钙离子通道基因midA/cchA和figA在构巢曲霉无性产孢中的功能研究

Asexual sporulation is a common reproductive mode for a diverse group of fungi that includes many industrially, agriculturally, and medically important species. The understanding of the regulatory mechanisms of asexual sporulation will benefit us in using or controlling those fungi. Asexual development in Aspergillus involves the formation of specialized multicellular structures called conidiophores which is controlled through a central regulatory pathway (CDPs) with the transcription factor (TF) brlA as the key gene. However, our knowledge of the connection between sporulation signals and CDPs is rather limited. Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. We found that the calcium channel genes located in the cytoplasm membrane played important roles on conidiation in Aspergillus nidulans, but the regulatory mechanisms of calcium based signaling system on conidiation were limited. In this project, we will focus on the relationships between asexual development and calcium based signaling system through studying the cross talk among high-affinity calcium channel genes midA/cchA and low-affinity calcium channel gene figA and the CDPs, screening the key candidate genes downstream of calcium based signaling system on A. nidulans conidiation by digital gene expression profile technology and clarify the regulatory mechanisms of calcium based signaling system on CDPs. Those results will deepen our understanding of the asexual sporulation regulatory mechanisms, especially in the sporulation signals transduction in vivo and provide the theoretical basis for us to use and modify those fungi.

无性产孢是工业、农业和医学上很多重要的丝状真菌最普遍的繁殖方式，研究其调控机理可以更好地控制和利用这些真菌。研究表明曲霉无性产孢是受以brlA为关键基因的产孢中心途径（CDPs）控制，但目前对于产孢中心途径如何与产孢信号偶联从而调控分生孢子产生的机制还不清楚。本项目基于前期发现的作为胞内第二信使的钙离子的通道基因在模式真菌构巢曲霉的无性产孢中发挥重要作用，将进一步围绕无性产孢的分子调控机理这一主题，以位于质膜上高亲和性钙离子通道基因midA/cchA和低亲和性钙离子通道基因figA为研究对象，研究midA/cchA和figA基因与产孢中心途径的关系；并结合数字基因表达谱技术筛选钙信号系统下游调控产孢的关键基因；阐明钙信号系统和产孢调控途径的偶联关系。本研究不仅会加深我们对丝状真菌产孢信号在胞内传递过程的认识，还将为有效地控制有害病菌或更好地利用有益真菌提供理论依据。

本项目研究发现由MidA和CchA组成的高亲和性钙离子吸收系统和以FigA为代表的低亲和性钙离子吸收系统在构巢曲霉产孢发育中发挥重要作用。将cchA/midA敲除后，在高钙条件下几乎不影响产生分生孢子和菌丝的生长，在低钙条件下会延缓构巢曲霉分生孢子的形成，但不能阻断产生分生孢子。酵母双杂交实验表明MidA和CchA形成复合体发挥作用。将figA敲除后任何条件均阻断分生孢子的产生，说明钙亲和性双元吸收系统在不同时间以及不同营养条件下发挥的功能不同。敲除菌株的分生孢子梗都出现了不同程度的缺陷或发育迟缓，相比野生型菌株，cchA和midA敲除菌株的分生孢子梗都能形成柄，泡囊，梗基，瓶梗，在瓶梗上产生少量的分生孢子。figA敲除菌株的分生孢子梗虽然也能形成柄，泡囊，梗基，瓶梗等结构，但是形态上比较野生型病态，瓶梗上有稀少的发育不完整的分生孢子，并且产生分生孢子的数量不会随着培养时间的延长而增加。构巢曲霉中从营养菌丝到分生孢子的形成受到严格的调控。brlA是产孢发育中的关键基因，其产物可以特异的激活形成分生孢子的基因。研究发现figA基因缺失导致brlA在产孢期间不能被激活，说明figA影响产孢主要是通过调控brlA实现的。figA还影响曲霉的有性生殖，figA敲除菌不能形成闭囊壳和子囊孢子，而将cchA和midA敲除后则不影响曲霉的有性生殖。本项目成功的将可以与钙离子特异结合的水母荧光蛋白在曲霉中进行了表达，可以实时监控曲霉胞内钙离子浓度的动态变化。研究表明将cchA/midA敲除后细胞应对外界钙刺激时胞内钙离子浓度降至野生型的70％，表明高亲和性钙离子吸收系统在钙离子由胞外泵入胞内的过程中发挥重要作用。将figA 敲除后细胞应对外界钙刺激时胞内钙离子浓度降至野生型的68％，与cchA/midA敲除菌相当，而将cchA/figA双敲除后胞内的钙离子浓度进一步下降，表明在构巢曲霉中FigA和CchA/MidA协同调控外源钙离子进入胞内。进一步研究发现FigA和CchA/MidA都受到钙调磷酸酶（Calcineurin）的反馈抑制。