CN112961842B - Soybean phytochrome chromophore synthesis gene GmHY2 and encoding protein and application thereof - Google Patents

Abstract

The invention relates to the technical field of molecular biology, and provides a soybean phytochrome chromophore synthesis gene GmHY2, coding proteins thereof and functions thereof, wherein the gene and the coding proteins thereof have high and low activities, can adjust leaf chlorophyll content and internode length, and can be used for adjusting plant photoperiod sensitivity, namely adjusting flowering time, solving the problem of flowering asynchronism in cross breeding, and providing germplasm resources and theoretical support for cultivating eurytopic soybean varieties.

Description

Soybean phytochrome chromophore synthesis gene GmHY2 and encoding protein and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a soybean phytochrome chromophore synthesis gene GmHY2 and an encoding protein and application thereof.

Background

The light is an important environmental factor in the growth and development of plants, and the photosynthetic efficiency, the photoperiod reaction and the like of the plants can be regulated and controlled by illumination. The photoperiod response is an important regulation link affecting plant flowering and maturation, and directly affects the yield, quality and adaptability of plants. The soybean belongs to short-day crops, namely, the soybean blooms obviously in advance under short-day conditions, the growth and development of the soybean are very sensitive to photoperiod response, the characteristic seriously influences the adaptability of soybean varieties and limits the planting range of the soybean. The soybean planted in northern China has long growth period and late maturation period, so that the change of the photoperiod reaction characteristic of the soybean is an important research direction for expanding the planting adaptability of the soybean.

In order to adapt to the change of the environment, a series of light receptors are formed in the evolution process to capture light signals, and then signal transduction reaction in vivo is initiated to regulate and control the growth and development of plants. In the photoperiod response of plants, the flowering time is controlled by first sensing a light signal through a photoreceptor, transmitting the light signal to a downstream biorhythm clock, and then activating or inhibiting the expression of downstream genes. So far, phytochromes (phytochromes) are the most widely and deeply studied class of photoreceptors, playing an important role in the photoperiod response, and the research on soybean phytochromes has focused on the phytochrome apolipoprotein. Therefore, the photoperiod sensitivity of the soybeans is reduced by changing the functions of the photosensitive pigments, and the method has important practical significance for cultivating the wide-adaptability soybean varieties.

Disclosure of Invention

In view of the above, the present invention aims to provide a soybean phytochrome chromophore synthesis gene GmHY2 and a protein P phi B synthase coded by the same, and applications of the gene GmHY2 and the protein P phi B synthase in plant photoperiod sensitivity regulation or related product preparation, wherein the regulation of the plant photoperiod sensitivity is specifically expressed by regulating flowering time early and late;

the invention also aims to provide the application of the soybean phytochrome chromophore synthetic gene GmHY2 and the protein P phi B synthase coded by the gene in regulating the chlorophyll content of plant leaves or in the preparation of related products;

the invention also aims to provide the application of the phytochrome chromophore synthesis gene GmHY2 and the protein P phi B synthase coded by the same in regulating the internode length of plants or in the preparation of related products;

it is another object of the present invention to provide methods for modulating photoperiod sensitivity of plants, methods for modulating chlorophyll content of leaves, methods for modulating internode length, or methods for modulating the three physiological characteristics described above simultaneously.

In order to achieve the above purpose, the invention provides the following technical scheme:

the sequence of the P phi B synthase for synthesizing the soybean phytochrome chromophore is shown as SEQ ID NO. 2 or an amino acid sequence which is shown as SEQ ID NO. 2 and has the same or similar functions and is substituted, deleted or added with one or more amino acids.

Meanwhile, the invention also provides a GmHY2 gene for encoding the P phi B synthase.

In a specific embodiment of the invention, the sequence of the phytochrome chromophore synthesis gene GmHY2 is shown as SEQ ID NO. 1 (whole gene sequence) or as SEQ ID NO. 3 (CDS sequence), and the sequence shown as SEQ ID NO. 3 is included as an exon by the sequence shown as SEQ ID NO. 1.

In addition, the invention also provides a biological material containing the gene, and the biological material is an expression vector, an expression cassette or a host.

The invention discovers a soybean phytochrome chromophore synthesis gene in soybean for the first time, named as GmHY2, and codes soybean P phi B (tetrapyrrole chromophore) synthetase; the research of the invention shows that the mutation of the phytochrome chromophore synthesis gene GmHY2 can reduce the soybean photoperiod sensitivity, namely, the soybean blooms in advance, and simultaneously can reduce the leaf chlorophyll content (leaf yellowing) and extend the internode; meanwhile, the vector capable of normally expressing the P phi B synthase is transferred into the GmHY2 mutant strain, so that the phenotype of the GmHY2 mutant strain is restored to be a wild type, and therefore, the soybean phytochrome chromophore synthetic gene GmHY2 and the P phi B synthase coded by the gene can be used for adjusting the photoperiod sensitivity of plants, adjusting the chlorophyll content of plant leaves and adjusting the internode length of the plants, can be used for solving the problem of flowering asynchronism in cross breeding, and can provide germplasm resources and theoretical support for cultivating a eurytopic soybean variety. The invention therefore provides the use of the P Φ B synthase, the GmHY2 gene or the biological material in any one or more of the following:

regulating plant photoperiod sensitivity, regulating plant leaf chlorophyll content, and regulating plant internode length.

