Method for excavating rice low-phosphorus adaptive gene by combining phenotype and transcriptomics
By combining systematic methods of phenotypic screening, gene localization and transcriptomics, the problem that traditional methods are difficult to fully explore the low-phosphorus adaptive genes in rice is solved, and precise gene mining and in-depth analysis are achieved, providing gene resources and theoretical basis for the cultivation of low-phosphorus-resistant rice varieties.
Patent Information
- Application Number
- CN202510405213.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-08-08
AI Technical Summary
Traditional gene mining methods are usually limited to a single phenotypic screening method or gene expression analysis, and it is difficult to fully and in-depth reveal the functions of rice low-phosphorus adaptive genes and their complex regulatory networks.
Combined with systematic methods of phenotype screening, gene localization, transcriptome sequencing and functional verification, including phenotype screening under low phosphorus stress, hybrid population construction, molecular marker and linkage analysis, RNA sequencing and analysis, and gene function verification, we accurately dig up rice low phosphorus adaptive genes.
The comprehensive and precise mining of low-phosphorus adaptive genes in rice has been achieved, rich gene resources have been provided, and the gene expression changes under low-phosphorus stress has been deeply analyzed, which has promoted the cultivation of low-phosphorus-resistant rice varieties and the sustainable development of agriculture.
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Figure CN120452537A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of plant molecular biology and genetics, and relates to a method for mining rice low-phosphorus adaptability genes by combining phenotype and transcriptomics. Background Art
[0002] Phosphorus, as a key nutrient element indispensable for plant growth and development, plays a vital role in various physiological activities of plants. However, in the natural soil environment, phosphorus mostly exists in a form that is difficult for plants to directly absorb and utilize. This makes crops often face the dilemma of low phosphorus stress during their growth, which in turn seriously limits the growth and development of crops and the increase in yield. As an important food crop widely grown around the world, the root structure of rice has a vital influence on the efficiency of phosphorus absorption. Research in recent years has gradually revealed that auxin plays a key role in regulating the root structure of rice in response to low phosphorus stress, but the specific molecular mechanism has not yet been fully understood.
[0003] Traditional gene mining methods are often limited to single phenotypic screening methods or rely solely on gene expression analysis. This one-sided approach makes it difficult to fully and deeply reveal the functions of low-phosphorus adaptability genes and the complex regulatory networks behind them. With the rapid development of modern biological technologies, the need for systematic and comprehensive exploration of low-phosphorus adaptability genes in rice is becoming increasingly urgent. Therefore, developing a new gene mining method that organically combines phenotypic analysis and transcriptomics technology is of great practical significance for promoting the breeding of low-phosphorus-tolerant rice varieties and deepening the understanding of plant low-phosphorus response mechanisms.
[0004] Therefore, the present invention, through a series of rigorous and systematic operational processes, starts from phenotypic screening and goes through key steps such as gene mapping, transcriptome sequencing and functional verification, and is committed to accurately discovering the adaptive genes of rice that play an important role under low-phosphorus stress conditions, providing a solid genetic resource foundation and reliable theoretical basis for the subsequent breeding of low-phosphorus-tolerant rice varieties. Summary of the Invention
[0005] The first purpose of the present invention is to provide a method for mining rice low-phosphorus adaptability genes by combining phenotypic and transcriptomics, overcoming traditional gene mining methods that are usually limited to a single phenotypic screening method or rely solely on gene expression analysis. This one-sided approach makes it difficult to fully and deeply reveal the functions of low-phosphorus adaptability genes and the complex regulatory networks behind them.
[0006] The second object of the present invention is to provide the use of the above method.
[0007] The present invention is achieved through the following technical solutions: 1. A method combining phenotypic and transcriptomics to mine low-phosphorus adaptability genes in rice, comprising the following steps: phenotypic screening under low-phosphorus stress; gene mapping and cloning; transcriptome sequencing and analysis; functional verification; and application of adaptability genes.
