SiDTH2 protein and its encoding gene for increasing crop biomass and extending its growth period

Abstract

The present invention discloses a SiDTH2 protein and a gene encoding the same for increasing crop biomass and extending its growth period. The sequence of the SiDTH2 protein disclosed in the present invention is sequence 1 in the sequence list, and the sequence of the encoding gene is sequence 2 in the sequence list. Experiments have shown that compared with the wild type, the plant height, ear length, number of stem nodes and heading period of the transgenic plants transformed with the SiDTH2 gene are significantly increased, indicating that the SiDTH2 gene and the protein encoded by the same can increase plant biomass, extend its growth period, and produce taller plants. The SiDTH2 gene and the protein encoded by the present invention have good application prospects.

Description

Technical Field

[0001] The invention relates to SiDTH2 protein and its coding gene in the field of biotechnology, which are used for increasing crop biomass and extending its growth period. Background Art

[0002] The regulation of plant responses to photoperiod plays an extremely important role in crop domestication and spread and migration. Changes in photoperiod length play a decisive role in plant development. It controls plant germination, growth, flowering period and other yield-related traits. Changes in plant flowering time are closely related to geographical latitude. When plants migrate to different latitudes, they often need to use different signal mechanisms to induce flowering due to differences in light length and temperature between latitudes to adapt to changes in different growth environments. Plants have their own internal biological clocks that can respond to day length fluctuations and circadian rhythms, thereby sensing seasonal changes to regulate flowering. According to the plant's flowering requirements for day length (24-hour photoperiod), plants can be divided into long-day (LD), short-day (SD) and day-neutral plants. When the photoperiod required for flowering exceeds the critical day length, it is a long-day plant, and when the photoperiod required for flowering is shorter than the critical day length, it is a short-day plant, while the flowering of day-neutral plants has nothing to do with day length.

[0003] The heading period is an important developmental transition process for plants from the growth stage to the reproductive stage, and is an important factor for crops to adapt to different ecological zones and ensure yield. The change in heading period is determined by multiple genes and environmental factors, among which the photoperiod plays a major role. Crops can adapt to a wider range of growth environments by reducing their sensitivity to the photoperiod through changes in themselves. An important goal of crop breeding is to cultivate varieties whose flowering is not sensitive to the photoperiod, thereby increasing the utilization rate of improved varieties.

[0004] Millet has a cultivation history of more than 10,000 years in my country. It is one of the main food crops in northern my country. It plays an important role in dryland agriculture and people's daily diet. It is an important food crop in arid and semi-arid areas of the world. Although millet is an important cereal crop, its genetic diversity and trait inheritance mechanism have not been well studied.

[0005] Millet is a short-day crop. Usually, long-day conditions have a significant inhibitory effect on the heading period of millet. The typical phenotype is that many millet varieties perform well in mid-latitudes, but have difficulty heading when introduced to the high-latitudes in the north; even if they can barely head after a long number of days of growth, the growth period is too long, and they cannot mature normally in the cold high-latitude areas in the north due to insufficient accumulated temperature. This should be mainly caused by the long daylight in the high-latitudes in the north inhibiting certain key genes in the millet heading regulation pathway. However, in the natural evolution and artificial breeding of millet, some millet varieties in the high-latitudes of the north can still grow normally and complete their entire life history. The molecular mechanism for the huge differences in millet heading traits at high latitudes is still unclear. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to increase the biomass of plants and prolong their growth period.

[0007] In order to solve the above technical problems, the present invention first provides any of the following applications of a protein or a substance for regulating the activity or content of the protein:

[0008] D1) Regulate plant biomass and growth period;

[0009] D2) preparing products for regulating plant biomass and growth period;

[0010] D3) Increase plant biomass and extend plant growth period;

[0011] D4) preparing products for increasing plant biomass and extending plant growth period;

[0012] D5) Regulate plant growth period;

[0013] D6) preparing products for regulating plant growth period;

[0014] D7) Extend the plant growth period;

[0015] D8) preparing products for prolonging plant growth period;

[0016] The protein is derived from millet and is named SiDTH2. SiDTH2 is as follows A1), A2) or A3):

[0017] A1) a protein whose amino acid sequence is sequence 1;

[0018] A2) a protein having the same function as the amino acid sequence shown in SEQ ID NO: 1 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0019] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0020] In order to facilitate purification of the protein in A1), a tag as shown in the following table may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.

