Gametophyte male sterility related gene OsPTDb and application

Abstract

The invention discloses a gametophyte male sterility related gene OsPTDb and application thereof. The amino acid sequence of the OsPTDb gene is as shown in SEQ ID NO. 1; the OsPTDb gene encodes an ascorbic acid oxidase which is an essential gene for male gametophyte development, the insemination of the male gametophyte cannot be completed and gametophyte male sterility is generated due to the abnormal function of the gene, and the OsPTDb gene plays an important role in the aspects of preventing transgene from escaping along with pollen, breeding sporophyte male nuclear sterility and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular breeding, in particular to a gametophyte male sterility related gene OsPTDb for preparing plant gametophyte male sterile material, and to the application of the gene. BACKGROUND

[0002] Male sterility is an important trait in hybrid vigor utilization. According to whether its fertility is controlled by the sporophyte (plant vegetative organ) genotype or the gametophyte (pollen) genotype, it can be divided into sporophyte sterility and gametophyte sterility. These two types of sterility play an important role in explaining pollen development and male sterility mechanisms. Cytokinesis sporophyte male sterility is a necessary genetic factor for the third generation of hybrid rice, while gametophyte male sterility plays an important role in the propagation of sporophyte male sterility. Therefore, identifying gametophyte male sterility genes, creating gametophyte male sterile materials and applying them have important theoretical and practical significance.

[0003] Due to its inherent genetic characteristics, gametophyte male sterility is difficult to find in the field. Currently, the sterile line propagation technology of the third generation of hybrid rice is based on artificially designed gametophyte male sterility elements. For example, the fusion pollen lethal element based on the pollen-specific promoter PG47, the chloroplast transit peptide and the maize amylase gene, the fusion pollen lethal element based on the pollen-specific promoter PG47, the mitochondrial transit peptide and the cell toxin protein orfH79 of the red lotus type sterility (Chen et, al., 2023). These gametophyte male sterility elements affect the limited function of pollen, and have a certain probability of transgenic escape, which makes the purity of the sterile lines based on these pollen lethal elements not ideal, and is easy to cause transgenic concern. Therefore, finding new male gametophyte sterility related genes and developing complete male sterile pollen lethal modules have important practical value.

[0004] Ascorbate oxidase is involved in the regulation of plant growth and development, reproduction, aging and response to environmental stress, and can regulate flowering time (Shen et al. 2009). There are multiple genes encoding ascorbate oxidase in rice, but their biological functions are not clear. Finding a gametophyte male sterility related gene from them and creating a complete gametophyte male sterile will help prevent transgenic escape and promote the development of the third generation of hybrid rice. SUMMARY

[0005] Therefore, one of the purposes of the present application is to provide a gametophytic male sterility related gene OsPTDb for preparing a plant gametophytic male sterile material; another purpose of the present application is to provide a gene combination for preparing a plant gametophytic male sterile material; a third purpose of the present application is to provide a method for preparing a plant gametophytic male sterile material; a fourth purpose of the present application is to provide an application of the gametophytic male sterility related gene OsPTDb in restoring mutant pollen fertility; and a fifth purpose of the present application is to provide an application of the gametophytic male sterility related gene OsPTDb in preventing transgene escape with male gametophytes or in breeding sporophytic cytoplasmic male sterility.

[0006] To achieve the above purposes, the present application provides the following technical solutions. 1. A gametophytic male sterility related gene OsPTDb for preparing a plant gametophytic male sterile material, wherein the amino acid sequence encoded by the gametophytic male sterility related gene OsPTDb is shown in SEQ ID NO. 1, or the amino acid sequence shown in SEQ ID NO. 1 is substituted, deleted or added with at least one amino acid and has an amino acid sequence with the same function as the OsPTDb protein.

[0007] Preferably, by down-regulating the expression of the OsPTDb gene, male gametes of the transgenic plant are killed.

[0008] 2. A gene combination for preparing a plant gametophytic male sterile material, wherein the gene combination comprises an OsPTDa gene and an OsPTDb gene, the genomic sequence of the OsPTDa gene is shown in SEQ ID NO. 12, and the amino acid encoded by the OsPTDb gene is shown in SEQ ID NO. 1.

[0009] 3. A method for preparing a plant gametophytic male sterile material, wherein male gametes of the transgenic plant are killed by simultaneously down-regulating the expression of the genes, i.e. the expression of the OsPTDa gene and the OsPTDb gene.

