Application and application method of OsELF3-2 gene in controlling heading stage of rice

Abstract

The invention discloses an application and an application method of an OsELF3-2 gene in controlling the heading stage of rice, the gene OsELF3-2 is from a rice variety Nipponbare, the cDNA sequence of the gene OsELF3-2 is shown as a sequence 1, and genetic transformation experiments show that overexpression of the OsELF3-2 gene inhibits heading of rice under short-day and long-day conditions; the invention finds that the OsELF3-2 gene is expressed as circadian rhythm expression, the transcriptional level of Ehd1 is inhibited by promoting the expression of Ghd7 and OsGI, and finally the expression of Hd3a / RFT1 is reduced to cause heading delay.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to an application of an OsELF3-2 gene in controlling the heading period of rice and an application method thereof. Background Art

[0002] Heading date is a key agronomic trait that controls a variety's regional adaptability. Precisely controlling flowering time is crucial for reproductive transformation, thereby impacting crop yield. In a suitable growing season, late heading results in a long vegetative period, promoting seed dry matter accumulation. However, heading too late may result in lower seed maturity at harvest. On the other hand, early heading is beneficial for crops with a short growing season, but premature heading shortens the vegetative period, resulting in reduced yield. Therefore, a balance between dry matter accumulation and stress avoidance is crucial for crop yield.

[0003] Heading in rice is determined by both genetics and the environment. Photoperiod sensitivity is a key factor in determining flowering time and regional adaptability, forming a complex network. Rice is a typical short-day crop that flowers early under short days. To date, over 35 genes / QTLs have been identified in the rice photoperiod flowering pathway. Three photoperiod flowering pathways exist in rice: the short-day-promoting pathway, which converges at Hd1 and Ehd1; and the long-day-promoting and long-day-repressing pathways, which primarily converge at Ehd1. Hd1, a homolog of the Arabidopsis flowering activator CONSTANS (CO), encodes a transcription factor containing two B-box domains and a CCT domain. It promotes flowering under short-day conditions by activating Hd3a, while repressing Hd3a under long-day conditions, thereby delaying heading. Ehd1 encodes a B-type response regulator that promotes heading under both short-day and long-day conditions by promoting the expression of Hd3a and RFT1.

[0004] Ehd1 serves as a rice flowering integrator. Numerous flowering repressors (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 Ehd1. Ghd7 encodes a CCT domain protein that plays a key role in the repression pathway. Ghd7 simultaneously receives multiple signals from other 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 homologous gene of Arabidopsis ELF3, which negatively regulates Ghd7 to promote flowering under long-day conditions.

[0005] As the rice homolog of the Arabidopsis thaliana ELF3 gene, OsELF3 plays a key role in regulating rice growth, development, and environmental adaptability. This gene family comprises two members, OsELF3-1 and OsELF3-2. OsELF3-1 primarily regulates the rice circadian clock and heading date, while OsELF3-2 regulates rice immune responses and heading date. As core regulators of the circadian clock system, these two genes coordinate rice's responses to environmental signals.

[0006] Studies have shown that both OsELF3-1 and OsELF3-2 interact with OsLUX and form two EC complexes with OsELF4, regulating the expression of downstream genes at the transcriptional level, thereby coordinating the plant's circadian rhythm and photoperiodic response. In addition to interacting with OsLUX, OsELF3-2 also interacts with the photoperiodic regulation inhibitor OsCOL5, participating in the flowering regulation of the rice photoperiod pathway. Summary of the Invention

[0007] In view of this, the present invention provides an application of the OsELF3-2 gene in controlling the heading period of rice and an application method.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The OsELF3-2 gene is used to delay the heading period 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 SEQ ID NO: 1.

[0010] The method for applying the OsELF3-2 gene in delaying the heading period of rice comprises constructing the OsELF3-2 gene into a vector pCUbi1390, transforming rice with the obtained recombinant vector, and screening positive transgenic rice plants.

[0011] Preferably, rice is transformed using Agrobacterium-mediated genetic transformation, and the transformed material undergoes co-cultivation-screening-differentiation-rooting-transgenic seedling hardening and transplanting to screen positive transgenic rice plants.

[0012] Preferably, the Agrobacterium is EHA105.

[0013] Compared with the prior art, the present invention has achieved the following technical effects:

[0014] (1) The present invention overexpresses the OsELF3-2 gene in rice, resulting in a significant late heading phenotype. This gene inhibits flowering under both short-day and long-day conditions.

[0015] (2) Under natural long-day conditions in Hangzhou, the transgenic positive plants reached heading about 47 days later and were taller than the wild-type plants. Under natural short-day conditions in Hainan, the heading period of the transgenic positive plants was about 63 days later than that of the wild-type plants. Analysis of the rhythmic expression pattern revealed that the OsELF3-2 gene exhibited a clear circadian rhythm of expression.