[0008] Furthermore, the phenotypic screening under low phosphorus stress includes: Culture Conditions: Healthy, plump rice seeds were selected and cultured in hydroponic environments with either low phosphorus (10 μM) or normal phosphorus (300 μM). During the culture process, environmental conditions such as light, temperature, and humidity were strictly controlled to ensure stability and consistency of the experimental environment.
[0009] Phenotypic Observation and Recording: Regularly and meticulously observe and accurately record rice root phenotypes, including changes in taproot length, lateral root growth, and root hair length. Close attention is also paid to aboveground phenotypes, such as changes in leaf inclination and plant height growth. Through long-term observation and statistical analysis of a large number of samples, mutants with significantly reduced responses to low-phosphorus stress, such as the lrlp1 mutant, have been identified.
[0010] Furthermore, the gene mapping and cloning includes: Hybrid population construction: Using map-based cloning technology, the selected mutants are hybridized with wild-type rice to construct F2 segregating populations. During the hybridization process, strict hybrid breeding practices are adhered to to ensure the quality and quantity of the hybrid offspring.
[0011] Molecular markers and linkage analysis: Molecular marker technology is used to perform genotyping on the F2 segregating population, and combined with genetic linkage analysis methods, the target gene can be accurately located to a specific region of the chromosome, such as the 242.5 kb region.
[0012] Gene identification: By sequencing and analyzing the located region and conducting functional complementation experiments, the target gene, such as the OsTAR2 gene, can be accurately identified and its mutation site determined.
[0013] Furthermore, the transcriptome sequencing and analysis includes: RNA extraction: RNA was extracted from wild-type and mutant root tissues under low-phosphate and normal-phosphate conditions, respectively, to ensure RNA integrity and purity.
[0014] High-throughput sequencing: High-throughput RNA sequencing (RNA-seq) technology is used to perform deep sequencing of the extracted RNA samples.
[0015] Bioinformatics analysis: Professional bioinformatics tools, such as HISAT2 for sequence alignment, StringTie for transcript assembly, and DESeq2 for differentially expressed genes (DEGs) analysis, were used to screen out genes that were significantly upregulated or downregulated under low-phosphorus conditions.
[0016] Gene function annotation: Through gene ontology (GO) and pathway analysis, we deeply identified gene networks related to phosphorus starvation response, auxin synthesis, and root development, providing clues for understanding gene functions and regulatory mechanisms.
[0017] Furthermore, the functional verification includes: Construction of gene knockout mutants: Using CRISPR / Cas9 technology, corresponding knockout mutants of target genes, such as OsTAR2 and OsYUC8, were constructed to verify their phenotypic and gene expression changes under low phosphorus stress.
[0018] Gene expression verification: qRT-PCR technology was used to verify the expression patterns of key genes, such as OsPHT1;2, OsPHT1;4, and OsSPX1, under low-phosphorus conditions to ensure the accuracy of gene expression data.
[0019] Auxin distribution observation: With the help of auxin reporter lines, such as DR5::VENUS, the distribution changes of auxin in root tissue under low phosphorus conditions are observed to further reveal the mechanism of action of auxin in low phosphorus response.
[0020] Furthermore, the application of the adaptive gene includes: Molecular marker-assisted breeding: Identified low-phosphorus adaptability genes, such as OsTAR2 and OsYUC8, will be used in molecular marker-assisted breeding to accelerate the breeding process of low-phosphorus-tolerant rice varieties through marker screening.
[0021] Application of gene editing and transgenic technology: Use gene editing or transgenic technology to optimize the expression of target genes to improve the adaptability and yield of rice in low-phosphorus soil environments.
[0022] The positive effects of adopting the above technical solutions: 1. Comprehensive and precise gene mining: Through a systematic phenotypic screening, gene mapping, transcriptome sequencing, and functional verification process, key genes related to rice's adaptability to low-phosphorus can be comprehensively and accurately mined. Compared with traditional methods, this greatly improves the efficiency and accuracy of gene mining.
