enhanced, the accumulation of nutrient substances is increased, and the F1 shows heterosis. Further transgenic research shows that the CCA1 overexpression can inhibit the growth of plants, and research on transgenic plants expressing the TOC1:: CCA1 shows that the expression level of CCA1 in transgenic plants expressing the TOC1:: CCA1 is improved by 3 times at noon, but the expression of downstream target genes PORA, PORB, AMY, DPE1 and GWD3 is inhibited to different degrees, so that the content of phytoalexin and starch is reduced by 14% and 17%. Further, the research of transgenic plants silenced by CCA1 gene proves that the growth of the plants is enhanced by the inhibition expression of CCA1, and the starch content of the transgenic plants is increased by 28%. In conclusion, the inhibition expression of CCA1 increases the content of chlorophyll and starch in plants, enhances the growth vigor and biomass accumulation of plants, and the plants show growth advantages.

In addition to the demonstration of the important role of the biological clock in heterosis in model plants, research on the mechanism of the biological clock is also gradually developed on crops, two homologous genes ZmCCA1a and ZmCCA1b which code for a biological clock core oscillator element ZmCCA1 exist in the corn genome, and overexpression of the ZmCCA1b gene can cause the imbalance of the corn biological rhythm, plant dwarfing and the reduction of the leaf chlorophyll content, and the biological clock system can play an important role in corn heterosis. Further, analysis by zmcc 1 immunoprecipitation sequencing found: the genes and networks regulated by the biological clock in the corn hybrid F1 are 3-6 hours earlier than those of the inbred line, and in the corn hybrid, the genes and networks regulated by the biological clock are advanced, so that the early growth advantage is established, and the foundation is laid for the later yield advantage.

In conclusion, the CCA1 plays a crucial role in regulating the growth and development of plants and improving the yield of the plants, and has great promotion and economic benefits on high-yield molecular breeding and agricultural production. Therefore, the wheat strong-dominance hybrid is used as an experimental material, and cloning and separating the gene TaCCA1-7D, which is a biological clock core factor, has very important significance for improving and increasing the yield of crops.

Disclosure of Invention

The invention aims to provide a two-line hybrid wheat yield heterosis related protein TaCCA 1-7D.

It is a further object of the present invention to provide a gene encoding TaCCA1-7D for heterosis related yield in plants as described above.

Another object of the present invention is to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide a transgenic cell line comprising the above gene.

Another object of the present invention is to provide the use of the above-mentioned plant yield heterosis-related protein TaCCA 1-7D.

The yield heterosis related protein TaCCA1-7D provided by the invention is derived from a wheat hybrid seed Jingmai 179, and the amino acid sequence of the protein is shown as SEQ ID NO:1 is shown.

The TaCCA1-7D protein consists of 718 amino acid residues. The 20 th to 31 th amino acid residues from the amino terminal end of SEQ ID NO.1 are DNA binding domains, and the 150 th and 180 th amino acid residues from SEQ ID NO.1 are phosphorylation site regions.

SEQ ID NO：1

MEINSSGEETMIKVRKPYTITKQRERWTEAEHKRFLEALKLYGRAWQRIEEHVGTKTAVQIRSHAQKFFTKLEKEAINNGTSPGQAHDIDIPPPRPKRKPNCPYPRKGCLSSETPTREVPKSSVSLSNSNAEMGSNGTLQLTCIQKLQRKELSENGSCSEVINIFREAPSASFSSSNKSSSNHGVSGGIEPTKTENKDIATMERKSTSIDVGKDVKDINDQEMERNNRVHISSNYDRSHEDCLDNSMKHMQLKPNTAETTYTGQHAASAPLYQMNKTGATGAPDPGTEGSHPDQTSDQVGGANGSMDCIHPTLLVDPKMGSSSTAQPFPHNYAGFAPTMQCHCNQDAYRSSLNMSSTFSNMLVSTLLSNPTVHAAARLAASYWPAADSNIPVGPNQEVFAENAQGRHIGSPPSMASVVAATVAAASAWWATQGLLPLFAPPMAFPFVPVPTASFPTADVQRATENFPVDNAPKECQVAQEQGQPEAMIVVASSVSDESGKGEVSPHTELNISPADKVETTPPTGAETSDAFGNKKKQDRSSCGSNTPSSSDVEAEHVPENQDQANDKTQQACCSNSSAGDMNHRRFRNISSTNDSWKEVSEEGRMAFDKLFSRGKLPQSFSPPQAEGLKVVPRGEQDEATTVTVDLNKSAAVMDHELDTLVGPRAATFPIELSHLNMKSRRTGFKPYKRCSVEAKENRVPASDEVGTKRIRLDSEPST

The coding gene of TaCCA1-7D has the sequence shown in SEQ ID NO: 2.

