Polyploid rice high seed gene osths-a1 and application thereof

Abstract

The application belongs to the technical field of rice genetic engineering, and particularly relates to a polyploid rice high-fruiting gene OsTHS-A1 and application thereof in improving the fruiting rate of polyploid rice. OsTHS-A1 is highly expressed during the meiosis of rice, the nucleotide sequence of the CDS region of the gene is shown in the sequence table SEQ ID NO. 1, and the amino acid sequence of the protein encoded by the gene is shown in the sequence table SEQ ID NO. 2. Function verification shows that the expression of the OsTHS-A1 gene in high-fruiting polyploid rice is inhibited, and the fruiting rate is significantly reduced; while the OsTHS-A1 gene is overexpressed in low-fruiting polyploid rice, and the fruiting rate is significantly improved. The fruiting rate of low-fruiting polyploid rice is effectively improved by overexpressing the OsTHS-A1 gene.

Description

Technical Field

[0001] The invention belongs to the technical field of rice genetic engineering and relates to a polyploid rice high-fruiting gene OsTHS-A1 and an application thereof. Background Art

[0002] Rice is one of the world's three major staple crops, providing a stable food source for more than half of the world's population (Chen et al. 2022). It is estimated that the world population will reach 9 billion by 2050. To meet the global food and nutritional needs by then, rice yields must increase by at least 60% (Khush 2013). However, after experiencing two major leaps in rice yield, from tall to dwarf rice and from conventional rice to hybrid rice, rice yields have long eluded significant breakthroughs. Polyploid rice, with its advantages of sturdy stems, large grains, and strong stress resistance, has the potential to significantly increase yields. However, its low seed set rate has long hindered its application. Therefore, improving the seed set rate of polyploid rice is key to its production and application.

[0003] Based on the principles of allopolyploidization in natural plants, Cai Detian et al. (2001) proposed a new breeding strategy: "Exploiting the dual advantages of distant hybridization and polyploidy to breed super rice." This strategy employed methods such as increasing kinship distance, reducing unstable chromosome pairings, and utilizing specialized genes to genetically improve the seed set rate of polyploid rice. Guided by this strategy, extensive interspecific hybridization, backcrossing, and selection between indica and japonica subspecies resulted in the development of polyploid meiotically stable (PMeS) lines. PMeS lines possess three notable characteristics: 1) Meiotic stability: bivalent pairings are predominant during prophase I of meiosis, with unstable pairings such as univalents and trivalents rarely occurring, and lagging chromosomes are rarely present during anaphase I. 2) High seed set: over 70% seed set. 3) Meiotic stability and high fruit set can be stably inherited, and hybrid offspring with low-fruit set autotetraploid indica or japonica rice varieties can produce high-fruit set rates (Cai Detian et al. 2007). The selection and breeding practice of PMeS lines show that the bottleneck problem of low fruit set rate that has long plagued polyploid rice breeding has been broken through, laying the foundation for the production and application of polyploid rice and significantly increasing rice yields (Song Zhaojian et al. 2023; Li Hangyu et al. 2024). High-fruit set PMeS lines are the key to realizing the production and application of polyploid rice. Therefore, a deep understanding of the meiotic stability and high fruit set mechanism of PMeS lines can provide a theoretical basis and practical guidance for polyploid rice breeding. Based on this, the present invention provides a polyploid rice high-fruit set gene OsTHS-A1 and its application. Summary of the Invention

[0004] In view of the defects of the prior art and the demand for improvement, the purpose of the present application is to provide a polyploid rice high seed setting gene OsTHS-A1 and its application.

[0005] To achieve the above-mentioned purpose, the present application provides a polyploid rice high seed setting gene OsTHS-A1, wherein the CDS region nucleotide sequence of the OsTHS-A1 gene is shown in SEQ ID NO. 1.

[0006] Further, the amino acid sequence of the protein encoded by the OsTHS-A1 gene is shown in SEQ ID NO. 2.

[0007] On the other hand, the application of the OsTHS-A1 gene in improving the seed setting rate of polyploid rice, wherein the CDS region nucleotide sequence of the OsTHS-A1 gene is shown in SEQ ID NO. 1.

[0008] Further, the CDS region nucleotide sequence of the OsTHS-A1 gene is shown in SEQ ID NO. 1.

[0009] Further, the OsTHS-A1 gene improves the seed setting rate of low seed setting polyploid rice through overexpression.

