Chimeric interference pollen lethal module as well as preparation method and application thereof

Abstract

The invention discloses a chimeric interference pollen lethal module as well as a preparation method and application thereof. The module consists of a pollen specific promoter, a chimeric interference element of an OsPTDa gene and an OsPTDb gene, and a plant endogenous terminator. After the elements are assembled, pollen can be killed. The pollen lethal module disclosed by the invention can accurately down-regulate the expression of male gametophyte development related genes OsPTDa and OsPTDb in the same time and space, so that pollen germination is abnormal, transgenic pollen cannot generate offspring, gametophyte male sterility is generated, and the pollen lethal module has important value in the aspects of preventing transgene from escaping along with pollen, breeding sporophyte male nuclear sterility and the like.

Description

Technical Field

[0001] This invention relates to the field of genetic breeding, specifically to a chimeric interference pollen lethal module, and also to the preparation method and application of the lethal module. Background Technology

[0002] Sporophytic male sterility is an essential genetic element for third-generation hybrid rice, and solving its reproductive problems is crucial for its successful application. Pollen lethality is a strategy for propagating sporophytic male sterility (Perez-Prat et al., 2002), and it also plays an important role in preventing transgenic escape and maintaining ecological security. Currently, successful examples of nuclear sterility propagation are mainly based on the pollen lethality strategy. Reproduction systems based on this strategy require multiple gene modules (Chenet et al., 2023). The nucleic acid sequences used in the pollen lethality module are too long, and the capacity of commonly used plant expression vectors is limited, making it very difficult to successfully load all modules into a single vector. This makes the miniaturization of each module an urgent need. Furthermore, the use of exogenous genes in staple crops is subject to public skepticism and strict regulation. Therefore, developing small pollen lethality modules that use little or no exogenous genes is essential. Compared to the influence of the full length of a gene on traits, RNAi uses shorter elements and sequences. For example, when using RNAi to interfere with genes related to endogenous pollen development, it is possible to construct short pollen lethal modules based entirely on endogenous genes. This is beneficial for loading more regulatory modules into the same sporophyte nuclear sterility propagation vector. Summary of the Invention

[0003] In view of this, one objective of the present invention is to provide a chimeric interference pollen lethal module; a second objective of the present invention is to provide a method for constructing the chimeric interference pollen lethal module; a third objective of the present invention is to provide the application of the chimeric interference pollen lethal module in the preparation of male gametophyte sterility-related plant materials or in the propagation of sporophyte male sterility; and a fourth objective of the present invention is to provide a method for propagating sporophyte male sterility plant materials using the pollen lethal module of the present invention.

[0004] To achieve the above objectives, the present invention provides the following technical solution: 1. A chimeric interference pollen lethal module, the module comprising an endogenous pollen-specific promoter, a chimeric interference element of OsPTDa and OsPTDb, and a plant endogenous terminator, wherein the sequence of the endogenous pollen-specific promoter is shown in SEQ ID NO.3; the sequence of the interference element is shown in SEQ ID NO.5; and the plant endogenous terminator is shown in SEQ ID NO.4.

[0005] Preferably, the interferometric element can simultaneously downregulate the expression of OsPTDa and OsPTDb.

[0006] 2. The method for constructing the chimeric interference pollen lethal module involves sequentially linking an endogenous pollen-specific promoter, chimeric interference elements of OsPTDa and OsPTDb, and a plant endogenous terminator into a vector containing anthocyanin synthesis-related genes to obtain the pollen lethal module.

[0007] 3. The application of the chimeric interference pollen lethality module in the preparation of plant materials related to male gametophyte sterility or in the propagation of sporophyte male sterility.

[0008] 4. A method for propagating sporophyte nucleus male-sterile plant material using the pollen lethal module, specifically including the following steps: (1) The OsSTRL3 gene was ligated into the pollen lethal module, and the resulting vector was then transferred into the sterile mutant osstrl3. Positive plants were screened. The nucleic acid sequence of the OsSTRL3 gene is shown in SEQ ID NO.6. (2) The positive strains obtained in step (1) were pollinated with the sterile mutant osstrl3 to obtain F1 strains that exhibited male sterility.