SEQ ID NO：2

1 ATGGAGATAA ATTCTTCGGG TGAGGAAACA ATGATAAAGG TGCGAAAGCC GTACACAATA

61 ACAAAACAGC GGGAGCGGTG GACCGAGGCA GAGCACAAAC GGTTCCTTGA AGCCCTCAAA

121 CTGTATGGCA GAGCTTGGCA GCGCATAGAA GAGCACGTTG GGACAAAGACGGCCGTGCAA

181 ATCAGAAGCC ATGCTCAGAA GTTCTTCACC AAGTTGGAAA AGGAAGCTAT CAATAATGGT

241 ACTTCTCCGG GACAAGCTCA TGATATAGAC ATACCTCCAC CACGGCCTAA AAGAAAACCT

301 AACTGTCCAT ATCCTCGAAA AGGTTGTCTC AGTTCTGAAA CACCCACCAG AGAAGTTCCA

361 AAATCAAGTG TAAGCTTGAG CAATAGCAAT GCAGAAATGG GGAGCAATGG AACTCTTCAG

421 CTCACCTGCA TTCAGAAACT TCAAAGGAAG GAGTTATCTG AAAATGGCAG TTGCTCAGAA

481 GTTATTAATA TCTTTCGAGA AGCACCATCT GCCTCATTTT CTTCCTCTAA CAAGAGCTCT

541 TCAAATCATG GTGTCTCTGG GGGAATTGAA CCGACTAAAA CAGAAAACAA AGATATTGCA

601 ACCATGGAAA GGAAATCTAC TTCCATTGAT GTGGGGAAGG ATGTAAAAGA TATTAATGAC

661 CAGGAAATGG AAAGGAACAA CAGAGTCCAC ATCAGTTCTA ATTATGACCG TTCTCATGAA

721 GATTGTTTGG ATAACTCAAT GAAACACATG CAGTTGAAGC CAAATACCGC GGAGACAACA

781 TACACGGGTC AACATGCTGC AAGTGCCCCA CTCTACCAAA TGAATAAGAC TGGGGCAACT

841 GGCGCTCCAG ACCCTGGAAC TGAAGGAAGT CATCCTGATC AAACAAGTGA TCAAGTGGGA

901 GGAGCTAATG GAAGCATGGA CTGCATCCAT CCAACACTTC TGGTGGATCC AAAAATGGGC

961 AGCAGTTCCA CAGCACAGCC CTTTCCCCAC AACTATGCAG GCTTTGCACC AACGATGCAA

1021 TGCCACTGCA ACCAAGATGC CTACAGGTCT TCTCTTAATA TGTCATCCAC CTTCTCCAAC

1081 ATGCTCGTTT CCACGTTGTT ATCAAACCCC ACAGTACATG CAGCCGCAAG GCTTGCAGCA

1141 TCATACTGGC CAGCAGCAGA CAGCAACATT CCTGTTGGTC CAAATCAAGA AGTTTTTGCT

1201 GAGAATGCTC AAGGAAGACA TATTGGTTCT CCTCCAAGCA TGGCTTCTGT GGTAGCAGCT

1261 ACAGTTGCTG CGGCTTCGGC ATGGTGGGCA ACACAAGGTC TTCTCCCTCT TTTTGCTCCC

1321 CCCATGGCTT TTCCATTTGT CCCAGTTCCT ACTGCTTCCT TTCCCACAGC GGATGTTCAG

1381 CGAGCTACAG AGAACTTCCC AGTGGACAAC GCACCGAAGG AATGCCAAGT AGCTCAGGAG

1441 CAAGGTCAAC CTGAAGCTAT GATAGTTGTA GCGTCTTCTG TATCCGACGA GAGTGGAAAA

1501 GGAGAGGTGT CTCCCCACAC TGAGTTAAAT ATATCTCCTG CTGATAAAGT TGAGACAACA

1561 CCTCCCACAG GAGCTGAAAC AAGTGATGCT TTCGGCAACA AGAAGAAGCA GGATCGCTCT

1621 TCATGTGGTT CCAACACGCC ATCAAGTAGT GATGTAGAGG CAGAACATGT TCCTGAGAAC

1681 CAAGATCAAG CCAACGACAA GACACAGCAA GCATGTTGCA GTAATTCTTC AGCTGGTGAC

1741 ATGAACCACC GCAGGTTTAG GAACATTTCA AGCACAAATG ATTCATGGAA GGAAGTTTCC

1801 GAAGAGGGTC GTATGGCTTT CGATAAACTG TTCAGTAGAG GAAAGCTTCC CCAAAGCTTT

1861 TCTCCTCCAC AAGCAGAAGG ATTGAAGGTG GTACCCAGGG GGGAACAAGA CGAAGCTACT

1921 ACGGTGACGG TCGACCTCAA CAAGAGTGCT GCAGTTATGG ACCATGAACT TGATACATTG

1981 GTTGGGCCAA GAGCTGCTAC CTTTCCCATT GAATTGTCAC ACCTGAATAT GAAATCCCGC

2041 CGGACAGGCT TCAAACCTTA CAAGAGGTGC TCGGTGGAAG CAAAGGAGAA TAGGGTGCCG

2101 GCTTCTGACG AGGTTGGTAC CAAGAGGATT CGTCTTGACA GCGAACCCTC CACGTGA

To facilitate purification of the protein TaCCA1-7D, the amino-terminal or carboxy-terminal end of the protein consisting of the amino acid sequence shown in SEQ ID NO:1 may be attached with the tags shown in Table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG
	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

According to the SEQ ID NO.1 sequence disclosed by the invention, the transcription factor TaCCA1-7D can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The expression cassette, the recombinant expression vector, the transgenic cell line and the recombinant bacteria containing the TaCCA1-7D gene belong to the protection scope of the invention.

