A nucleotide sequence for delaying plant flowering and its application

Abstract

It was found in the present invention that when the 130th base of the CDS sequence of the HY5 gene in the model plant Arabidopsis thaliana mutates from C to G, overexpression or heterologous expression of the HY5<supgt;C130G< / supgt; nucleotide sequence with this point mutation can delay the flowering of transgenic Arabidopsis thaliana and rice, but overexpression of the CDS sequence of the HY5 gene without the point mutation has no effect on flowering, and its application was disclosed. The nucleotide sequence of HY5<supgt;C130G< / supgt; is shown as SEQ ID NO:1. Utilizing the important discovery of the present invention, by constructing an expression vector of "CaMV 35S-HY5<supgt;C130G< / supgt;" through chemical synthesis or site-directed mutagenesis and transforming plants such as Arabidopsis thaliana and rice, transgenic plants with delayed flowering can be cultivated, which have broad application values.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to the use of molecular biological techniques to obtain a HY5 C130G Nucleotide sequences and their use in transgenic plants. Background Art

[0002] Flowering is the transition stage from vegetative growth to reproductive growth of plants, which is crucial for plant reproduction. The early or late flowering period of crops has a great impact on their yield, quality and regional distribution. As a key agronomic trait, flowering period has also attracted much attention in the encounter of the flowering periods of parents in hybrid breeding. In urban greening and festival celebrations, agronomic measures are usually used to make flowers bloom earlier or later to meet market demand. Mastering the technology of flowering period control can bring huge economic and social benefits. For this reason, the regulatory mechanism of plant flowering time has always been a research hotspot in many disciplines such as plant molecular genetics, crop breeding and molecular biology. In recent years, scientists have made great progress by studying the regulatory mechanism of flowering in different plants, especially in the research on flowering regulation of the model plant Arabidopsis. It has been preliminarily found that the vernalization pathway, photoperiod pathway, autonomous pathway and gibberellin pathway are the main pathways for regulating flowering in higher plants. These pathways are both independent and interconnected, forming a complex flowering regulation network for higher plants.

[0003] Flowering of plants is not only regulated by intrinsic genetic factors and hormone levels, but also by external environmental conditions such as temperature and light duration. External environmental conditions mainly regulate flowering of plants by changing physiological metabolism or hormone levels in the body to promote or inhibit the expression of flowering genes. At the molecular level, the balanced expression of flowering time and inflorescence meristem genes determines the time of flower primordium formation and flowering of plants. There are many important components that positively and negatively regulate flowering in the model plant Arabidopsis, such as Arabidopsis SUMO protease ASP1, which positively regulates flowering time by regulating the stability of FLC, a key inhibitor of plant flowering; REM16 encodes a B3 domain transcription factor of Arabidopsis, which promotes flowering of Arabidopsis by directly binding to the promoter of FT, a key gene for flowering determination. In addition to the positive regulatory factors that regulate flowering, multiple flowering inhibitors have been identified in Arabidopsis. Most of these inhibitors are inflorescence meristem genes or floral organ development genes. When the genes encoding flowering inhibitors mutate, the mutants will show an early flowering phenotype and regulate the flowering time of plants in different ways. For example, FLC encodes a DNA binding protein with transcription factor activity, which can bind to the promoter of downstream flowering genes to inhibit the expression of flowering genes. When FLC is downregulated, it can also indirectly activate the expression of FT and SOC1, and finally upregulate the expression of floral meristem genes to initiate flowering; DREB2C, as an important transcription factor, can bind to FLC. Overexpression of DREB2C upregulates the expression of FLC, resulting in delayed flowering of transgenic Arabidopsis. For the important crop rice, many key genes regulating heading and flowering have been identified, such as Hd1, Hd3a, OsGI, Ehd3, Ghd7, RFT1 and Edh1. These genes are mainly involved in regulating rice heading and flowering through two main pathways: Hd1-Hd3a and Ghd7-Ehd1-Hd3a / RFT1. Hd1 is a key gene controlling rice heading and belongs to a homologous component of the Arabidopsis CO family. Hd1 regulates rice heading and is closely related to the photoperiod, promoting rice heading under short-day conditions but inhibiting rice heading under long-day conditions. RFT1 is another important gene that regulates rice heading and flowering. It has a high homology with the amino acid sequence of Hd3a and can positively regulate rice heading and flowering under long-day conditions. The key flowering regulation genes identified in the model plants Arabidopsis and rice can provide a theoretical basis and important gene resources for the directional regulation of flowering time in crops, vegetables and flowers. For example, the results of the molecular function analysis of the model plant Arabidopsis FLC can be applied to the molecular breeding of flowering regulation in important cruciferous crops such as rapeseed and radish. Scientists can find homologous genes with the model plant Arabidopsis FLC in these crops or vegetables, and use the gene editing technology developed in recent years to edit the important cis-acting sites on FLC to regulate its expression, so as to achieve precise control of flowering time and quickly obtain ideal germplasm resources.

