map‐based cloning

  • 文章类型: Journal Article
    籽粒白垩是一种不良性状,对水稻的籽粒产量和品质有负面影响。然而,白垩的调节机制很复杂,目前尚不清楚.我们确定了白腹率(WBR)的正调节剂。WBR7基因编码蔗糖合酶3(SUS3)。WBR7的弱功能等位基因有利于提高籽粒产量和品质。在水稻驯化的过程中,WBR7编码区的功能性G/A变异导致GT-4糖基转移酶结构域中的E541K氨基酸取代,与WBR7G(品种Beilu130中的等位基因)相比,导致WBR7A(品种Jin23B中的等位基因)的分解活性显着降低。NIL(J23B)和敲除线NIL(BL130)KO表现出低于NIL(BL130)和NIL(J23B)COM的WBR7分解活性,导致较少的蔗糖在传导器官中的分解和代谢。这导致更多的蔗糖运输到胚乳,增强胚乳中储存成分的合成并导致WBR降低。更多的蔗糖也被输送到花药,为花粉成熟和发芽提供足够的基质和能量供应,最终导致结实率增加和谷物产量增加。我们的发现阐明了通过调节蔗糖代谢和分配来提高水稻产量和品质的机制。并为改善稻米品质提供了有价值的等位基因。
    Grain chalkiness is an undesirable trait that negatively regulates grain yield and quality in rice. However, the regulatory mechanism underlying chalkiness is complex and remains unclear. We identified a positive regulator of white-belly rate (WBR). The WBR7 gene encodes sucrose synthase 3 (SUS3). A weak functional allele of WBR7 is beneficial in increasing grain yield and quality. During the domestication of indica rice, a functional G/A variation in the coding region of WBR7 resulted in an E541K amino acid substitution in the GT-4 glycosyltransferase domain, leading to a significant decrease in decomposition activity of WBR7A (allele in cultivar Jin23B) compared with WBR7G (allele in cultivar Beilu130). The NIL(J23B) and knockout line NIL(BL130)KO exhibited lower WBR7 decomposition activity than that of NIL(BL130) and NIL(J23B)COM, resulting in less sucrose decomposition and metabolism in the conducting organs. This caused more sucrose transportation to the endosperm, enhancing the synthesis of storage components in the endosperm and leading to decreased WBR. More sucrose was also transported to the anthers, providing sufficient substrate and energy supply for pollen maturation and germination, ultimately leading to an increase rate of seed setting and increased grain yield. Our findings elucidate a mechanism for enhancing rice yield and quality by modulating sucrose metabolism and allocation, and provides a valuable allele for improved rice quality.
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  • 文章类型: Journal Article
    叶片形状被以为是作物育种中最显著的农艺性状之一。然而,棉花叶片形态发生的分子基础在很大程度上仍然未知。在这项研究中,通过使用叶片向上卷曲的天然棉花突变体Cu的遗传作图和分子研究,因果基因GHCU被成功鉴定为叶片变平的关键调节因子。使用CRISPR在棉花和烟草中敲除GHCU或其同源物导致异常的叶片形状。进一步发现GHCU促进HD蛋白KNOTTED1样(KNGH1)从正面向背面结构域的转运。GHCU功能的丧失将KNGH1限制在近轴表皮区域,导致与后轴边界相比,近端边界的生长素响应水平较低。生长素分布的这种空间不对称性产生了cu突变体的向上卷曲的叶片表型。通过分析单细胞RNA测序和时空转录组数据,证实生长素生物合成基因在近轴-后轴表皮细胞中不对称表达。总的来说,这些发现表明,GHCU通过促进KNGH1的细胞间运输并因此影响生长素响应水平,在调节叶片变平中起着至关重要的作用。
    Leaf shape is considered to be one of the most significant agronomic traits in crop breeding. However, the molecular basis underlying leaf morphogenesis in cotton is still largely unknown. In this study, through genetic mapping and molecular investigation using a natural cotton mutant cu with leaves curling upward, the causal gene GHCU is successfully identified as the key regulator of leaf flattening. Knockout of GHCU or its homolog in cotton and tobacco using CRISPR results in abnormal leaf shape. It is further discovered that GHCU facilitates the transport of the HD protein KNOTTED1-like (KNGH1) from the adaxial to the abaxial domain. Loss of GHCU function restricts KNGH1 to the adaxial epidermal region, leading to lower auxin response levels in the adaxial boundary compared to the abaxial. This spatial asymmetry in auxin distribution produces the upward-curled leaf phenotype of the cu mutant. By analysis of single-cell RNA sequencing and spatiotemporal transcriptomic data, auxin biosynthesis genes are confirmed to be expressed asymmetrically in the adaxial-abaxial epidermal cells. Overall, these findings suggest that GHCU plays a crucial role in the regulation of leaf flattening through facilitating cell-to-cell trafficking of KNGH1 and hence influencing the auxin response level.