The existing plant expression vector can be used for constructing a recombinant expression vector containing the TaCCA1-7D gene.

The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can lead polyadenylic acid to the 3 'end of the mRNA precursor, and the untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopaline synthase Nos genes) and plant genes all have similar functions.

When TaCCA1-7D is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before transcription initiation nucleotide, such as cauliflower mosaic virus (CaMV)35S promoter and Ubiquitin promoter (Ubiquitin) of corn, and the promoter can be used alone or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

It is another object of the present invention to provide a method for cultivating a high-yielding plant.

The method for cultivating high-yield plants provided by the invention is to introduce any one of the recombinant expression vectors containing the TaCCA1-7D gene into plant cells to obtain a new high-yield wheat material.

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the TaCCA1-7D gene provided by the invention into plant cells, and a high-yield transgenic cell line and a transgenic plant can be obtained. The expression vector carrying the encoding gene can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation, etc., and the transformed plant tissues can be cultivated into plants. The plant host to be transformed may be either a monocotyledonous or dicotyledonous plant, such as: arabidopsis, wheat, arabidopsis, rice, corn, cucumber, tomato, poplar, turfgrass, alfalfa and the like.

According to the invention, the Jingmai 179 with strong yield heterosis is taken as an experimental material, the TaCCA1-7D protein related to yield heterosis and the coding gene thereof are obtained and are introduced into wheat, and the yield of plants is obviously improved. The yield heterosis related TaCCA1-7D protein and the coding gene thereof have very important theoretical and practical significance for improving and enhancing the yield of wheat and accelerating the high-yield molecular breeding process.

The invention is further described with reference to the following drawings and specific embodiments.

Drawings

FIG. 1 shows the cloning and vector construction of wheat TaCCA1-7D gene, wherein, A, the amplification of target gene TaCCA1-7D full length CDS; b, identification of positive clone after TaCCA1-7D full-length CDS is connected to an overexpression vector, wherein

lanes

3, 5 and 6 represent positive clones, and templates of '1' and '2' are water and no-load plasmids respectively; the templates of "3", "4", "5" and "6" are recombinant plasmids.

FIG. 2 shows that the expression of TaCCA1-7D shows a circadian pattern, wherein P1, P2 and F1 represent parent 1, parent 2 and hybrid, respectively, and MVP represents the average value of the expression of TaCCA1-7D in parent 1 and parent 2; white boxes represent day and black boxes represent night.

FIG. 3 shows the PCR identification of TaCCA1-7D overexpressing transgenic plants, in which 1 is positive control, 2-7, 9-16 are positive plants, and 8 is negative control.

