剪接体蛋白SAPH在拟南芥耐热性中的功能研究

The environmental factors play important roles in the regulation of pre-mRNA splicing. However, the mechanism of how splicing is regulated by various stresses is poorly understood. In our preliminary research, a gene encoding a spliceosome-associated protein was identified through EMS mutagenesis, mutant screening by a heat-inducible LUC reporter gene system, and map-based cloning. The gene was designated SAPH (spliceosome-associated protein in heat stress). SAPH pre-mRNA can produce two alternative splicing isoforms upon temperature conditions. At normal temperature (22℃) conditions, SAPH-I, the isoform containing the right open reading frame is produced while at high temterature (38℃), an alternative mini-exon from intron 4 is spliced into the mRNA to form the isoform SAPH-II, which results in the frame-shift and introduces a premature stop codon (PTC). The saph mutant is more tolerant to heat stress than wild type and seed germination is less sensitive to ABA. Under normal temperature conditions, the mutant plants are smaller than wild type plants. The LUC reporter gene, driven by the heat stress-inducing promoter from HSP18.2, is acitvated in mutant plants. From these data, it is hypothysized that the functional SAPH is essential to regulate the expression of heat-inducible genes in Arabidopsis. SAPH regulates the expression of its target genes through inhibiting the transcription initiation or (and) controlling the splicing of pre-mRNA of the target genes. Furthermore, the function of SAPH gene is auto-regulated by SAPH protein through temperature-dependent alternative splicing, which mediates the regulation of downstream target genes by the environmental factors. This project aims to verify the hypothesis by further experiments. To test the hypothesis, we will analyze the expression and splicing patterns of SAPH by RT-PCR, Q-PCR and reporter gene methods, evaulate the effects of different splicing isoforms on phenotypes of complementary transgenic plants, probe the interacting protein patners of SAPH by yeast two hybrid, immuno-precipitation and BiFC , and identify target genes of SAPH by RNA sequencing. The proposed research will elucidate the role and molecular mechanism of SAPH in heat stress response and thermotolerance of Arabidopsis, and will be very helpful to comprehensively elucidate the molecular mechanism of heat stress response and thermotolerance and to uncover the functions of alternative splicing in abiotic stress response and tolerance.

环境对mRNA剪接有重要调控功能，但作用机理尚不清楚。前期研究中我们克隆到一个编码剪接体蛋白的基因SAPH，其mRNA剪接受温度调控，在22℃下形成的mRNA有正常开放阅读框,在38℃高温下形成的mRNA因含有34bp的可变外显子而移码、产生提前终止密码。saph突变体在正常温度下比野生型矮小，种子萌发对ABA不敏感。在热胁迫下突变体中由热激启动子驱动的LUC报告基因的表达上调，突变体对热胁迫耐受性增加。据此我们推测：SAPH是热激基因表达的调控因子，通过影响靶基因的转录或（和）剪接而调控其表达；SAPH通过温度依赖的可变剪接反馈调节自身功能，介导温度对下游靶基因的调控。本项目拟进一步探测SAPH的表达与剪接特点，分析不同剪接异构体的功能，鉴定SAPH的互作蛋白及靶基因，以揭示SAPH在拟南芥耐热性中的功能。项目对于阐明热激基因的表达调控机制、理解可变剪接在逆境反应中的功能有重要意义。