A nucleotide sequence for inhibiting rice panicle germination and its application

Abstract

The present invention discloses a nucleotide sequence for inhibiting rice panicle germination and its application. The nucleotide sequence is shown in SEQ ID NO: 1. The nucleotide sequence is used to construct a "CaMV 35S-OsHY5L2" fusion gene, which is transformed into rice. The nucleotide sequence can effectively inhibit rice panicle germination under high humidity conditions during the mature harvest period. The method provides an important gene resource for inhibiting rice panicle germination through molecular genetic improvement methods and has potential application value.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to a nucleotide sequence for inhibiting rice panicle germination and application thereof. Background Art

[0002] Rice occupies an important position among my country's grain crops, and its yield is closely related to national food security. Rice panicle sprouting, induced by high humidity during the rice harvest period, seriously reduces rice quality. Rice panicle sprouting increases the activity of hydrolytic enzymes such as endosperm α-amylase, accelerating the degradation of storage substances such as starch and protein in the embryo and endosperm, deteriorating rice quality. [3] Therefore, isolating and identifying the key genes that regulate rice panicle germination and revealing the molecular mechanism of their regulation will provide important gene resources for inhibiting rice panicle germination through molecular genetic improvement methods and have potential application value.

[0003] Seed dormancy is closely linked to ear germination, with premature breaking of seed dormancy being the primary cause of ear germination. Plant hormones, light, and temperature, among other factors, regulate seed dormancy through complex pathways (Finkelstein et al., 2008; Holdsworth et al., 2008). Abscisic acid (ABA) and gibberellins (GA) are the primary hormones influencing seed germination. GA breaks seed dormancy and promotes germination, while ABA promotes dormancy and inhibits germination (Seo et al., 2006; Yano et al., 2009). Therefore, the balance between GA and ABA at the metabolic level and in their signaling pathways plays a crucial role in regulating seed dormancy. Plants regulate seed dormancy by altering the ABA:GA ratio (Finkelstein et al., 2008). Light, as a key environmental signal, regulates plant physiological processes such as photomorphogenesis, dormancy and germination, phototropism, shade shelter, and flowering. Previous studies have shown that the phytochrome B (phyB) photoreceptor plays a crucial role in light-induced regulation of seed dormancy and germination. PIF1 is a key transcription factor that inhibits phyB-mediated seed germination, while RVE1 is a key regulatory factor controlling seed dormancy and germination. Genetic analysis has shown that RVE1's inhibition of seed germination requires the involvement of PIF1, and conversely, PIF1's inhibitory effect is also partially dependent on RVE1. Furthermore, PIF1 and RVE1 synergistically control seed germination by regulating the expression of downstream genes in the ABA and GA signaling pathways (Sohn et al., 2020). These studies, integrating key nodes in the light and GA synthesis and signaling pathways, provide new insights into the interaction between plant hormones and light signals in regulating seed dormancy and spike germination.

[0004] HY5, a member of the bZIP transcription factor family, is an important regulatory factor in plant photomorphogenesis, mainly involved in regulating many physiological processes such as seed germination, root development, floral organs, fruits, and plant hormone transport.

[22] In the model plant Arabidopsis thaliana, HY5 accumulates under light conditions but is degraded by the COP-SPA complex in the dark (Bhatnagar et al., 2020). During seedling development, HY5 protein levels are tightly regulated by the E3 ubiquitin ligase COP1, which ubiquitinates HY5 and targets it for proteasomal degradation (Hardtke et al., 2000). HY5 can also regulate photomorphogenesis by modulating various plant hormones. For example, HY5 positively regulates photomorphogenesis by inhibiting IAA and GA signaling. However, ethylene in the light increases COP1 accumulation, leading to decreased HY5 stability and promoting hypocotyl elongation. In the dark, CTK reduces COP1 accumulation, increases HY5 protein stability, and promotes photomorphogenesis (Wang et al., 2018). Most of the above studies on the function of HY5 have been conducted in the model plant Arabidopsis thaliana, but there are fewer reports on the functions of genes homologous to Arabidopsis HY5 in crops such as rice, wheat, and maize. HY5L2 is a rice homolog of HY5 in Arabidopsis. HY5L2 is involved in regulating rice plant growth, and OsBBX14 regulates photomorphogenesis by upregulating HY5L2 expression (Bai et al., 2019). Although studies have shown that HY5L2 plays an important role in regulating plant photomorphogenesis, its involvement in regulating rice panicle germination under high-humidity conditions has not yet been reported. This study, using transgenic rice overexpressing HY5L2, found that under high-humidity conditions, HY5L2 transgenic rice lines exhibited significantly enhanced ability to inhibit panicle germination, with a significantly lower percentage of grains germinating than wild-type controls. This important finding has significant application in molecular genetic modification of rice panicle germination. Summary of the Invention

[0005] The present invention aims to isolate and clone the rice OsHY5L2 gene, construct a "CaMV 35S-OsHY5L2" fusion gene using the open reading frame (ORF) sequence of the gene, and transform the gene into rice, so that the panicle germination of the transgenic rice overexpressing OsHY5L2 is significantly inhibited under high humidity conditions at the mature harvest stage, and provide an application of the OsHY5L2 gene in transgenic rice.