[0021] Table: Sequence of tags

[0022] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL

[0023] The protein in A2) is a protein having 75% or more identity with the amino acid sequence of the protein shown in SEQ ID NO: 1 and having the same function. The 75% or more identity is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0024] The protein in A2) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0025] The coding gene of the protein in A2) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in sequence 2, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in the above table to its 5′ end and / or 3′ end. Among them, the DNA molecule shown in sequence 2 encodes the protein shown in sequence 1.

[0026] The present invention also provides any of the following applications of SiDTH2-related biomaterials:

[0027] D1) Regulate plant biomass and growth period;

[0028] D2) preparing products for regulating plant biomass and growth period;

[0029] D3) Increase plant biomass and extend plant growth period;

[0030] D4) preparing products for increasing plant biomass and extending plant growth period;

[0031] D5) Regulate plant growth period;

[0032] D6) preparing products for regulating plant growth period;

[0033] D7) Extend the plant growth period;

[0034] D8) preparing products for prolonging plant growth period;

[0035] The biological material is any one of the following B1) to B7):

[0036] B1) Nucleic acid molecule encoding SiDTH2;

[0037] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0038] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0039] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0040] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0041] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0042] B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

[0043] In the above application, the nucleic acid molecule in B1) may be the following b11) or b12) or b13) or b14) or b15):

[0044] b11) The coding sequence is a cDNA molecule or a DNA molecule of sequence 2 in the sequence list;

[0045] b12) cDNA molecules or DNA molecules shown in Sequence 2 in the sequence listing;

[0046] b13) a cDNA molecule or a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b11) or b12) and encodes SiDTH2;

[0047] b14) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) and encodes SiDTH2.

[0048] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0049] A person skilled in the art can easily mutate the nucleotide sequence encoding the SiDTH2 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the SiDTH2 protein isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the SiDTH2 protein and have the function of the SiDTH2 protein.

[0050] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 73% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 1 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0051] In the above application, the stringent conditions may be as follows: 50°C, in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 2×SSC, 0.1% SDS at 50°C; or rinse in 7% SDS, 0.5M NaPO at 50°C. 4 Hybridize in a mixed solution of 1 mM EDTA and rinse in 1×SSC, 0.1% SDS at 50°C; or rinse in 7% SDS, 0.5 M NaPO at 50°C. 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 0.5×SSC, 0.1% SDS at 50°C; or rinse in 7% SDS, 0.5M NaPO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 0.1×SSC, 0.1% SDS at 50°C; or rinse in 7% SDS, 0.5M NaPO 4The membrane can be hybridized in a mixed solution of 1mM EDTA and rinsed in 0.1×SSC, 0.1% SDS at 65°C. It can also be hybridized in a solution of 6×SSC, 0.5% SDS at 65°C, and then washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS. It can also be hybridized in a solution of 2×SSC, 0.1% SDS at 68°C and washed twice for 5 minutes each time, and then hybridized in a solution of 0.5×SSC, 0.1% SDS at 68°C and washed twice for 15 minutes each time. It can also be hybridized and washed in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS at 65°C.

[0052] The aforementioned 73% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0053] In the above application, the expression cassette (SiDTH2 gene expression cassette) containing the nucleic acid molecule encoding the SiDTH2 protein described in B2) refers to a DNA capable of expressing the SiDTH2 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the SiDTH2 gene, but also a terminator for terminating transcription of the SiDTH2 gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of the cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Pat. No. 5,187,267); tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al. (1996). 985)Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0054] The recombinant vector containing the SiDTH2 gene expression cassette can be constructed with an existing expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301 or pCAMBIA1391-Xb (CAMBIA company), etc. The plant expression vector can also include the 3' end non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the non-translated region transcribed at the 3' end of the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0055] In the above application, the vector may be a plasmid, a cosmid, a phage or a viral vector. Specifically, the plasmid may be pCUbi1390.

