Polyploidy rice high-seed-setting gene OsTHS-A1 and application thereof

Abstract

The invention belongs to the technical field of rice gene engineering, and particularly relates to a polyploidy rice high-seed setting gene OsTHS-A1 and application thereof in increasing the seed setting rate of polyploidy rice. The OsTHS-A1 is highly expressed in a rice meiosis period, a nucleotide sequence of a CDS region of the gene is shown as SEQ ID NO.1 in a sequence table, and an amino acid sequence of a protein coded by the gene is shown as SEQ ID NO.2 in the sequence table. Functional verification shows that expression of the OsTHS-A1 gene in the high-seed-setting polyploidy rice is inhibited, and the seed setting rate is remarkably reduced; when the OsTHS-A1 gene is excessively expressed in the low-fructification polyploidy rice, the maturing rate is remarkably increased. Through over-expression of the OsTHS-A1 gene, the setting rate of the low-setting polyploidy rice is effectively improved.

Description

Technical Field

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

[0002] Rice is one of the three major food crops in the world, 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 in 2050. By then, to meet the food and nutrition needs of the global population, rice production must increase by at least 60% (Khush 2013). However, after experiencing two leaps from tall stalks to dwarf stalks and from conventional rice to hybrid rice, rice yield has not made a major breakthrough for a long time. Polyploid rice has the advantages of thick stalks, large grains, and strong stress resistance. It has the potential to significantly increase production, but it has long been plagued by the problem of low fruiting rate and has been difficult to apply. Therefore, improving the fruiting rate of polyploid rice is the key to the production and application of polyploid rice.

[0003] According to the principle of allopolyploidization of plants in nature, Cai Detian et al. (2001) proposed a new breeding strategy of "using the dual advantages of distant hybridization and polyploidy to breed super rice", and adopted methods such as "increasing the distance of kinship, reducing the unstable pairing of chromosomes, and using special genes" to improve the fruiting rate of polyploid rice from the genetic mechanism. Under the guidance of this strategy, through extensive hybridization, backcrossing and selection between indica and japonica subspecies, polyploid meiosis stability (PMeS) lines were selected. PMeS lines have three significant characteristics: 1) Meiotic stability, with bivalent pairing as the main form in prophase I of meiosis, and rarely appearing unstable pairing forms such as monovalent and trivalent, and rarely appearing lagging chromosomes in late phase I. 2) High fruiting, with a high fruiting rate of more than 70%. 3) Meiotic stability and high fruitfulness can be stably inherited, and hybrid offspring with low-fruitfulness homologous tetraploid indica or japonica rice varieties can produce hybrid offspring with high fruitfulness rate (Cai Detian et al. 2007). The selection and breeding practice of PMeS lines show that the bottleneck problem of low fruitfulness rate that has long plagued polyploid rice breeding has been broken through, laying a foundation for the production and application of polyploid rice and significantly increasing rice yield (Song Zhaojian et al. 2023; Li Hangyu et al. 2024). High-fruitfulness PMeS lines are the key to realizing the production and application of polyploid rice. Therefore, in-depth understanding of the meiotic stability and high fruitfulness 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-fruitfulness gene OsTHS-A1 and its application. Summary of the invention

[0004] In view of the defects and improvement needs of the prior art, the object of the present invention is to provide a polyploid rice high-fruiting gene OsTHS-A1 and its application.

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

[0006] Furthermore, 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 OsTHS-A1 gene is used for improving the fruit setting rate of polyploid rice. The nucleotide sequence of the CDS region of the OsTHS-A1 gene is shown in SEQ ID NO.1.

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

[0009] Furthermore, the OsTHS-A1 gene improves the fruit setting rate of low-fruiting polyploid rice through overexpression.

[0010] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects:

[0011] The present invention clones the high-fruiting gene OsTHS-A1 from the high-fruiting polyploid rice PMeS strain, and uses overexpression and inhibition expression genetic transformation techniques to transform low-fruiting and high-fruiting polyploid rice, respectively, to verify its function in regulating the fruiting rate of polyploid rice, and uses the gene to improve the fruiting rate of low-fruiting polyploid rice; in the present invention, the OsTHS-A1 gene is highly expressed during the rice meiosis period, which may affect the meiosis of the polyploid rice, thereby affecting the pollen fertility and activity, and then affecting the fruiting rate. Therefore, the expression of the OsTHS-A1 gene can stabilize the meiotic behavior of the polyploid rice, improve the pollen fertility and activity, and thus effectively improve the fruiting rate of the low-fruiting polyploid rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 is 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 cells; S6: meiosis period of pollen mother cells; 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 with inhibited expression. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

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

[0021] Based on the genome BSA sequencing analysis and positioning of the extreme populations of polyploid rice F2 with high and low fruiting, and the quantitative PCR screening of candidate genes, a candidate gene for high fruiting in polyploid rice was identified. The polyploid rice high fruiting PMeS line A1-4x was used as the material to extract the total RNA of its panicle during meiosis, and DNase I was used to remove genomic DNA contamination. The ABclonalRNA reverse transcription kit was used to reverse transcribe it into cDNA. Specific primers were designed according to the BLAST sequence alignment results on the NCBI website, and Gimmico Q6 PCR Master Mix (1.1×) was used for amplification.

