甘蓝型油菜一液胞铁转运蛋白基因（BnVIT-L2）参与铁胁迫下植物侧根发育的分子机制

Iron stress affects root development and reduces biological yield. In previous experiments, we identified a vacuolar iron transporter protein gene (BnVIT-L2) from Brassica napus. Its over-expression could enhance the resistance to iron stress and detoxification in transgenic Arabidopsis thaliana. Further studies showed that the number and length of lateral roots, auxin content and the expression level of auxin transporter PIN2 and AUX1 genes in over-expressing BnVIT-L2 transgenic plants were significantly higher than those in wild-type control under iron stress. We hypothesize that BnVIT-L2 may participate in the interaction of PIN2 and AUX1 genes to regulate the development of lateral roots under iron stress. This subject will use B. napus and A. thaliana as materials, and use the transgenic, mutation analysis, molecular hybridization, microscopic and slice observation, report gene display, transcriptome sequencing, bioinformatics and qPCR methods to investigate the molecular mechanism of the BnVIT-L2 gene involved in PIN2 and AUX1 auxin signaling pathway regulating lateral root development under iron stress and enhancing plant resistance and detoxification ability. This topic will enrich and develop people's understanding of the development of lateral roots of iron stress, which will have a wide application prospect in genetic engineering of agriculture.

铁胁迫影响根系发育并造成生物产量降低。前期实验中，我们发现：过量表达一甘蓝型油菜液胞铁转运蛋白BnVIT-L2基因可以增强转基因拟南芥抗铁胁迫水平及解毒能力。进一步研究表明：铁胁迫下过量表达BnVIT-L2基因植株的侧根数量及长度、生长素含量及生长素运输蛋白PIN2和AUX1基因的表达水平明显高于野生型对照。我们猜测：BnVIT-L2参与PIN2和AUX1基因互作调控植物铁胁迫下侧根的发育。本课题利用甘蓝型油菜和拟南芥为材料，拟采用转基因、突变分析、分子杂交、显微及切片观察、报告基因显示、转录组测序、生物信息学及qPCR等技术方法，研究BnVIT-L2基因参与的PIN2和AUX1生长素信号途径调控铁胁迫下侧根的发育进而增强植株抗逆水平及解毒能力的分子机制。本课题将丰富并发展人们对铁胁迫侧根发育的认识，在农业基因工程中具有广泛的应用前景。

本课题首次在甘蓝型油菜中克隆了一液泡铁转运蛋白基因（BnVIT-L2），该基因在多种组织器官中表达，且被铁胁迫诱导。BnVIT-L2定位于液泡膜上，具有结合铁等二价金属离子的能力。铁胁迫处理下，转BnVIT-L2基因拟南芥植株（BnVIT-L2/At）与野生型Col相比，其侧根数量及根长显著增加，叶绿素及铁含量较高，表明该基因能提高转基因拟南芥在铁胁迫下的适应能力。另外，与对照K407相比，过量表达BnVIT-L2转基因油菜植株（BnaVIT-L2-OE）在不同浓度铁胁迫下的侧根数目、叶片面积等都显著增加，也说明其具有生物强化作用。Ribo-seq测序发现铁胁迫下BnaVIT-L2-OE和K407油菜间翻译水平的差异主要富集在二价金属离子转运、碳代谢、ROS等信号通路上。DAB、NBT染色结果表明：与K407相比，BnaVIT-L2-OE的染色程度较浅，积累的活性氧量少，推测：BnaVIT-L2-OE对铁胁迫有更高的耐受性，通过活性氧清除机制来减少ROS的积累，从而减轻氧化损伤，促进植物生长。通过16S扩增子测序分析来研究根际微生物多样性发现：与对照相比，生长BnaVIT-L2-OE植株土壤的根际微生物中含有固氮作用菌属的相对较高，且土壤根际微生物物种组成更接近无铁胁迫下的微生物物种组成，这可能是由于BnaVIT-L2能够将铁转运到液泡储存，减少根系分泌物，从而使根际微生物物种组成差异性变小。该项目将为铁胁迫下VIT功能研究提供一定的借鉴。