[0056] B3) The recombinant vector may specifically be OX-SiDTH2. The OX-SiDTH2 is a recombinant vector obtained by replacing the DNA fragment between the KpnI and BamHI recognition sequences of pCUbi1390 with the SiDTH2 gene shown in Sequence 2 in the sequence table. The OX-SiDTH2 can express the SiDTH2 protein shown in Sequence 1 in the sequence table, and the expression of the SiDTH2 gene is driven by the 35S promoter.

[0057] In the above application, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes EHA105.

[0058] In the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

[0059] The present invention also provides any of the following methods:

[0060] X1) A method for cultivating plants with increased biomass and prolonged growth period, comprising expressing SiDTH2 in a recipient plant, or increasing the content or activity of SiDTH2 in the recipient plant, to obtain a target plant with increased biomass and prolonged growth period compared with the recipient plant;

[0061] X2) A method for increasing plant biomass and extending plant growth period, comprising expressing SiDTH2 in a recipient plant, or increasing the content or activity of SiDTH2 in the recipient plant, to obtain a target plant having increased biomass and extended growth period compared with the recipient plant, thereby achieving an increase in plant biomass and an extension of growth period;

[0062] X3) A method for cultivating a plant with an extended growth period, comprising expressing SiDTH2 in a recipient plant, or increasing the content or activity of SiDTH2 in the recipient plant, to obtain a target plant with an extended growth period compared with the recipient plant;

[0063] X4) A method for extending the growth period of a plant, comprising expressing SiDTH2 in a recipient plant, or increasing the content or activity of SiDTH2 in the recipient plant, to obtain a target plant having a longer growth period than that of the recipient plant, thereby achieving an extension of the growth period of the plant.

[0064] In the above methods, methods X1) to X4) can be achieved by introducing a gene encoding SiDTH2 into the recipient plant and expressing the gene encoding SiDTH2.

[0065] In the above method, the coding gene may be the nucleic acid molecule described in B1).

[0066] In the above method, the coding gene of SiDTH2 can be modified as follows before being introduced into the recipient plant to achieve better expression effect:

[0067] 1) Modification and optimization according to actual needs to achieve efficient gene expression; for example, the codons of the coding gene of SiDTH2 of the present invention can be changed to conform to the plant preference while maintaining the amino acid sequence of the coding gene of SiDTH2 of the present invention according to the codons preferred by the recipient plant; during the optimization process, it is best to maintain a certain GC content in the optimized coding sequence to best achieve high-level expression of the introduced gene in the plant, wherein the GC content can be 35%, more than 45%, more than 50% or more than about 60%;

[0068] 2) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, using a sequence known to be effective in plants;

[0069] 3) Connected to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression, and also depends on the target species; for example, a tissue or organ-specific expression promoter, depending on the stage of development at which the receptor is required; although it has been demonstrated that many promoters derived from dicotyledons can function in monocotyledons and vice versa, ideally, dicotyledon promoters are selected for expression in dicotyledons, and monocotyledon promoters are selected for expression in monocotyledons;

[0070] 4) Connecting with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to work in plants can be connected with the gene of the present invention;

[0071] 5) Introduction of enhancer sequences, such as intron sequences (e.g. from Adhl and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).

[0072] The coding gene of SiDTH2 can be introduced into a recipient plant using a recombinant expression vector containing the coding gene of SiDTH2. The recombinant expression vector can specifically be the OX-SiDTH2.

[0073] The recombinant expression vector can be introduced into plant cells by conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).).

[0074] The target plant is understood to include not only the first generation plant in which the SiDTH2 protein or its encoding gene is changed, but also its progeny. For the target plant, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus, complete plants and cells.

[0075] The present invention also provides a product having any of the following functions D1)-D4), wherein the product contains SiDTH2 or the biological material:

[0076] D1) Regulate plant biomass and growth period;

[0077] D2) Increase plant biomass and extend plant growth period;

[0078] D3) Regulate the growth period of plants;

[0079] D4) Prolong the plant growth period.

[0080] In the above, the plant may be M1) or M2) or M3):

[0081] M1) monocots or dicots;

[0082] M2) Gramineae;

[0083] M3) Millet or rice.

[0084] In the above, the biomass can be reflected in plant height, ear length and / or number of stem nodes. The growth period can be reflected in heading period.

[0085] SiDTH2 or the biological material also falls within the protection scope of the present invention.

