Application and method of oself3-2 gene in controlling heading date of rice
By overexpressing the OsELF3-2 gene in rice and using Agrobacterium-mediated genetic transformation, the problem of controlling the heading period of rice was solved, resulting in delayed heading, increased plant height, enhanced dry matter accumulation, and improved crop yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA NAT RICE RES INST
- Filing Date
- 2025-05-29
- Publication Date
- 2026-07-24
AI Technical Summary
Existing technologies make it difficult to precisely control the heading stage of rice, which makes it difficult to balance the dry matter accumulation and yield, thus affecting crop yield.
Overexpression of the OsELF3-2 gene in rice was achieved by constructing the OsELF3-2 gene into a vector through Agrobacterium-mediated genetic transformation. Positive transgenic rice plants were screened, and the OsELF3-2 gene inhibited rice heading under both short-day and long-day conditions.
Delaying the heading date of rice resulted in a significantly late heading phenotype, increased plant height, and expression of the OsELF3-2 gene in various tissues. By promoting the expression of Ghd7 and inhibiting Ehd1, the OsELF3-2 gene enriched the photoperiodic flowering regulatory network of rice.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering, and in particular to the application of the OsELF3-2 gene in controlling the heading stage of rice and its application method. Background Technology
[0002] Heading date is a crucial agronomic trait for controlling a variety's regional adaptability. Precise control of flowering time is essential for the reproductive transformation of a variety, thus affecting crop yield. During the appropriate growing season, late heading leads to a longer vegetative growth period, promoting dry matter accumulation in the seeds; however, excessively late heading may result in lower seed maturity at harvest. Conversely, for crops with short growing seasons, early heading is beneficial, but premature heading shortens the vegetative growth period, leading to yield reduction. Therefore, maintaining a balance between dry matter accumulation and stress avoidance is vital for crop yield.
[0003] Rice heading is determined by both genetics and environment. Photoperiod sensitivity is a crucial factor in determining flowering time and regional adaptability, forming a complex network. Rice is a typical short-day crop, flowering earlier under short-day conditions. To date, over 35 genes / QTLs have been cloned in the rice photoperiodic flowering pathway. There are three photoperiodic flowering pathways in rice: a short-day promoting pathway, converging with Hd1 and Ehd1; and a long-day promoting and inhibiting pathway, primarily converging with Ehd1. Hd1 is a homolog of the Arabidopsis flowering activator CONSTANS(CO), encoding a transcription factor containing two B-box domains and one CCT domain. Under short-day conditions, it promotes flowering by activating Hd3a, while under long-day conditions, it inhibits Hd3a, thus delaying heading. Ehd1 encodes a B-type response regulator that promotes heading under both short-day and long-day conditions by enhancing the expression of Hd3a and RFT1.
[0004] Ehd1 is an integrative factor for rice flowering, and many flowering inhibitors (such as Ghd7, DTH8 / Ghd8, OsCOL4, and Hd16 / EL1) and flowering promoters (such as RID1 / OsID1 / Ehd2, Ehd3, Ehd4, OsMADS50, OsMADS51, Hd17 / OsELF3, and OsFKF1) converge on it. Ghd7 encodes a CCT domain protein that plays an important role in the repression pathway. Ghd7 simultaneously receives signals from multiple genes, such as Hd16 / EL1, Ehd3, Hd17 / OsELF3, and OsFKF1, to regulate Ehd1 expression. Under both short-day and long-day conditions, Ehd2 and Ehd4 positively regulate Ehd1 expression. Hd17 / OsELF3 is a homolog of Arabidopsis thaliana ELF3, which negatively regulates Ghd7 under long-day conditions to promote flowering.
[0005] As a homolog of the Arabidopsis thaliana ELF3 gene in rice, OsELF3 plays a crucial role in regulating rice growth, development, and environmental adaptability. This gene family comprises two members, named OsELF3-1 and OsELF3-2. OsELF3-1 is mainly involved in regulating the rice circadian rhythm and heading stage, while OsELF3-2 is mainly involved in regulating the rice immune response and heading stage. As core regulators of the circadian rhythm system, they coordinate the rice's response to environmental signals.
[0006] Studies have shown that both OsELF3-1 and OsELF3-2 can interact with OsLUX and form two EC complexes with OsELF4, regulating the expression of downstream genes at the transcriptional level, thereby coordinating the diurnal rhythm and photoperiodic response of plants. In addition to interacting with OsLUX, OsELF3-2 also interacts with the photoperiodic regulatory repressor OsCOL5, participating in the flowering regulation of the rice photoperiodic pathway. Summary of the Invention
[0007] In view of this, the present invention provides the application of the OsELF3-2 gene in controlling the heading period of rice and its application method.