FIG. 4 shows statistical analysis of transgenic wheat seedling expression and panicle length traits, wherein a is the seedling phenotype of TaCCA1-7D overexpression plants; b is the expression condition of TaCCA1-7D mRNA in the real-time fluorescent quantitative PCR detection over-expression material; c is the statistical result of the seedling biomass of TaCCA1-7D overexpression plants; d is the phenotype of TaCCA1-7D overexpression plant ears; e is the statistical result of the panicle length of the TaCCA1-7D overexpression plant; f is the statistical result of the grain number of each ear of the TaCCA1-7D overexpression plant.

FIG. 5 shows statistical analysis of transgenic wheat grain traits, wherein a is TaCCA1-7D overexpression plant grain length phenotype; b is TaCCA1-7D overexpression plant grain width phenotype; c is the statistical result of the seed length phenotype of TaCCA1-7D overexpression plants; d is the statistical result of the TaCCA1-7D overexpression plant grain width phenotype; e is the statistical result of the seed weight of the TaCCA1-7D overexpression plant.

Detailed Description

The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

Example 1: cDNA clone of TaCCA1-7D gene related to wheat yield heterosis

Wheat seedlings which grow for about 30 days are subjected to drought treatment for 5 hours, and Trizol is used for extracting the total RNA of the wheat. The full-length sequence of TaCCA1-7D gene was obtained at 2157bp using 5 'RACE Kit (5' RACE System for Rapid Amplification of cDNA Ends Kit) (GIBCOBRL, CAT. NO.18374-058) and 3 'RACE Kit (3' RACE System for Rapid Amplification of cDNA Ends Kit) (GIBCOBRL, CAT. NO.18373-019) (FIG. 1, A).

Trizol is used for extracting total RNA of wheat seedlings, and superscript II (invitrogen) reverse transcriptase is used for reverse transcription to obtain cDNA. Primers P1 and P2 were designed based on the coding region sequence of TaCCA1-7D gene. PCR amplification was performed using the cDNA obtained by reverse transcription as a template, and primers P1 and P2. The sequences of primers P1 and P2 are as follows:

P1：5’-ATGGAGATAAATTCTTCGGGT-3’，

P2：5’-TCACGTGGAGGGTTCGCTGTCAAGAC-3’。

the PCR product was subjected to 0.8% agarose gel electrophoresis to obtain a band having a molecular weight of about 2.1kb, which was consistent with the expected result. The fragment was recovered using agarose gel recovery kit (TIANGEN). The recovered fragment was ligated to pGEM-T Easy (Promega), and the ligation product was transformed into E.coli DH 5. alpha. competent cells by the method of Cohen et al (Proc Natl Acad Sci, 69:2110), and positive clones were selected based on ampicillin resistance marker on pGEM-T Easy vector to obtain recombinant plasmid containing the recovered fragment. The nucleotide sequence of the T7 and SP6 promoter sequences on the recombinant plasmid vector is taken as a primer for nucleotide sequence determination, and the sequencing result shows that the Open Reading Frame (ORF) of the amplified TaCCA1-7D gene is the deoxyribonucleotide from the 5' end 1 to the 2157 position of SEQ ID No. 2, and the coding amino acid sequence is the protein of SEQ ID No. 1. The recombinant vector containing the gene TaCCA1-7D shown in the sequence SEQ ID No. 2 is named as pTE-TaCCA 1-7D.

The sequences of the TaCCA1-7D genes are aligned, and homologous protein genes are not found in wheat, so that the TaCCA1-7D gene is proved to be a novel gene.

Further amplification was performed in the wheat genome using primers P1 and P2, and the results showed that the genomic sequence of the gene was identical in size to the cDNA length and contained no intron sequence.

And (3) constructing a 35S-TaCCA1-7D recombinant expression vector. Taking cDNA obtained by reverse transcription of total RNA of wheat as a template, and carrying out PCR amplification by using a specific primer containing SmaI and SpeI linker sequences; then SmaI and SpeI double-enzyme digestion PCR products are recovered, and the digestion products are inserted between SmaI and SpeI digestion sites behind the CaMV 35S promoter of the vector pBI121 in the forward direction to obtain a recombinant vector p35S:: TaCCA1-7D (figure 1, B).