[0086] Experiments have shown that compared with the wild type, the plant height, ear length, number of stem nodes and heading period of transgenic plants transformed with the SiDTH2 gene were significantly increased, indicating that the SiDTH2 gene and the protein it encodes can increase plant biomass, extend its growth period, and produce taller plants, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The relative expression level of SiDTH2 gene was detected. WT, OX1, OX5, and OX6 represent foxtail millet Ci846, Line 1, Line 5, and Line 6, respectively.

[0088] Figure 2 These are the statistical results of plant height, ear length, number of stem nodes and heading period of different strains. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0090] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were set up for three repeated experiments, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0091] Example 1: DTH2 can regulate plant height and heading period of crops

[0092] The present invention discovered a protein related to plant height derived from Jingu 21, named SiDTH2, whose sequence is sequence 1 in the sequence list, and whose CDS sequence in Jingu 21 is sequence 2 in the sequence list. The DNA fragment shown in sequence 2 is recorded as SiDTH2 gene.

[0093] 1. Construction of recombinant vector

[0094] 1. Transgenic vector: pCUbi1390 (can be obtained from the BioVector Plasmid Vector Bacteria Cell Gene Collection Center, http: / / www.biovector.net / product / 305997.html , item number: pcubi1390).

[0095] 2. Amplification of SiDTH2 gene coding region

[0096] Total RNA was extracted from the leaves of Jingu 21, and reverse transcribed to obtain cDNA. The obtained cDNA was used as a template and PCR amplification was performed using the following amplification primers to obtain PCR products. Amplification primers:

[0097] F: 5′-TTACTTCTGCACTAGGTACCATGGGAGGTACCCATCAGCAACCG-3′ (the bold part is the homologous segment to the vector)

[0098] R: 5′-GAATTCCCGGGGATCCTCATCTATCTCCAGCTCCTTCCCA-3′ (the bold part is the homologous segment to the vector).

[0099] 3. Construction of recombinant vector

[0100] 1) Linearized vector: The vector pCUbi1390 was double-digested with KpnI and BamHI, and the linearized vector backbone was recovered by purification using 1% agarose gel.

[0101] 2) Vector ligation: The linearized vector backbone was ligated with the PCR product obtained in step 2 using the pEASY-uni seamless cloning and assembly kit (Beijing Quanshijin Biotechnology Co., Ltd.), and the obtained recombinant vector with the correct sequence was recorded as OX-SiDTH2.

[0102] OX-SiDTH2 is a recombinant vector obtained by replacing the DNA fragment between the KpnI and BamHI recognition sequences of pCUbi1390 with the SiDTH2 gene shown in sequence 2 in the sequence table. The recombinant vector can express the SiDTH2 protein shown in sequence 1 in the sequence table. The expression of the SiDTH2 gene is driven by the 35S promoter.

[0103] 4. Transformation of Agrobacterium: Introduce OX-SiDTH2 into Agrobacterium EHA105 to obtain the recombinant bacterium EHA105 / OX-SiDTH2.

[0104] 2. Genetic transformation and phenotypic identification of millet

[0105] Millet Ci846 was used as the recipient plant and the Agrobacterium-mediated genetic transformation method was used to transform millet Ci846 using the recombinant bacteria EHA105 / OX-SiDTH2 obtained in step 1, and 24 independent transformation events were obtained.

[0106] The T1 transgenic seedlings were planted in a greenhouse, and 8 transgenic plants were selected to detect the relative expression of the SiDTH2 gene in the leaves using quantitative PCR. The results showed that the expression of the SiDTH2 gene in 6 different transformation events increased significantly. The transformed plants with increased expression were self-pollinated to obtain T2 seeds, of which three lines were Line 1, Line 5 and Line 6 ( Figure 1 ). The cells with no significant change in SiDTH2 gene expression were selected as negative controls.

[0107] The primers used for quantitative PCR were: 5'-TGATGTCACCCTCAGCTATTAAGG-3' and 5'-ATTCTCCCGGGACACATCAAAAT-3'; SiACTIN gene was used as an internal reference, and the internal reference primers were 5'-TATCGTTCAAACAGATTTACGGCCT-3' and 5'-TAGAGAAGAAGTGACGAAGCCTTG-3'.