[0010] Overall, the above technical solutions conceived by the present application can achieve the following beneficial effects:

[0011] The present application clones a high seed setting gene OsTHS-A1 from a high seed setting polyploid rice PMeS line, and uses overexpression and suppression expression genetic transformation technology to transform low seed setting and high seed setting polyploid rice, respectively, to verify its function in regulating the seed setting rate of polyploid rice, and to use the gene to improve the seed setting rate of low seed setting polyploid rice; the OsTHS-A1 gene in the present application is highly expressed during the meiosis period of rice, which may affect the meiosis of polyploid rice, thereby affecting pollen fertility and activity, and further affecting the seed setting rate. Therefore, the meiosis behavior of polyploid rice can be stabilized by the expression of the OsTHS-A1 gene, the pollen fertility and activity are improved, and thus the seed setting rate of low seed setting polyploid rice is effectively improved. BRIEF DESCRIPTION OF DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments or prior art of the present application, the drawings needed in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0013] Figure 1 The structure of the OsTHS-A1 gene;

[0014] Figure 2 The expression pattern of OsTHS-A1 gene was analyzed. S4: differentiation period of stamen and pistil primordium; S5: formation period of pollen mother cell; S6: meiosis period of pollen mother cell; S7: pollen grain content filling period;

[0015] Figure 3 It is an overexpression vector of OsTHS-A1 gene;

[0016] Figure 4 It is an RNA interference vector of OsTHS-A1 gene;

[0017] Figure 5 To detect the expression level of OsTHS-A1 gene in overexpressed transformed plants;

[0018] Figure 6 To detect the expression level of OsTHS-A1 gene in the transformed plants. DETAILED DESCRIPTION

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0020] Example 1: Cloning and sequence analysis of the OsTHS-A1 gene

[0021] Based on genomic BSA sequencing and mapping of extreme polyploid rice F2 populations with high and low fruiting potential, and quantitative PCR screening of candidate genes, a candidate gene for high fruiting potential in polyploid rice was identified. Total RNA was extracted from meiotic panicles of the high-fruiting polyploid rice PMeS line A1-4x, and genomic DNA contamination was removed using DNase I. The RNA was then reverse-transcribed into cDNA using the ABclonal RNA Reverse Transcription Kit. Specific primers were designed based on BLAST sequence alignments from the NCBI website, and amplification was performed using Gimmico Q6 PCR Master Mix (1.1×).

[0022] The primer sequences are:

[0023] OsTHS-A1-F: 5'-CTCGGCTGGCTACATCTTCT-3'

[0024] OsTHS-A1-R:5'-GTCCCGTTGCAGGAGACAAT-3'

[0025] The full-length CDS sequence of the gene (1194 bp) was cloned and named OsTHS-A1. The gene structure is shown in Figure 1 The OsTHS-A1 gene is located on rice chromosome 3. The full-length CDS sequence is shown in SEQ ID NO. 1; it encodes 397 amino acids, as shown in SEQ ID NO. 2. The protein has a molecular weight of 44486.98 and an isoelectric point (PI) of 3.23.

[0026] Example 2: Analysis of OsTHS-A1 gene expression pattern

[0027] The spatiotemporal expression patterns of the OsTHS-A1 gene in A1-4x were analyzed, primarily in roots, stems, leaves, and panicles during the four stages of stamen primordium differentiation (S4), pollen mother cell formation (S5), pollen mother cell meiosis (S6), and pollen grain filling (S7). Total RNA was extracted from each tissue and reverse transcribed into cDNA. The expression of the OsTHS-A1 gene was quantitatively analyzed by qRT-PCR using an Agilent Stratagene Mx3000P real-time fluorescence quantitative PCR instrument. The primer sequences were as follows:

[0028] QP-OsTHS-A1-F:5'-GGGAATCTACCCTTGTATGGC-3'

[0029] QP-OsTHS-A1-R: 5'-AATAAGTTGTGGCTGGGAGTG-3'

[0030] The sequences of the internal reference gene primers are:

[0031] UBQ5-F:5'-ACCACTTCGACCGCCACTACT-3'

[0032] UBQ5-R:5'-ACGCCTAAGCCTGCTGGTT-3'

[0033] The results of spatiotemporal expression analysis showed that the expression level of OsTHS-A1 gene was generally low in roots, stems, and leaves during the three stages except the root stage during the pollen mother cell formation period; the expression level was low in the young panicle during the stamen primordium differentiation period and the pollen grain filling period; the expression level was high in the root and young panicle during the pollen mother cell formation period; the expression level was highest in the young panicle during the pollen mother cell meiosis period, and it was the highest expression level in all tissues during the four stages ( Figure 2 ).

[0034] Example 3: Functional verification of the OsTHS-A1 gene

[0035] To verify the function of the OsTHS-A1 gene from both the overexpression and expression inhibition directions, an OsTHS-A1 gene overexpression vector and an RNA interference vector were constructed. The low-fruiting polyploid rice Nipponbare-4x and the high-fruiting polyploid rice A1-4x were transformed into transformed plants, and the phenotypes of the transformed plants were investigated to verify the gene function.