[0004] HY5 was first discovered as a positive regulator of photomorphogenesis in higher plants. With further in-depth research, scientists have found that HY5 not only plays an important role in photomorphogenesis, but also plays an important regulatory function in the entire life activities of plants. Light as a signal mainly affects the growth and development of plants through photoreceptors and downstream signal transduction components. At present, there are at least three types of receptors that receive light signals, namely, photosensitive pigments that absorb red and far-red light, cryptochromes that absorb blue light and UV-A, and receptors that absorb UV-B. HY5 can sense light signals through the above-mentioned photoreceptors and transmit them to downstream target genes, thereby regulating the photomorphogenesis, anthocyanin synthesis, root development, and chlorophyll synthesis of plants. Most of the studies published so far use HY5 loss-of-function mutants as experimental materials to explore their biological functions. For example, hy5 mutants grow under normal light conditions and show dark morphogenesis phenotypes with elongated hypocotyls and reduced anthocyanin content, but there are few reports on the changes in Arabidopsis phenotypic caused by overexpression of HY5. Recent studies have shown that when the HY5 gene of Arabidopsis thaliana mutates, its loss-of-function mutant hy5 exhibits an early flowering phenotype, but no research has been conducted to determine whether overexpression of Arabidopsis thaliana HY5 affects the flowering of transgenic plants. Our research group found that when the Arabidopsis thaliana HY5 gene is overexpressed, the transgenic Arabidopsis thaliana strains have no significant phenotypic differences from the wild type, and have no significant effect on flowering time. However, when the 130th base of the open reading frame sequence (CDS) of the HY5 gene mutates from C to G, overexpression and heterologous expression of the HY5 with a point mutation in Arabidopsis and rice, respectively, C130G The transgenic plants all showed a delayed flowering phenotype.

[0005] For crops, flowering is one of the important agronomic traits. Delaying the flowering of plants can increase the yield and quality of crops by extending the growth period. In hybrid breeding, delayed flowering is conducive to the flowering period of parents, thereby improving the efficiency of hybrid breeding. By delaying the flowering of economic plants such as vegetables and flowers, it can also bring greater market economic value to the production of off-season vegetables and flowers. Therefore, the discovery and identification of important gene resources for regulating delayed plant flowering is of great practical significance for the high-quality and sustainable development of agriculture. The present invention uses molecular biological technology to discover overexpression and heterologous expression of HY5 in the dicotyledonous plant Arabidopsis and the monocotyledonous plant rice, respectively. C130G The nucleotide sequence significantly delays the flowering of transgenic plants, providing useful genetic resources for creating new transgenic crop varieties with delayed flowering through molecular breeding methods, and has broad application value. Summary of the invention

[0006] The present invention aims to clone the sequence HY5 in which the 130th base of the CDS of the model plant Arabidopsis thaliana HY5 gene is mutated from C to G.C130G , and construct "CaMV 35S-HY5 C130G The fusion gene of " is used to transform Arabidopsis and rice, providing a nucleotide sequence for delaying plant flowering and its application in transgenic plants.