[0108] The T2 seeds of Line 1, Line 5 and Line 6 were sown in the field in early May, with no less than 30 plants in each line. Foxtail millet Ci846 (WT) and negative control were used as controls to investigate phenotypic data such as heading date, plant height, number of stem nodes and ear length.

[0109] The heading period refers to the number of days from the second day after seedling to the day when half of the plants in the family head.

[0110] Plant height refers to the length from the base of the main stem to the base of the ear at maturity.

[0111] The number of nodes is the number of internodes from the first elongation node at the base of the main stem to the panicle node at maturity.

[0112] Ear length is the length of the main stem ear from the first yard at the base to the tip at maturity.

[0113] Some results are shown in Table 1. Figure 2 As shown. The results showed that there were no significant differences in plant height, ear length, number of stem nodes and heading period between millet Ci846 and the negative control; for plant height, the plant heights of Line 1, Line 5 and Line 6 were significantly higher than that of millet Ci846; for ear length, the ear lengths of Line 1, Line 5 and Line 6 were significantly higher than that of millet Ci846; for number of stem nodes, the number of stem nodes of Line1, Line 5 and Line 6 were significantly higher than that of millet Ci846; for heading period, the heading period of Line1, Line 5 and Line 6 was significantly higher than that of millet Ci846. This indicates that the SiDTH2 gene and the SiDTH2 protein it encodes can simultaneously increase the plant height, ear length and number of stem nodes of millet, and can also extend its heading period.

[0114] Table 1. Phenotypic test results

[0115] Plant Plant height(cm) Ear length (cm) Number of stem nodes Heading period (days) WT 138.57±7.25 7.09±0.70 12.00±0.82 53.71±2.69 Line 1 158.08±9.20* 7.53±0.77* 13.92±1.56* 63.00±3.05* Line 5 161.75±7.80* 7.35±0.72* 14.25±0.96* 61.25±1.71* Line 6 157.80±7.19* 7.62±0.64* 14.00±1.00* 63.80±2.77*

[0116] In Table 1, * indicates a significant difference compared with WT, p < 0.05.

[0117] 3. Construction and phenotypic identification of transgenic rice

[0118] Rice Kitaake was used as the recipient plant, and the Agrobacterium-mediated genetic transformation method was used to transform rice Kitaake using the recombinant bacteria EHA105 / OX-SiDTH2 obtained in step 1 to obtain 128 transgenic strains.

[0119] The relative expression level of the SiDTH2 gene in the transgenic lines was detected by the quantitative PCR method in step 2, and the SiDTH2 gene overexpression lines with significantly increased SiDTH2 gene expression compared with rice Kitaake were selected, and the heading date and plant height of the T0 generation overexpression lines were counted.

[0120] The heading period refers to the number of days from the second day after seedling to the day when half of the plants in the family head. The plant height refers to the length from the base of the main stem to the base of the ear at maturity.

[0121] The results showed that the heading period of rice Kitaake was 53 days, and the heading period of SiDTH2 gene overexpression strain was 58 days, which was significantly different from the two. The average plant height of rice Kitaake was 53 cm, and the plant height of SiDTH2 gene overexpression strain was 62 cm, which was significantly different from the two. That is, the SiDTH2 gene overexpression strain showed a phenotype of late heading and increased plant height. This shows that the SiDTH2 gene and the SiDTH2 protein it encodes can increase the plant height of rice and extend its heading period at the same time.

[0122] Transgenic experiments on millet and rice have shown that the SiDTH2 gene and the SiDTH2 protein it encodes can extend the growing period of crops, increase biomass, and produce taller plants.