According to the application of the invention, the invention also provides an inhibitor of soybean GmHY2 gene or a protein coded by the gene, wherein the inhibitor can reduce the level and/or activity of endogenous P phi B synthase in a plant, so that the inhibitor can play a role in reducing the sensitivity of soybeans to photoperiod, reducing the chlorophyll content of plant leaves and increasing the internode length of the plant, so the invention also provides the application of the inhibitor in any one or more of the following items:

reducing photoperiod sensitivity of plants, preparing products for reducing photoperiod sensitivity of plants, reducing chlorophyll content of plant leaves, preparing products for reducing chlorophyll content of plant leaves, increasing internode length of plants and preparing products for increasing internode length of plants.

The inhibitor can be selected from any one or more than two of the following items:

i) An expression vector for knocking out or knocking down the GmHY2 gene;

ii) a recombinant host comprising i);

iii) An agent (including a nucleic acid molecule agent) for inhibiting the expression of the GmHY2 gene;

v), an agent that inhibits P.PHIB synthase activity;

vi) inactivating mutation in the GmHY2 gene.

Meanwhile, the invention also provides a method for regulating the photoperiod sensitivity of the plant and/or the chlorophyll content of the plant leaves and/or the internode length of the plant, if the photoperiod sensitivity of the target plant and/or the chlorophyll content of the plant leaves and/or the internode length of the plant are/is reduced, the GmHY2 gene of the target plant or the protein P phi B synthase coded by the gene are inhibited or inactivated;

if the photoperiod sensitivity of the target plant and/or the chlorophyll content of a plant leaf and/or the internode length of the plant are to be improved or restored, the expression level of the GmHY2 gene of the target plant or the protein P phi B synthase coded by the gene is improved or restored;

the sequence of the P phi B synthase is shown as SEQ ID NO. 2 or is an amino acid sequence which is substituted, deleted or added with one or more amino acids and has the same or similar functions in the amino acid sequence shown as SEQ ID NO. 2; the sequence of the GmHY2 gene is a nucleotide sequence for coding P phi B synthase. Preferably, the method for inhibiting or inactivating the phytochrome chromophore synthesis gene GmHY2 of the target plant or the protein P phi B synthase encoded by the gene is to use the inhibitor of the invention, or any mutagenesis means for mutationally inactivating the GmHY2 gene of the target plant, such as chemical mutagenesis and physical mutagenesis means.

Preferably, the method for increasing the expression level or recovering the expression of the GmHY2 gene of the target plant or the protein P phi B synthase coded by the GmHY2 gene is to recover the overexpression or expression of the GmHY2 gene of the target plant or the protein P phi B synthase coded by the GmHY2 gene by using the biological material, or any means for increasing the expression level of the GmHY2 gene of the target plant or the protein P phi B synthase coded by the GmHY2 gene of the target plant.

The plant of the invention is preferably leguminous plant, and in the specific embodiment of the invention, soybean is used as a verification material for new functions of genes.

The photoperiod reaction is an important regulation and control link of plant flowering, and the capability of reducing photoperiod sensitivity in the invention refers to breeding improvement on the existing germplasm so as to obtain a new variety with better wide adaptability. Or the ability of applying a preparation capable of reducing a suppressor of GmHY2 gene expression or reducing the activity of GmHY2 protein to a plant so as to reduce photoperiod sensitivity.

According to the technical scheme, the soybean phytochrome chromophore synthesis gene GmHY2, the coding protein thereof and the functions of the gene and the coding protein are provided, the activity of the gene and the coding protein thereof is high and low, the chlorophyll content and the internode length of leaves can be adjusted, meanwhile, the gene and the coding protein are also used for adjusting the photoperiod sensitivity of plants, namely adjusting the flowering time, the problem of flowering asynchronism in cross breeding can be solved, and germplasm resources and theoretical support can be provided for cultivating a eurytopic soybean variety.

Drawings

The genome and the transcription sequence of the soybean GmHY2 gene are shown in figures 1-3, wherein: bold indicates exon sequences, italics indicates intron sequences, ATG indicates promoter, TGA indicates terminator;

FIG. 4 shows the difference in leaf color phenotype between wild-type Williams82 and the Gmlh1-1,2,3 mutant; * P < 0.001, and the length of the scale is 5 cm;

FIG. 5 shows the phenotypic differences in internode length and flowering time of wild type Williams82 and Gmlh1-1,2,3 mutants; * P < 0.001, length of scale 5 cm, and scale of detail drawing 1 cm;

FIG. 6 shows the expression vector pCAMBIA3301T-GmHY2p with GmHY2 sense: a GmHY2 map;

FIG. 7 shows that the phenotype and flowering time of the Gmlh1-1 mutant plant expressing the GmHY2 gene are recovered to normal; * P < 0.001, length of scale 5 cm;

FIG. 8 shows the difference in flowering-time for the wild-type Williams82 and the Gmlh1-1 mutant under different sun exposure; LD long day, SD short day.

