化感水稻灵敏性抑制靶标稗草的分子调控机制研究

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.

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。

稗草是稻田恶性杂草之一，严重影响水稻生长与产量。在稻-稗竞争过程中，强化感潜力水稻能够灵敏感应稗草胁迫并增强合成化感物质抑制邻近稗草的生长。本项目研究了化感水稻灵敏响应稗草胁迫的分子机制，通过比较化感水稻‘PI312777’和非化感水稻‘Lemont’对稗草胁迫的响应灵敏性差异，分析‘PI312777’和‘Lemont’的酚酸类化感物质合成关键基因PAL的转录调控方式，结果表明PAL基因表达灵敏度是两种水稻响应稗草胁迫灵敏性差异的原因之一。与‘Lemont’相比，‘PI312777’的OsPAL2;3基因具有更高的转录水平，其基因启动子活性也更高。超表达OsPAL2;3后，水稻中的OsC4H、OsCCA、OsCOL、OsOMT等酚酸类合成途径中的基因也增强表达，OsPAL2;3 蛋白与转酮醇酶、碳酸酐酶、果糖二磷酸醛缩酶、ATP 合酶α亚基、ATP 合酶β亚基等相互作用，调控水稻的苯丙氨酸代谢途径，提高了原儿茶酸和香草酸的含量，使得‘PI312777’对稗草的抑制率增加11.11%，‘Lemont’对稗草的抑制率增加5.56%；过表达OsPAL2;1、OsPAL2;2，‘PI312777’的抑草率分别增加3.12%，1.11%；进一步研究了OsPAL2;3的表达调控机制，发现OsPAL2;3基因表达受OsWHIRLY转录因子的负调控；此外，转录因子OsMYB57通过转录调控OsMAPK11的表达，MAPK11可与PAL2;3相互作用，调控酚酸类化感物质合成。因此，OsPAL2;3的表达水平对PI312777的酚酸类化感物质合成具有正调控作用，该基因在稗草胁迫下的化感水稻PI312777中灵敏性增强表达，有利于增强化感水稻对稗草的抑制作用。项目研究结果为利用现代分子遗传学技术提高水稻对稗草的化感响应提供候选基因。