[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0009] The application of the OsELF3-2 gene in delaying the heading stage of rice: overexpression of the OsELF3-2 gene inhibited rice heading under both short-day and long-day conditions; the cDNA sequence of the OsELF3-2 gene is shown in Sequence 1.
[0010] The method for applying the OsELF3-2 gene to delay the heading stage of rice involves constructing the OsELF3-2 gene into the vector pCUbi1390, transforming rice with the obtained recombinant vector, and screening for positive transgenic rice plants.
[0011] Preferably, rice is transformed using Agrobacterium-mediated genetic transformation. The transformed material undergoes co-culture, screening, differentiation, rooting, and hardening-up of transgenic seedlings before transplanting, and positive transgenic rice plants are selected.
[0012] Preferably, the Agrobacterium is EHA105.
[0013] The present invention achieves the following technical effects compared to the prior art:
[0014] (1) This invention achieves a significant late heading phenotype by overexpressing the OsELF3-2 gene in rice. This gene inhibits flowering under both short-day and long-day conditions.
[0015] (2) Under natural long-day conditions in Hangzhou, compared with wild-type plants, transgenic positive plants were about 47 days later in heading and taller; under natural short-day conditions in Hainan, transgenic positive plants were about 63 days later in heading than wild-type plants; through rhythmic expression pattern analysis, it was found that the OsELF3-2 gene showed obvious diurnal rhythmic expression.
[0016] (3) Tissue expression studies have found that the OsELF3-2 gene is expressed in all tissues and has a high expression level during the grain-filling stage. qRT-PCR experiments have found that the OsELF3-2 gene is located upstream of the rice flowering inhibitor Ghd7. By promoting the expression of Ghd7, it inhibits the expression of Ehd1 and ultimately inhibits flowering. This result further enriches the photoperiodic flowering regulation network of rice. Attached Figure Description
[0017] Figure 1 The phenotype of the OsELF3-2 gene overexpressing plant in Example 1 of this invention;
[0018] In this diagram, A represents the heading phenotype of Nipponbare (left) and OsELF3-2 overexpressing transgenic negative plant (right) under natural long-day conditions; B represents the heading phenotype of Nipponbare (left) and OsELF3-2 overexpressing transgenic negative plant (right) under natural short-day conditions; and C represents the heading time of OsELF3-2 overexpressing plants under natural short-day and natural long-day conditions.
[0019] Under NLD and NSD conditions, the number of plants with Nip and OsELF3-2 overexpression at the heading stage was n=40.
[0020] Figure 2 Analysis of the expression pattern of the OsELF3-2 gene in Example 2 of this invention;
[0021] In Figures A, B, and C, AB represents the rhythmic expression pattern of the OsELF3-2 gene under long-day and short-day conditions. Figure C shows the expression level of the OsELF3-2 gene in different tissues at three different time stages, analyzed using qRT-PCR. The transcriptional level of the OsELF3-2 gene in Figures A, B, and C is the ratio to that of the rice UBQ (Ubiquitin) gene. White and black boxes represent the light and dark periods, respectively. The mean ± sd was obtained from three biological replicates and three technical replicates.
[0022] Figure 3The rhythmic expression patterns of Hd1, Ehd1, Hd3a, RFT1, Ghd7, Ehd2, Ehd4, and OsGI in Nipponbare and OsELF3-2 overexpressing plants under long-day (A, C, E, G, I, K, M, O) and short-day (B, D, F, H, J, L, N, P) conditions in Examples 3 and 4 of this invention are shown. White and black boxes represent photoperiod and darkperiod, respectively. The mean ± sd was obtained from three biological replicates and three technical replicates. Detailed Implementation
[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] This invention discloses the application of the OsELF3-2 gene in delaying the heading stage of rice. Overexpression of the OsELF3-2 gene inhibits rice heading under both short-day and long-day conditions. The cDNA sequence of the OsELF3-2 gene is shown in Sequence 1.
[0025] There are two Arabidopsis thaliana ELF3 homologs in rice: OsELF3-1 and OsELF3-2. OsELF3-1 has been studied in terms of heading date, while OsELF3-2 has been studied less extensively. OsELF3-2 (accession number in the RAP-DB database is Os01g0566100, and accession number in the RGAP database is LOC_Os01g38530.1).
