A transcriptional repressor and its applications
By knocking out transcriptional repressors in rice through cross-species analysis and gene editing technology, the problem of inefficient regulatory factor discovery in traditional methods has been solved, the repression of target gene expression has been relieved, and crop growth, development and yield regulation under stress have been improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2026-04-03
AI Technical Summary
Traditional forward genetics methods have limited efficiency in identifying regulatory factors of complex traits. The persistent or excessive activation of some transcriptional repressors has become a negative regulatory bottleneck that limits crop adaptability, and it is difficult to remove their restriction on target pathways through gene editing technology.
By constructing a whole-genome ATAC-seq map of *Pennisetum oleraceum*, combined with open chromatin maps and H3K27me3 histone modification data of rice, transcriptional repressors were identified across species. Using CRISPR/Cas9 gene editing technology, the target ACR region upstream of LOC_Os08g06320 was specifically knocked out in rice. A vector was constructed and transformed into rice callus tissue to verify the function of the repressor.
Successfully relieving the inhibitory effect of transcriptional repressors significantly increased the expression levels of related target genes, verifying the function of repressors in regulating gene expression and providing a new breeding strategy to improve crop productivity and adaptability under abiotic stress.
Smart Images
Figure CN121022862B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology and crop genetic improvement technology, and specifically relates to a transcriptional repressor and its application. Background Technology
[0002] Plants have developed complex and sophisticated gene regulatory networks over long periods of evolution to adapt to changing external environments. Transcription factors (TFs), as core components of these networks, specifically recognize cis-acting elements, precisely regulating the expression intensity and spatiotemporal patterns of target genes, and coordinating the balance between growth, development, and environmental response. In this process, the activation or repression of transcription factors often occurs synergistically with transcriptional repressors, which act as "brakes" in gene expression, helping to suppress unnecessary or costly gene expression. By inhibiting the expression of certain developmental pathway genes or genes related to excessive stress responses, transcription factors and repressors work together to maintain cellular homeostasis and regulate resource allocation. However, the persistent or excessive activation of some repressors can also become a "negative regulatory bottleneck" limiting the expression of beneficial genes, hindering the release of plant potential. Therefore, in-depth analysis of the molecular mechanisms of transcription factors in signal recognition, transcription complex assembly, and downstream gene regulation, identifying key negative regulatory factors, and removing their restrictions on target pathways through gene editing and other means are important strategies for improving crop adaptability.
[0003] Traditional forward genetics methods have limited efficiency in identifying regulators of complex traits (such as heat resistance and yield balance). In recent years, the development of epigenomics technologies has provided powerful tools for systematically analyzing gene expression regulatory networks. Among these, techniques such as ATAC-seq can efficiently identify accessible chromatin regions (ACRs) across the entire genome. These regions mark active cis-regulatory elements (such as enhancers and promoters). Repressive histone modifications (such as H3K27me3) are often associated with long-term gene silencing or fine-tuning. Integrating the dynamic changes of ACRs with the distribution of key repressive histone modifications allows for more precise identification of genomic regions that play core regulatory roles (especially negative regulation) under normal conditions and their associated transcription factors.
[0004] This invention focuses on an important forage grass with outstanding drought and heat resistance—American foxtail grass ( Pennisetum glaucumBy constructing a whole-genome ATAC-seq map of its leaves, we systematically analyzed the dynamic changes in ACR under normal and heat stress conditions. Combined with motif enrichment analysis, we successfully identified a motif that was significantly enriched with transcriptional repressors in differentially expressed ACRs, indicating that its ACRs may contain repressors.
[0005] To overcome the technical bottleneck of difficult genetic transformation of *Napierella foenum-graecum* and to further verify the function of this transcriptional repressor, we adopted a strategy of cross-species conservation analysis and pattern system validation. Through preliminary integrated analysis of *Napierella foenum-graecum* ATAC-seq data, and further combining this with open chromatin maps and H3K27me3 histone modification data from rice, we found that the aforementioned ACR region is conserved in both species. Furthermore, this ACR is located within the H3K27me3 histone modification signal, and H3K27me3 also covers a downstream rice gene (LOC_Os08g06320) of this ACR. This suggests that the ACR region may contain a repressor, potentially regulating the expression of the downstream LOC_Os08g06320 gene.