Detailed Description

The invention discloses a phytochrome chromophore synthesis gene GmHY2 and an encoding protein and application thereof, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The genes, proteins and applications of the present invention have been described in terms of preferred embodiments, and it will be apparent to those skilled in the art that modifications or appropriate variations and combinations of the genes, proteins and applications described herein can be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The invention also provides a mutant phytochrome chromophore synthesis gene GmHY2, the sequence of which is mutated in any one or more of the following nucleic acid sequences shown in SEQ ID NO. 3:

mutation of 329 th A to G, 763 th G to A, 930 th G to deletion, 764 th G to A.

The mutant gene is used for replacing the original photochromic pigment chromophore synthesis gene GmHY2 by a molecular biological means, and plants with expected properties can be obtained.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The GmHY2 gene for coding the P phi B synthase comprises genomic DNA, cDNA, recombinant DNA or mRNA and hnRNA for coding GmHY2 protein; or a nucleic acid molecule which is reverse complementary to the above DNA, cDNA, recombinant DNA or mRNA.

The GmHY2 gene can be modified or optimized according to actual needs, so that the gene expression is more efficient; for example, (1) the codon of the GmHY2 gene of the present invention can be changed to conform to the preference of the recipient plant while maintaining the amino acid sequence thereof according to the preference of the recipient plant. (2) Or modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, the modification is carried out using a sequence known to be effective in plants. (3) Linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; (4) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

In the present invention, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The host may be a fungus, a bacterium, an algae, or a cell.

The invention is further illustrated by the following examples.

Example 1: isolation and structural analysis of GmHY2 gene in soybean

(1) Isolation of GmHY2 Gene

Total RNA was extracted from a soybean variety Williams82, and first strand cDNA was synthesized using the total RNA as a template and Oligo (T) 17 as a primer. PCR amplification was performed using the first strand of cDNA as a template, and a forward primer (5'-ATGGGTTTTAGAATTAGCGGT-3') and a reverse primer (5'-CCTAAAATATTCAATAAATTCTCCTC-3'), respectively. A990 bp cDNA fragment of the GmHY2 gene is obtained and is connected with a pEASY-Blunt vector (gold Corp. Va.) and named as Blunt-GmHY2.

The gene sequence of the obtained GmHY2 gene is shown as SEQ ID NO. 3, and the total length of CDS of the GmHY2 gene is 990bp; the amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 2, and the total number of amino acids is 329.

(2) Structural analysis of GmHY2 gene

Extracting DNA from young leaves of soybean variety Williams82, and amplifying to obtain GmHY2 genome segment with genome sequence shown in SEQ ID NO 1 and total length of 7475bp and containing 9 exons and 8 introns (as shown in FIGS. 1-3) by using the genome DNA as a template.

Example 2: separation, screening and phenotype identification of soybean GmHY2 gene mutant

With Williams82 as background, the invention constructs a soybean mutant library by EMS mutagenesis, utilizes the platform to screen 4 strains of GmHY2 protein to have nonsynonymous mutation, and respectively names the proteins as Gmlh1-1,2,3,4. The A at the 329 th site in the nucleic acid sequence shown as SEQ ID NO. 3 of the Gmlh1-1 mutant is mutated into G, the mutation causes the error shearing of a transcript, and the 110 th site in the amino acid sequence shown as SEQ ID NO. 2 of the encoded protein sequence begins to have frame shift mutation; the 763 rd G of the nucleotide sequence of the Gmlh1-2 mutant is shown as SEQ ID NO. 3 is mutated into A, and the 255 th glycine (G) of the amino acid sequence shown as SEQ ID NO. 2 is mutated into glutamic acid (E); the nucleotide sequence of the Gmlh1-3 mutant is G deletion at the 930 th position in the nucleic acid sequence shown by SEQ ID NO. 3, and the coding protein sequence of the Gmlh1-3 mutant is the 310 th position in the amino acid sequence shown by SEQ ID NO. 2 and begins to generate frame shift mutation; the 764 th G of the Gmlh1-4 mutant with the nucleotide sequence shown in SEQ ID NO. 3 is mutated into A, and the 255 th glycine (G) of the amino acid sequence shown in SEQ ID NO. 2 is mutated into glutamic acid (E). Since Gmlh1-4 and Gmlh1-2 are two adjacent base mutations and finally cause the same amino acid mutation, only three mutants of Gmlh1-1,2,3 are subjected to related verification experiments.

(1) GmHY2 gene regulated chlorophyll synthesis

Compared with wild type Williams82, the Gmlh1-1,2,3 mutant has yellowing of leaves, and the chlorophyll content of 15-day Williams82 and Gmlh1-1,2,3 leaves is detected. The pigment content of the Gmlh1-1,2,3 mutant was significantly reduced compared to Williams 82. Wherein the content of chlorophyll a in the Gmlh1-1,2,3 mutant is 51%, 70% and 74% of Williams82 respectively; chlorophyll b content was 39%, 52%, 55% of Williams82 (see fig. 4).

