水稻叶形建成调控基因NL7功能分析及其热胁迫响应机制研究

In rice (Oryza sativa L.), leaves on a stem are the primary source of the carbohydrates production and its spatial extension posture is important components of ideal plant architecture, which played a significant role in grain yield of rice. In our previous study, we identified a narrow leaf mutant, named nl7, which was obtained from EMS-induces mutagenesis of indica rice variety Shuhui 527. The major difference between the wild-type and mutant plants in leaf shape and photosynthetic indexes was investigated. The candidate gene underlying nl7 has been identified by map-based cloning. In this study, we are aimed to explore the NL7-mediated molecular mechanism on leaf morphogenesis by using the technology of genetics and molecular biology. Then, the NL7-mediated regulatory pathway was constructed by integrating the Y2H, BiFC, Co-IP and Pull-down with Transcriptome, proteome and metabolome analyses, and CRISPR/Cas9 was used to analyze the genetic function of NL7 interacting genes. Further, in view that NL7 encodes a heat shock protein, its response to heat stress was investigated. These results would greatly contribute to the rice breeding of ideal plant architecture and stress tolerance.

叶片是水稻进行光合作用的主要场所，是理想株型重要组成部分，与水稻的产量表现密切相关。本研究利用前期EMS诱变籼稻品种蜀恢527获得的窄叶突变体nl7，在完成性状考察、生理测定和图位克隆并确定候选基因NL7的基础上，拟借助遗传学、形态学和分子生物学技术，探索NL7基因在叶片形态及植株形态建成中的分子生物学作用，解析其分子调控机制；根据生物信息学分析预测该基因编码一个热激蛋白，结合蛋白质组、代谢组和转录组结果进行联合分析，并利用酵母双杂交、BiFC、Co-IP、Pull-down等技术对热胁迫调控基因间互作关系进行分析，借助CRISPR/Cas9技术对与NL7具有互作关系的基因进行遗传功能分析，解析水稻热胁迫调控通路及其响应机制，为水稻理想株型育种及抗逆育种提供理论依据。

适宜的叶片形态是理想株型的重要组成部分，与水稻产量和抗逆性等息息相关。植物热激蛋白是一组非常保守的蛋白质分子家族，主要起分子伴侣、热防御和稳定细胞结构等功能，然而有关热激蛋白协同调控叶片形态发育和逆境适应性的机制研究还鲜有报道。本项目前期鉴定了一个水稻窄叶突变体nl7，该突变体伴有多蘖、植株矮小、产量减少的表型。nl7突变体叶片的大小维管束数目的减少是导致叶片窄化的主要原因。图位克隆结果表明NL7编码了一个含DnaJ结构域的热激蛋白，主要在细胞质和细胞核中表达。互补和基因编辑植株表明NL7就是调控水稻叶片宽度和分蘖的目标基因。nl7突变体耐旱能力增强，且ABA合成和流速基因的表达受到显著诱导，但对高温处理，没有显著表型。转录组测序分析表明NL7基因影响生长素的生物合成和独脚金内酯合成途径相关基因的表达。酵母双杂交、BiFC、Co-IP结果表明NL7蛋白能够与光合系统放氧复合物蛋白和碳同化电子传递蛋白相互作用，且均在叶绿体中互作。研究结果表明，NL7编码的热激蛋白通过介导叶绿体的光合系统放氧和碳同化过程，影响叶片光合作用速率，调控水稻产量形成及对逆境胁迫的适应性。因此，项目研究成果可以为水稻株型和抗逆协同改良提供重要基因资源和材料，为选育具理想株型的高产高抗水稻品种提供新思路。