[0006] LOC_Os08g06320 The gene belongs to the rice AHL (AT-hook motif nuclear-localized) family. The AHL family is widely distributed in plants, possessing an AT-hook DNA-binding motif and a conserved PPC / DUF296 domain. It can regulate chromatin state and gene expression through DNA-protein and protein-protein interactions, thereby influencing plant growth, development, and stress responses. Previous studies have identified and analyzed the entire rice AHL gene family, revealing that the AHL family not only participates in embryonic development, floral organ formation, and hormone regulation, but also plays an important role in responses to abiotic stresses such as high temperature, drought, and salinity. As a member of the AHL family, LOC_Os08g06320 The encoded protein possesses a typical AT-hook structure, suggesting that it participates in the developmental process and environmental adaptation regulation of rice by modulating gene expression. LOC_Os08g06320 Its functional regulation can be used to improve rice yield or enhance stress resistance, and has broad application prospects.
[0007] Based on this discovery, we designed a rigorous functional validation protocol: using CRISPR / Cas9 gene editing technology, we specifically targeted and knocked out... LOC_Os08g06320The target ACR region (approximately 500 bp) upstream was modified by H3K27me3. Through constructing editing vectors, Agrobacterium-mediated transformation of rice callus, screening and molecular identification of mutant plants, we successfully obtained homozygous mutant plants with the target sequence knocked out. By systematically comparing the phenotypes of these knockout mutants with wild-type rice under the same growth conditions, we were able to directly verify the effect of this repressor's loss of function on gene expression in a model system.
[0008] This study innovatively combined open chromatin mapping analysis of forage grass (American foxtail grass) with multidimensional epigenomic data from rice (ATAC-seq + H3K27me3 ChIP-seq). Through cross-species conservation mining, a key transcriptional repressor located in the ACR region and marked by H3K27me3 was successfully identified and located. Utilizing rice's mature and efficient genetic transformation and gene editing system, we precisely knocked out its function and visually validated the function of this repressor in a model crop, providing practical and theoretical references for future research in this area. This strategy has universal significance for improving the productivity and adaptability of various crops under abiotic stress environments. Summary of the Invention
[0009] To gain a deeper understanding of the regulatory mechanisms of transcriptional repressors on plants, this invention provides a transcriptional repressor and its applications. By inhibiting its function through gene editing or knockout, it provides a new regulatory mechanism for the regulatory pathways of crop growth, development, or yield formation. In addition, this invention also provides a method for constructing a vector to increase the repressor sequence. By editing the repressor into the vector, downstream target genes are precisely inhibited, providing a new method for gene expression.
[0010] To achieve the above objectives, the technical solution provided by this invention is as follows:
[0011] This invention provides a transcriptional repressor, the base sequence of which is shown in SEQ ID NO.1 and is named AHL17-SUP.
[0012] This invention also provides an example diagram of a vector construction that can effectively utilize the repressor sequence, the example being... Picture 3 As shown, the vector contains the base sequence SEQ ID NO.1.
[0013] The present invention also provides three knockout mutants that knock out transcription repressors in rice, the specific knockout base sequences of which are shown in SEQ ID NO2, SEQ ID NO.3 and SEQ ID NO.4.
[0014] Furthermore, the present invention also provides a knockout mutant plant of a transcriptional repressor, wherein the knockout mutant plant uses CRISPR / Cas9 technology to randomly knock out SEQ ID NO.1 to obtain a knockout vector, and uses Agrobacterium-mediated transformation to transform the knockout vector into rice callus tissue to obtain transgenic rice plants.
[0015] Furthermore, the present invention also provides a method for detecting target genes in knockout mutant plants and normal plants ( LOC Os08g06320 The expression level primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.
[0016] Furthermore, this invention provides a transcriptional repressor and its application in breeding and other fields.
[0017] The present invention has the following beneficial effects:
[0018] This invention provides a transcriptional repressor and its application. Using rice as a model plant, the repressor is knocked out, and the related target gene ( LOC_Os08g06320 The expression level of the gene significantly increased, indicating that the suppression of target gene expression was relieved after the repressor was knocked out, verifying that the element has a clear repressive activity at the transcriptional level. This discovery provides a new research direction for rice breeding and has important breeding significance and development prospects. Compared with existing technologies, this invention is the first to identify and verify a gene with transcriptional repressive activity, which plays an important role in plant functional regulation of yield, growth and development, and breeding research, and has significant biological and applied value.