(2) GmHY2 gene for regulating soybean internode elongation and flowering time

Compared with wild type Williams82, the soybean Gmlh1-1,2,3 mutant has the advantages that the internodes of the main stem are elongated and the flowering time is advanced. Sowing the seeds in the test field of Jilin Changchun in month 5, counting the flowering time of the R1 stage (the first flower of any node of the plant is marked as the beginning flowering stage), the flowering time of Williams82 is 55 days, and the flowering time of the Gmlh1-1,2,3 mutant is 44-46 days, which is obviously earlier than that of the wild type. Statistics were performed on the average internode length of the mutant and wild type at the maturation stage, and the results showed that the mutant had an average internode length of 5.5-6.0cm, which was higher than that of wild type Williams82 (4.3 cm) (see FIG. 5).

Example 3 construction of sense expression vector for Soybean GmHY2 Gene

DNA of soybean Williams82 leaves is extracted, a promoter (shown as SEQ ID NO: 4) and the full length of a gene (shown as SEQ ID NO: 1) of the GmHY2 are amplified and are positively connected into a plant expression vector pCAMBIA3301T (CAMBIA research center) to obtain a GmHY2 sense expression vector, the full length of the vector is 18.8Kbp (shown as figure 6), the resistance in escherichia coli is kanamycin resistance, and the resistance in plants is glufosinate ammonium (Bar) resistance.

Example 4: agrobacterium mediated transformation of leguminous plants

In this example, an explant of a Gmlh1-1 mutant containing a sense expression vector of a GmHY2 regulatory gene was obtained by a method of transforming soybean cotyledon nodes by an agrobacterium-mediated method. In this example, the reason why the promoter and the gene of GmHY2 were introduced into the Gmlh1-1 mutant in their entire length was to complement the function of the mutated GmHY2 gene, and if the phenotype of the gene was restored to match that of the wild type, the function of the gene could be verified.

(A) Obtaining of Soybean explants

Selecting the Gmlh1-1 mutant mature soybean seeds with smooth surfaces, no damage, no scab and no crack, and sterilizing for 14h by a chlorine method. Ventilating the sterilized seeds on a super clean bench to completely volatilize chlorine, and germinating in a germination culture medium for 6h. Removing 1/2 hypocotyl from soybean, cutting soybean along the hypocotyl longitudinally, and using the remaining hypocotyl as acceptor material for Agrobacterium mediated transformation.

(B) Transformation of soybean

Adopting secondary agrobacterium infection by an agrobacterium-mediated method, and carrying out dark culture on a co-culture medium at 22 ℃ for 5d; culturing in SI-I culture medium under strong light for 7 days; cutting off big buds of the explant (Gmlh 1-1 mutant), and culturing in SI-II culture medium under strong light for 14d; cutting out cotyledon and hypocotyl of the explant, and subculturing every 14d in SE medium; shearing off about 3cm of cluster buds, and placing the cluster buds into a rooting culture medium for rooting; and (4) transferring the plant with developed root growth in the RM rooting culture medium into soil for planting. 30 resistant plants were selected by Bar resistance test. The pods started to mature after 5 months of cultivation in the greenhouse and harvest was complete after 6 months.

(C) Genetically modified plants which can be inherited

T to be harvested ₁ When seeds were planted in the greenhouse, it was observed that the transfer of wild type GmHY2 gene into Gmlh1-1 mutants (Com-GmHY 2#1 and Com-GmHY2# 2) resulted in restoration of leaf color, internode length and flowering time of transgenic plants, consistent with wild type Williams82 (see FIG. 7).

The results fully indicate that the GmHY2 gene and the protein encoded by the gene are functional genes and proteins for reducing the photoperiod sensitivity (early flowering), leaf yellowing and internode elongation of plants.

Example 5: soybean Gmlh1-1 mutant with reduced photoperiod sensitivity

To verify the biological function of the GmHY2 gene, wild type Williams82 and the Gmlh1-1 mutant were grown separately and placed in a light climatic chamber for long (16 h light/8h dark) and short (12 h light/12h dark) day treatment at 25 ℃ to record the time the first flower was open (R1) at any locus. The results show that under the short-day treatment, the flowering time of the wild type Williams82 is 29 days, and the flowering time of the Gmlh1-1 mutant is 28 days; however, under the long-day treatment, the wild type Williams82 flowering for 54 days, and the Gmlh1-1 mutant flowering for 45 days (see FIG. 8). Therefore, after the GmHY2 gene is mutated, the sensitivity of soybeans to the photoperiod is reduced, and the flowering time is obviously advanced.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> institute of geography and agroecology of northeast China academy of sciences

<120> soybean phytochrome chromophore synthesis gene GmHY2, and coding protein and application thereof