[0006] The present invention provides an open reading frame (ORF) sequence of the OsHY5L2 gene capable of inhibiting panicle germination of transgenic rice under high humidity conditions during the mature harvest period, wherein the nucleic acid sequence is selected from:

[0007] (a) the nucleotide sequence shown in SEQ ID NO: 1;

[0008] (b) As an example of a specific application, the present invention provides a method for cloning the OsHY5L2 gene, and the specific steps are as follows:

[0009] (1) Extract total RNA from rice plants and reverse transcribe it into cDNA;

[0010] (2) Using cDNA as a template, the open reading frame (ORF) sequence of the OsHY5L2 gene was amplified by PCR;

[0011] (3) Recover the PCR amplification product.

[0012] The present invention also provides a method for overexpressing the rice OsHY5L2 gene to inhibit panicle sprouting in transgenic rice under high humidity conditions during the mature harvest period. Specifically, the open reading frame (ORF) sequence of the cloned OsHY5L2 gene is used to construct a "CaMV35S-OsHY5L2" fusion gene for rice transformation, thereby obtaining a transgenic rice line constitutively expressing the OsHY5L2 gene. The target gene in the fusion gene can be any target gene required for basic research or for improving traits such as rice panicle sprouting inhibition.

[0013] As an example of specific application, the present invention provides a construction of a "CaMV 35S-OsHY5L2" fusion gene and its application in inhibiting panicle germination under high humidity conditions during the mature and harvest period of transgenic rice. The specific operation process is as follows:

[0014] (1) The open reading frame (ORF) sequence fragment of the OsHY5L2 gene amplified by PCR was digested with Bsa I;

[0015] (2) The pBWA(V)HS overexpression vector plasmid was digested with Bsa I to recover the large vector fragment;

[0016] (3) The open reading frame (ORF) sequence fragment of the OsHY5L2 gene obtained in the first step and the large fragment of the pBWA(V)HS vector obtained in the second step were mixed and ligated under the catalysis of T4 ligase to complete the construction of the "CaMV 35S-OsHY5L2" fusion gene on the pBWA(V)HS vector.

[0017] The designed PCR amplification primer sequences for the open reading frame (ORF) sequence of the OsHY5L2 gene are as follows, wherein the upstream and downstream primers introduce Bsa I restriction sites:

[0018] Upstream primer: 5'-CAGT GGTCTCACAACATGCAACGAGATCACCGGAG-3'

[0019] Downstream primer: 5'-CAGT GGTCTC ATACAG-CTGTCTCCGCCGGCACTGC-3'

[0020] The "CaMV 35S-OsHY5L2" fusion gene in the application is constructed and constitutively expressed in transgenic rice. The operation process is as follows: rice callus tissue is transformed by Agrobacterium infection, seeds of the obtained resistant seedlings are harvested and propagated, DNA is extracted from leaves of the resistant seedlings for PCR detection, and RNA is extracted from the propagated offspring lines for real-time fluorescence quantitative PCR detection. Homozygous transgenic rice lines overexpressing OsHY5L2 are screened, and the panicle germination ratio of these lines under high humidity conditions at the mature harvest period is measured.

[0021] Beneficial effects of the present invention:

[0022] Experimental results showed that transforming the cloned OsHY5L2 into rice significantly suppressed the rate of panicle sprouting in transgenic rice lines overexpressing OsHY5L2 under high humidity conditions at the mature harvest stage. The CaMV 35S-OsHY5L2 fusion gene construct of this invention can be used as a genetic resource to generate transgenic rice lines with significantly suppressed panicle sprouting through genetic modification methods, and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 Figure 1 is a PCR molecular identification diagram of the integration of the fusion gene in resistant strains of rice (variety: Nipponbare, NIP) transformed with the "CaMV 35S-OsHY5L2" fusion gene. M: DL10000, 1, 2, 3, and 4 represent PCR amplification diagrams using DNA from wild-type rice (NIP) and three resistant strains transformed with OsHY5L2 as templates, respectively.

[0025] Figure 2 This is an analysis of the relative expression levels of the OsHY5L2 gene in transgenic rice lines transformed with the "CaMV 35S-OsHY5L2" fusion gene. NIP, OEHY5L2-1, OEAOsHY5L2-2, and OEOsHY5L2-3 represent wild-type rice and three independent transgenic rice lines transformed with the OsHY5L2 gene, respectively. *P﹤0.05.