[0007] The present invention provides a nucleotide sequence HY5 for delaying plant flowering C130G The nucleotide sequence is shown in SEQ ID NO: 1. As a specific application example, HY5 was synthesized by whole gene synthesis. C130G Sequence, using synthetic HY5 C130G Sequence construction "CaMV 35S-HY5 C130G ” fusion gene and transformed Arabidopsis and rice to obtain Arabidopsis and rice plants that constitutively expressed HY5 C130G The transgenic plants. C130G The sequence can be any nucleotide sequence required for basic research, transformation technology, or improvement of delayed flowering traits in crops or flowers.

[0008] As a specific application example, the present invention provides a "CaMV 35S-HY5 C130G "Construction of fusion gene and its application in delaying flowering of transgenic Arabidopsis and rice. The specific operation process is as follows:

[0009] (1) In HY5 C130G The Nco I and BstE II restriction site sequences were introduced into the upstream and downstream of the nucleotide sequence, respectively. The sequence of the Nco I restriction site introduced upstream was CCATGGTA (before the ATG start codon), and the sequence of the BstE II restriction site introduced downstream was GGTTACC (after the TGA stop codon). The HY5 with restriction sites was synthesized by a biological company. C130G Nucleotide sequence;

[0010] (2) Synthesis of HY5 with restriction sites by double digestion with Nco I and BstE II C130G Nucleotide fragments and recovery;

[0011] (3) Double digestion of the pCAMBIA1301 expression vector plasmid with Nco I and BstE II to recover the large vector fragment;

[0012] (4) Mixing the HY5 recovered in the second step C130G The nucleotide fragment and the large fragment of the pCAMBIA1301 vector recovered in the third step are ligated under the catalysis of ligase to complete the "CaMV 35S-HY5 C130G "Construction of fusion genes.

[0013] The "CaMV 35S-HY5 C130G The construction of the fusion gene was performed in transgenic Arabidopsis and rice for constitutive expression. The operation process was as follows:

[0014] For the transformation of Arabidopsis, the conventional bud dipping method was used to transform Arabidopsis (ecotype Col-0), and the harvested seeds were screened for hygromycin resistance and molecularly identified to obtain the transformed "CaMV 35S-HY5 C130G "The homozygous transgenic Arabidopsis strains with the fusion gene were tested and counted for the bolting and flowering time of the transgenic Arabidopsis. For the transformation of rice, the conventional Agrobacterium-infected rice callus method was used to transform rice (variety: Nipponbare). After the harvested seeds were propagated, the homozygous transgenic rice strains were obtained through hygromycin resistance screening and molecular identification. Finally, the heading and flowering time of the transgenic rice were tested and counted.

[0015] Positive effects of the present invention:

[0016] The experimental results showed that the sequence HY5 was cloned by mutating the 130th base of CDS from C to G. C130G Transformation of Arabidopsis and rice can significantly delay the flowering of transgenic plants. The fusion gene construction of the present invention "CaMV35S-HY5 C130G "It can be used as a genetic resource in agricultural production or through molecular breeding to breed new transgenic plant varieties with delayed flowering, and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 The HY5 fusion gene "CaMV 35S-HY5" without point mutation was transformed into Arabidopsis, and the flowering time of the transgenic Arabidopsis lines was not significantly affected. A: Bolting phenotypes of wild-type Arabidopsis and transgenic Arabidopsis transformed with the "CaMV 35S-HY5" fusion gene; B: Relative expression levels of the HY5 gene in wild-type Arabidopsis and transgenic Arabidopsis transformed with the "CaMV 35S-HY5" fusion gene; C: Bolting time of wild-type Arabidopsis and transgenic Arabidopsis transformed with the "CaMV 35S-HY5" fusion gene; OEHY5-1, OEHY5-2 and OEHY5-3 represent three independent transgenic lines, *P﹤0.05.