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aaatgagaga gttatatgtt aggccttttt atttgtaggc ctaaaacttg aactttagta 840

ggcttaccct ttggccgacc ctgaggaaga ggagaaaaag aaattgaatg aactgctcta 900

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gttttaatat ctcattgaaa agtgagaaca tcagtcaatt tggaattttt tcaaaggtta 1860

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ctttgcaaat cgaagtggga atcattcagt tttaaattaa aaaataagcc agcctctgat 2760

ctatgtggat atatactttc aagaggtaca cacaacatag aacttttatg tcttcatttg 2820

ggtgtggtaa atgcatatgt ttcattgcct agtaatacac accaaagtag gtgtatcaag 2880

tatcaaccag cctcagcagc aggtatttga ttgtacttgg taaaagaaaa taccatattt 2940

cattagttgt aaatttactt gcataatcac ttgaaaagga tgtttcattc ttttgtaaaa 3000

tgggttcatg tctcttttat ggtatattcc cgattgaata aaagcatgca tttccagaca 3060

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atagtagttg ttttactcca gccctttggt tttgctattt tttttctatg aacctttaaa 3240

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agattttagg attgaaagca tctttgattg actatttgaa attatgttat atataccaaa 3480

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caataaaaaa aattagatct gaagttgatg tatactttaa tttgagttta taggcttttg 3600

gatgattggt attgactctt catccaatct tagaaagaac tatttggtga agctgttata 3660

aacttataat aaatgagaaa aaaaatgcct ttctttatca gtaaatagaa actcaaggat 3720

ttaatgtgtg ttgcatgtct actaaaattt gccacctact cagctgtgga agaatattat 3780

agtgtcatcc ttttagataa tctactgtta gctttcttag gtcgcattac ttctccctca 3840

atttaattgt gtttagtcat attgtcagtg catgtgcaaa tttaatggta cttgggtgtt 3900

tatacttgtc caaaattaac ataatggaaa agaccaagct ttggcaagaa ataaagccta 3960

aagcaaatcc atgaaaaatc tgtgacagtc tgtagtgctc atttctcccc aatattgttt 4020

acaccttaaa cgtcttggaa atctaatatt tgttcaccga catcactggt aatgtgataa 4080

agtttttccc cactgtgcta tttatagctt caaaattatt taagaacaaa ttactgtatg 4140

taatgcagag cactaacctt gaattaattg tgccagggac cttaacccct tgcatgatat 4200

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atatgctgag gtaactgcaa atattttagg gaaaattata aggattattg gtagaagtat 4320