[0026] Figure 3A is the phenotypic diagram of panicle germination of wild-type rice (variety: Nipponbare, NIP) and transgenic rice lines transformed with the "CaMV 35S-OsHY5L2" fusion gene (OEHY5L2-1, OEAHY5L2-2, and OEHY5L2-3), scale bar = 1 cm; B is the panicle germination ratio of wild-type rice (NIP) and transgenic rice lines transformed with the "CaMV 35S-HY5L2" fusion gene under high humidity conditions at the mature harvest stage, *P < 0.05. DETAILED DESCRIPTION

[0027] This section will describe in detail the specific embodiments of the present invention. The preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the accompanying drawings is to supplement the description of the text part of the specification with graphics, so that people can intuitively and vividly understand each technical feature and the overall technical solution of the present invention, but it should not be understood as a limitation on the scope of protection of the present invention.

[0028] Example 1: Cloning of the Nipponbare rice OsHY5L2 gene

[0029] (1) Total RNA from rice plants was extracted using the guanidine isosulfate-phenol method.

[0030] (2) Specific primers were designed based on the known open reading frame (ORF) sequence of the OsHY5L2 gene of Nipponbare rice, and Bsa I restriction enzyme sites were introduced into the upstream and downstream primers.

[0031] Upstream primer: 5'-CAGT GGTCTC ACAACATGCAACGAGATCACCGGAG-3'

[0032] Downstream primer: 5'-CAGT GGTCTC ATACAG-CTGTCTCCGCCGGCACTGC-3'

[0033] (3) Take 1 μg RNA as the template for reverse transcription and use P 2853 ImProm-II RT TM Reverse transcription was performed, P 2853 The primer sequences, reverse transcription procedure, and reverse transcription system are as follows.

[0034] P 2853 Primer sequence: 5'-GCGAATTCTTTTTTTTTTTTTTTTT-3'

[0035] Reverse transcription procedure:

[0036] 72℃5min; 25℃5min; 42℃60min; 80℃20min; keep warm at 4℃.

[0037] Reverse transcription system:

[0038]

[0039]

[0040] (4) Using the reverse transcribed cDNA as a template, PCR amplification was performed using the above primers to obtain the open reading frame (ORF) sequence fragment of the OsHY5L2 gene.

[0041] PCR reaction program: 94°C for 3 min; 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min; and insulation at 4°C.

[0042] PCR reaction system:

[0043]

[0044] (5) Recover the target DNA fragment by agarose gel electrophoresis.

[0045] (6) The recovered PCR product was subjected to DNA sequencing, and its nucleic acid sequence is shown in Sequence Table 1.

[0046] Example 2: Construction of the "CaMV 35S-OsHY5L2" fusion gene using the pBWA(V)HS expression vector

[0047] (1) The pBWA(V)HS plasmid vector was extracted from Escherichia coli and the large fragment of the vector was recovered after digestion with Bsa I.

[0048] (2) The open reading frame (ORF) sequence fragment of the OsHY5L2 gene recovered in Example 1 was digested with Bsa I, and the digested fragment was recovered by agarose gel electrophoresis.

[0049] (3) The two fragments recovered above were ligated under the catalysis of T4 ligase to complete the construction of the "CaMV 35S-OsHY5L2" fusion gene on the pBWA(V)HS expression vector.

[0050] (4) Transform the ligation mixture into E. coli DH5α competent cells as follows:

[0051] E. coli DH5α competent cells were prepared according to conventional CaCl2 induction and transformation methods. 10 μl of the ligation product was used to transform the competent cells, which were then evenly spread on a plate containing Amp, X-gal, and IPTG and cultured in an inverted manner at 37°C for 12 h.

[0052] (5) PCR reaction was performed using the plasmid as a template to identify the "CaMV 35S-OsHY5L2" fusion gene in the plasmid. The size of the amplified fragment was 1371 bp. The primers used were as follows:

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

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

[0055] (6) Plasmids were extracted from positive clones and transformed into Agrobacterium EHA105 using conventional methods to obtain engineered Agrobacterium for rice transformation.

[0056] Example 3: Preparation of transgenic rice

[0057] (1) The "CaMV 35S-OsHY5L2" fusion gene constructed in Example 2 was transformed into rice (variety: Nipponbare). The specific transformation method was to infect rice callus with Agrobacterium tumefaciens. DNA was extracted from rice plant leaves with hygromycin resistance. PCR was used to identify that the CaMV 35S-OsHY5L2 fusion gene was integrated into the wild-type rice genome. The size of the amplified fragment was 488 bp. The primers used were as follows:

[0058] Upstream primer: 5'-ACGGTGTCGTCCATCACAGTTTGCC-3'

[0059] Downstream primer: 5'-GCGACGTCTGTCGAGAAGTTTC-3'

[0060] PCR reaction program: 94°C for 3 min; 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min; and insulation at 4°C.