[0009] The beneficial effects of this invention are as follows: This invention provides a chimeric interference pollen lethal module, which utilizes the chimeric interference of two endogenous redundant genes in rice to construct a small and efficient pollen lethal module, avoiding the use of exogenous genes and fragments, and using fewer exogenous gene fragments than existing technologies; the interference of the pollen lethal module adopts the direct connection of the interference arms of the two genes to form a chimeric interference element, which is placed between a promoter and a terminator, realizing the simultaneous interference of two genes with a single interference element, which saves a significant amount of vector space for loading other modules. Therefore, the sporophytic nuclear sterility reproduction system constructed using the pollen lethal module of this invention reduces concerns about exogenous genes and is easier to regulate through transgenic technology; the pollen lethal module of this invention leaves more space for the addition of other regulatory modules, which is beneficial for achieving more regulatory targets with a single vector. Attached Figure Description

[0010] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 This is a schematic diagram of the PMCOS1-IM180-Tmco interferometer module (LA represents the left arm of the interferometer, IC represents the intron of OsCPL1 / OsMYB76, and RA represents the right arm of the interferometer). Figure 2The effect of the pollen lethal module on pollen germination and transgene transfer (A: normal germination of wild-type Zhonghua 11 pollen; B: pollen tubes of chimeric interference progeny exploded after about half of the pollen had just germinated; C: pollen of chimeric interference progeny did not transfer transgene components (F1 without purple coleoptile line); the leftmost is the maternal parent without anthocyanin color; the grain is a transgene-positive plant (purple grain tip). Figure 3 Map-based cloning and verification of the sporophyte nucleus male sterility gene (A: gene location; B: left: mutant spikelet, right: complementary positive spikelet; C: mutant anthers and pollen iodine staining; D: complementary positive plant pollen iodine staining; E: top: mutant non-fertile spike, bottom: complementary positive plant fertile spike; F: left: mutant shriveled grain, right: complementary positive purple-tipped fertile grain).

[0011] Figure 4 For propagation verification and purity identification of sterile lines (A: Left: transgenic positive spikelet of propagation vector (purple at the tip of the spikelet), right: spikelet of male sterile mutant (no purple at the tip of the spikelet); B: Left: transgenic positive anther of propagation vector (yellow and fertile), right: anther of male sterile mutant (white and sterile); C: For observation of fertility of offspring, all showed white sterile anthers). Detailed Implementation

[0012] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al.'s *Molecular Cloning Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Various vectors known in the art, such as commercially available vectors including plasmids and granules, can be used in the embodiments. The relevant full-length nucleotide sequence or fragments thereof can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this embodiment, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This usually involves cloning the sequence into a vector, transferring it into a cell, and then isolating the sequence from the proliferated host cell using conventional methods.

[0013] Example 1: Design and Construction of the Interferometric Module Pollen development is a crucial aspect of reproductive development, playing a vital role in plant genetic recombination and generational succession. To ensure normal pollen function, long-term evolution has resulted in numerous functionally redundant genes that work together to guarantee normal pollen development. OsPTD1 / OsPTDa (Chen et al., 2021) and OsPTDb are two such functionally redundant genes, both encoding ascorbic acid oxidase, which may redundantly regulate pollen development. The genomic sequence of OsPTDa is shown in SEQ ID NO.1, and the genomic sequence of OsPTDb is shown in SEQ ID NO.2. To obtain a short pollen lethal module based on rice endogenous genes, the following design was made: the interference arms of the two genes were directly connected to form a chimeric interference arm, and their complementary sequences were linked through the first intron of OsCPL1 / OsMYB76 (Du et al., 2022) to form the interference element IM180 of the two pollen development genes; then this interference element was placed between the promoter PMCOS1 and the terminator Tmco of OsPTDa to form a pollen lethal module PMCOS1-IM180-Tmco. Figure 1 ).

[0014] The specific construction process is as follows: The promoter of OsPTDa was selected, common restriction enzyme sites were removed, and EcoRI-BglII and BamHI restriction sites were added to both ends to obtain the optimized promoter PMCOS1, the sequence of which is shown in SEQ ID NO.3; Blast alignment was performed on the Gramene website (https: / / www.gramene.org / ) to screen for interference-specific target sequences of OsPTDa and OsPTDb, and these sequences were directly tandem to obtain the left arm of the dual-gene chimeric interference. Then, reverse complementation was performed to obtain the right arm of the chimeric interference. The left and right arms were connected using the first intron of OsMYB76 to obtain the interference element. Common restriction enzyme sites were then removed, and EcoRI-BglII and BamHI restriction sites were added to both ends to obtain the optimized interference element IM180, the sequence of which is shown in SEQ ID NO.5; The terminator of OsPTDa was selected, common restriction enzyme sites were removed, and EcoRI-BglII and BamHI restriction sites were added to both ends to obtain the optimized terminator Tmco, the sequence of which is shown in SEQ ID NO.5. Shown in NO.4.

[0015] PMCOS1, IM180, and Tmco were artificially synthesized. Then, using isosigmasynthesis technology, Tmco, IM180, and PMCOS1 were sequentially loaded into the vector pBOsMYB76RM containing anthocyanin synthesis-related genes (pBOsMYB76RM was constructed by replacing the multiple cloning site and hygromycin resistance gene Hyg in pOsMYB76RM with the synthesized multiple cloning site FSVM (SEQ ID NO.7) and glufosinate resistance gene Bar (SEQ ID NO.8). The construction process of pOsMYB76RM is described in Chinese Patent Publication No. CN112239762A). The process was as follows: Tmco was digested with EcoRI + BamHI, and the target fragment was recovered for later use; simultaneously, pBOsMYB76RM was digested with EcoRI + BglII, and the backbone was recovered for later use. The target fragment and the backbone were then ligated. IM180 and PMCOS1 were then sequentially ligated in the same manner as described above. This yielded the vector pRNAi-PTDab-3, completing the assembly of the pollen lethality module.