[0036] 1. Construction of genetic transformation vector

[0037] The overexpression vector used in the present invention is pBWA(v)HS, which is modified based on the pCAMBIA1300s skeleton. The vector carries the right border (RB) and left border (LB) of TD NA, and can insert foreign genes into the host plant genome through Agrobacterium-mediated transformation. The vector contains the strong CaMV 35S promoter, which can efficiently express the inserted gene. The total cDNA of the polyploid rice high-fruiting PMeS variety A1-4x was used as a template, and the full-length cDNA of OsTHS-A1 was amplified using primers OsTHS-A1-F and OsTHS-A1-R. The full-length cDNA was ligated to the TA cloning vector Seamless Cloning Kit (Beijing Qingke Biotechnology Co., Ltd.) was used to transform into competent E. coli, screen the positive clones and send them for sequencing. First, the target fragment that was sequenced correctly was double-digested with restriction endonucleases Not I and EcoR I and recovered from the gel. Then, it was ligated into pBWA(v)HS using T4 DNA ligase to finally obtain the overexpression vector OsTHS-A1-O E ( Figure 3 ).

[0038] The RNA interference vector used in the present invention is pBWA(v)HS. Using the TA cloning vector into which the target fragment was transferred as a template, RNAi-F1 (5'-CAGTGGTCTCACAACAAGTTCCGACCTGCTCTAAACGAGG-3') and RNAi-R1

[0039] (5'-GATGGTCTCACAGGCTCTCCATAACAAAAAGACTGCTGCCAGC-3') amplified a 245 bp cDNA fragment, in which the restriction endonuclease Eco31 I enzyme cutting site is "GGTCTC". This fragment was ligated into the TA cloning vector The cells were transformed into competent E. coli using the Seamless Cloning Kit, and positive clones were screened and sent for sequencing. The target fragments that were sequenced correctly were digested with restriction endonuclease Eco31 I and recovered from the gel to obtain the hairpin forward strand. The same method was used to generate the target fragments using RNAi-F2 (5'-GATGGTCTCACAGGCTCTCCATAACAAAAAGACTGCTGCCAGC-3') and RNAi-R2.

[0040] (5'-CAGTGGTCTCACAACAAGTTCCGACCTGCTCTAAACGAGG-3') was used to obtain the reverse strand of the hairpin. The forward strand and the reverse strand of the hairpin were connected using T4 DNA ligase through the middle loop structure (Wuhan Boyuan Biotechnology Co., Ltd.), and the gel was recovered and detected. After the sequencing was correct, it was ligated to the linearized pBWA(v)HS plasmid obtained by digestion with the restriction endonuclease Eco31 I, and the positive clones were selected for sequencing verification, and finally the RNA interference vector OsTHS-A1-RNAi ( Figure 4 ).

[0041] 2. Genetic transformation

[0042] Agrobacterium-mediated transfection of OsTHS-A1-O E and OsTHS-A1-RN A i into the low-fruiting polyploid rice Nipponbare-4x and the high-fruiting polyploid rice A1-4x, respectively, was performed. The specific steps are as follows:

[0043] (1) Callus induction and pre-culture

[0044] Remove the husks from mature seeds of Nipponbare-4x and A1-4x, excise the embryos, treat with 75% alcohol for 1 minute, and then disinfect by soaking in 0.1% mercuric chloride (HgCl2) for 30 minutes, shaking every 5 minutes. After disinfection, rinse five times with sterile distilled water. Inoculate the embryos into induction medium and incubate in the dark at 28°C until callus forms. Select yellow, granular, and vigorous callus and transfer it to pre-culture medium for 5–8 days.

[0045] (2) Agrobacterium activation and suspension culture

[0046] Agrobacterium EHA105 carrying the OsTHS-A1-O E or OsTHS-A1-RN A i vector was cultured on LB solid medium of the corresponding resistance at 28°C. The activated Agrobacterium was transferred to a suspension culture medium and cultured on a shaker at 28°C, 200 rpm, for 3 hours. The OD600 value was detected to be approximately 0.5.

[0047] (3) Agrobacterium suspension infection and co-cultivation

[0048] In a super-clean bench, the cultured callus was transferred into Agrobacterium suspension for 3-5 min of infection; the callus was taken out and dried with sterile filter paper, and then transferred into co-culture medium for dark culture for 2-3 d, and then transferred into a culture box for culture.

[0049] (4) Water washing and screening

[0050] The callus after co-culture was transferred into a sterile triangular flask, washed with sterile distilled water for more than 6 times until the washed water became clear, 200 mL of sterile distilled water containing 500 mg / L Cn was added, and soaked at room temperature for 30 min. The callus was taken out and dried with sterile filter paper, and then transferred into a screening medium, and dark cultured at 26°C for 15 d.