[0061] The PCR reaction system is as follows:

[0062]

[0063] PCR test results such as Figure 1 As shown, the target bands could be amplified in the genomes of the three tested resistant seedling lines (lanes 2, 3 and 4), indicating that the exogenous OsHY5L2 fragment had been integrated into the genome of the wild-type rice.

[0064] (2) Real-time fluorescence quantitative RT-PCR detection of transgenic plants: The transgenic rice lines identified by PCR were propagated, and total RNA of the transgenic rice lines was extracted and reverse transcribed into cDNA according to the method of Example 1. The relative expression level of OsHY5L2 was detected by real-time fluorescence quantitative RT-PCR using the following primers, PCR reaction procedure and reaction system:

[0065] Detection primers for OsHY5L2 gene:

[0066] Upstream primer: 5'-GAGATAGTAGGGAGGGTGCCGG-3'

[0067] Downstream primer: 5'-CCTCAGCAATCTTTTGAGGCGC-3'

[0068] Detection primers for OsUBI internal standard gene:

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

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

[0071] PCR reaction procedure:

[0072] 95℃: 2 min; 95℃: 10 s, 60℃: 30 s, 40 cycles.

[0073] The PCR reaction system is as follows:

[0074]

[0075] Real-time fluorescence quantitative RT-PCR results Figure 2 As shown in the data, the expression levels of the OsHY5L2 gene in three transgenic rice lines (OEHY5L2-1, OEAHY5L2-2, and OEHY5L2-3) were significantly increased compared with those in wild-type rice (NIP), indicating that OEHY5L2-1, OEAHY5L2-2, and OEHY5L2-3 are three transgenic rice lines overexpressing the OsHY5L2 gene.

[0076] Example 4: Detection of the proportion of panicle germination at the mature harvest stage of transgenic rice lines overexpressing the OsHY5L2 gene

[0077] A pot experiment was conducted in wild-type rice (NIP) and three homozygous transgenic lines overexpressing the OsHY5L2 gene (OEHY5L2-1, OEAHY5L2-2, and OEHY5L2-3). Forty-five days after heading, the rice plants, which were at the mature harvest stage, were transferred to an artificial climate chamber under high humidity conditions for 5 days (photoperiod: 12 h light / 12 h dark, temperature: 32°C during the day, 27°C at night). Distilled water was sprayed on the rice panicle every 3 hours until water droplets dripped from the top of the panicle. The germination ratio of the grains in the panicle (number of germinated grains / total number of grains) was counted every day. The phenotypic scanning results are shown in Figure 3. Figure 3 As shown in A, the statistical results of the proportion of grain germination in the ear are as follows Figure 3 As shown in B, the experimental results showed that the panicle germination ratio of the three transgenic rice lines overexpressing the OsHY5L2 gene was significantly reduced compared with the wild-type Nipponbare NIP. The reduction ranges on the 2nd, 3rd, 4th, 5th, 6th and 7th days of high humidity treatment were 27.66%-31.02%, 51.85%-64.35%, 46.71%-67.09%, 40.77%-60.31%, 36.71%-56.09% and 37.93%-54.95%, respectively.

[0078] The results of the above examples demonstrate and confirm that the OsHY5L2 gene provided by the present invention negatively regulates panicle germination in transgenic rice lines. Overexpression of this gene significantly inhibits panicle germination in transgenic rice plants under high humidity conditions at the mature harvest stage. Therefore, the fusion gene construct "CaMV 35S-OsHY5L2" of the present invention can be used as a gene resource to inhibit panicle germination in rice through molecular genetic modification methods, and has important application value.

[0079] Under the premise that no conflict occurs, those skilled in the art may freely combine and superimpose the above-mentioned additional technical features.

[0080] The above descriptions are only preferred embodiments of the present invention. Any technical solution that achieves the purpose of the present invention by substantially the same means shall fall within the scope of protection of the present invention.

Claims

1. A method for inhibiting rice ear germination <h2 style=";text-align:left;direction:ltr">OsHY5L2 The application of genes is characterized by: Using this gene to construct <h2 style=";text-align:left;direction:ltr"> CaMV <h2 style=";text-align:left;direction:ltr"> 35S-OsHY5L2 " fusion gene, which is transformed into Nipponbare rice to inhibit the panicle germination of transgenic Nipponbare rice lines under high humidity conditions during the mature harvest period, <h2 style=";text-align:left;direction:ltr"> OsHY5L2 The coding sequence of the gene is shown in SEQ ID NO: 1.

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