[0016] Example 2: Verification of the transgenic pollen lethal module The pRNAi-PTDab-3 plasmid was transformed into anthocyanin-free Zhonghua 11 using Agrobacterium tumefaciens-mediated transformation. Pollen germination of the positive plants with purple valley tips was observed during flowering. It was found that half of the pollen from the positive plants germinated abnormally, with pollen tubes exploding shortly after germination. Figure 2 This will seriously affect its fertilization and seed setting; purple valley tip positive plants were selected as male parents to pollinate colorless fragrant 1A to produce F1 seeds. After the seeds matured, the presence of purple coleoptile lines in the F1 generation was investigated. It was found that the hybrid F1 generation had no purple coleoptile lines at all. Figure 2 (B), which is a typical characteristic of gametophyte male sterility. Therefore, the constructed pollen lethal module can kill pollen containing transgenic components, block the transmission of transgenics to offspring with pollen, produce ideal gametophyte male sterility, and can be used to prevent transgenics from escaping with pollen and to reproduce sporophyte nuclear male sterility.

[0017] Example 3: Propagation of sporophyte nucleo-male sterile osstrl3 using a pollen lethality module and identification of the purity of the sterile line. (1) Discovery and map-based cloning of the osstrl3 male sterile mutant: osstrl3 was discovered by the inventor in 2018 in the Jinghong rice experimental field of Yunnan University in Xishuangbanna. Its wild type is japonica rice Zhonghua 11. In order to clone the relevant gene, the BSA method was used to locate the target gene on chromosome 3. Figure 3 (A) Through next-generation sequencing, a 135bp deletion and an 11bp insertion mutation were found in the third exon of the Loc_Os03g53950 gene in the OsSTRL3 mutant, resulting in an amino acid frameshift in the encoded protein. After cloning the gene and transforming it into the mutant, the mutant's physical characteristics were restored to normal. Figure 3 BF).

[0018] (2) The normal OsSTRL3 gene (SEQ ID NO.6) was inserted between AscI and SbfI of pRNAi-PTDab-3 to obtain the propagation vector pOsSTRL3-RNAi-PTDab-3.

[0019] (3) pOsSTRL3-RNAi-PTDab-3 was transformed into the sterile mutant osstrl3, and a total of 41 positive plants of purple valley tip were screened out. All positive plants showed normal pollen and fertility.

[0020] (4) Positive plants were used to pollinate the sterile mutant osstrl3, and a total of 235 F1 seeds were obtained. After planting, 201 plants survived. Fertility was observed during the flowering period, and it was found that all F1 plants showed male sterility, and the tips of the plants were all without purple color. Figure 4 ).

[0021] The above situation illustrates that the pollen lethal module designed in this invention has produced a sporophytic male sterile line with a sterility rate of 100%.

[0022] 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 chimeric interference pollen lethal module, characterized in that: The module includes an endogenous pollen-specific promoter, a chimeric interference element of OsPTDa and OsPTDb, and a plant endogenous terminator. The sequence of the endogenous pollen-specific promoter is shown in SEQ ID NO.3; the sequence of the interference element is shown in SEQ ID NO.5; and the plant endogenous terminator is shown in SEQ ID NO.

4.

2. The chimeric interference pollen lethal module according to claim 1, characterized in that: The interferometric element can simultaneously downregulate the expression of OsPTDa and OsPTDb.

3. The method for constructing the chimeric interference pollen lethal module as described in claim 1 or 2, characterized in that: By sequentially linking the endogenous pollen-specific promoter, the chimeric interference elements of OsPTDa and OsPTDb, and the plant endogenous terminator into a vector containing genes related to anthocyanin synthesis, a pollen lethal module was obtained.

4. The application of the chimeric interference pollen lethal module as described in claim 1 or 2 in the preparation of plant materials related to male gametophyte sterility or in the propagation of sporophyte male sterility.

5. A method for propagating sporophytic male-sterile plant material using the pollen-lethal module described in claim 1 or 2, characterized in that, Specifically, the steps include the following: (1) The OsSTRL3 gene was ligated into the pollen lethal module, and the resulting vector was then transferred into the sterile mutant osstrl3. Positive plants were screened. The nucleic acid sequence of the OsSTRL3 gene is shown in SEQ ID NO.

6. (2) The positive strains obtained in step (1) were pollinated with the sterile mutant osstrl3 to obtain F1 strains that exhibited male sterility.

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