Plant gene function analysis method

Abstract

The invention discloses a plant gene function analysis method (FAGEN), and belongs to the technical field of molecular genetics and functional genomics. The method comprises the following steps: constructing a target gene mutant by using a gene editing technology; hybridizing the mutant and the corresponding wild type as a common parent with a plurality of parents with different genetic backgrounds to construct a pair F1 group; an F2 group is obtained through selfing; and determining phenotypic data of target characters of F1 and F2 populations, and comparing phenotypic differences of filial generations of wild types and mutants under different genetic backgrounds to judge the functions of the target genes. The method has the advantages of simplicity in operation, flexibility in design, short construction period and low cost, and gene functions can be efficiently and accurately analyzed under various genetic backgrounds.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and functional genomics, and specifically relates to a method for analyzing plant gene function. Background Technology

[0002] Gene function refers to the biological role that a gene plays in an organism, including the products it encodes and the physiological, biochemical, or developmental processes in which these products participate.

[0003] Gene function analysis techniques mainly include gene knockout, RNA interference (RNAi), gene editing, overexpression, and reporter gene analysis. Gene knockout studies the function of a target gene by completely disrupting it, providing clear results but potentially masking the phenotype due to compensatory effects or lethality. RNAi achieves gene silencing by degrading mRNA; it is simple to operate but suffers from off-target effects and inconsistent efficiency. CRISPR-Cas9 can precisely edit genes and is applicable to various organisms, but it carries the risk of off-target effects. Overexpression studies functional gains by increasing the gene dose, but may lead to artifacts due to non-physiological expression.

[0004] Genetic background refers to the entire genome composition of an individual or strain, excluding the target gene, including variations in other genes, non-coding regulatory sequences, and epigenetic modifications. The same gene may exhibit significantly different phenotypes or even completely opposite functions under different genetic backgrounds; this phenomenon is called the genetic background effect.

[0005] Studying gene function under different genetic backgrounds faces multiple challenges: First, modifying genes or structural variations in the background genome may interfere with the phenotypic presentation of the target gene through epistatic effects or compensatory mechanisms, leading to significant differences in the expression of the same gene in different strains. Second, phenotypic plasticity and pleiotropic effects complicate phenotype-genotype relationships, making it difficult to distinguish the contributions of genetic and environmental factors. Technically, constructing near-isogenic lines is time-consuming, and gene editing efficiency is affected by background-dependent chromatin states. During data analysis, background-specific differences in omics results and limitations of computational models increase the difficulty of functional interpretation. Furthermore, differences in the genetic backgrounds used by different laboratories may make it difficult to directly compare research results.

[0006] Therefore, there is an urgent need in this field for a new method that can rapidly, efficiently, and reliably analyze gene function in a variety of genetic backgrounds. Summary of the Invention

[0007] In view of the above, it is necessary to provide a method for analyzing plant gene function that is simple to operate, has a short cycle, is low in cost, and can efficiently analyze gene function in multiple genetic backgrounds.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for analyzing plant gene function includes the following steps: (a) Select the target gene and construct an edited mutant of the gene using gene editing technology; (b) Using the edited mutant and its corresponding wild-type plant as common parents, they were hybridized with multiple parents with different genetic backgrounds to construct multiple pairs of F1 populations; (c) Self-cross or cross-cross the F1 population to construct the F2 population; (d) Measure the phenotypic data of the target trait in the F1 and F2 populations, and determine the function of the target gene by comparing the phenotypic differences between wild-type and mutant offspring under different genetic backgrounds.

[0009] Furthermore, in this invention, the gene editing technology includes the CRISPR / Cas9 system.

[0010] In this invention, the multiple parents with different genetic backgrounds further include japonica rice and indica rice varieties.

[0011] In this invention, the target gene is further defined as the OsTT2 gene.

[0012] In this invention, the target trait is further defined as rice grain length and / or grain width.

[0013] In this invention, further, the common parent in step (b) is the wild type of *Donglanmomi* and its OsTT2 gene-edited mutant cr2.

[0014] In this invention, the F2 population is further obtained by self-pollination of the F1 population.

[0015] The present invention also provides a kit for carrying out the method, comprising a target sequence for constructing a gene editing vector, amplification primers, and genotype identification markers.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Precise analysis of genetic background effects: The innovative Dual-NAM population design of this invention directly addresses the problem of genetic background interference in gene function analysis. By simultaneously constructing two nested hybridization populations—"wild type × multiple maternal parents" and "mutant × multiple maternal parents"—parallel comparison of target genes under multiple genetic backgrounds is achieved. This method effectively overcomes the phenotypic misjudgment caused by epistatic effects or compensatory mechanisms in traditional single-background analysis, and significantly improves the accuracy and reliability of gene function analysis.