[0010] Preferably, the method for down-regulating the expression of the OsPTDa gene and the OsPTDb gene is to construct a recombinant expression vector for simultaneously inhibiting the expression of the OsPTDa gene and the OsPTDb gene, to transfect plants by means of Agrobacterium mediation, and to screen transgenic positive plants.

[0011] Preferably, the recombinant expression vector is constructed as follows: the multiple cloning site of pOsMYB76RM and the hygromycin resistance gene Hyg are replaced by a multiple cloning site FSVM and a glufosinate ammonium resistance gene Bar to obtain a pBOsMYB76RM vector, the sequence of the Bar gene is shown as SEQ ID NO. 7, and the sequence of the multiple cloning site FSVM is shown as SEQ ID NO. 9; Then, interference elements IM2-1 and I802 of the OsPTDa and OsPTDb genes are synthesized, and tRNA-Gly is used as a separation sequence, the sequences of the IM2-1 and I802 are shown as SEQ ID NO. 11 and SEQ ID NO. 4, respectively; a promoter PMCOS1 shown as SEQ ID NO. 5 and a Tnos1 terminator shown as SEQ ID NO. 6 are synthesized, and then Tnos1, IM2-1, I802 and PMCOS1 are connected into the pBOsMYB76RM vector by enzyme digestion and ligation to obtain an OsPTDa and OsPTDb gene simultaneous inhibition vector pRNAi-PTDab-1.

[0012] 4. Application of the gametophytic male sterility related gene OsPTDb in restoring pollen fertility of mutants, wherein the mutants are pollen sterility mutants caused by variation of the OsPTDb gene or a homologous gene.

[0013] 5. Application of the gametophytic male sterility related gene OsPTDb in preventing transgene escape with male gametophytes or in breeding sporophytic cytoplasmic male sterility.

[0014] Preferably, a sporophytic male fertile gene Y is linked with the pollen-specific driven OsPTDb and OsPTDa gene simultaneous interference fragments, and then the sporophytic male fertile gene Y is introduced into a sterile mutant to screen transgenic positive plants to obtain pollen fertile plants.

[0015] Preferably, the obtained transgenic positive plants are used as the father to pollinate the sporophytic male fertile gene Y sterile mutant, so that the sporophytic cytoplasmic male sterility is reproduced.

[0016] The application has the beneficial effects that the application provides a gametophyte male sterility related gene OsPTDb for preparing a plant gametophyte male sterile material, the OsPTDb is an endogenous gene of rice, and gametophyte sterility can be generated without the help of exogenous genes or fragments by down-regulating the expression of the OsPTDb, so that a plant variety with transgenic components not drifting with pollen can be cultivated (if the method is used in other species, a gametophyte male sterility related gene in the target species can be selected); meanwhile, the down-regulation of the expression of redundant genes of the OsPTDb can generate complete gametophyte male sterility, which is beneficial to the breeding of spore nucleus sterility with standard purity and the relief of the worry about transgenic escape. The gametophyte male sterility prepared based on the OsPTDb gene and the redundant gene OsPTDa of the OsPTDb is more complete than the current technology and is easier to pass the transgenic supervision. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to make the objectives, technical solutions and beneficial effects of the application clearer, the application provides the following drawings for description: Figure 1 Knocking out the fertility of OsPTDa and OsPTDb of Zhonghua 11 (A is the wild type of Zhonghua 11; B is the fertility rate slightly lower after knocking out the OsPTDa; C is the fertility rate seriously decreased after knocking out the OsPTDb; D indicates that the aaBb type single plant is hybridized with the wild type in F1, and the OsPTDb gene mutation site (44 bp is deleted) is detected: 1 is the mutant (aaBb) of the father, 2 is the wild type of the mother, and 3-15 are single plants of the hybrid F1, and the mutant band is not transmitted to F1).

[0018] Figure 2 Schematic diagram of FSVM for polyclonal site modification.

[0019] Figure 3 Pollen germination and purple line transmission of OsPTDa and OsPTDb double interference (A is a positive transgenic T0 seedling (right, purple at the base), B is normal pollen germination of the wild type, C is pollen half unable to normally germinate caused by interference, D is the leftmost positive plant with a purple line, the rest are half and half of the selfed offspring with or without a purple line, E is the rightmost two plants, which are respectively colorless 1A and the positive plant with a purple line, and the rest are hybrid F1 generation without a purple line).