[0016] (3) Tissue expression studies found that the OsELF3-2 gene was expressed in all tissues, and its expression level was higher during the filling period. qRT-PCR experiments found that the OsELF3-2 gene was located upstream of the rice flowering inhibitor Ghd7, and inhibited the expression of Ehd1 by promoting the expression of Ghd7, ultimately inhibiting flowering. This result further enriched the rice photoperiod flowering regulation network. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The phenotype of the OsELF3-2 gene overexpressing plant in Example 1 of the present invention;

[0018] Among them, A represents the heading date phenotype of Nipponbare (left) and OsELF3-2 overexpressing transgenic negative plant (right) under natural long-day conditions, B represents the heading date phenotype of Nipponbare (left) and OsELF3-2 overexpressing transgenic negative plant (right) under natural short-day conditions, and C represents the heading date of OsELF3-2 overexpressing plants under natural short-day and natural long-day conditions.

[0019] The number of plants surveyed at the heading stage under NLD and NSD conditions with Nip and OsELF3-2 overexpression was n=40.

[0020] Figure 2 This is the expression pattern analysis of the OsELF3-2 gene in Example 2 of the present invention;

[0021] Figures AB represent the rhythmic expression patterns of the OsELF3-2 gene under long-day and short-day conditions, and Figure C shows the expression levels of the OsELF3-2 gene in various tissues during the three periods analyzed by qRT-PCR. In Figures A, B, and C, the transcript levels of the OsELF3-2 gene are expressed relative to those of the rice UBQ (Ubiquitin) gene. White and black boxes indicate the light and dark periods, respectively. Means ± SD are obtained from three biological replicates and three technical replicates.

[0022] Figure 3Figure 3 shows the 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 the present invention. The white and black boxes indicate the light period and dark period, respectively. The mean ± sd was obtained from three biological replicates and three technical replicates. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] The present invention discloses the application of the OsELF3-2 gene in delaying the heading period 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 ELF3 homologs in rice, OsELF3-1 and OsELF3-2. OsELF3-1 has been studied in terms of heading date, while OsELF3-2 has been less studied. OsELF3-2 (RAP-DB database accession number: Os01g0566100, RGAP database accession number: LOC_Os01g38530.1)

[0026] The OsELF3-2 gene, which controls rice heading date, is derived from the rice variety Nipponbare. Its cDNA sequence is shown in SEQ ID NO: 1, which is 3139 bp long, and its coding region (CDS) sequence is shown in SEQ ID NO: 2. Studies have shown 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] The present invention also discloses an application method of the OsELF3-2 gene in delaying the heading period of rice. The OsELF3-2 gene is constructed into a vector pCUbi1390, the obtained recombinant vector is used to transform rice, and positive transgenic rice plants are screened.

[0028] Rice is transformed using Agrobacterium-mediated genetic transformation. The transformed material undergoes co-cultivation-screening-differentiation-rooting-transgenic seedling training and transplanting to screen positive transgenic rice plants.

[0029] The Agrobacterium was EHA105.

[0030] Sequence 1:

[0031]

[0032] Sequence 2:

[0033]

[0034] Example 1: Overexpression of the OsELF3-2 gene delays heading

[0035] 1. Construction of overexpression vector

[0036] To achieve the phenotype of rice overexpressing the OsELF3-2 gene, an overexpression vector was constructed in this example. The specific construction method is as follows: Wild-type rice varieties (Nipponbare, whose entire genome has been sequenced) were grown until they were approximately two weeks old. RNA was extracted using a plant RNA mini kit (purchased from Tiangen Biotechnology Co., Ltd.), then reverse-transcribed into cDNA. The full-length cDNA sequence of OsELF3-2 was amplified by PCR using primers 1390-OsELF3-2-BamHI-F and 1390-OsELF3-2-BamHI-R. The primer sequences are shown in Table 1. This fragment was then recombined into the BamHI site of plasmid pCUbi1390 (purchased from Fermentas, Canada) via homologous recombination.

[0037] Table 1: Vector primer sequences

[0038]