[0019] Figure 2 The Arabidopsis HY5 gene CDS base 130 was mutated from C to G.C130G Fusion gene "CaMV35S-HY5 C130G " was transformed into Arabidopsis thaliana, and the flowering time of its transgenic Arabidopsis lines was significantly delayed. A: Wild-type Arabidopsis thaliana and transformed with "CaMV 35S-HY5 C130G "The bolting phenotype of transgenic Arabidopsis thaliana with fusion gene; B:HY5 C130G In wild-type Arabidopsis and transformed with CaMV 35S-HY5 C130G " Relative expression levels of transgenic Arabidopsis thaliana containing the fusion gene; C: Wild-type Arabidopsis thaliana and transformed with "CaMV 35S-HY5 C130G Bolting time of transgenic Arabidopsis thaliana with fusion gene; OEHY5 C130G -1, OEHY5 C130G -2 and OEHY5 C130G -3 represents three independent transgenic lines, *P﹤0.05.

[0020] Figure 3 The Arabidopsis HY5 gene CDS base 130 was mutated from C to G. C130G Fusion gene "CaMV35S-HY5 C130G " Transformed rice (variety: Nipponbare), the flowering time of its transgenic rice lines was significantly delayed. A: Wild-type rice and transformed "CaMV 35S-HY5 C130G "Heading phenotype of transgenic rice with fusion gene; B:HY5 C130G In wild-type rice and transformed with CaMV 35S-HY5 C130G " Relative expression levels of transgenic rice containing the fusion gene; C: Wild-type Arabidopsis and transformed with "CaMV 35S-HY5 C130G "The heading time of transgenic rice with fusion gene; OEHY5 C130G -R1, OEHY5 C130G -R2 and OEHY5 C130G -R3 represents three independent transgenic lines, *P﹤0.05. DETAILED DESCRIPTION

[0021] The following will describe the implementation methods of the present invention in detail with reference to the accompanying drawings and embodiments, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0022] Example 1: Construction of the Arabidopsis HY5 fusion gene "CaMV 35S-HY5" without point mutation

[0023] (1) The total RNA of wild-type Arabidopsis thaliana was extracted by conventional Trizol method. Specific primers were designed based on the known CDS sequence of Arabidopsis thaliana HY5, and Bsa I and Eco31 I restriction sites were introduced into the upstream and downstream primers, respectively.

[0024] Upstream primer: 5'-CAGTGGTCTCACAACATGCAGGAACAAGCGACTAG-3'

[0025] Downstream primer: 5'-CAGTGGTCTCATACAAAGGCTTGCATCAGCATTAG-3'

[0026] (2) Using the extracted RNA as template, P 2853 As primer for reverse transcription, P 2853 The primer sequences, reverse transcription procedure and reverse transcription system are as follows.

[0027] P 2853 Primer sequence: 5'-GCGAATTCTTTTTTTTTTTTTTTTTT-3'

[0028] Reverse transcription procedure:

[0029] 72℃5min; 25℃5min; 42℃60min; 80℃20min.

[0030] Reverse transcription system:

[0031]

[0032] (3) Using the reverse transcribed cDNA as a template, PCR amplification was performed using the above primers to obtain the CDS fragment of the HY5 gene.

[0033] PCR reaction program: 94°C for 5 min; 94°C for 30 s, 50°C for 45 s, 72°C for 30 s, 30 cycles; 72°C for 10 min, 16°C for 30 min.

[0034] PCR reaction system:

[0035]

[0036] (4) The pBWA(V)HS vector was double-digested with Bsa I and Eco31 I to recover the large fragment, and the HY5 gene CDS fragment was double-digested with Bsa I and Eco31 I and recovered. The recovered vector and the HY5 CDS fragment were ligated with ligase to complete the construction of the "CaMV 35S-HY5" fusion gene.

[0037] Enzyme Digestion and Ligation System:

[0038]

[0039]

[0040] (5) According to the conventional CaCl 2 Induction and transformation method: Prepare E. coli DH5α competent cells, transform the ligation mixture into competent cells, and invert and culture at 37°C for 12 hours. After a single clone is generated, extract the plasmid and transform Agrobacterium GV3101 using conventional methods to prepare for the transformation of Arabidopsis thaliana.