[0026] The OsELF3-2 gene for controlling heading date in rice, provided by this invention, is derived from the rice variety Nipponbare. Its cDNA sequence is shown in Sequence 1, with a length of 3139 bp, and its coding region (CDS) sequence is shown in Sequence 2. Studies have found that under short-day and long-day conditions, the OsELF3-2 gene inhibits the expression of Ehd1, Hd3a, and RFT1 by upregulating Ghd7 and OsGI, ultimately delaying heading.
[0027] This invention also discloses a method for applying the OsELF3-2 gene in delaying the heading stage of rice. The OsELF3-2 gene is constructed into the vector pCUbi1390, and the resulting recombinant vector is used to transform rice, and positive transgenic rice plants are screened.
[0028] Rice was transformed using Agrobacterium-mediated genetic transformation. The transformed material underwent co-culture, screening, differentiation, rooting, and hardening-up of transgenic seedlings before transplanting. Positive transgenic rice plants were then selected.
[0029] Agrobacterium is EHA105.
[0030] Sequence 1:
[0031]
[0032] Sequence 2:
[0033]
[0034] Example 1: Overexpression of OsELF3-2 gene delays heading
[0035] 1. Construction of overexpression vectors
[0036] To obtain the phenotype of rice overexpressing the OsELF3-2 gene, an overexpression vector was constructed in this embodiment. The specific construction method is as follows: Wild-type rice variety Nipponbare (whose whole genome has been sequenced) was planted for approximately two weeks. RNA was extracted using a plant RNA mini kit (purchased from Tiangen Biotech Co., Ltd.), and then reverse transcribed into cDNA. The full-length cDNA sequence of OsELF3-2 was obtained by PCR amplification using primers 1390-OsELF3-2-BamHI-F and 1390-OsELF3-2-BamHI-R. The primer sequences are shown in Table 1. Then, this fragment was recombined into the BamHI site of plasmid pCUbi1390 (purchased from Fermentas, Canada) using homologous recombination.
[0037] Table 1: Vector Primer Sequences
[0038]
[0039] 2. Agrobacterium-mediated genetic transformation of rice
[0040] The correctly sequenced recombinant plasmids were transformed into callus tissue of Nipponbare rice using the Agrobacterium-mediated rice genetic transformation system mediated by strain EHA105 (purchased from CAMBIA). Following callus induction, subculture, pre-culture, infection, co-culture, screening for hygromycin-resistant callus, differentiation, rooting, hardening, and transplantation, transgenic plants were obtained. The Agrobacterium-mediated genetic transformation system for japonica rice mainly followed the method reported by Hiei et al., with slight modifications.
[0041] 3. Detection of transgenic plants and verification of gene function
[0042] The results showed that overexpression of the OsELF3-2 gene in rice led to a significant late heading phenotype. Under natural long-day conditions in Hangzhou, PCR molecular detection was performed using primers 1390-F and 1390-R. The sequencing primer sequences are shown in Table 2. Compared with wild-type plants, transgenic positive plants headed approximately 47 days later and were taller. Figure 1 As shown in Figure A. Under the natural short-day conditions in Hainan, the phenotype of the transgenic positive plants was the same as that in Hangzhou, exhibiting delayed heading (approximately 63 days later) and increased plant height, as shown in Figure A. Figure 1 As shown in B. The heading date data of transgenic positive plants under natural long-day conditions in Hangzhou and natural short-day conditions in Hainan are as follows. Figure 1 As shown in C.
[0043] Table 2: Sequencing Primer Sequences
[0044]
[0045] Example 2: Spatiotemporal expression pattern of the OsELF3-2 gene
[0046] To investigate the rhythmic expression pattern of OsELF3-2, we studied the expression level of the OsELF3-2 gene using quantitative real-time PCR (qRT-PCR). The primer sequences were OsELF3-2-qRTF and OsELF3-2-qRTR. Leaves were collected every 4 hours under short-day (10 hours light, 14 hours dark) and long-day (14 hours light, 10 hours dark) conditions in a 48-hour cycle. Under LD conditions, OsELF3-2 expression exhibited a pattern of initial depletion followed by accumulation after both light and dark periods, reaching its lowest point after 4 hours of light and 6 hours of darkness. Figure 2 As shown in Figure A; under SD conditions, the expression level of OsELF3-2 gradually decreased after darkness, reaching its lowest level 4 hours after transitioning from darkness to light, and then gradually accumulated over time, as shown in Figure A. Figure 2 As shown in B. These results demonstrate that OsELF3-2 transcriptional levels exhibit significant rhythmic expression.
