野生稻化感作用QTLs反复回交过程的基因表达差异研究

Rice Allelopathy is an important genetic ecology phenomenon of resistance to weeds , which is of great significance to the ecological management weeds. The genetic diversity of wild rice germplasm and its prominent resistance to weed were important gene pools for the exploitation and utilization of allelopathy. However, the relationship of allelopathic genotype with allelochemicals and the regulation mechanism have not been clear. After the National Natural Science Foundation Project of China completed recently, the genetic background and allelopathic individuals in the F2 population of Oryza Longistaminata / Oryza sativa have been identified, and the QTL locus associated with allelopthic traits were located on 1, 2 and 11 chromosomes. On the basis, this project first repeated backcross the allelopathic QTLs to Asian cultivated rice, and every time backcross combined with the molecular assisted selection (MAS) of located marker. Secondly, the allelochemical components of the root exudates were qualitatively and quantitatively analyzed by GC-MS and HPLC techniques in the process of molecular assisted selection. Finally, these gene expression characteristics and results of allelopathic genes in rice root system will be detected and analyzed by RNAseq sequencing. The results of the study will explore the association of allelopathic genotypes with allelochemicals and their phenotypes in this wild rice germplasm. This project will explain the molecular genetic mechanism of the quantitative characters of the wild rice allelopathy to the cultivated rice at the apparent level, and to lay the foundation for the fine localization and molecular selection of allelopathic QTL, for promoting genetic improvement of modern cultivated rice to increase weed-suppression ability.

水稻化感作用是抗杂草胁迫的重要遗传生态学现象，对稻田杂草生态管理具有重要意义。野生稻种资源的遗传多样性及其较强的抗杂草能力，是发掘和利用化感性状的重要基因库。然而，化感作用基因型与化感物质组分的关系及其调控机制一直不清楚。项目前期研究已明确长雄野生稻（Oryza longistaminata）遗传背景的化感作用表型和目标性状植株，并将3个化感作用QTL定位在遗传连锁图上。在此基础上，本项目首先对携带化感QTLs的植株结合已定位标记反复回交到栽培稻；其次，对QTLs的植株反复回交过程的中选材料，采用GC-MS和HPLC分析比较根系分泌物的化感物质组分；最后，采用RNAseq测序研究比较水稻根系化感基因的表达差异。研究结果将探明野生稻化感作用基因型与化感物质组分及其表型的关联性，在表观水平阐释野生稻化感数量性状转移到栽培稻的分子遗传机理，并为化感作用QTL精细定位和分子辅助选择奠定基础。

为发掘和利用野生稻种质资源的化感作用及其基因，通过遗传改良现代栽培稻以提高水稻对杂草的抗（耐）能力。本项目通过野生稻 O. longistaminata 化感作用 QTLs 反复回交过程的表型与和基因表达差异研究，获得如下重要结果：（1）将该化感野生稻与栽培稻杂交、回交并结合分子辅助选择，成功用MAS 方法将其QTLs 转移到亚洲栽培稻中，选育出化感作用指数大于0.60的BC1、BC2和 BC3目标性状材料。（2）采用 GC-MS 和HPLC分析化感目标性状材料的化感物质组分，明确酚酸类和萜烯类物质的代谢差异。（3）比较目标性状BC1、BC2和BC3植株根系分泌物，明确酚酸类物质含量分别为化感野生稻的0.92倍、0.87倍和0.85倍；而萜类物质含量则分别为该野生稻的1.07倍、1.22倍和1.34倍；（4）对化感物质抑制杂草根/芽生长的生物活性测定，证实酚酸类物质对根生长的抑制率高，其中反肉桂酸毒力最强（ED50为43.92mg/L）；而萜类物质则对芽生长的抑制率高，其中倍萜烯则的毒力最强（ED50为65.54 mg/L）。（5）对化感受体植物的光合生理测定，证实酚酸类和萜类物质组合下显著影响杂草物的光合作用系统PSⅡ，其中萜烯作用导致稗草POD和SOD酶活性显著降低，而酚酸类物质酸则明显引起CAT和ATP酶的降低。（6）对化感作用目标性状材料进行RNAseq测序评估，检测统计了样本基因、覆盖度和差异表达情况。（7）FQ-PCR检测发现BC1、BC2和BC3中选材料的苯丙氨酸解氨酶（PAL）均较化感野生稻下调1.32~2.16倍，而萜类物质合成酶关键基因较化感野生稻上调，其中如二甲羟戊酸激酶（MK）上调达4.92~9.47倍。这些研究结果加深了对野生稻化感作用性状转移到栽培稻中的分子机制认识，为选育化感抗杂草的水稻新品种（品系））提供了科学依据。