[0017] (2) Simple operation and significantly shortened cycle: This invention only requires the construction of F1 and F2 generation populations, which significantly shortens the breeding cycle by more than 50% (from ≥3 years to 1-2 years) compared with the traditional backcross breeding method (usually requiring 6-8 generations), and reduces costs by more than 60%. Its advantages are: ① Simplified operation, no need for multiple generations of backcrossing and cumbersome genotype screening, avoiding the problem of background residue in the construction of near-isogenic lines; ② Flexible design, the coverage of genetic background can be dynamically controlled by adjusting the number of maternal parents (such as the use of 27 maternal parents to cover the variation of the indica and japonica subspecies in this implementation case), adapting to the genetic diversity needs of different species.

[0018] (3) High-throughput and high-efficiency analysis: A single experiment can simultaneously analyze the functional differences of the target gene in multiple genetic backgrounds. Taking the OsTT2 gene as an example, through paired F1 population analysis of 27 genetic backgrounds, it was found that the gene significantly regulates grain length in 20 backgrounds and significantly regulates grain width in 18 backgrounds, which greatly improves the analysis efficiency compared with the traditional single-background method. This method can also accurately identify background-specific functional variations (such as the loss of function caused by the natural nonsense mutation of OsTT2 in P4 material), providing a high-resolution analysis tool for the genetic analysis of complex traits.

[0019] (4) Standardized kits facilitate widespread application: The kits developed for the FAGEN method (including primers designed for specific targets, vector construction components, and SSR linkage markers) help standardize the analysis process. The kits support genotyping of the F2 population at the seedling stage, enabling large-scale and rapid sample testing. The editing modules (such as CRISPR vectors / targets) and genotyping modules (such as SSR primers) are compatible, making it possible to extend the application of this method to other crops (such as maize). Attached Figure Description

[0020] Figure 1 A schematic diagram comparing the seed phenotypes of wild-type Donglan Momi rice and the OsTT2 gene-edited line; Figure 2 This is a flowchart illustrating the experimental design of the method of the present invention; Figure 3 This is a schematic diagram showing the statistical analysis results of phenotypic differences in grain length and grain width among 27 paired F1 populations; Figure 4 This is a schematic diagram showing the statistical analysis results of phenotypic differences in grain length and grain width among 27 paired F2 populations. Detailed Implementation

[0021] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Example 1: Validation of OsTT2 gene editing (1) OsTT2 affects rice grain shape Li et al. (Theoretical and Applied Genetics, 2020, 133: 2387–2399) showed in Japonica rice C7 that the minor gene SG3 negatively regulates rice grain length but does not affect grain width. Li et al. (Frontiers in Plant Science, 2022, 13: 880919) found in Japonica rice M494 and Indica rice Z9B that the major gene SMG3 regulates cell elongation and proliferation, acting as a negative regulator of both grain length and width. In this invention, we identified a rice grain shape gene, OsTT2, which is located at the same gene locus as SG3 and SMG3.

[0023] (2) Gene identification and target design Based on the Nipponbare genome sequence, the OsTT2 gene (LOC_Os02g47720) of the rice variety Donglan Heimi Rice (DLMM) was sequenced. Based on the sequencing results, CRISPR / Cas9 editing targets were designed on exons 1 and 2, respectively. Target 1: 5'- CCT ACATCGCCAAGCACGGGGAG-3' (negative chain), denoted as SEQ ID NO: 1.

[0024] Target 2: 5'-ATCAAGAGGGGCAACATCTC CGG -3' (positive chain), denoted as SEQ ID NO: 2.

[0025] Target1 is located within the SANT domain, and Target2 is located within the Myb-like DNA-binding domain of OsTT2. The underlined portion represents the PAM sequence.

[0026] (3) Vector construction and genetic transformation The target sequence described above was ligated into a CRISPR / Cas9 vector to construct the recombinant vector CRISPR / Cas9-OsTT2. PCR amplification was performed using primers: Pbw2-F (5'-GCAACGCTCTGTCATCGTTACAAT-3'), designated SEQ ID NO: 3, and Pbw2-R (5'-GCGATTAAGTTGGGTAACGCCAGGG-3'), designated SEQ ID NO: 4. Sequencing confirmed the correct insertion of the sgRNA sequence into the expression vector. The validated recombinant vector was then transformed into Agrobacterium EHA105 competent cells.

[0027] Agrobacterium-mediated transformation of rice (Nishimura et al., Nature Protocols, 2006, 1: 2796-2802) was used to transform callus tissues of Donglanmomi (DLMM) and Guanyangmomi (GYMM), respectively. After resistance selection and regeneration culture, T0 generation transgenic plants were obtained. T1 and T2 generation seeds were obtained through self-pollination, and homozygous edited lines were screened. Detailed phenotypic analysis was subsequently performed on the Donglanmomi (DLMM) background edited lines.

[0028] (3) Phenotypic analysis A genetic analysis was conducted on homozygous T2 generation plants, using DLMM as an example. The results are as follows: The wild type of Donglan Momi (DLMM WT) has a grain length of 9.16 mm and a grain width of 3.30 mm. The length of the Cr2 gene-edited line was 7.05 mm and the width was 3.79 mm. The length of the Cr3 gene-edited line was 7.12 mm and the width was 3.81 mm.