[0047] To investigate the spatial expression pattern of OsELF3-2, we used qRT-PCR to detect the expression levels of OsELF3-2 in various tissues of rice, such as leaves, leaf sheaths, panicles, and stems. Figure 2 As shown in Figure C. Primers OsELF3-2-qRTF and OsELF3-2-qRTR were used. Results showed that OsELF3-2 was expressed in all tissues of rice, and was expressed at three stages: booting, heading, and grain-filling, with the highest expression level at the grain-filling stage. During the booting stage, the highest expression level was found in leaves; during the heading stage, the highest expression level was found in leaf sheaths, followed by leaves; and during the grain-filling stage, the highest expression level was found in panicles, followed by stems, and then leaves.
[0048] Example 3: Downregulating Ehd1 in the OsELF3-2 gene to inhibit flowering
[0049] The photoperiodic response and rhythmic expression characteristics of OsELF3-2 suggest that this gene may be involved in the regulation of photoperiodic flowering. To investigate the role of OsELF3-2 in the photoperiodic flowering regulation pathway in rice, we used qRT-PCR to detect the expression of related genes involved in the rice photoperiodic pathway in Nipponbare and OsELF3-2 overexpressing transgenic negative plants under short-day and long-day conditions. Primer sequences are shown in Table 3. Leaves were selected from rice plants treated with short-day for 40 days and long-day for 50 days, respectively.
[0050] Hd1 expression levels in OsELF3-2 overexpressing plants were significantly higher than those in WT plants, especially under long-day conditions. However, the rhythmic expression of Hd1 in OsELF3-2 overexpressing plants and WT plants was similar. Figure 3 As shown in Figure AB, OsELF3-2 is located upstream of Hd1 and promotes its expression. The expression level of Ehd1 in OsELF3-2 overexpressing plants was significantly lower than that in WT plants. Figure 3 As shown in CD, the expression levels of Hd3a and RFT1 were also significantly reduced in overexpressing plants, such as Figure 3 As shown in EH, OsELF3-2 is located upstream of Ehd1, Hd3a and RFT1 and inhibits their expression. The qRT-PCR primer sequences are shown in Table 3.
[0051] Table 3: qRT-PCR primer sequences
[0052]
[0053] Example 4: The OsELF3-2 gene is located upstream of the Ghd7 gene.
[0054] To further investigate the photoperiodic flowering pathway involved in OsELF3-2-Ehd1, we studied the expression levels of upstream regulatory genes of Ehd1. We examined the expression levels of upstream regulatory factors of Ehd1, such as Ghd7, Ehd2, Ehd4, and OsGI. The results showed that in OsELF3-2 overexpressing plants, the transcription levels of Ghd7 and OsGI were significantly increased, such as... Figure 3 As shown in IJ and OP, the expression levels of other Ehd1 regulators, such as Ehd2 and Ehd4, were unaffected. Figure 3 As shown in IN. These results indicate that OsELF3-2 functions upstream of Ghd7 and OsGI, delaying heading by promoting the expression of Ghd7 and OsGI and inhibiting the expression of Ehd1, Hd3a, and RFT1.
[0055] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.
Claims
1. The application of the OsELF3-2 gene in delaying the heading stage of rice, characterized by, Overexpression of the OsELF3-2 gene inhibited rice heading under both short-day and long-day conditions; the cDNA sequence of the OsELF3-2 gene is shown in the sequence SED ID NO:
1.
2. The method for applying the OsELF3-2 gene to delay the heading stage of rice, characterized in that, The OsELF3-2 gene was constructed into the vector pCUbi1390, and the resulting recombinant vector was used to transform rice. Positive transgenic rice plants were screened. The cDNA sequence of the OsELF3-2 gene is shown in the sequence SED ID NO:
1.
3. The method for applying the OsELF3-2 gene in delayed heading stage of rice according to claim 2, characterized in that, Rice was transformed using Agrobacterium-mediated genetic transformation. The transformed material underwent co-culture, screening, differentiation, rooting, and hardening-up of transgenic seedlings before transplanting. Positive transgenic rice plants were then selected.
4. The method for applying the OsELF3-2 gene in delaying the heading stage of rice according to claim 3, characterized in that, The Agrobacterium was EHA105.