[0051] (5) Second screening

[0052] The dry and Agrobacterium-contaminated callus particles from the first screening medium were selected and transferred into a new screening medium. The callus was dark cultured at 26°C for 25 d, and the growth condition of the callus was observed frequently during the process, and once no growth of the callus was found, the callus was timely transferred into a new screening medium.

[0053] (6) Differentiation, rooting, transplanting and positive verification

[0054] The callus with good growth in the screening medium was selected and transferred into a differentiation medium, and dark cultured in a 26°C culture box for 5-7 d, and then cultured at 28°C under normal light for 30 d until seedlings were differentiated. The seedlings were transferred into a rooting medium, and cultured under normal light at 28°C for 15-25 d. After rooting was completed, the seedlings were hardened for 3-5 d, and then transplanted into a field. The transplanted plants were grown in the field for 2-3 weeks, and then the leaves of the transplanted plants were taken for positive identification.

[0055] The finally obtained overexpression transplanted plants in the application were named as NIP-OE, and 16 positive seedlings were obtained. The NIP-OE meiosis stage young panicles were taken, total RNA was extracted, and the expression amount of the OsTHS-A1 gene in the transplanted plants was detected by qRT-PCR technology. The results showed that the expression amount of the OsTHS-A1 gene in the transplanted plants NIP-OE was significantly higher than that of wild type Nipponbare-4x (NIP-WT), and was 2.86 times of that of NIP-WT. Figure 5

[0056] ​The finally obtained expression-inhibited transformed plant was named A1-RN A i, and a total of 9 positive seedlings were obtained. The young spikelets of A1-RN A i in the meiotic stage were taken, total RNA was extracted, and the expression level of the OsTHS-A1 gene in the transformed plants was detected by qRT-PCR technology. The results showed that the expression level of the OsTHS-A1 gene in the transformed plant A1-RN A i was significantly reduced compared with the wild type A1-4x (A1-WT), which was 0.52 times that of A1-WT ( Figure 6 ).

[0057] 3. Phenotypic Analysis of Transformed Plants

[0058] The phenotypes of transformed plants with overexpression and suppression of the OsTHS-A1 gene were investigated to understand the effect of the OsTHS-A1 gene on the seed setting rate of polyploid rice and to determine the function of the OsTHS-A1 gene.

[0059] Agronomic trait assessments showed that NIP-OE plants exhibited higher plant height, panicle number, panicle length, grain length, grain width, and total number of grains per panicle than NIP-WT plants, although the differences were not significant. However, the number of filled grains per panicle and seed set rate were significantly higher than those of NIP-WT plants. The seed set rate of NIP-WT plants was only 9.55%, while that of NIP-OE plants was 42.24%, significantly higher than that of the wild type, indicating that overexpression of the OsTHS-A1 gene can effectively improve the seed set rate of polyploid rice. Plant height, panicle number, panicle length, grain length, grain width, total number of grains per panicle, filled grains per panicle, and seed set rate of A1-RNAi plants were all lower than those of A1-WT plants, with significant differences in plant height, total number of grains per panicle, filled grains per panicle, and seed set rate. The fruit setting rate of A1-WT plants was 86.48%, while that of A1-RNAi plants was only 54.15%, which was significantly lower than that of the wild type, indicating that the inhibition of OsTHS-A1 gene expression will lead to a decrease in the fruit setting rate of polyploid rice.

[0060] Observations of pollen fertility, viability, and meiotic behavior of pollen mother cells in A1-RNAi and A1-WT plants revealed that the fertile and viable pollen rates of A1-WT plants were 85.39% and 88.65%, respectively, while those of A1-RNAi plants were 55.51% and 40.27%, respectively, significantly lower than those of the wild type. A1-RNAi plants exhibited a high incidence of unstable pairings, such as univalents and trivalents, during prophase I of meiosis, and a high incidence of lagging chromosomes during metaphase I and anaphase I. In contrast, A1-WT plants, belonging to the PMeS lineage, exhibit meiotic stability, with bivalent pairing predominantly occurring, with few abnormalities such as univalents, trivalents, and lagging chromosomes.

[0061] The applicant declares that the present invention uses the above-described embodiments to illustrate the products and detailed preparation methods of the present invention. However, the present invention is not limited to the above-described products and detailed preparation methods. This does not mean that the present invention must rely on the above-described products and detailed preparation methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for the raw materials of the products of the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0062] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above embodiments. Within the technical concept of the present invention, various simple variations can be made to the technical solution of the present invention. These simple variations all fall within the scope of protection of the present invention.

[0063] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0064] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. The application of the OsTHS-A1 gene in improving the seed setting rate of polyploid rice is characterized in that: The nucleotide sequence of the CDS region of the OsTHS-A1 gene is shown in SEQ ID NO.

1. The OsTHS-A1 gene improves the seed setting rate of low-seed polyploid rice through overexpression.

Images