[0023] 2. Providing rich genetic resources: A series of low-phosphorus adaptability genes identified, such as OsTAR2 and OsYUC8, provide rich and valuable genetic resources for the breeding of low-phosphorus-tolerant rice varieties, which helps promote the genetic improvement of rice varieties.
[0024] 3. In-depth analysis of molecular mechanisms: In-depth analysis of changes in rice gene expression and regulatory networks under low-phosphorus stress provides important clues to reveal the molecular mechanisms of plant response to low-phosphorus, and helps to further improve the theoretical system of plant stress biology.
[0025] 4. Promote sustainable agricultural development: The cultivated low-phosphorus-tolerant rice varieties can maintain good growth and yield in low-phosphorus soil environments, reduce the use of phosphorus fertilizers, lower agricultural production costs, and alleviate environmental pollution, which is of great significance for promoting sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Comparison of the phenotypes of the wild type and two mutants under low-P conditions; Figure 2 Analysis of differentially expressed genes (DEGs) between the wild type WT and different mutants under phosphorus-sufficient and phosphorus-deficient conditions, including (A) the number of differentially expressed genes between different treatments. (B) Venn diagram of differentially expressed genes between the wild type WT and different mutants. Figure 3 shows the enrichment of gene functions corresponding to differentially expressed genes, including (A) WT wild type-specific and (B) wild type and two mutants-shared gene function enrichment. DETAILED DESCRIPTION
[0027] The present invention is further described below by way of examples. It should be understood that these examples are for illustrative purposes only and are in no way intended to limit the scope of the present invention.
[0028] Sources of biological materials in the present invention: 1. All gene information in this invention was obtained from the Rice Genome Annotation Project (MSU-RGAP); rice plants were generated from wild-type (WT) Nipponbare (NIP). Example
[0029] This example illustrates the implementation of phenotypic screening under low phosphorus stress.
[0030] Seed pretreatment: Select several plump, pest-free rice seeds and disinfect them by soaking them in 75% ethanol for 3-5 minutes. Then rinse them 3-5 times with sterile water. Place the disinfected seeds in a 30°C incubator to accelerate germination for 24-48 hours. Once the seeds appear white, proceed to the next step of the experiment.
[0031] Hydroponic cultivation: Prepare several hydroponic containers and add complete nutrient solutions containing either low phosphorus (10μM) or normal phosphorus (300μM). Sow the germinated seeds evenly on the nylon mesh in the hydroponic containers, sowing 20-30 seeds per container. Place the hydroponic containers in a light incubator with a light intensity of 3000-5000 lux, a light duration of 16 hours per day, a temperature of 28°C, and a humidity of 70%.
[0032] Phenotypic observation and recording: Starting from the 7th day after sowing, the main root length and lateral root length of the rice were measured every 3 days using a ruler, the root hair length was measured using a microscope, and the leaf inclination and plant height were recorded. The observation was continued for 4-6 weeks, and the data were statistically analyzed ( Figure 1 ), and screened out mutants with significant phenotypic differences. Example
[0033] This example illustrates the implementation of gene mapping and cloning.
[0034] Hybridization: A mutant identified through phenotypic screening is selected as the female parent, and a wild-type rice plant is used as the male parent. During the flowering phase, the female plant is emasculated to remove immature stamens. The female plant is then pollinated with pollen from the male plant. After pollination, the seeds are bagged to prevent contamination from other pollen. Once mature, the resulting F1 seeds are harvested.
[0035] F2 population construction: Sow F1 seeds in a hydroponic environment with a normal phosphorus nutrient solution. After the F1 plants grow to the flowering stage, self-pollinate and harvest F2 seeds. Sow a large number of F2 seeds in a hydroponic environment with a low phosphorus nutrient solution to construct an F2 segregating population.