[0123] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. <110> Beijing Agricultural Biotechnology Research Center <120> SiDTH2 protein and its encoding gene for increasing crop biomass and extending its growth period <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 755 <212> PRT <213> Millet (Setaria italica) <400> 1 Met Gly Gly Thr His Gln Gln Pro Gly Val Asp Gly Gln Ser Val Arg 1 5 10 15 Arg Asp Ala Arg Leu Leu Gly Asn Ser Ala Val Asp Val Ala Glu Gly 20 25 30 Asn Gly Leu Gln Gln Gly Asp Ser Asp Leu Arg Ser Ser Asp Pro Asp 35 40 45 Gly Leu Leu Ser Gly Pro Thr Thr Gly Leu Gln Gln Gly Asp Thr Glu 50 55 60 Asn Gln Gln Gln Gln Gln Val Cys Trp Glu Arg Phe Leu Gln Lys Lys 65 70 75 80 Thr Ile Lys Val Leu Leu Val Glu Ser Asp Asp Ser Thr Arg Gln Val 85 90 95 Val Ser Ala Leu Leu Arg His Cys Met Tyr Glu Val Ile Pro Ala Glu 100 105 110 Asn Gly Gln Gln Ala Trp Ser Tyr Leu Glu Asp Met Gln Asn Ser Ile 115 120 125 Asp Leu Val Leu Ala Glu Val Phe Met Pro Gly Val Ser Gly Ile Ser 130 135 140 Leu Leu Ser Arg Ile Met Ser His Asn Ile Cys Lys Asn Ile Pro Val 145 150 155 160 Ile Met Met Ser Ser Asn Asp Ala Met Gly Thr Val Phe Lys Cys Leu 165 170 175 Ser Lys Gly Ala Val Asp Phe Phe Val Lys Pro Ile Arg Lys Asn Glu 180 185 190 Leu Lys Asn Leu Trp Gln His Val Trp Arg Arg Cys His Ser Ser Ser 195 200 205 Gly Ser Gly Ser Glu Ser Gly Ile Gln Arg Gln Lys Cys Ala Lys Ser 210 215 220 Lys Ser Gly Asp Glu Ser Asp Asn Ser Gly Ser Asn Asp Asn Asp Glu 225 230 235 240 Asp Asp Glu Ala Ser Met Gly Leu Asn Ala Arg Asp Gly Ser Asp Asn 245 250 255 Gly Ser Gly Thr Gln Ala Arg Ser Ser Trp Thr Lys Arg Ala Val Glu 260 265 270 Ile Asp Ser Pro Gln Ala Met Ser Pro Asp Gln Leu Ala Asp Pro Pro 275 280 285 Asp Ser Thr Cys Ala Gln Val Val His Pro Lys Ser Glu Ile Cys Ser 290 295 300 Asn Arg Trp Leu Pro Gly Thr Asn Asn Arg Asn Cys Lys Lys Gln Lys 305 310 315 320 Asp Thr Asn Asp Asp Phe Lys Glu Lys Asp Leu Asp Ile Gly Ala Pro 325 330 335 Arg Lys Leu Asn Thr Asp Ser Gln Cys Ser Pro Asn Glu Arg Pro Met 340 345 350 Lys Pro Ala Asp Gly Arg Arg Glu Tyr Pro Pro Glu Asn Asn Ser Asn 355 360 365 Glu Ser Met Met Glu Asn Leu Glu Glu Ala Thr Val Arg Ala Ala Asp 370 375 380 Leu Ile Gly Ser Met Ala Lys Asn Met Asp Ala Gln Gln Ala Ala Arg 385 390 395 400 Gly Ala Asp Ala Pro Asn Cys Ser Ser Lys Val Pro Glu Gly Lys Asp 405 410 415 Met Asn Arg Gly Asn Val Leu Pro Leu Leu Glu Leu Ser Leu Lys Arg 420 425 430 Ser Arg Ser Ser Ala Asp Gly Ala Asn Thr Ile Gln Asp Glu Gln Arg 435 440 445 Asn Val Leu Arg Arg Ser Asp Pro Ser Ala Phe Thr Arg Tyr His Thr 450 455 460 Ser Ala Val Ser Asn Gln Gly Gly Thr Gly Phe Val Gly Ser Cys Ser 465 470 475 480 Pro His Asp Asn Ser Ser Glu Ala Met Lys Thr Asp Ser Thr Tyr Asn 485 490 495 Met Lys Ser Asn Ser Asp Ala Ala Met Ile Lys Gln Gly Ser Asn Gly 500 505 510 Ser Ser Asn Asn Asn Asp Met Gly Ser Thr Thr Lys Asn Val Val Thr 515 520 525 Lys Pro Thr Thr Asn Lys Glu Arg Val Met Ser Pro Ser Ala Ile Lys 530 535 540 Ala Asn Ala His Thr Ser Ala Phe His Pro Val Gln His Trp Thr Val 545 550 555 560 Pro Ala Asn Ala Ala Gly Lys Ala Lys Ala Asp Glu Met Val Asn Asn 565 570 575 Ala Thr Lys Asn Gly His Pro Gly Glu Val Gln Ser Asn Leu Val Gln 580 585 590 His Pro Arg Pro Ile Leu Tyr Val His Phe Asp Val Ser Arg Glu Asn 595 600 605 Gly Gly Ser Gly Ala Pro Gln Cys Gly Ser Ser Asn Val Phe Asp Pro 610 615 620 Pro Leu Glu Gly Gln