[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, and the full-length CDS sequence is shown in SEQ ID NO.1; it encodes 397 amino acids, and the sequence is shown in SEQ ID NO.2. The relative molecular mass of the protein is 44486.98, and the isoelectric point (PI) is 3.23.

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

[0027] The spatiotemporal expression pattern of OsTHS-A1 gene in A1-4x was analyzed, mainly including the root, stem, leaf and panicle tissues corresponding to the four periods of stamen primordium differentiation (S4), pollen mother cell formation (S5), pollen mother cell meiosis (S6) and pollen grain content filling (S7). Total RNA was extracted from each tissue, reverse transcribed into cDNA, and the expression of OsTHS-A1 gene was quantitatively analyzed (qRT-PCR) using AgilentStratagene Mx3000P real-time fluorescence quantitative PCR instrument. The primer sequences were:

[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 in the other three periods except the root during the pollen mother cell formation period; the expression level was low in the panicle during the stamen primordium differentiation period and the pollen grain content filling period; the expression level was high in the root and panicle during the pollen mother cell formation period; the expression level was highest in the panicle during the pollen mother cell meiosis period, and it was the highest expression level in all tissues in the four periods ( Figure 2 ).

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

[0035] In order to verify the function of OsTHS-A1 gene from the two directions of overexpression and inhibition of expression, the OsTHS-A1 gene overexpression vector and RNA interference vector were constructed, and the low-fruiting polyploid rice Nipponbare-4x and the high-fruiting polyploid rice A1-4x were transformed, respectively, to obtain transformed plants, examine the phenotypes of the transformed plants, and 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 with pCAMBIA1300s as the skeleton. The vector carries the right border (RB) and the left border (LB) of TDNA, and can insert foreign genes into the host plant genome through Agrobacterium-mediated transformation. The vector contains the strong promoter of CaMV 35S, which can efficiently express the inserted gene. The total cDNA of the polyploid rice high-fruiting PMeS line A1-4x is used as a template, and the full-length cDNA of OsTHS-A1 is amplified with primers OsTHS-A1-F and OsTHS-A1-R. The full-length cDNA is connected to the TA cloning vector Seamless Cloning Kit (Beijing Qingke Biotechnology Co., Ltd.), transformed into E. coli competent cells, screened positive clones and sent for sequencing. First, the correctly sequenced target fragment was double-digested with restriction endonucleases Not I and EcoR I and recovered from the gel, and then ligated into pBWA(v)HS with 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 is transferred as a template, RNAi-F1 (5'-CAGTGGTCTCACAACAAGTTCCGACCTGCTCTAAACGAGG-3') and RNAi-R1

[0039] (5'-GATGGTCTCACAGGCTCTCCATAACAAAAAGACTGCTGCCAGC-3') amplified to obtain a 245 bp cDNA fragment, in which the restriction endonuclease Eco31 I cleavage site is "GGTCTC". This fragment was ligated to the TA cloning vector Seamless Cloning Kit, transfer to E. coli competent cells, screen positive clones and send for sequencing. The correctly sequenced target fragment was digested with restriction endonuclease Eco31 I and recovered from the gel to obtain the hairpin forward strand. In the same way, RNAi-F2 (5'-GATGGTCTCACAGGCTCTCCATAACAAAAAGACTGCTGCCAGC-3') and RNAi-R2

[0040] (5'-CAGTGGTCTCACAACAAGTTCCGACCTGCTCTAAACGAGG-3') to obtain the reverse strand of the hairpin. Through the middle loop structure (Wuhan Boyuan Biotechnology Co., Ltd.), the hairpin forward strand and the hairpin reverse strand were connected using T4 DNA ligase, and gel recovery was detected. After the sequencing was correct, it was connected to the linearized pBWA(v)HS plasmid obtained by 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 method was used to introduce OsTHS-A1-O E and OsTHS-A1-RN A i into low-fruiting polyploid rice Nipponbare-4x and high-fruiting polyploid rice A1-4x, respectively. The specific operation steps are as follows:

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

[0044] Remove the husks of mature seeds of Nipponbare-4x and A1-4x, cut off the embryos, treat with 75% alcohol for 1 minute, and then soak and disinfect with 0.1% mercuric chloride (HgCl2) for 30 minutes, shaking every 5 minutes. After disinfection, wash with sterile distilled water 5 times, inoculate the embryos into the induction medium, and culture in the dark at 28℃ until callus grows. Select yellow, granular, and vigorous callus, transfer to the pre-culture medium, and culture for 5 to 8 days.

[0045] (2) Agrobacterium activation and suspension culture

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

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

[0048] In an ultra-clean workbench, transfer the cultured callus tissue to the Agrobacterium suspension and infect for 3 to 5 minutes; remove the callus tissue, dry it with sterile filter paper, then transfer it to the co-culture medium, culture it in the dark for 2 to 3 days, and then transfer it to the incubator for culture.