[0041] Example 2: Preparation of transgenic Arabidopsis transformed with “CaMV 35S-HY5” and analysis of flowering-related phenotypes

[0042] (1) The successfully constructed "CaMV 35S-HY5" fusion gene was transformed into Arabidopsis thaliana according to the conventional bud infection method. 0 Seeds are treated with 50 mg l -1 After hygromycin resistance screening, DNA from resistant plants was extracted using conventional methods, and PCR was used to identify whether the exogenous HY5 fragment had been integrated into the Arabidopsis genome. The primers used were as follows:

[0043] Upstream primer: 5'-GGAGAGAACACGGGGGAC-3'

[0044] Downstream primer: 5'-AGCTGGTCAGTCTTCGGG-3'

[0045] PCR reaction program: 94°C for 5 min; 94°C for 30 s, 50°C for 45 s, 72°C for 30 s, 30 cycles; 72°C for 10 min, 16°C for 30 min.

[0046] The PCR reaction system is as follows:

[0047]

[0048] (2) The transgenic lines that were positive after resistance screening and PCR identification were propagated, and T was extracted using the conventional Trizol method. 3 Total RNA of transgenic Arabidopsis thaliana lines was used for reverse transcription of cDNA, and the relative expression level of HY5 was detected by real-time fluorescence quantitative PCR using the following primers, PCR reaction program and reaction system:

[0049] Detection primers for HY5 gene:

[0050] Upstream primer: 5'-GGCTGAAGAGGTTGTTGAGGAAC-3'

[0051] Downstream primer: 5'-ACCACCTCCTCTCTTGTTTCCTG-3'

[0052] Detection primers for ACTIN2 internal standard gene:

[0053] Upstream primer: 5'-CAAACGAGGGCTGGAACAAGACT-3'

[0054] Downstream primer: 5'-CTGTTGACTACGAGCAGGAGATGG-3'

[0055] PCR reaction procedure:

[0056] 95℃: 2min; 95℃: 10s, 60℃: 30s, 40 cycles.

[0057] The PCR reaction system is as follows:

[0058]

[0059] Real-time fluorescence quantitative PCR results Figure 1 As shown in Figure B, the relative expression level of the HY5 gene was significantly increased in three independent Arabidopsis transgenic lines (OEHY5-1, OEHY5-2 and OEHY5-3), indicating that the above three independent transgenic lines are Arabidopsis lines overexpressing the HY5 gene.

[0060] (3) Analysis of flowering-related phenotypes of transgenic Arabidopsis lines transformed with the “CaMV 35S-HY5” fusion gene: Seeds of wild-type Arabidopsis and the three transgenic Arabidopsis lines overexpressing the HY5 gene were sterilized with 1% sodium hypochlorite and placed in 1 / 2MS medium for germination and growth (temperature 23°C, light intensity 90 μE m -2 s -1 , photoperiod 16h light / 8h dark), during which the stem length of 1cm after bolting was used as the sign of bolting and flowering, and the bolting and flowering time of wild-type and transgenic Arabidopsis was recorded and counted. Figure 1 As shown ( Figure 1 A and C), the bolting time of the three transgenic Arabidopsis transformed with the "CaMV35S-HY5" fusion gene was not significantly different from that of the wild type, indicating that overexpression of the HY5 gene without point mutation in the model plant Arabidopsis had no significant effect on flowering.

[0061] Example 3: A fusion gene "CaMV35S-HY5" in which the 130th base of the CDS of the Arabidopsis HY5 gene is mutated from C to G C130G "Build

[0062] (1) Sequence Table 1 shows the sequence HY5 of the Arabidopsis thaliana HY5 gene CDS sequence in which the 130th base is mutated from C to G. C130G , in HY5C130G The Nco I and BstE II restriction site sequences were introduced into the upstream and downstream of the nucleotide sequence, and the sequence of the upstream Nco I restriction site was CCATGG TA (before the ATG start codon), the sequence of the downstream BstE II restriction site is GGTTACC (after the TGA stop codon), and handed it over to a biotechnology company to synthesize HY5 with restriction enzyme cleavage sites C130G Nucleotide sequence.

