玉米ZmHsf17基因可变剪接在高温响应中的功能及分子机制研究

Maize is one of the important food crops in China，and high temperature and drought are the major disasters in maize production. The heat shock transcription factors (Hsfs) have been reported extensively to play crucial roles in plant heat stress signal transduction and heat stress resistance. Under high temperature stress treatment, we found the ZmHsf17 gene had three different splicing, then we cloned them from maize and named them as ZmHsf17-I, ZmHsf17-II, ZmHsf17-III, respectively. Whereas, up to now, the function and molecular mechanism of ZmHsf17 gene are still not clear. In this study, we will analyze the expression patterns of these three different splicing of ZmHsf17 in maize different tissues and under different stress treatments, as well as detect their transcriptional activation activity and the HSE element binding ability. Then, over-expression of three different splice genes in maize to obtain different types of transgenic plants, subsequently, we will observe the difference of their phenotypes in response to heat stress, and analyze their directly binding downstream genes through ChIP-Seq assay, and check the expression level of stress related genes in different transgenic plants. This study will contribute to find the novel function of different splicing of ZmHsf17 gene, and throw light on the molecular mechanism of ZmHsf17 in response to heat stress.

玉米是我国重要的粮食作物，高温是目前玉米生产主要灾害之一，已经有大量报道植物热激转录因子( heat stress transcription factors，Hsfs)在植物热胁迫信号转导过程中具有重要作用。在高温诱导下克隆了玉米ZmHsf17全长基因，发现其有三个不同的剪接体，命名为ZmHsf17-I、ZmHsf17-II、ZmHsf17-III，其基因功能及可变剪接机制尚不清楚。本项目分析ZmHsf17基因三个不同剪接体的组织和诱导表达模式，转录激活活性与HSE元件结合能力，然后过表达三个不同剪接体基因，CRISPR/Cas9敲除该基因，获得不同类型转基因株，研究不同转基因株的表型差异，利用ChIP-Seq分析不同剪接体结合下游基因，RNA-Seq分析不同转基因株抗逆相关基因的表达，从而明晰ZmHsf17基因及其不同剪接体功能和调控网络，该研究将对玉米耐高温分子机制具有重要的意义。

玉米是世界三大粮食作物之一，种植广泛，适应性强，具有重要的食用和饲用价值。然而，全球变暖带来的高温、干旱等非生物胁迫严重影响了其生长发育、产量及品质。本研究中，我们克隆了玉米ZmHsf17基因的两个不同转录本，鉴定了它们在高温胁迫中的功能和可变剪接介导的调控机制。通过基因克隆发现，ZmHsf17基因在热诱导条件下，产生全长转录本ZmHsf17Ⅰ 和截短转录本ZmHsf17Ⅱ。ZmHsf17Ⅰ 定位在细胞核中，具有转录激活活性，活性中心位于AHA基序中；ZmHsf17Ⅰ 通过诱导热响应相关基因的表达应对热胁迫，提高转基因拟南芥和玉米的耐热性。对获得的阳性ZmHsf17Ⅰ 转基因株系进行RNA-seq和ChIP-seq分析，获得9个在热胁迫中ZmHsf17Ⅰ 直接结合调控的候选靶标。ZmHsf17Ⅱ 定位在细胞核与细胞膜上，没有转录激活活性。EMSA实验表明ZmHsf17Ⅱ不结合热休克元件HSE，但是可以与ZmHsf17Ⅰ 形成异源二聚体，抑制ZmHsf17Ⅰ 的DNA结合活性。拟南芥杂交系的耐热分析表明ZmHsf17Ⅰ 转基因拟南芥提高的耐热能力被ZmHsf17Ⅱ 减弱。这些结果表明，玉米中ZmHsf17基因的可变剪接产生了一种自我控制的调节方式，ZmHsf17Ⅰ 负责提高玉米的耐热能力，ZmHsf17Ⅱ 通过竞争性形成异二聚体降低ZmHsf17Ⅰ 同二聚体的DNA结合能力，减弱ZmHsf17Ⅰ 的功能，从而调控玉米耐热和减弱热胁迫反应。这一发现拓展了对高温响应机制的理解，为利用基因工程的方法进行玉米抗高温育种提供了基础。