缺铁诱导可变剪切的功能分析及其调控机制研究

Sophisticated regulatory mechanisms allow plants to tightly control the homeostasis of iron, ensuring sufficient amount for optimal growth while preventing excess accumulation. Alternative splicing (AS) is an important mechanism involved in regulating the gene expression. In recent years, the increasing transcriptome analysis of alternative splicing using RNA sequencing has revealed that AS is highly pervasive in plant during development and stress responses. However, the functions of most splice variants and the regulation of AS are largely unknown. Our previous study using RNA-seq technology identified iron deficiency-induced AS. In this proposal, we will identify the functional AS variants using genetic approaches and address its physiological functions in response to iron deficiency in plants. Further experiments will carry out to understand how the potential AS control transcript and expression levels under iron starvation conditions. Regulation of AS reflects the interactions between positive and negative Cis-elements in the precursor messenger RNA and a range of trans-factors. In order to elucidate the mechanisms that control iron deficiency-dependent AS changes, we will investigate known Cis-elements and search conserved motifs that significantly enriched in iron deficiency-induced AS. Known plant arginine/serine-rich (SR) proteins and heterogenous nuclear ribonucleo proteins (hnRNPs) that involved in alternative splice site regulation will be examined by loss-of-function analysis to uncover the trans-factors that affect AS changes under iron deficiency conditions. This study will shed light on the significance and regulation of AS in response to stress signals in plants, expand our knowledge to understand the fine-turning mechanism of gene activity in cellular iron homeostasis, and open new avenues to enhance plant performance.

铁是植物必需的微量元素，缺铁会造成作物减产和品质下降，而过量会对细胞有毒害。植物通过复杂的调控网络来严格控制铁的平衡。作为调控真核基因表达的重要机制，可变剪接（Alternative Splicing, AS）参与调控植物铁平衡的研究在国内外尚属空白。前期通过分析拟南芥转录组数据，发现缺铁诱导产生特异性AS，推测AS可能参与缺铁响应过程，但对缺铁诱导AS的功能和产生机制还不清楚。本项目采用分子生物学、遗传学和生物信息学等方法，进一步验证和克隆缺铁诱导差异表达的AS异构体，分析其生理功能和分子特性，并研究调控AS的顺式元件和反式因子。通过本项目，有望挖掘功能性AS异构体，阐明其在缺铁响应中的生理功能和可能的作用机制，并鉴定缺铁诱导AS的调控因子，揭示缺铁引发AS变化的分子机制。本项目的实施，将弥补当前对植物中AS认识的不足，完善对铁平衡精细调控系统的理解，为培育富铁农林作物品种提供新思路。

由于元素自身的化学属性，铁在植物体内既有不可或缺的功能，又受到严格的调控，以满足正常生长发育的需求，同时又要避免累积过多造成的损害。因此，植物发展出精细复杂的调控系统来应对土壤中可利用铁的动态变化。本项目主要调查可变剪接（Alternative Splicing, AS）如何参与调控植物的铁平衡。首先分析了缺铁诱导产生AS的62个基因，发现有12个基因产生了新剪接异构体，缺铁条件下，部分基因的剪接模式发生改变；接着克隆了缺铁诱导变化最显著的GSTL3的2个新的AS异构体，AS-I1和AS-I6，后者由于引入了提前终止密码子而可能编码C-末端结构域缺失得截短蛋白；不同缺素处理实验表明AS-I1和AS-I6受缺铁特异诱导，且处理48小时后表达持续增强，显示GSTL3的可变剪接与缺铁应答密切相关；获得了分别过量表达GSTL3的组成型（CS）和两个可变剪接异构体的纯合系拟南芥植株，RT-PCR结果表明，AS-I6的过表达引起组成型剪接本的含量升高；但是在正常和缺铁条件下，野生型和各个过表达植株的根长、叶绿素含量以及铁还原酶活性，均未发现明显差异。.SR蛋白是调控可变剪接的重要因子，通过筛选拟南芥SR 基因家族成员的108 个T-DNA 插入突变体，获得了16 个SR基因的缺失突变体纯合系；分析正常和缺铁条件下SR家族基因的表达谱发现，在15 个SR 基因中检测到了80-90个转录本，显示AS 极大地增加了SR 基因转录本的丰富度；尽管缺铁对SR 基因的表达谱没有明显影响，但发现特定SR 基因的转录谱可能受到另外SR 基因的影响。更重要的是，定量PCR检测sr突变体中AS-I1，AS-I6，CS中表达量，发现SR34和RSZ21可能参与调控GSTL3基因的可变剪接；进一步检测了各个sr单突变体在正常和缺铁条件下的表型，所有测试材料在正常和缺铁条件下都与野生型表现相似；为了检测SR基因间是否存在功能冗余，进行了SR亚家族四个基因的突变体sr34 sr34a sr34b sr30在不同生长条件下的表型观察，结果发现，sr34 sr34a sr34b sr30在正常条件下与野生型一样，但在高铁环境下表现为植株较小，叶片颜色更暗，与铁累积造成毒害的症状相似，表明SR亚家族可能参与缺铁诱导的可变剪接调控。进一步的实验还在进行中。.