[0020] Figure 4 Preparation and breeding of osabcg15 male sterile mutant (A is the anther of the male sterile 1A with small and white anthers after knocking out the OsABCG15, B is the anther of the male sterile 1A with normal fertile anthers after the male sterile 1A transferring the pOsABCG15-RNAi-PTDab, C is the anther of the male sterile 1A, followed by the positive fertile anther, and the rest are the anthers of the F1 generation obtained by hybridization and breeding, all of which are male sterile). DETAILED DESCRIPTION

[0021] The present application is further described below to enable those skilled in the art to better understand and implement the present application, but the examples are not intended to limit the present application. If specific conditions and experimental methods are not described in detail, they can generally be carried out according to general conditions and methods, such as those described in the fourth edition of Molecular Cloning Experiment Guide or the fifth edition of Concise Molecular Biology Experiment Guide (Science Press), or according to the steps provided by the manufacturer. In the examples, various carriers known in the art can be selected.

[0022] Example 1, verification of knockout of OsPTDb gene In order to find some gametophytic male sterility related genes, an androspore sterility related gene OsPTD1 (Chen et al., 2021) / OsPTDa (used in the present application, because OsPTD1 is repeated with other article gene nomenclature) was identified by interference strategy. The genomic sequence of OsPTDa gene is shown in SEQ ID NO. 12. Although the interference of OsPTDa produced severe gametophytic male sterility, the abortion was not complete, and the transgenic had a certain pollen transmission rate. After searching gramene (https: / / www.gramene.org / ), it was found that OsPTDa encodes ascorbate oxidase, and there is a paralogous gene OsPTDb in rice, which also encodes ascorbate oxidase, and is likely to also participate in pollen development, resulting in incomplete gametophytic male sterility by interfering with OsPTDa alone. The amino acid sequence encoded by OsPTDb gene is shown in SEQ ID NO. 1, the sequence of CDS is shown in SEQ ID NO. 2, and the genomic sequence is shown in SEQ ID NO. 3. Zhonghua 11 OsPTDa and OsPTDb genes were knocked out alone: artificial synthesis of OsPTDa and OsPTDb single knockout elements, the sequences are shown in SEQ ID NO. 14 and SEQ ID NO. 15 respectively, let the company perform synthesis directly load into gene editing vector pHR2-Cas9 (Yang Jiaxin et al., 2025), obtain OsPTDa and OsPTDb gene knockout vectors OsPTDaK and OsPTDbK, and transfer them into Zhonghua 11 alone.

[0023] The results show that the single knockout of OsPTDa has a certain effect on the seed setting rate, and the single knockout of OsPTDb seriously affects the seed setting rate (Fig. 1, A-C). Figure 1 , A-C).

[0024] The self-crossing of the F1 generation AaBb of the homozygous mutant OsPTDa (genotype AA) and the homozygous mutant OsPTDb (genotype BB) of OsPTDa (genotype aa) cannot separate aabb genotype individuals, and the aaBb in the F2 generation cannot obtain ab type male gametes when pollinated by normal materials 1P, and cannot obtain offspring of ab type male gametes Figure 1 , D), the above results show that the mutant male gametes of OsPTDb cannot produce offspring by fertilization. This is the typical genetic characteristic of gametophytic male sterility, and therefore, OsPTDb is a typical gametophytic male sterility related gene, which can be used to prepare pollen lethal modules.

[0025] Example 2, simultaneous inhibition of OsPTDa and OsPTDb expression to prepare complete male gametophytic sterility In order to obtain a complete gametophytic male sterility module, the strategy of simultaneously interfering with OsPTDa and OsPTDb is used to inhibit the expression of the target gene, and in order to improve the interference efficiency and reduce the use of foreign genes, the promoter is the OsPTDa itself promoter, and the specific operation is as follows: Vector modification: In order to facilitate the loading of related target genes or DNA fragments, in order to accurately and directly identify transgenic positive strains and male gametophytic sterility, the GUS gene is replaced with the modified OsMYB76R gene related to anthocyanin synthesis in rice, and the multiple cloning site of the vector pCAMBIA1301 is modified. The modified vector is named pOsMYB76RM, and the specific process is described in Chinese Patent No. CN112239762A. In order to facilitate the loading and use of more regulatory elements and to screen transgenes by herbicide, the newly synthesized multiple cloning site FSVM ( Figure 2 ) and the glufosinate resistance gene Bar are used to replace the multiple cloning site and the hygromycin resistance gene Hyg in pOsMYB76RM, respectively, to obtain the newly modified vector pBOsMYB76RM. The sequence of the Bar gene is shown in SEQ ID NO. 7, the sequence of the OsMYB76R gene is shown in SEQ ID NO. 8, the sequence of the multiple cloning site FSVM is shown in SEQ ID NO. 9, and the sequence of the plant expression vector pBOsMYB76RM is shown in SEQ ID NO. 10.