The primer sequences are as follows:

TaCCA1-7D[SmaI]5’-TCCCCCGGGGATGGAGATAAATTCTTCGGGT-3’

TaCCA1-7D[SpeI]5’-GGACTAGTTCACGTGGAGGGTTCGCTGTCAAGAC-3’

example 2: TaCCA1-7D gene expression pattern analysis

Expression analysis is carried out on the TaCCA1-7D gene, and the gene is found to present regular rhythm expression characteristics in parent and hybrid seedling-stage tissues. Among them, the expression level of hybrid F1 is significantly higher than that of parents, indicating that the gene plays a very important regulatory role at the level of hybrid yield and heterotic molecules (FIG. 2).

Example 3: enhancement of yield heterosis in plants with the TaCCA1-7D gene

1) Obtaining transgenic wheat

And (3) respectively transforming the constructed recombinant expression vector p35S: TaCCA1-7D into Agrobacterium tumefaciens EHA105 by a freeze-thaw method, transforming wheat by the Agrobacterium tumefaciens EHA105 of the TaCCA1-7D in p35S, and screening by an MS culture medium containing 100mg/L kanamycin to obtain a positive transgenic plant. And performing further identification and screening on the positive transgenic plants obtained by screening by using PCR (polymerase chain reaction), wherein a pair of primers used by the PCR is P3 and P4.

P3 (upstream primer) 5'-TGCCACTGCAACCAAGATGC-3',

p4 (downstream primer): 5'-ATGGCGTGTTGGAACCACATGA-3'.

PCR identification is carried out on 35S: TaCCA1-7D transgenic wheat, a positive transgenic plant can obtain a band about 350bp through PCR amplification, and a result shows that 35S: TaCCA1-7D wheat 14 strains are obtained (figure 3).

At the same time, the pBI121 empty vector was introduced into wheat in the same manner as above, and 5 lines of wheat were obtained as a control (T for transgenic wheat obtained by screening)₂Generation representation).

2) TaCCA1-7D transgenic wheat yield identification

And (3) carrying out yield identification on TaCCA1-7D transgenic strains OE-1, OE-2 and OE-6, such as seedling biomass, spike length, grain size and the like. Wherein, fieldier is the receptor control. The results show that: control WT leaf biomass was significantly greater than the transgenic lines and spike length was also significantly longer than the 3 transgenic lines (fig. 4). The grain length and width comparison shows that the transgenic strains OE-1, OE-2 and OE-6 are shorter than the control WT in length and width (FIG. 5). The results show that the TaCCA1-7D overexpression reduces the yield level of the transgenic wheat, causes the disorder of a biological clock system, further causes the disorder of the metabolism level of the transgenic line, and causes the problem of the growth and development of the transgenic line. The above results further confirm from the side that the expression level of the gene plays a crucial role in maintaining the biological clock system of wheat hybrids.

Sequence listing

<110> agriculture and forestry academy of sciences of Beijing City

<120> two-line hybrid wheat yield heterosis related protein TaCCA1-7D, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 718

<212> PRT

<213> wheat (Triticum aestivuml.)