Ala Ala Asn Tyr Gly Met His Gly Ser Asn Ser 625 630 635 640 Gly Ser Asn Asn Gly Thr Asn Gly Gln Asn Arg Ser Thr Ala Ala Ala 645 650 655 Asn Ala Glu Arg Thr Asn Thr Glu Ile Ala Asn Gly Ala Ile Asn Lys 660 665 670 Ser Gly Pro Gly Gly Gly Asn Gly Ser Gly Ser Val Ser Gly Asn Asp 675 680 685 Thr Tyr Val Lys Arg Met Ala Pro Thr Pro Arg Glu Ala Gln Leu Met 690 695 700 Lys Tyr Arg Glu Lys Lys Lys Asp Arg Asn Phe Gly Lys Lys Val Arg 705 710 715 720 Tyr Gln Ser Arg Lys Arg Leu Ala Asp Gln Arg Pro Arg Val Arg Gly 725 730 735 Gln Phe Val Lys Gln Ala Val Gln Asp Gln Gly Gly Trp Glu Gly Ala 740 745 750 Gly Asp Arg 755 <210> 2 <211> 2268 <212> DNA <213> Foxtail millet (Setaria italica) <400> 2 atgggaggta cccatcagca accgggtgtg gatggacaat ccgttagaag agatgccaga 60 ctgcttggga acagcgcggt ggatgtggct gagggcaatg ggctccagca aggggacagc 120 gacttgagga gcagtgaccc tgatggcctg ctcagcgggc caaccactgg tctgcagcag 180 ggtgacacgg aaaatcagca gcagcaacag gtctgctggg agcgcttcct ccagaagaag 240 actatcaaag tcttgctcgt ggagagtgat gattcaacca ggcaggtggt cagtgccctg 300 ctacgtcact gcatgtacga agttatccct gccgaaaatg gtcagcaagc atggagttat 360 cttgaagata tgcaaaacag catagatctt gttttggctg aggtttttat gcctggtgtt 420 tctggaattt ctctgctaag taggatcatg agccacaata tttgcaagaa tattccagtg 480 attatgatgt cttcgaatga tgctatgggt actgtcttta aatgtttgtc aaaaggtgct 540 gttgactttt ttgtcaagcc tatacgtaag aatgaactta agaacctttg gcagcatgtg 600 tggagacggt gccacagctc cagtggtagt ggaagtgaaa gtggcattca gagacagaag 660 tgtgccaaat caaaaagtgg tgatgaatct gataacagtg gtagcaatga caacgacgag 720 gacgatgagg caagcatggg acttaatgca agggatggca gtgataatgg cagtggcact 780 caagcgcgga gctcgtggac taagcgtgct gtggagatcg acagtccaca ggctatgtct 840 ccagatcagt tagctgatcc accagatagc acatgtgcac aagtggtcca cccaaaatca 900 gagatatgta gcaacagatg gctaccaggt acaaacaaca gaaactgcaa gaagcaaaaa 960 gacactaatg atgactttaa ggagaaggac ttggatatag gtgcccctag aaaattaaat 1020 acggatagtc aatgttcccc aaatgagagg cccatgaagc cagcagatgg tcgtcgcgag 1080 tacccaccag aaaacaattc caatgagtca atgatggaaa atttagagga ggctactgtt 1140 cgagctgctg atctgattgg ttcaatggcc aaaaacatgg atgcccagca ggcagcaaga 1200 ggtgcagatg cccctaattg ctcttccaaa gtgccagaag ggaaagatat gaaccgtggt 1260 aatgttttgc cattacttga attgagttg agaggtca gatcatctgc agatgtgcc 1320 atacaatcc aagatgaca gcggaatgta ttaagacgat cagatccctc agcattcaca 1380 aggtaccata catctgcggt ttccaatcaa ggcgggacag gatttgtggg gagctgttca 1440 ccgcatgata acagctctga ggctatgaa acggactcta cttacacat gaagtcaat 1500 tcagatgctg ctatgataaaaaggctcc aatggcagta gcaatacaa tgacatgggt 1560 tccactaca agaatgttgt vakaaaacct actacaata aggagagt gatgtcaccc 1620 tcagctatta aggctaatgc acacacatca gcattcatc ctgtgcagca ctggacggtt 1680 ccagctaatg ctgcaggaaa agcaaggct gatgaatgg tcacaatgc actaagaat 1740 gtcaccctg gtgaagtgca gagcaacctc gtgcacacc ctcgtccaat tcttacgtc 1800 cattttgatg tgtcccggga gatggtggg tcaggagcac cgcaatgtgg ttcctccac 1860 gtatttgatc ctccacttga agttcaagct gccaactatg gtatgcacgg gagcactca 1920 ggcagtaaca atggaaccaa tgggcagaat agaagcacag ctgctgcaaa tgctgaacgg 1980 acaaacacgg agattgctaa tggggccatc aacaaaagtg gacctggagg tggcaatggg 2040 agtggaagtg tcagcggcaa tgacacatat gtgaaacgga tggccccgac accacgagaa 2100 gcacagctga tgaaatatag agaaaaag aaagatcgga actttgggaa aaaggtgcgg 2160 taccagagca gaagaggtt ggccgatcag cggccccggg tccgaggaca gtttgtgaag 2220 caagctgtgc aagatcaggg cggatgggaa ggagctggag atagatga 2268