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  • 文章类型: Journal Article
    叶片衰老是关系到籽粒产量潜力和营养品质的重要生理过程。花后的绿叶持续时间(GLD)直接反映了普通小麦的叶片衰老过程,并表现出较大的基因型差异;但是,潜在的基因调控机制仍然缺乏。这里,我们通过基于图的克隆将TaNAM-A1鉴定为籽粒灌浆期间GLD的主要基因座qGLD-6A的因果基因。转基因测定和TILLING突变体分析表明,TaNAM-A1在调节叶片衰老中起关键作用,并影响穗长和晶粒尺寸。此外,系统评估了TaNAM-A1三种单倍型之间的功能差异。与野生型TaNAM-A1a相比,具有TaNAM-A1d的小麦品种(在TaNAM-A1的编码DNA序列中包含两个突变)表现出更长的GLD和与产量相关的性状。所有三种单倍型在激活参与大分子降解和矿物质营养再动员的基因的表达方面都有功能。TaNAM-A1a的活性最强,TaNAM-A1d的活性最弱。TaNAM-A1还调节衰老相关转录因子TaNAC-S-7A和TaNAC016-3A的表达。TaNAC016-3A通过蛋白质相互作用增强TaNAM-A1a的转录激活能力,从而促进衰老过程。我们的研究为在各种地理气候条件下小麦育种的叶片功能期和籽粒产量形成的微调提供了新的见解。
    Leaf senescence is an essential physiological process related to grain yield potential and nutritional quality. Green leaf duration (GLD) after anthesis directly reflects the leaf senescence process and exhibits large genotypic differences in common wheat; however, the underlying gene regulatory mechanism is still lacking. Here, we identified TaNAM-A1 as the causal gene of the major loci qGLD-6A for GLD during grain filling by map-based cloning. Transgenic assays and TILLING mutant analyses demonstrated that TaNAM-A1 played a critical role in regulating leaf senescence, and also affected spike length and grain size. Furthermore, the functional divergences among the three haplotypes of TaNAM-A1 were systematically evaluated. Wheat varieties with TaNAM-A1d (containing two mutations in the coding DNA sequence of TaNAM-A1) exhibited a longer GLD and superior yield-related traits compared to those with the wild type TaNAM-A1a. All three haplotypes were functional in activating the expression of genes involved in macromolecule degradation and mineral nutrient remobilization, with TaNAM-A1a showing the strongest activity and TaNAM-A1d the weakest. TaNAM-A1 also modulated the expression of the senescence-related transcription factors TaNAC-S-7A and TaNAC016-3A. TaNAC016-3A enhanced the transcriptional activation ability of TaNAM-A1a by protein-protein interaction, thereby promoting the senescence process. Our study offers new insights into the fine-tuning of the leaf functional period and grain yield formation for wheat breeding under various geographical climatic conditions.
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