attttagaaa aagttaatca ctcgactgca taacaatgtg aaaagttttg ttaataacag 4380

ctacttattg acctaattag ccacatgagt gatgcattgt tctaaaactg gatttacttt 4440

taatatgaat tctatatttt ttacttttcc ttttagtatt gattcattga agtcctcatg 4500

gatgcatctc cgtttttctg taaattatag agattctttc ttaaaggacc tagtgagttg 4560

acttaacaca gactttatgc agctttttcc atggggaggg aagctcacaa gtgagtccat 4620

aaattttttt tcaccaattg tcatctggac aaagtttacc tcaaacccag aaaaatatga 4680

tattctgtat tccgcattta gggaatatta caaggtaata gccccagcat catgatcaac 4740

tttatgaatg acaattcata ttttgctaaa tactctctcc gttcctatat ataagacctt 4800

tgaactaatt cacactcttt aataaaattg gttaatatag ttagtgacat taaatttgtc 4860

aataatttat atttttttat aattttcctt aatgttttta aaattaatca tctctccctt 4920

tccacctaat ttcttttcgc ctaattaatt aatgtttggg gatggaataa tgattatgta 4980

atttgtggct actcatcttc tccaatccta ataagagagt ttatctcatt aatttccagc 5040

atttattttt cttggctaaa aagtttgtta attaattaag ggtattttag taaaaaaaat 5100

aatacaagag acaattagaa aagggtctta taaaaaggga caagaaattt tctgaaaatg 5160

gtcttatata taaggatgga gggagtatta atacatttag tgaactttaa taaagttgac 5220

cttgtacatg tgtaattttg ttatcttgat gtctgttacc ctggcaggta tggttgaaat 5280

tgatatgcaa agcagataaa gagacagatg aatctcagat tttccacaac ctcgaagcac 5340

aacatagata tctaacatgg agagttgaaa aggtagttca catttgctaa aattttcttt 5400

gacagccaat cataaatatg attggtgatc tagccataag caaacttata tttgtcccag 5460

gtataattgg tttaggattt tctaattgat caaggatctt cactcattca gctagcatcg 5520

gttgaagata atttagatga taatattaac aaagatggtt ctgtttgaat atgatgtctt 5580

caaaaaacca acctaacttc agttattttc tgcattcaaa tctcaacaat aaaattggca 5640

aataagtttg aaatgaagtt ttgaacttca aactatataa acaagggaca gccaatccta 5700

tcaggacttt aatttagaat gtatagggcc tgtttggata actctctaaa aaccactcta 5760

ggagagaaat agaaggaaaa atgaaaaagc ttctccataa tctatttcta attcagtttt 5820

aatggacctg gttcatggac atacttgaaa gtaggatcca ggacaaggtg ttttgaagaa 5880

gctgattggt gacacacttg ccaaggtagg ccaaatttgt gctcgattta tttaattcct 5940

ctacatccta gttccacaga tcctcacttc ctgttttata ctaggatatg ctgagaagct 6000

ttctctttaa tggagttgat gaactaggaa gcaaaacatt caatgattat tttccacgct 6060

actgctgtca agagggaact ctaaataaaa aaggcaatgt tattgggaag tcctttgaaa 6120

atcgcccatg gaatgctaga ggagaattta ttggtaacag attttcaaat tggcctgtct 6180

aattgctgct ttcttttcat ctctgcttgg cgtgtttttt atcctgctca tctgttatca 6240

ttctgtttat gtcctgcggc cttctattag ggtcttacaa gtaaaaaact atgctgtgat 6300

caatggattc tttcacctgc cttttcttct tttatattaa aattggtcct gcatatttat 6360

ctatggcatt ttgcatgtat atgttctttg attacttgaa gttgcttaac tggcagtttg 6420

atttgagatg cgagagacat tgttgaagcc agtaggactt gaattctatt attcatgatt 6480

aagaatgaga tgatttcagt aatgaaatag ttgtatcgtt tagcaagcta gcaaatattt 6540

ttgtagaagc aagcacttac aagcatacta tatgtagcac aagaaatctt cagaattata 6600

atgatatgtt ggggggtgct tgaaaaaaaa aattggactt gtgagaaagt agttcagaac 6660

ttttgtaagt tttttttttc aatttatttt aacaagattt acagagaaac tggttgaagt 6720

aagttttttt ttaacctatt tcagtaattc agttaatttt atgaacaaat ttttatttgg 6780

taggagttta ttaaataagt ggttaatgaa attctttatt taaatgcatc cttagtttat 6840

agtaattttt aaggatcctt cagctggatt ggcgttgatg tgtagctttc aggccaaggt 6900

atacttaccg cggtgctgac atttgatctt tagaatattt taggtgaatg actgtacact 6960

ggagaagctt gtgcacacgt ttgcagtcaa acttttacaa ggcgtgccac tggttatatt 7020

ttcggctata gaggttctgc aagcatacaa aagaccgctg taatttttgt agacaagtta 7080

tgccataaga tgcttgtaat atattatgga aagaattttc tgtaacaaat ctcaggctca 7140

gcacctatgg ctctacatgt ttgtaatatt aacattcgta ttgtatcagt caaaaaaaca 7200

ttcgtattgg aagattctag ttatacatta actgcagccg tgtccgatta gcttcaagtc 7260

atagtagaaa tttaagtgta atattttacc ttctgtaaaa taaagtttaa tgttttacct 7320

tttgtaaaaa aaaatgtaat attttagcat gcagtttaga ttaatttgta taacccaaaa 7380

attgtcactc gatttaattt ttgggttaaa tcaagatttt ttttaatacg attgggctct 7440

ttatttggca tgctggctaa attgggtctc tttgt 7475

<210> 2

<211> 329

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Phe Arg Ile Ser Gly Ser Ser Ser Ser Ser Cys Phe Cys Leu