[0063] (2) The synthesized target gene fragment was double-digested with Nco I and BstE II and recovered. The pCAMBIA1301 vector was double-digested with Nco I and BstE II and the large fragment was recovered. The recovered vector large fragment and HY5 C130G The nucleotide fragments were connected at 16°C overnight under the action of ligase to complete the "CaMV 35S-HY5 C130G "Construction of fusion genes.

[0064] Connection system:

[0065]

[0066] (3) According to the conventional CaCl 2 Induction and transformation method: Prepare E. coli DH5α competent cells, transform the ligation mixture into competent cells, and invert and culture at 37°C for 12 hours. After a single clone is generated, extract the plasmid and transform Agrobacterium GV3101 and EHA105 using conventional methods to prepare for Arabidopsis transformation (GV3101) and rice transformation (EHA105).

[0067] Example 4: Transformation of "CaMV 35S-HY5 C130G Preparation of transgenic Arabidopsis and rice and analysis of flowering-related phenotypes

[0068] (1) The successfully constructed "CaMV35S-HY5 C130G The fusion gene was transformed into Arabidopsis and rice, DNA of resistant plants was extracted by conventional methods, and exogenous HY5 was identified by PCR. C130G Whether the fragment has been integrated into the genome of Arabidopsis and rice, the primers used are as follows:

[0069] Upstream primer: 5'-GCGATAAAGGAAAGGCCATCG-3'

[0070] Downstream primer: 5'-ACCACCTCCTCTCTTGTTTCCTG-3'

[0071] PCR reaction program: 94°C for 3 min; 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 10 min.

[0072] The PCR reaction system is as follows:

[0073]

[0074] (2) The positive transgenic lines that were screened for resistance and identified by PCR were propagated, and the total RNA from the leaves of transgenic Arabidopsis and rice plants was extracted by conventional Trizol method and reverse transcribed into cDNA. The following primers, PCR reaction procedures and reaction systems were used for real-time fluorescence quantitative PCR detection of HY5 C130G Relative expression levels:

[0075] HY5 C130G Detection primers:

[0076] Upstream primer: 5'-GGCTGAAGAGGTTGTTGAGGAAC-3'

[0077] Downstream primer: 5'-ACCACCTCCTCTCTTGTTTCCTG-3'

[0078] Detection primers for ACTIN2 internal standard gene (detection of transgenic Arabidopsis):

[0079] Upstream primer: 5'-CAAACGAGGGCTGGAACAAGACT-3'

[0080] Downstream primer: 5'-CTGTTGACTACGAGCAGGAGATGG-3'

[0081] Detection primers for OsUBI internal standard gene (detection of transgenic rice):

[0082] Upstream primer: 5'-AACCAGCTGAGGCCCAAGA-3'

[0083] Downstream primer: 5'-ACGATTGATTTAACCAGTCCATGA-3'

[0084] PCR reaction procedure:

[0085] 95℃: 2min; 95℃: 10s, 60℃: 30s, 40 cycles.

[0086] The PCR reaction system is as follows:

[0087]

[0088] Real-time fluorescence quantitative PCR results Figure 2 B and 3B, HY5 C130G The relative expression levels of three independent transgenic Arabidopsis thaliana (OEHY5 C130G -1, OEHY5 C130G -2 and OEHY5 C130G -3) and transgenic rice (OEHY5 C130G -R1, OEHY5 C130G -R2 and OEHY5 C130G -R3) lines, indicating that the above transgenic Arabidopsis and transgenic rice lines are HY5 C130G overexpression strains.