[0029] Compared to the wild type, the OsTT2 gene-edited lines showed a significant decrease in grain length and an increase in grain width. Figure 1 The above results, combined with editing experiments in the GYMM genetic background, preliminarily indicate that OsTT2 has a regulatory function on rice grain type under different genetic backgrounds.

[0030] Example 2: Implementation of the FAGEN method This invention, using grain length and width as examples, develops a novel method for identifying gene function—functional analysis using gene editing and nested crossing population (FAGEN)—to verify the function of OsTT2 in different genetic backgrounds. The specific experimental procedure is as follows: (1) Parental selection and population building Twenty-seven rice materials (12 japonica and 15 indica) were screened using molecular markers to serve as female parents. Using the wild-type Donglanmo rice and its gene-edited line cr2 as common male parents, each was crossed with one of the 27 female parents to construct 27 paired F1 populations. Each F1 population was then self-crossed to obtain the corresponding 27 paired F2 populations.

[0031] (2) Phenotypic survey and data analysis Measure the data values ​​of the target trait in the F1 and F2 populations and analyze the phenotypic variation patterns (e.g. Figure 2 ).

[0032] Example 3: Application of the FAGEN method (1) F1 representative type analysis In the 27 pairs of F1 groups: The differences in grain length between the 20 pairs of F1 populations reached a significant level; The difference in grain width between the 18 groups and the F1 population reached a significant level; The F1 populations produced by crossing only the two maternal parents, P4 and P25, with wild-type Donglanmo rice and cr2 showed no significant differences in grain length and grain width. Figure 3 ).

[0033] (2) F2 representativeness verification In the 27 paired F2 groups: Only the P1 population showed significant differences in grain length and grain width; The remaining 26 pairs of F2 populations showed no significant differences in grain length and grain width. Figure 4 ).

[0034] The above results indicate that the OsTT2 gene has a regulatory function on rice grain length and width under different genetic backgrounds. The FAGEN method can effectively analyze the expression of gene function under different genetic backgrounds.

[0035] Example 4: Reagent kit for implementing the FAGEN method This embodiment provides a kit for implementing the FAGEN method described in Examples 1-3. The kit contains the following components, which work synergistically to efficiently and accurately create gene-editing mutants and genotype their hybrid offspring to eliminate genetic background interference and accurately analyze gene function: Gene editing components: These include primer pairs for amplifying target sequences. The target sequences are designed to target key functional domains of the target gene to effectively disrupt gene function. For example, for the rice OsTT2 gene, target 1 (5'-CCTACATCGCCAAGCACGGGGAG-3') located in its SANT domain coding region and target 2 (5'-ATCAAGAGGGGCAACATCTCCGG-3') located in its Myb-like DNA-binding domain coding region are provided.

[0036] Vector construction components: include expression vectors suitable for the CRISPR / Cas9 system (e.g., pBUN421-Cas9 empty vector) for constructing gene editing and recombinant vectors corresponding to the above targets.

[0037] Validation components: These include primer pairs used to verify successful vector construction, such as primers Pbw2-F (5'-GCAACGCTCTGTCATCGTTACAAT-3') and Pbw2-R (5'-GCGATTAAGTTGGGTAACGCCAGGG-3'). This ensures the reliability of subsequent editing experiments.

[0038] Genotyping component: Contains molecular marker primer pairs for identifying the genotype of the target gene in the F2 population. The markers are SSR markers that are closely linked to the target gene (genetic distance ≤1 cM), used to rapidly and accurately identify the allelic type (wild type / mutant type) of the target gene in F2 individuals, thereby eliminating the interference of genetic background noise in phenotypic analysis and accurately revealing the function of the target gene.

[0039] The above embodiments are merely examples of several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A method for plant gene function analysis, characterized by, The method comprises the following steps: (a) selecting a target gene and constructing an editing mutant of the gene by using a gene editing technology; (b) using the editing mutant and its corresponding wild type plant as common parents, crossing them with a plurality of parents of different genetic backgrounds respectively to construct a plurality of pairs of F1 populations; (c) selfing or intercrossing the F1 populations to construct F2 populations; (d) determining the phenotype data of the target traits in the F1 and F2 populations, comparing the phenotype differences between the wild type and the mutant offspring of different genetic backgrounds, and determining the function of the target gene according to the phenotype differences.

2. The method of claim 1, wherein, The gene editing technology comprises a CRISPR / Cas9 system.

3. The method of claim 1, wherein, The parents of different genetic backgrounds comprise japonica rice and indica rice varieties.

4. The method of claim 1, wherein, The target gene is an OsTT2 gene.

5. The method of claim 1, wherein, The target traits are rice grain length and / or grain width.

6. The method of claim 1, wherein, In step (b), the common parents are Donglanmi wild type and its OsTT2 gene editing mutant cr2.

7. The method of claim 1, wherein, The F2 population is obtained by selfing the F1 population.

8. A kit for carrying out the method of any one of claims 1 to 7, characterized in that The target point sequence, amplification primer and genotype identification marker for constructing a gene editing vector are provided.

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