[0026] Construction and verification of OsPTDa and OsPTDb gene simultaneous inhibition module (1) Screening of OsPTDa and OsPTDb gene interference target The coding frame sequences of the OsPTDa and OsPTDb genes were found on the Gramene website (https: / / www.gramene.org / ). Each gene's coding sequence of about 200 bp was BLASTed on the aforementioned website, and fragments with good specificity were selected as interference targets.

[0027] (2) Design and synthesis of OsPTDa and OsPTDb gene interference elements The specific targets and complementary sequences selected from each gene were used as the left and right arms of the interference, respectively. The first intron of the OsActin1 gene was used as the interference stem-loop of the OsPTDa gene, and the first intron of the OsMYB76 gene was used as the interference stem-loop of the OsPTDb gene. The interference elements IM2-1 (with EcoRI-Bgl II at the front end and BamHI at the rear end) and I802 (with EcoRI-Bgl II at the front end and BamHI at the rear end) of the OsPTDa and OsPTDb genes were artificially synthesized in this order. Both IM2-1 and I801 have tRNA-Gly sequences at the very front end for precise splicing. The sequences of IM2-1 and I802 are shown in SEQ ID NO.11 and SEQ ID NO.4, respectively.

[0028] (3) Construction of a module that simultaneously inhibits OsPTDa and OsPTDb genes By removing the commonly used restriction enzyme sites in the OsPTDa gene promoter through base substitution, and adding EcoRI-Bgl II and BamHI restriction sites to both ends, the promoter PMCOS1 was artificially synthesized, with its sequence shown in SEQ ID NO.5; the terminator Tnos1 (with EcoRI-Bgl II at the front and BamHI at the end) was artificially synthesized, with its sequence shown in SEQ ID NO.6; Tnos1, IM2-1, I802, and PMCOS1 were loaded into the aforementioned vector pBOsMYB76RM using isosigmazyme technology to obtain the OsPTDa and OsPTDb gene simultaneous repression vector pRNAi-PTDab-1. The ligation process of each element is as follows: Tnos1 was digested with EcoRI + BamHI, and the target fragment was recovered for later use; at the same time, pBOsMYB76RM was digested with EcoRI + BglII, and the backbone was recovered for later use; the target fragment and the backbone were ligated; IM2-1, I802, and PMCOS1 were sequentially ligated into the vector in the aforementioned manner.

[0029] (4) Verification of the simultaneous inhibition effect of OsPTDa and OsPTDb genes pRNAi-PTDab-1 was transformed into a non-purple fragrant 1P to obtain a transgenic positive strain. Figure 3A); During the flowering period, pollen germination was observed, and it was found that about half of the pollen germinated abnormally, with premature explosion of the pollen tubes, while the other half germinated normally. Figure 3 (B and C), indicating that the simultaneous suppression of the two genes had a serious impact on pollen germination; positive plants were self-pollinated and seeds were harvested, and the separation of purple lines from the coleoptiles was observed after sowing, and whether the ratio of purple lines met 1:1 ( Figure 3 D); When a purple-positive plant was pollinated with a colorless fragrant 1P, the F1 generation showed no purple lines. Figure 3 This inheritance pattern (E) is precisely the genetic characteristic of gametophyte male sterility. Results from pollen germination and purple line transmission experiments demonstrate that the gametophyte male sterility module designed using a strategy that simultaneously interferes with the OsPTDa and OsPTDb genes completely eliminates the fertilization capacity of transgenic pollen, preventing the transmission of transgenic components through pollen and the production of transgenic offspring. This module can be used to reproduce high-purity sporophyte nuclear male sterility.