[0049] (4) Washing and screening

[0050] The co-cultured callus was transferred to a sterile Erlenmeyer flask, washed with sterile distilled water for more than 6 times until the washed water became clear, and then 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 to the screening medium and cultured in the dark at 26°C for 15 days.

[0051] (5) Second screening

[0052] Select dry callus particles without Agrobacterium contamination from the first screening medium and transfer them to a new screening medium. Culture them in the dark at 26°C for 25 days. During this process, frequently observe the growth of the callus. If no growth signs are found in the callus, transfer them to a new screening medium in time.

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

[0054] Select callus tissue with good growth in the screening medium and transfer it to the differentiation medium. Incubate it in the dark at 26℃ for 5-7 days, then at 28℃ and normal light for 30 days until the seedlings differentiate. Transfer the seedlings to the rooting medium and culture them at 28℃ for 15-25 days. After rooting, harden the seedlings for 3-5 days and transplant them to the field. After growing in the field for 2-3 weeks, take the leaves of the transformed plants for positive identification.

[0055] The overexpression transformed plant finally obtained by the present invention was named NIP-OE, and a total of 16 positive seedlings were obtained. The young spikelets of NIP-OE in the meiotic stage were taken, and the total RNA was extracted. The expression level of the OsTHS-A1 gene in the transformed plant was detected by qRT-PCR technology. The results showed that the expression level of the OsTHS-A1 gene in the transformed plant NIP-OE was significantly increased compared with the wild type Nipponbare-4x (NIP-WT), which was 2.86 times that of NIP-WT ( Figure 5 ).

[0056] The finally obtained expression inhibition transformed plant of the present invention was named A1-RN A i, and a total of 9 positive seedlings were obtained. The young spikelets of A1-RN A i in meiosis stage were taken, total RNA was extracted, and the expression level of OsTHS-A1 gene in the transformed plant was detected by qRT-PCR technology. The results showed that the expression level of OsTHS-A1 gene in the transformed plant A1-RN A i was significantly lower than that of 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 OsTHS-A1 gene were investigated to understand the effect of OsTHS-A1 gene on the fruit setting rate of polyploid rice and determine the function of OsTHS-A1 gene.

[0059] The results of agronomic trait investigation showed that the plant height, panicle number, panicle length, grain length, grain width, and total number of grains per panicle of NIP-OE plants were higher than those of NIP-WT plants, but the differences were not significant, while the number of filled grains per panicle and the seed setting rate were significantly higher than those of NIP-WT plants. The seed setting rate of NIP-WT plants was only 9.55%, while that of NIP-OE plants was 42.24%, which was significantly higher than that of the wild type, indicating that overexpression of the OsTHS-A1 gene can effectively improve the seed setting rate of polyploid rice. The plant height, panicle number, panicle length, grain length, grain width, total number of grains per panicle, filled grains per panicle, and seed setting rate of A1-RNAi plants were lower than those of A1-WT plants, among which the differences in plant height, total number of grains per panicle, filled grains per panicle, and seed setting rate reached a significant level. 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 would lead to a decrease in the fruit setting rate of polyploid rice.

[0060] The results of observation on pollen fertility and activity and meiotic behavior of pollen mother cells of A1-RNAi and A1-WT plants showed that the fertile pollen rate and active pollen rate of A1-WT plants were 85.39% and 88.65%, respectively, while the fertile pollen rate and active pollen rate of A1-RNAi plants were 55.51% and 40.27%, respectively, which were significantly lower than those of the wild type. In the prophase I of meiosis, A1-RNAi plants had more unstable pairing forms such as univalents and trivalents, and more lagging chromosomes in metaphase I and anaphase I; while A1-WT plants belonged to the PMeS strain, which had meiotic stability, and their meiosis was mainly bivalent pairing, and rarely had abnormal phenomena such as univalents, trivalents and lagging chromosomes.

[0061] The applicant declares that the present invention illustrates the product and detailed preparation method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned product and detailed preparation method, that is, it does not mean that the present invention must rely on the above-mentioned product and detailed preparation method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention and addition of auxiliary materials, selection of specific methods, etc., all fall within the protection scope and disclosure scope 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 in the above embodiments, and the technical solution of the present invention can be subjected to a variety of simple modifications within the technical concept of the present invention. These simple modifications all belong to the protection scope 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 suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0064] In addition, 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. Polyploid rice high-fruiting gene OsTHS-A1, characterized by: The nucleotide sequence of the CDS region of the OsTHS-A1 gene is shown in SEQ ID NO.

1.

2. The polyploid rice high-fruiting gene OsTHS-A1 according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the OsTHS-A1 gene is shown in SEQ ID NO.

2.

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

1.

4. The use of the OsTHS-A1 gene in improving the fruit setting rate of polyploid rice according to claim 3, characterized in that: The amino acid sequence of the protein encoded by the OsTHS-A1 gene is shown in SEQ ID NO.

2.

5. Use of the OsTHS-A1 gene according to claim 1 or 2 in improving the fruit setting rate of polyploid rice, characterized in that: The OsTHS-A1 gene improves the fruit setting rate of low-fruiting polyploid rice through overexpression.

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