[0036] Molecular marker and linkage analysis: 100-200 plants with distinct phenotypic differences are selected from the F2 segregating population, and their genomic DNA is extracted. Based on the rice genome sequence, primers for molecular markers such as SSR and SNP are designed and synthesized. Genotyping of these plants is performed through PCR amplification and electrophoresis analysis. Genetic linkage analysis is performed using software such as JoinMap to localize the target gene to a specific region of the chromosome.
[0037] Gene identification: The gene within the targeted region is sequenced and analyzed, compared with the wild-type gene sequence to identify the mutation site. An expression vector containing the wild-type target gene is constructed and transformed into the mutant for functional complementation experiments. If the transformed plant returns to a normal phenotype, the gene is confirmed to be the target gene. Example
[0038] This example illustrates the implementation of transcriptome sequencing and analysis.
[0039] RNA Extraction: Fresh root tissue was collected from wild-type and mutant rice plants grown under low- and normal-phosphate conditions for 4-6 weeks and immediately frozen in liquid nitrogen. Total RNA was extracted from the root tissue using Trizol reagent, and RNA integrity and purity were assessed by agarose gel electrophoresis and a Nanodrop spectrophotometer.
[0040] RNA-seq library construction and sequencing: Using the extracted high-quality RNA as a template, construct an RNA-seq library according to the instructions of the Illumina sequencing library construction kit. The constructed library is subjected to high-throughput sequencing on the Illumina sequencing platform to obtain raw sequencing data.
[0041] Bioinformatics analysis: HISAT2 software was used to align the raw sequencing data with the rice reference genome, and unaligned reads were removed. StringTie software was used to assemble transcripts and calculate gene expression levels ( Figure 2 DESeq2 software was used to analyze differentially expressed genes, screening for genes with a fold difference greater than 2 and a P value less than 0.05 between low-P conditions and normal-P conditions. Gene Ontology (GO) and KEGG pathway analyses were performed using online tools such as DAVID to determine the functions of the differentially expressed genes and the metabolic pathways they participated in (Figure 3). Example
[0042] This embodiment illustrates the implementation of functional verification.
[0043] CRISPR / Cas9 gene knockout mutant construction: Specific sgRNA sequences are designed for target genes, such as OsTAR2 and OsYUC8. The sgRNA sequences are ligated to CRISPR / Cas9 expression vectors and transformed into Agrobacterium. Using Agrobacterium-mediated genetic transformation, the constructed vectors are introduced into rice callus tissue. After screening, differentiation, and regeneration, gene knockout mutant plants are obtained.
[0044] qRT-PCR verification of gene expression: Root tissues of wild-type and mutant plants grown under low-phosphorus and normal-phosphorus conditions were selected, RNA was extracted and reverse-transcribed into cDNA. Specific primers were designed based on the sequences of the target gene and the reference gene. Amplification reactions were performed using a qRT-PCR instrument. ΔΔ The relative expression levels of target genes were calculated using the Ct method to verify their expression patterns under low-phosphorus conditions.
[0045] Auxin distribution observation: A vector containing the auxin reporter line DR5::VENUS was transformed into wild-type and mutant rice to generate transgenic plants. Transgenic plants were cultured under low- and normal-phosphate conditions. Root tissue was collected and the distribution of auxin in the root tissue was observed using a laser confocal microscope. Photos were taken and analyzed. Example
[0046] This example illustrates the implementation of adaptive gene application.
[0047] Marker-assisted breeding: Based on identified low-phosphorus adaptability genes, such as OsTAR2 and OsYUC8, molecular markers closely linked to them are developed. During rice breeding, these markers are used to screen hybrid offspring, rapidly identifying plants harboring the target gene and accelerating the selection of low-phosphorus-tolerant rice varieties.
[0048] Application of gene editing and transgenic technologies: Using CRISPR / Cas9 gene editing technology, endogenous low-phosphorus adaptability genes in rice can be optimized and edited to alter their expression levels or protein structure. Alternatively, transgenic technology can be used to introduce exogenous low-phosphorus adaptability genes into rice, causing them to be overexpressed. Gene-edited or transgenic rice plants are then subjected to low-phosphorus stress treatment to identify plants that grow well and increase yield under low-phosphorus conditions, allowing them to be further developed into new low-phosphorus-tolerant rice varieties.