[0019] This invention also provides an example diagram of constructing a vector for controlling gene expression. By inserting the repressor sequence of this invention into the vector and transferring it into the target plant, the expression level of the relevant gene can be artificially regulated. This discovery provides a new method and approach for artificially controlling the expression of target genes, and has significant breeding significance and development prospects. Compared with existing technologies, this invention is the first to propose a vector construction method that adds a repressor sequence, which plays an important role in artificially controlling the expression of relevant genes and has significant biological and applied value. Attached Figure Description
[0020] Picture 1 : Knockout qRT-PCR detection results.
[0021] Picture 2 : Rice target gene in control group and knockout body ( LOC_Os08g06320 Comparison of expression levels.
[0022] Picture 3 Example diagram of the construction of this transcriptional repressor vector. Detailed Implementation
[0023] The rice material used in this invention is derived from the rice variety with the USDA application name "Nipponbare".
[0024] Example 1: Construction of target transcriptional repressor knockout mutant plants
[0025] This invention, through preliminary experiments, combined analysis of single-cell open chromatin information and H3K27me3 histone data in rice to identify a repressor located in the rice gene sequence. Using CRISPR / Cas9 technology, a 500bp region of open chromatin covered by H3K27me3 in rice was randomly knocked out (see SEQ ID NO. 1). Based on this sequence, a target was designed and a knockout vector was constructed to obtain gene knockout mutants. The verified mutant vector was transformed into Agrobacterium, and rice callus tissue was infected. The callus tissue was cultured until the seedlings reached approximately 2-3 cm in height, and mutant plants were screened. The mutants were then cultured under the same conditions as wild-type plants.
[0026] The results are as follows Picture 1 As shown, a total of 7 samples were detected as knockouts of the target transcriptional repressor, namely sample numbers 02, 19, 35, 38, 41, 42, and 43. Based on expression characteristics and data quality, samples 02 (mutant 1), 19 (mutant 2), and 35 (mutant 3) were selected as representative materials for expression verification experiments of this invention; ysx served as the control treatment in this experiment, further demonstrating the reliability of the previous experimental results.
[0027] Example 2: Knockout of target genes related to repressors in mutant plants ( LOC_Os08g06320 ) expression level verification
[0028] Select LOC_Os08g06320 This gene serves as the subject of research on downstream gene expression levels regulated after knockout of this repressor. LOC_Os08g06320 The gene belongs to the AHL family. AHLs play a key role in regulating stress resistance in a variety of plants, and also play an important role in controlling the growth and development of rice.
[0029] Fresh leaf samples were collected from rice mutant plants and immediately stored at -80°C.
[0030] Total RNA was extracted using a high-purity RNA extraction kit (TransGen Biotech, China).
[0031] cDNA synthesis and real-time quantitative PCR (qRT-PCR) were performed using a one-step cDNA first-strand synthesis premix (Tiangen Biotech, China) and a qPCR-specific premix (Novizan Biotech, China), respectively.
[0032] Suppressor-related target genes ( LOC_Os08g06320 The qPCR amplification primers for [the gene] were SEQ ID NO.5 and SEQ ID NO.6, with actin as the internal reference gene. The specific primer sequences are shown in Table 1. Quantitative data were generated using a Bio-Rad CFX96 Touch instrument (Bio-Rad, USA). Relative gene expression levels were calculated using a 22 -ΔΔCT The method was used for calculation, and all experiments were performed in triplicate.
[0033] Table 1 Primers and internal control primers used for qPCR detection
[0034]
[0035] To study the effects of knocking out this repressor on related target genes ( LOC_Os08g06320 The effect of expression levels was investigated. Knockout mutants (mutant 1, mutant 2, mutant 3) were constructed and cultured under the same conditions as wild-type plants, followed by qRT-PCR expression analysis. The results are as follows: Picture 2 As shown, this gene is stably expressed in wild-type rice, and its expression level increases significantly after knockout, indicating that this transcriptional repressor may be involved in regulating the gene expression network and physiological response of rice under adverse conditions, and plays an important regulatory role in rice growth, development or yield formation.
Claims
1. A transcriptional repressor, characterized in that: The base sequence of the repressor is shown in SEQ ID NO.
1.
2. A vector containing the transcriptional repressor as described in claim 1.