Claims

1. Any of the following applications of protein: D1) Increase plant biomass and extend plant growth period; D2) Preparation of products for increasing plant biomass and extending plant growth period; D3) Extend the plant growth period; D4) Preparation of products for prolonging the growth period of plants; The protein is as follows A1) or A2): A1) The protein whose amino acid sequence is sequence 1; A2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of A1); The biomass is embodied in plant height, ear length and / or stem node number; The growth period is reflected in the heading period; The plant is millet or rice.

2. Any of the following uses of the biological material related to the protein of claim 1: D1) Increase plant biomass and extend plant growth period; D2) Preparation of products for increasing plant biomass and extending plant growth period; D3) Extend the plant growth period; D4) Preparation of products for prolonging the growth period of plants; The biological material is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); The biomass is embodied in plant height, ear length and / or stem node number; The growth period is reflected in the heading period; The plant is millet or rice.

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is the DNA molecule shown in Sequence 2 in the sequence table.

4. Any of the following methods: X1) A method for cultivating plants with increased biomass and prolonged growth period, comprising expressing the protein of claim 1 in a recipient plant, or increasing the content of the protein of claim 1 in the recipient plant, to obtain a target plant with increased biomass and prolonged growth period compared with the recipient plant; X2) A method for increasing plant biomass and extending plant growth period, comprising expressing the protein of claim 1 in a recipient plant, or increasing the content of the protein of claim 1 in the recipient plant, to obtain a target plant having increased biomass and extended growth period compared with the recipient plant, thereby achieving an increase in plant biomass and an extension of growth period; X3) A method for cultivating a plant with an extended growth period, comprising expressing the protein of claim 1 in a recipient plant, or increasing the content of the protein of claim 1 in a recipient plant, to obtain a target plant with an extended growth period compared with the recipient plant; X4) A method for extending the growth period of a plant, comprising expressing the protein of claim 1 in a recipient plant, or increasing the content of the protein of claim 1 in a recipient plant, to obtain a target plant having a longer growth period than that of the recipient plant, thereby extending the growth period of the plant; The biomass is embodied in plant height, ear length and / or stem node number; The growth period is reflected in the heading period; The recipient plant is millet or rice.

5. The method according to claim 4, characterized in that: The methods X1) to X4) are achieved by introducing a gene encoding the protein of claim 1 into the recipient plant and expressing the gene encoding the protein.

6. The method according to claim 5, characterized in that: The encoding gene is the nucleic acid molecule described in claim 2 or 3.

Images