CRD1调控水稻冠根发育的分子机理研究

Root is an important vegetative organ of rice. In addition to the rice capacity of tolerance to drought, waterlogging, salt alkali or leanness, the growing condition and activity of crown roots were closely related to rice plant type and fertility. The fibrous root system in rice comprises bulk of crown roots, which play important roles in nutrient and water uptake. However, little is known on the molecular mechanism of crown root development. We got a crown root deficient mutant, termed crown root deficient mutant1 (crd1), which produced fewer and less crown roots, by screening T-DNA rice mutant database. The crown root phenotypes of crd1 could be fully complemented by the CRD1 gene, and promoter fusion transgenetic plants showed CRD1 specially expressed in the crown root and the base of shoots where crown root primordia were produced, indicated that CRD1 was involved in regulating crown root development in rice. On the basis of previous work, various of approaches including in situ, Microarray, Yeast two-hybrid assays and Coimmunoprecipitation assay will be performed to study of the function of CRD1 during crown root development, and to uncover the molecular mechanism of crown root development. The result of the study will provide useful information for root genetic improvement in rice.

根是水稻重要的营养器官，水稻的耐旱、耐涝、耐盐能力以及株型、育性差异都与其根系特别是冠根的生长活力密切相关，但人们仍缺乏对冠根发育机理的认识。为了深入开展冠根发育机理研究，我们筛选了T-DNA水稻突变体库并获得一个冠根缺陷突变体，命名为crown root deficient mutant1 (crd1)，表现为冠根长度变短且冠根数目减少。此外，还表现为植株矮小、不能生殖生长。前期研究结果，CRD1基因组DNA可以恢复crd1冠根的突变表型，且CRD1在冠根和冠根原基特异表达，证实CRD1参与了对冠根发育的调控，但其调控的分子机理并不清楚。在前期研究的基础上，本课题拟以原位杂交、芯片分析、酵母双杂交和免疫共沉淀等技术为手段，以CRD1如何调控冠根发育功能为研究内容，揭示CRD1调控冠根发育的分子机理。本研究结果，将为改良水稻根系，培育营养高效利用及抗逆增强型水稻新品种提供重要理论依据。

根系是植物进行物质交换和水分、养分吸收的场所，根系的发育状况同作物抗逆、株形和产量等性状密切相关。水稻根系为须根系，主要由冠根组成。虽然目前已经克隆到一些水稻冠根发育调控基因，但是人们对冠根发育的调控机制仍不清楚。为探明水稻冠根发育的机制，本课题开展了水稻冠根发育调控基因CRD1和CRD2的研究。前期获得一个T-DNA插入突变体crd1，表现为冠根数目减少，长度变短。本课题中，我们创建了CRD1基因编辑转基因植株，并成功鉴定到不同编辑位点的纯合子crd1cr1和crd1cr2。但是，不同于前期结果，crd1cr1和crd1cr2植株冠根数目和长度与野生型根系相比差异并不显著。为了检验T-DNA突变体crd1冠根缺陷表型是否由环境因素所致，我们分析了环境因素对crd1cr1和crd1cr2植株冠根发育的影响。结果证实，冷胁迫条件下，crd1cr1和crd1cr2表现出显著的冠根发育缺陷。启动子融合结果表明，CRD1在水稻冠根发生处受冷胁迫诱导表达。这些说明，CRD1调控了冷胁迫下水稻冠根的发育。借助RT-PCR，我们从水稻冠根中分离到16条CRD1转录本。基于这些可变剪接位点信息，最终鉴定出冠根发生处受冷胁迫诱导的转录本，并创建了过量表达转基因材料。利用冷胁迫处理野生型和crd1cr1，我们开展了转录组分析。结果指示CRD1通过调控激素代谢、MAPK的活性、分生组织生长等过程，参与了冷胁迫下水稻冠根的发育。此外，CRD1互作蛋白的筛选和磷酸化实验正在开展中。针对CRD2研究，CRISPR基因敲除和过量表达植株根系表型、表达模式分析结果表明，CRD2调控了包括冠根在内的水稻根系发育。CRD2定位于细胞质膜、生长素能够部分恢复CRISPR基因敲除植株根系表型、过量表达植株对NPA处理不敏感等结果表明，CRD2调控了水稻根系生长素极性运输。CRD2基因敲除植株根系连同根茎交接处转录组分析表明，CRD2通过影响生长素在根系中的分布，改变了ERF3、DRO1、SPL3、qRT9等水稻根系发育调控基因的表达，调控了水稻根系发育。CRD1和CRD2调控水稻冠根发育研究的开展，将为改良水稻根系提供重要理论依据和有价值的基因资源。

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