1 5 10 15

Gln Arg Thr Leu Leu Pro Pro Leu Thr Ala Ile Ala Thr Ser Thr Arg

20 25 30

Gly Phe Lys Arg Arg Ser Asn Cys Ile Pro Ser Cys Ser Val Ser Tyr

35 40 45

Arg Lys Phe Val Glu Phe Ala Leu Asp Glu Thr Thr Leu His Thr His

50 55 60

Leu Ile Pro Ser Pro Leu Gln Glu Lys Tyr Asn Phe Met Asn Ser Lys

65 70 75 80

Asp Gly Lys Gly Thr Leu Ser Met Leu Ser Phe Glu Gly Ala Lys Ile

85 90 95

Arg Leu Leu Arg Ser Leu Ile Ile Glu Thr Glu Thr Met Gln Val Leu

100 105 110

Asp Phe Thr Val Phe Pro Lys Ala Glu Tyr Asp Ile Pro Ile Phe Cys

115 120 125

Ala Asn Phe Phe Thr Ser Ala Lys Thr Asn Ile Val Val Leu Asp Leu

130 135 140

Asn Pro Leu His Asp Ile Ile Asn Gln His Glu Tyr Lys Glu Lys Tyr

145 150 155 160

Phe Lys Ser Leu Ile Pro Leu Gly Leu Lys Tyr Ala Glu Leu Phe Pro

165 170 175

Trp Gly Gly Lys Leu Thr Ser Glu Ser Ile Asn Phe Phe Ser Pro Ile

180 185 190

Val Ile Trp Thr Lys Phe Thr Ser Asn Pro Glu Lys Tyr Asp Ile Leu

195 200 205

Tyr Ser Ala Phe Arg Glu Tyr Tyr Lys Val Trp Leu Lys Leu Ile Cys

210 215 220

Lys Ala Asp Lys Glu Thr Asp Glu Ser Gln Ile Phe His Asn Leu Glu

225 230 235 240

Ala Gln His Arg Tyr Leu Thr Trp Arg Val Glu Lys Asp Pro Gly Gln

245 250 255

Gly Val Leu Lys Lys Leu Ile Gly Asp Thr Leu Ala Lys Asp Met Leu

260 265 270

Arg Ser Phe Leu Phe Asn Gly Val Asp Glu Leu Gly Ser Lys Thr Phe

275 280 285

Asn Asp Tyr Phe Pro Arg Tyr Cys Cys Gln Glu Gly Thr Leu Asn Lys

290 295 300

Lys Gly Asn Val Ile Gly Lys Ser Phe Glu Asn Arg Pro Trp Asn Ala

305 310 315 320

Arg Gly Glu Phe Ile Glu Tyr Phe Arg

325

<210> 3

<211> 990

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggtttta gaattagcgg ttcctcctct tcatcatgct tttgtttgca acgtacgctt 60

cttcctccgt taacagcaat agccacttcc actcgtggct tcaagaggag gagtaattgc 120

ataccgagtt gttcagtatc ttatcgcaag tttgttgagt ttgctttgga tgaaaccaca 180

ctccacactc acttgatccc ttcgccttta caggaaaagt acaatttcat gaattccaag 240

gatggtaaag gaactcttag tatgctatca tttgaaggtg ccaaaattag gcttctacga 300

agtttgatca ttgagacaga aacaatgcag gttttggatt ttactgtctt tccaaaagca 360

gaatatgaca tacccatatt ttgtgctaac tttttcacct ctgctaaaac aaacattgtt 420

gtgttggacc ttaacccctt gcatgatatc atcaatcagc atgagtacaa ggagaagtac 480

tttaaaagct taattcctct cggccttaaa tatgctgagc tttttccatg gggagggaag 540

ctcacaagtg agtccataaa ttttttttca ccaattgtca tctggacaaa gtttacctca 600

aacccagaaa aatatgatat tctgtattcc gcatttaggg aatattacaa ggtatggttg 660

aaattgatat gcaaagcaga taaagagaca gatgaatctc agattttcca caacctcgaa 720

gcacaacata gatatctaac atggagagtt gaaaaggatc caggacaagg tgttttgaag 780

aagctgattg gtgacacact tgccaaggat atgctgagaa gctttctctt taatggagtt 840

gatgaactag gaagcaaaac attcaatgat tattttccac gctactgctg tcaagaggga 900

actctaaata aaaaaggcaa tgttattggg aagtcctttg aaaatcgccc atggaatgct 960

agaggagaat ttattgaata ttttaggtga 990

<210> 4

<211> 2957

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtattttgca tacgacgttt aagtttttaa attcgaatat agcaggattt ttattagact 60