[0030] Example 3: Using simultaneous downregulation of OsPTDa and b genes to reproduce sporophyte nucleus male sterility aroma 1A To determine whether sporophytic nuclear male sterility can be propagated using the OsPTDa and OsPTDb genes, the colorless, fragrant 1P OsABCG15 male sterility mutant, fragrant 1A, was first obtained based on the OsABCG15 gene (Wu et al., 2014). Then, a propagation vector for fragrant 1A was constructed using the aforementioned gametophytic male sterility vector pRNAi-PTDab-1 and validated, as detailed below: 1. Obtaining Chengxiang 1A: The OsABCG15 gene was knocked out using CRISPR / Cas9 technology. Using the constructed knockout vector TDK (an artificially synthesized knockout element of OsABCG15, the sequence of which is shown in SEQ ID NO.13), the company that performed the synthesis directly loaded it into the gene editing vector pHR2-Cas9 (Yang Jiaxin et al., 2025), obtaining the OsABCG15 gene knockout vector TDK. TDK was then transferred into Chengxiang 1P, and the transgenic male-sterile mutant Chengxiang 1A was obtained from the transgenic offspring. Figure 4 A).

[0031] 2. The normal OsABCG15 gene was ligated into the AscI and SbfI of pRNAi-PTDab-1 to obtain the vector pOsABCG15-RNAi-PTDab-1.

[0032] 3. pOsABCG15-RNAi-PTDab-1 was transformed into Chengxiang 1A, and a total of 17 purple positive plants were screened out. All positive plants showed normal pollen fertility. Figure 4 B).

[0033] 4. Using positive-positive plants to pollinate Chengxiang 1A, a total of 1126 F1 seeds were obtained. After sowing and transplanting, a total of 1008 surviving plants were obtained, all of which exhibited male sterility. Figure 4 C).

[0034] Under natural conditions, the method with the highest sterility rate in propagating Xiang 1A is sibling crossbreeding, with a sterility rate of 50%. However, the sterility rate we obtained using the propagation vector designed in this invention reached 100%, indicating that the OsPTDb gene and its homolog OsPTDa have important application value in preventing transgene escape and in propagation with sporophyte nucleus male sterility.

[0035] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A gametophyte male sterility-related gene OsPTDb, characterized in that: The amino acid sequence encoded by the gametophyte male sterility-related gene OsPTDb is shown in SEQ ID NO.1, or the amino acid sequence shown in SEQ ID NO.1 is modified by substituting, deleting, or adding at least one amino acid and has the same amino acid sequence as the OsPTDb protein.

2. The application of the gametophyte male sterility-related gene OsPTDb as described in claim 1 for preparing plant gametophyte male sterility or propagating sporophyte nuclear sterility materials, characterized in that: By downregulating or mutating the expression of the OsPTDb gene as described in claim 1, the male gametes of the transgenic plant are rendered lethal.

3. A gene combination for preparing male-sterile material from plant gametophytes, characterized in that: The gene combination includes the OsPTDa gene and the OsPTDb gene, the genomic sequence of the OsPTDa gene is shown in SEQ ID NO.12; the amino acid encoded by the OsPTDb gene is shown in SEQ ID NO.

1.

4. A method for preparing male-sterile plant gametophyte material, characterized in that, Simultaneous downregulation of the gene combination described in claim 3 leads to lethality of the male gametes of the transgenic plant, wherein the gene combination is the OsPTDa gene and the OsPTDb gene.

5. The method according to claim 4, characterized in that: The method for downregulating the OsPTDa and OsPTDb genes as described in claim 3 is to construct a recombinant expression vector that simultaneously inhibits the expression of the OsPTDa and OsPTDb genes, transfect plants through Agrobacterium-mediated transformation, and then screen for transgenic positive plants.

6. The application of the gametophyte male sterility-related gene OsPTDb as described in claim 1 in restoring pollen fertility in mutants, characterized in that: The mutant is a pollen sterility mutant resulting from mutations in the OsPTDb gene or its homologous genes.

7. The application of the gametophyte male sterility-related gene OsPTDb as described in claim 1 in preventing transgenes from escaping with male gametophytes or in reproducing sporophytes with nuclear male sterility.

8. The application according to claim 7, characterized in that: Linking a sporophyte male fertility gene Y with an interference fragment of the pollen-specific driven OsPTDb gene, then transferring it into a sterile mutant of the sporophyte male fertility gene Y, and screening for transgenic positive plants, yielded pollen-fertile plants.

9. The application according to claim 8, characterized in that: By pollinating the male-sterile mutant of the sporophyte male fertility gene Y with the obtained transgenic positive plant as the male parent, the sporophyte nuclear male sterility can be reproduced.

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