沈阳农业大学
辽宁省“兴辽英才计划”项目(XLYC2002073;XLYC2007169)
LiaoNing Revitalization Talents Program, China (No. XLYC2002073;No. XLYC2007169)
水稻是重要的粮食作物,盐碱是造成水稻产量下降的主要非生物胁迫之一,提高水稻品种的耐盐碱水平进而扩大种植面积是增加总产和确保粮食安全的重要举措。本研究在粳稻品种笹锦(WT)的γ射线诱导突变体库中筛选到一个耐碱性显著下降的突变体,并通过图位克隆将目标基因定位在11号染色体P2和P3之间一段87.7 kb的区间内,该区间内共有9个开放阅读框(ORF),序列比对发现突变体在ORF9的第三个外显子处有4个碱基的缺失,造成基因翻译提前终止,因此推测ORF9为耐碱性降低的候选基因,并命名为Alkaline Tolerance 11 (AT11)。AT11编码一个可能的多肽转运蛋白(peptide transporter, PTR)。转基因实验发现AT11的CRISPR/Cas9敲除突变体耐碱性减弱,而其过表达系耐碱性显著增强。AT11在水稻各组织中均有表达,在穗部表达量最高。酵母双杂交一对一验证试验和免疫共沉淀实验发现AT11可以与水稻异三聚体G蛋白γ亚基GS3互作。双分子荧光互补实验结果说明AT11与GS3在质膜上互作。转录组测序发现WT与AT11的CRISPR/Cas9敲除突变体中大量差异表达基因在色素结合、光合作用相关条目显著富集。在PH=9.0的大田条件下进行测产发现,AT11过表达植株可以显著提高产量,说明AT11能够提高水稻在盐碱地上的生产力,具有潜在耐盐碱育种应用前景。
Rice is a significant staple crop, with salinity being a primary abiotic stress leading to reduced yields. Improving rice alkaline tolerance to expand cultivation areas is vital for boosting total production and securing food supply. In this study, a mutant with significantly reduced alkaline tolerance was identified from the γ-ray induced mutation library of the Geng/japonica rice variety Sasanishiki (WT). The target gene was located in an 87.7 kb region between P2 and P3 on chromosome 11, containing nine open reading frames (ORFs) through map-based cloning. Sequence comparison revealed a deletion of four bases in the third exon of ORF9 in the mutant, leading to premature termination of the gene. Therefore, ORF9 is hypothesized to be the candidate gene responsible for reduced alkaline tolerance and has been named Alkaline Tolerance 11 (AT11). AT11 encodes a putative peptide transporter (PTR). Transgenic experiments showed that CRISPR/Cas9 knockout lines of AT11 weakens alkaline tolerance, whereas its overexpression significantly enhances alkaline tolerance. AT11 is expressed in all tissues of rice, with the highest expression in the panicle. Yeast two-hybrid and Immunoprecipitation assays revealed that AT11 can interact with GS3, the heterotrimeric G-protein γ subunit in rice. Bimolecular fluorescence complementation experiments show that AT11 and GS3 interact on the plasma membrane. Transcriptome sequencing revealed that the differentially expressed genes between WT and AT11 knockout lines were significant enriched in pigment binding and photosynthesis. Yield testing under field conditions at pH 9.0 showed that overexpressing AT11 significantly increases yield, demonstrating that AT11 can enhance the productivity of rice on saline-alkaline soils, with potential salt alkaline resistance broad breeding application prospects.