[0089] (3) Transformation of CaMV 35S-HY5 C130G Analysis of flowering-related phenotypes of transgenic Arabidopsis thaliana with fusion genes: Wild-type Arabidopsis thaliana and the three HY5 C130G The seeds of the Arabidopsis thaliana strain with the gene were sterilized with 1% sodium hypochlorite and placed in a culture medium for germination and growth (temperature 23°C, light intensity 90 μE m -2 s -1 , photoperiod 16h light / 8h dark), during which the stem length of 1cm after bolting was used as the sign of bolting and flowering, and the flowering time of wild-type and transgenic Arabidopsis was recorded and counted. Figure 2 As shown ( Figure 2 A and C), three transformations of "CaMV 35S-HY5 C130G The bolting time of transgenic Arabidopsis thaliana with fusion gene was significantly delayed, indicating that overexpression of HY5 in the model plant Arabidopsis C130G Flowering is significantly delayed.

[0090] (4) Transformation of "CaMV 35S-HY5 C130G Analysis of flowering-related phenotypes of transgenic rice with fusion genes: Wild-type rice (variety: Nipponbare) and the three HY5 C130G The seeds of the transgenic rice lines were germinated at 35°C in the dark for 48 hours, the germinating rice seeds were grown in culture soil, and then potted according to the conventional method (the growth environment was natural conditions). The heading of the rice plants was used as a sign of entering reproductive growth, and the heading time of wild-type and transgenic rice was recorded and counted. The experimental results are as follows Figure 3 As shown ( Figure 3 A and C), three transformations of "CaMV 35S-HY5 C130G The heading time of transgenic rice with the fusion gene was significantly delayed, indicating that heterologous expression of HY5 in monocotyledonous rice C130G Flowering is significantly delayed.

[0091] The above-mentioned example results show and confirm that although the overexpression of the CDS sequence of the model plant Arabidopsis HY5 gene has no effect on flowering, when the 130th base of its CDS sequence is mutated from C to G, overexpression or heterologous expression of the HY5 with point mutation C130G The nucleotide sequence can delay flowering of transgenic Arabidopsis and rice. Therefore, by utilizing the important discovery of the present invention, by constructing "CaMV 35S-HY5 C130G "The fusion gene can be used to transform plants such as Arabidopsis and rice to cultivate new transgenic plant varieties with delayed flowering, which has a wide range of application value.

[0092] The above description is only for the best embodiment of the present invention, but it should not be understood as limiting the claims. The present invention is not limited to the above embodiments, and its specific structure is allowed to be changed. All changes made within the scope of protection of the independent claims of the present invention are within the scope of protection of the present invention.

Claims

1. Arabidopsis HY5, a plant that delays flowering C130G The mutant gene is characterized in that The mutant gene is a mutant gene in which the 130th base of the Arabidopsis HY5 gene is mutated from C to G. The nucleotide sequence of the mutant gene is shown in the sequence table SEQ ID NO:

1.

2. The Arabidopsis thaliana HY5 for delaying plant flowering as claimed in claim 1 C130G The application of mutant genes is characterized by: The mutant gene was used to construct the "CaMV 35S-HY5 C130G " is transformed into the dicotyledonous plant Arabidopsis and the monocotyledonous plant rice to delay the flowering of the transgenic Arabidopsis and rice. The construction process of the fusion gene is as follows: (1) In HY5 C130G The Nco I and BstE II restriction site sequences were introduced into the upstream and downstream of the nucleotide sequence, respectively. The sequence of the Nco I restriction site before the upstream ATG start codon was CCATGGTA, and the sequence of the BstE II restriction site after the downstream TGA stop codon was GGTTACC. The HY5 with restriction site was synthesized by a biological company. C130G Nucleotide sequence; (2) Synthesis of HY5 with restriction sites by double digestion with Nco I and BstE II C130G Nucleotide fragments and recovery; (3) Double digestion of the pCAMBIA1301 expression vector plasmid with Nco I and BstE II to recover the large vector fragment; (4) Mixing the HY5 recovered in the second step C130G The nucleotide fragment and the large fragment of the pCAMBIA1301 vector recovered in the third step are ligated under the catalysis of ligase to complete the "CaMV 35S-HY5 C130G "Construction of fusion genes.

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