<400> 1

Met Glu Ile Asn Ser Ser Gly Glu Glu Thr Met Ile Lys Val Arg Lys

1 5 10 15

Pro Tyr Thr Ile Thr Lys Gln Arg Glu Arg Trp Thr Glu Ala Glu His

20 25 30

Lys Arg Phe Leu Glu Ala Leu Lys Leu Tyr Gly Arg Ala Trp Gln Arg

35 40 45

Ile Glu Glu His Val Gly Thr Lys Thr Ala Val Gln Ile Arg Ser His

50 55 60

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

65 70 75 80

Thr Ser Pro Gly Gln Ala His Asp Ile Asp Ile Pro Pro Pro Arg Pro

85 90 95

Lys Arg Lys Pro Asn Cys Pro Tyr Pro Arg Lys Gly Cys Leu Ser Ser

100 105 110

Glu Thr Pro Thr Arg Glu Val Pro Lys Ser Ser Val Ser Leu Ser Asn

115 120 125

Ser Asn Ala Glu Met Gly Ser Asn Gly Thr Leu Gln Leu Thr Cys Ile

130 135 140

Gln Lys Leu Gln Arg Lys Glu Leu Ser Glu Asn Gly Ser Cys Ser Glu

145 150 155 160

Val Ile Asn Ile Phe Arg Glu Ala Pro Ser Ala Ser Phe Ser Ser Ser

165 170 175

Asn Lys Ser Ser Ser Asn His Gly Val Ser Gly Gly Ile Glu Pro Thr

180 185 190

Lys Thr Glu Asn Lys Asp Ile Ala Thr Met Glu Arg Lys Ser Thr Ser

195 200 205

Ile Asp Val Gly Lys Asp Val Lys Asp Ile Asn Asp Gln Glu Met Glu

210 215 220

Arg Asn Asn Arg Val His Ile Ser Ser Asn Tyr Asp Arg Ser His Glu

225 230 235 240

Asp Cys Leu Asp Asn Ser Met Lys His Met Gln Leu Lys Pro Asn Thr

245 250 255

Ala Glu Thr Thr Tyr Thr Gly Gln His Ala Ala Ser Ala Pro Leu Tyr

260 265 270

Gln Met Asn Lys Thr Gly Ala Thr Gly Ala Pro Asp Pro Gly Thr Glu

275 280 285

Gly Ser His Pro Asp Gln Thr Ser Asp Gln Val Gly Gly Ala Asn Gly

290 295 300

Ser Met Asp Cys Ile His Pro Thr Leu Leu Val Asp Pro Lys Met Gly

305 310 315 320

Ser Ser Ser Thr Ala Gln Pro Phe Pro His Asn Tyr Ala Gly Phe Ala

325 330 335

Pro Thr Met Gln Cys His Cys Asn Gln Asp Ala Tyr Arg Ser Ser Leu

340 345 350

Asn Met Ser Ser Thr Phe Ser Asn Met Leu Val Ser Thr Leu Leu Ser

355 360 365

Asn Pro Thr Val His Ala Ala Ala Arg Leu Ala Ala Ser Tyr Trp Pro

370 375 380

Ala Ala Asp Ser Asn Ile Pro Val Gly Pro Asn Gln Glu Val Phe Ala

385 390 395 400

Glu Asn Ala Gln Gly Arg His Ile Gly Ser Pro Pro Ser Met Ala Ser

405 410 415

Val Val Ala Ala Thr Val Ala Ala Ala Ser Ala Trp Trp Ala Thr Gln

420 425 430

Gly Leu Leu Pro Leu Phe Ala Pro Pro Met Ala Phe Pro Phe Val Pro

435 440 445

Val Pro Thr Ala Ser Phe Pro Thr Ala Asp Val Gln Arg Ala Thr Glu

450 455 460

Asn Phe Pro Val Asp Asn Ala Pro Lys Glu Cys Gln Val Ala Gln Glu

465 470 475 480

Gln Gly Gln Pro Glu Ala Met Ile Val Val Ala Ser Ser Val Ser Asp

485 490 495

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

500 505 510

Pro Ala Asp Lys Val Glu Thr Thr Pro Pro Thr Gly Ala Glu Thr Ser

515 520 525

Asp Ala Phe Gly Asn Lys Lys Lys Gln Asp Arg Ser Ser Cys Gly Ser

530 535 540

Asn Thr Pro Ser Ser Ser Asp Val Glu Ala Glu His Val Pro Glu Asn

545 550 555 560

Gln Asp Gln Ala Asn Asp Lys Thr Gln Gln Ala Cys Cys Ser Asn Ser

565 570 575

Ser Ala Gly Asp Met Asn His Arg Arg Phe Arg Asn Ile Ser Ser Thr

580 585 590

Asn Asp Ser Trp Lys Glu Val Ser Glu Glu Gly Arg Met Ala Phe Asp

595 600 605

Lys Leu Phe Ser Arg Gly Lys Leu Pro Gln Ser Phe Ser Pro Pro Gln

610 615 620

Ala Glu Gly Leu Lys Val Val Pro Arg Gly Glu Gln Asp Glu Ala Thr

625 630 635 640

Thr Val Thr Val Asp Leu Asn Lys Ser Ala Ala Val Met Asp His Glu

645 650 655

Leu Asp Thr Leu Val Gly Pro Arg Ala Ala Thr Phe Pro Ile Glu Leu

660 665 670

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

675 680 685

Arg Cys Ser Val Glu Ala Lys Glu Asn Arg Val Pro Ala Ser Asp Glu

690 695 700

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

705 710 715

<210> 2

<211> 2157

<212> DNA

<213> wheat (Triticum aestivuml.)