[0039] 2. Agrobacterium-mediated genetic transformation of rice

[0040] The recombinant plasmid, whose sequencing results were correct, was then transformed into callus tissue of Nipponbare rice using the Agrobacterium tumefaciens strain EHA105 (purchased from CAMBIA). Transgenic plants were obtained through a process of callus induction, subculture, pre-culture, infection, co-cultivation, selection of hygromycin-resistant calli, differentiation, rooting, hardening, and transplantation. The Agrobacterium tumefaciens-mediated transformation system for japonica rice primarily employed the method reported by Hiei et al., with minor 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 resulted in 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 about 47 days later and had higher plant heights. Figure 1 As shown in A. Under the natural short-day conditions in Hainan, the phenotype of the transgenic positive plants was the same as that in Hangzhou, showing delayed heading, about 63 days later, and increased plant height, as shown in Figure 1 B. Under the natural long-day conditions in Hangzhou and the natural short-day conditions in Hainan, the heading date of transgenic positive plants is shown in 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 used real-time fluorescence quantitative PCR (qRT-PCR) to investigate the expression level of the OsELF3-2 gene. The primer sequences were OsELF3-2-qRTF and OsELF3-2-qRTR. Leaves were collected every 4 hours during a 48-hour cycle under short-day (10 hours light, 14 hours dark) and long-day (14 hours light, 10 hours dark) conditions in a light incubator. Under LD conditions, the expression level of OsELF3-2 showed an expression pattern of first consumption and then accumulation after darkness and light, reaching a low point after 4 hours of light and 6 hours of darkness. Figure 2 As shown in A; Under SD conditions, the expression level of OsELF3-2 gradually consumed after darkness, and the expression level of OsELF3-2 was the lowest 4 hours after darkness turned to light, and then gradually accumulated over time, as shown in Figure 2 As shown in B. These results indicate that OsELF3-2 transcription level exhibits obvious 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 rice tissues, such as leaves, sheaths, panicles, and stems. Figure 2 C. Using primers OsELF3-2-qRTF and OsELF3-2-qRTR, the results showed that OsELF3-2 was expressed in all rice tissues and was expressed at the booting, heading, and grain filling stages, with the highest expression during the grain filling stage. Expression was highest in leaves during the booting stage, and in leaf sheaths during the heading stage, followed by leaves. During the grain filling stage, expression was highest in the panicle, followed by the stem, and then the leaves.

[0048] Example 3: OsELF3-2 gene downregulates Ehd1 to inhibit flowering

[0049] OsELF3-2's photoperiod-responsive and rhythmic expression suggest that it may be involved in photoperiodic flowering regulation. To investigate the role of OsELF3-2 in the photoperiodic flowering regulation pathway in rice, we used qRT-PCR to examine the expression of genes involved in the rice photoperiod pathway in Nipponbare and OsELF3-2-overexpressing transgenic negative lines under short-day and long-day conditions. Primer sequences are shown in Table 3. Leaves were collected from rice plants treated with short-day (40 days) and long-day (50 days).

[0050] The expression level of Hd1 in OsELF3-2 overexpressing plants was significantly higher than that in WT plants, which was more obvious under long-day conditions. However, the rhythmic expression of Hd1 in OsELF3-2 overexpressing plants and WT plants was similar, such as Figure 3 As shown in AB, it shows that OsELF3-2 is located upstream of Hd1 and promotes its expression. The expression level of Ehd1 in OsELF3-2 overexpressing plants is 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 the overexpression plants. Figure 3 As shown in EH, it was shown that OsELF3-2 was located upstream of Ehd1, Hd3a, and RFT1 and inhibited their expression, where the qRT-PCR primer sequences are shown in Table 3;

[0051] Table 3: qRT-PCR primer sequences

[0052]

[0053] Example 4: OsELF3-2 gene is located upstream of Ghd7 gene

[0054] To further investigate the photoperiodic flowering pathway involved in OsELF3-2-Ehd1, we investigated the expression levels of Ehd1 upstream regulatory genes. We examined the expression levels of Ehd1 upstream regulatory factors such as Ghd7, Ehd2, Ehd4, and OsGI. We found that the transcription levels of Ghd7 and OsGI were significantly increased in OsELF3-2 overexpressing plants. Figure 3 As shown in IJ and OP, the expression levels of other Ehd1 regulatory factors, such as Ehd2 and Ehd4, were not affected. Figure 3 These results indicate that OsELF3-2 acts upstream of Ghd7 and OsGI, promoting the expression of Ghd7 and OsGI and inhibiting the expression of Ehd1, Hd3a, and RFT1, thereby delaying heading.

[0055] The above description is merely a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of the OsELF3-2 gene in delaying the heading period of rice, characterized in that: 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 SEQ ID NO:

1.

2. The method for using the OsELF3-2 gene to delay the heading period of rice is characterized by: The OsELF3-2 gene was constructed into the vector pCUbi1390, and the obtained recombinant vector was used to transform rice, and positive transgenic rice plants were screened.

3. The method for using the OsELF3-2 gene in delaying the heading period of rice according to claim 2, characterized in that: Rice is transformed using Agrobacterium-mediated genetic transformation. The transformed material undergoes co-cultivation-screening-differentiation-rooting-transgenic seedling training and transplanting to screen positive transgenic rice plants.

4. The method for using the OsELF3-2 gene in delaying the heading period of rice according to claim 3, characterized in that: The Agrobacterium is EHA105.

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