化感水稻灵敏性抑制靶标稗草的分子调控机制研究

稗草是严重影响水稻生产的生物因素之一。化感水稻能灵敏响应稗草胁迫并提高苯丙氨酸解氨酶(PAL)基因表达促进合成酚酸类化感物质抑制稗草生长，非化感水稻则灵敏度低，但这种现象的内在机制仍未知，推测这与特定PAL成员基因表达的转录调控作用有关。针对此，本项目拟克隆上述两种水稻PAL家族各成员基因的启动子，比较其序列组成及活性差异，研究调控启动子活性的转录因子，并进一步获得关键转录因子的调控蛋白；与此同时，构建PAL各成员基因融合eYFP的超表达载体、以及PAL各成员基因的基因编辑载体分别遗传转化化感水稻，对比未转化的野生型植株，明确PAL各成员基因对水稻化感潜力的调控能力，并研究PAL酶的互作蛋白，综合揭示化感水稻抑草时灵敏启动PAL基因表达，并与相关蛋白共同调控合成酚酸类化感物质的过程，为促进栽培上利用水稻化感抑草作用提供理论支持。

Barnyardgrass (Echinochloa crus-galli) is a pernicious weed which causes tremendous worldwide losses of rice yields. Allelopathic rice sensitively responses to the accompanied barndyardgrass and the gene expression of phenylalanine ammonia-lyase (PAL) is up-regulated to accelerate the biosynthesis of the phenolic acids allelochemicals to inhibit the growth of target barnyardgrass, however, it is less sensitivity in the non-allelopathic rice. The underlying mechanism of the bio-interactions between rice and target barnyardgrass is still unclear and it is speculated to relative to the different capacity of gene transcriptional regulation of particular PAL gene member(s). In this project, the promoters of PAL multigenes family will be cloned and sequenced from allelopathic rice PI312777 and non-allelopathic rice Lemont respectively, to reveal the difference of the sequence and the capacity of expression promotion and the transcription factors that binding on these promoters. Proteins that interact with the key transcription factors will also be precipitated and identified. Thereafter, genes encode PAL will be cloned and fused with eYFP gene respectively to construct the recombinant plant expression vectors. Meanwhile, the CRISPR-Cas9 vectors for PAL gene editing will be also constructed, and the recombinant vectors will be transformed into rice respectively basing on Agrobacterium meditation. The positive transgenic plant would be screened and collected for further getting homozygosity. These homozygosity of transgenic lines will be co-cultured with barnyardgrass to evaluate the change of allelopathic potential, in comparison with the wild type under the same treatment. Moreover, the rice tissue will be sampled for protein extraction. The proteins that interact with PAL enzyme will be obtained by using GFP-Trap Co-immunoprecipitation method. These proteins would be identified and analyzed of the biological function, with the aims to select the most relevant proteins interacting with PAL enzyme in the regulation of phenolic acids biosynthesis, which results in the susceptive response from allelopathic rice to inhibit barnyardgrass growth. Basing on this project, it is expected to indicate the underlying process and molecular mechanism on the sensitive response of allelopathic rice to increase PAL gene expression and promote the biosynthesis of phenolic acids allelochemicals to inhibit barnyardgrass growth, which will promote the using of rice allelopathy to inhibit the weed growth in the agricultural field.