<400> 2

atggagataa attcttcggg tgaggaaaca atgataaagg tgcgaaagcc gtacacaata 60

acaaaacagc gggagcggtg gaccgaggca gagcacaaac ggttccttga agccctcaaa 120

ctgtatggca gagcttggca gcgcatagaa gagcacgttg ggacaaagac ggccgtgcaa 180

atcagaagcc atgctcagaa gttcttcacc aagttggaaa aggaagctat caataatggt 240

acttctccgg gacaagctca tgatatagac atacctccac cacggcctaa aagaaaacct 300

aactgtccat atcctcgaaa aggttgtctc agttctgaaa cacccaccag agaagttcca 360

aaatcaagtg taagcttgag caatagcaat gcagaaatgg ggagcaatgg aactcttcag 420

ctcacctgca ttcagaaact tcaaaggaag gagttatctg aaaatggcag ttgctcagaa 480

gttattaata tctttcgaga agcaccatct gcctcatttt cttcctctaa caagagctct 540

tcaaatcatg gtgtctctgg gggaattgaa ccgactaaaa cagaaaacaa agatattgca 600

accatggaaa ggaaatctac ttccattgat gtggggaagg atgtaaaaga tattaatgac 660

caggaaatgg aaaggaacaa cagagtccac atcagttcta attatgaccg ttctcatgaa 720

gattgtttgg ataactcaat gaaacacatg cagttgaagc caaataccgc ggagacaaca 780

tacacgggtc aacatgctgc aagtgcccca ctctaccaaa tgaataagac tggggcaact 840

ggcgctccag accctggaac tgaaggaagt catcctgatc aaacaagtga tcaagtggga 900

ggagctaatg gaagcatgga ctgcatccat ccaacacttc tggtggatcc aaaaatgggc 960

agcagttcca cagcacagcc ctttccccac aactatgcag gctttgcacc aacgatgcaa 1020

tgccactgca accaagatgc ctacaggtct tctcttaata tgtcatccac cttctccaac 1080

atgctcgttt ccacgttgtt atcaaacccc acagtacatg cagccgcaag gcttgcagca 1140

tcatactggc cagcagcaga cagcaacatt cctgttggtc caaatcaaga agtttttgct 1200

gagaatgctc aaggaagaca tattggttct cctccaagca tggcttctgt ggtagcagct 1260

acagttgctg cggcttcggc atggtgggca acacaaggtc ttctccctct ttttgctccc 1320

cccatggctt ttccatttgt cccagttcct actgcttcct ttcccacagc ggatgttcag 1380

cgagctacag agaacttccc agtggacaac gcaccgaagg aatgccaagt agctcaggag 1440

caaggtcaac ctgaagctat gatagttgta gcgtcttctg tatccgacga gagtggaaaa 1500

ggagaggtgt ctccccacac tgagttaaat atatctcctg ctgataaagt tgagacaaca 1560

cctcccacag gagctgaaac aagtgatgct ttcggcaaca agaagaagca ggatcgctct 1620

tcatgtggtt ccaacacgcc atcaagtagt gatgtagagg cagaacatgt tcctgagaac 1680

caagatcaag ccaacgacaa gacacagcaa gcatgttgca gtaattcttc agctggtgac 1740

atgaaccacc gcaggtttag gaacatttca agcacaaatg attcatggaa ggaagtttcc 1800

gaagagggtc gtatggcttt cgataaactg ttcagtagag gaaagcttcc ccaaagcttt 1860

tctcctccac aagcagaagg attgaaggtg gtacccaggg gggaacaaga cgaagctact 1920

acggtgacgg tcgacctcaa caagagtgct gcagttatgg accatgaact tgatacattg 1980

gttgggccaa gagctgctac ctttcccatt gaattgtcac acctgaatat gaaatcccgc 2040

cggacaggct tcaaacctta caagaggtgc tcggtggaag caaaggagaa tagggtgccg 2100

gcttctgacg aggttggtac caagaggatt cgtcttgaca gcgaaccctc cacgtga 2157

Claims

1. The application of the plant yield heterosis related protein TaCCA1-7D in the following aspects,

the encoding gene of the plant yield heterosis related protein TaCCA1-7D is overexpressed, so that the biomass, the ear length and the seed length and width of wheat in the seedling stage are reduced, and the amino acid sequence of the plant yield heterosis related protein TaCCA1-7D is shown as SEQ ID NO:1 is shown.