agagtagttt acttactttt aaaattacta aactaatata gtttattttt aaaaaaaatc 120

aatatggtta agtcttgctt ttatatatta tttatttatt ttttactaaa tgccaaaaac 180

atttatgact gttcaaaatt accagacatt aattactatt ataaactgta aaaaaaattg 240

cataaaaagt ttaaaggaag ccatattcat gaagtgcgac ttacttatgt tatgtggtta 300

aattttaaaa actacatagt acactagttt agtaatttta aaaacaaaat gcacaaattt 360

ggtaaaaaga caaaaacaaa gatcgccacc ggagatcatg cctattttgc tctgaagtac 420

gtccaattgg gtatctctct ttgtgtatta ctcctttttt tcttttgtct aaaagaataa 480

actaataaag tattgtgtgg ttattaatgg tgtatcaata ttatatatac aaggttataa 540

ttaattagtg tcagagaaca gatattatag cataataatg atttgtgatc aattagcact 600

agtatctagt gataaaagct ttaaacaaca tccacagaac aagcaaacaa gcaagtaaaa 660

ggtaaacaga aagatgaggg ggaaagcaaa caatgcaatg acagccaggg ctctaaatca 720

acgctatctc ctatctactc tcttggctag aagtgggtaa ttgtggaatc cgatctttat 780

ttgaaaattt agctaaatgg gttgtgtttt tcttcttgtg tctttgcttt tgcatgggat 840

tgaggactaa tttattaatt gctcattatt cgttcccaat gatcaagggg agggaagaaa 900

cttcctcaat ctggacacta aattcctagc ctgctatcac aaatcttggg tacgtgagtg 960

agaagctagc tctgacaagg catctttaaa acactgatgt taagtataaa aacaaataac 1020

tatgtggctc agaggatgcc aacaagtcat aacaagcaca agggaagaag ttacattaag 1080

tggatccaaa attaaacttt gttaaccata aaattattaa ttcattagtg ttaaattgtc 1140

ccacggtaaa attataactg gataagcatt caattcccaa gctgtccaaa agagagtcag 1200

agttgttttg cattgcagct tattacatac taatgtatgc actagtttat ggcagtttta 1260

actttaagcc ctaatcattc aagctgatgg aaagtggaca aaaaactgtt ttgtgcgaat 1320

aaaaactgca ccaacagaaa gggcagtgcc gggacattat tatatgaata aaaattgtgt 1380

aggcaagggg ctgatatatg ctaggaaatt accaactaag agaagattta cttgaattat 1440

ttatgtctta ttttcatata tttttaaaat atgtattagg ttaattttga tgtgcatcaa 1500

aaagatcaaa taatttcggt ttgatctata tgataaaatt ttttaattca attataagtt 1560

ttgagttgaa agtattcaat aaaatgtcat taaataatgt aaaaataggt ttaactaatt 1620

tgagtcaata ggaaaaagaa gagggtaatt tgcaaagcat gaacctgagc aaattttagg 1680

acttgtcacg ttagtccgag atctattaag gtaagaggag aatctcttac ctgttctcgc 1740

gtctaaaatt gggtcatagt ttattccgcg aattgtatat tttatccttg ttttttgaaa 1800

atttttatct taataaatta atttaactta ttttattttt taatttttaa aaaacacatt 1860

caaatttaac ttttgtgtta gttgagttaa catgacacat taaaataatt acactttttt 1920

attctttagc aaatcttacc ttcaaatttt tctacttttt atgaatttta acccaaattt 1980

tttacattta tattagcttg tcacgtcaac tcaacaatta gaattcttgg tgccaatttc 2040

tacccctttg tgagtgtttt gtctattcca gccagtcaac aaactttatt atttataatc 2100

atataatgat aaagagtaaa ctaacgttag aatttttctt cctctgattc taactccaca 2160

cacacagcac actcactcag cacactcatt tgccatcatt aactaattaa aatctccgga 2220

tgtagtagga agattgagat tagttacttg ttcacgtttg gattttcatt cacacaaatt 2280

ggatgtacat ctgagaaata actagaaatg cgtgtttgga atttctttaa gagaaaatga 2340

attatgttta gattgtgaaa gttataatcg ttagcttttg acatttgaaa cttggaaaat 2400

cacatctaag aaaccaacaa actagctttg atacgttttg atgtaaaatt tggtaaaaca 2460

tggatgagtg attaggatcg aaacatgcac ttaatttgaa ttgtgatagt gataaggaaa 2520

gaaagaaatc acaatatttt gtatttgcac acacttgcac tagttatatt aattattttt 2580

gtcttttttc ctcttatctc ttttaaggta taaacacatt ttaatttaaa gtctctcggt 2640

atatttcttt aaataataat ttgttataac aaatttttgt taaaaatttt actagcaacg 2700

tcacataaat tgatataatg aatacttggt accaatatta ttatgatgga aagttaataa 2760

tgattggtaa ttagttttat tcatttggta attgaatttg aaaagtgaaa ggctactgaa 2820

tagtgtatga gagacaggag ggtaatttgg actttggagg cagctgcatt tgggccatat 2880

gaagaggccc ataggctgtt aatatacgtg aagggtggac cacacagttg agaaatccaa 2940

agcatcctaa aagtaga 2957

Claims (5)

1. A method for modulating photoperiod sensitivity and/or chlorophyll content of plant leaves and/or internode length of plants, characterized in that if the photoperiod sensitivity and/or chlorophyll content of plant leaves and/or internode length of plants are to be reduced, the target plants are treatedGmHY2The gene or the protein P phi B synthase coded by the gene is inhibited or inactivated;

if the photoperiod sensitivity and/or the chlorophyll content of plant leaves and/or the internode length of plants are to be improved or restored, the target plants are made to beGmHY2The gene or the protein P phi B synthase coded by the gene improves the expression quantity or restores the expression;

the sequence of the P phi B synthase is shown as SEQ ID NO. 2; the above-mentionedGmHY2The sequence of the gene is a nucleotide sequence for coding P phi B synthase;

the plant is soybean.

2. The method of claim 1, wherein the plant is grownGmHY2The gene or the coded protein P phi B synthase is inhibited or inactivated byGmHY2Inhibitors of genes or their encoded proteins, or any plant of interestGmHY2Mutagenesis means for inactivating gene mutation.

3. The method of claim 1, wherein the plant is grownGmHY2The method for increasing expression quantity or recovering expression of gene or its coded protein P phi B synthase is characterized by that it adopts the gene containingGmHY2Biological material of gene and target plantGmHY2Overexpression or restoration of expression of the gene or its encoded protein PhiB synthase, or any increase in the target plantsGmHY2A gene or its encoded protein P phi B synthase expression level.

4. Mutant soybean photosensitive pigment chromophore synthesis geneGmHY2The sequence is mutated in the nucleic acid sequence shown in SEQ ID NO. 3 as follows:

mutation of 329 th A to G, 763 th G to A, 930 th G to deletion, 764 th G to A.

5. The soybean phytochrome chromophore synthesis gene mutated as claimed in claim 4GmHY2The encoded protein.

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