[0049] Through the above specific implementation methods, the method of combining phenotypic and transcriptomics to mine rice low-phosphorus adaptability genes can be effectively implemented, providing strong technical support for the cultivation of low-phosphorus-tolerant rice varieties and the sustainable development of agricultural production.
[0050] This study developed an innovative, systematic method combining phenotypic analysis and transcriptomics to precisely identify genes that regulate rice's adaptation to low-phosphorus stress. This method efficiently identifies rice individuals with significant phenotypic variation under low-phosphorus conditions. Advanced gene mapping techniques are used to locate the relevant genes, followed by comprehensive analysis of gene expression changes using transcriptome sequencing. Finally, functional validation confirms the role of key genes, providing a rich and critical genetic resource for developing new low-phosphorus-tolerant rice varieties and laying a solid theoretical foundation.
Claims
1. A method for mining rice low-phosphorus adaptability genes by combining phenotypic and transcriptomics analysis, characterized by: The following steps are involved: Phenotypic screening under low phosphorus stress; gene mapping and cloning; transcriptome sequencing and analysis; functional verification; application of adaptive genes.
2. The method according to claim 1, characterized in that The phenotypic screening under low phosphorus stress includes: Culture condition setting: Select healthy and plump rice seeds and place them in low-phosphorus and normal-phosphorus hydroponic environments for cultivation; Phenotypic observation and recording: Regularly and carefully observe and accurately record the root phenotype of rice, including changes in the length of the main root, the growth length of the lateral roots, and the specific length of the root hairs; at the same time, pay close attention to the aboveground phenotype, such as changes in leaf inclination and the growth trend of plant height; and identify mutants with significantly reduced response to low phosphorus stress.
3. The method according to claim 1, characterized in that The gene positioning and cloning include: Hybrid population construction: Using map-based cloning technology, the screened mutants are hybridized with wild-type rice to construct F2 segregating populations; Molecular markers and linkage analysis: Use molecular marker technology to genotype the F2 segregating population and combine it with genetic linkage analysis to accurately locate the target gene to a specific region of the chromosome; Gene identification: The target gene can be accurately identified by sequencing and analyzing the located region and conducting functional complementation experiments.
4. The method according to claim 1, wherein The transcriptome sequencing and analysis include: RNA extraction: RNA was extracted from wild-type and mutant root tissues under low-phosphate and normal-phosphate conditions to ensure RNA integrity and purity. High-throughput sequencing: High-throughput RNA sequencing (RNA-seq) technology is used to perform deep sequencing on the extracted RNA samples; Bioinformatics analysis: Using professional bioinformatics tools, we screened out genes that were significantly up-regulated or down-regulated under low-phosphorus conditions; Gene function annotation: Gene ontology and pathway analysis were used to deeply identify gene networks related to phosphorus starvation response, auxin synthesis, and root development.
5. The method according to claim 1, wherein The functional verification includes: Construction of gene knockout mutants: Using CRISPR / Cas9 technology, corresponding knockout mutants are constructed for target genes; Gene expression verification: qRT-PCR technology was used to verify the expression pattern of key genes under low-phosphorus conditions; Auxin distribution observation: With the help of auxin reporter system, the distribution changes of auxin in root tissue under low phosphorus conditions were observed.
6. The method according to claim 1, characterized in that Applications of the adaptive genes include: Molecular marker-assisted breeding: The identified low-phosphorus adaptability genes are applied to molecular marker-assisted breeding to accelerate the breeding process of low-phosphorus-tolerant rice varieties through marker screening. Application of gene editing and transgenic technology: Gene editing or transgenic technology is used to optimize the expression of target genes.
7. Use of the method according to claim 1 in cultivating high-quality low-phosphorus-tolerant rice varieties.