灰盖鬼伞菌扩张蛋白 (expansin) 诱导菌柄细胞壁扩张的机制及生理功能研究

One of the characteristics of the growth and development of mushroom fruit bodies is the stipe elongation growth, which is mainly resulted from manifold cell elongation. Some researchers proposed that the cell elongation of the stipe was the result of enzymatic hydrolysis of matrix polysaccharides in cell walls, however, others proposed that the cell elongation of the stipe was resulted from creep of the polymers due to continuous breakage of unstable hydrogen bonds among the glucan chains by turgor pressure. Recently we purified an expansin-like protein from snail stomach juice, which could reconstitute heat-inactivated stipe wall extension of F. velutipes or Coprinopsis cinerea without the hydrolytic activity. We proposed that, like higher plant wall extension, stipe wall extension may be mediated by some endogenous expansin-like proteins that facilitate polymer slippage for stipe wall extension under stress by disrupting hydrogen binding among wall polymers. In the preliminary experiment, we isolated and purified a expansin like protein (Cc expansin1) which induced stipe wall extension from Coprinopsis cinerea. Protein blast analysis from C. cinerea genomic data explores that this cell wall protein family contains 16 homologues members. Knockdown of Cc expansin1 gene by dsRNA-inducing gene silencing inhibited formation of primordia and elongation growth of fruiting bodies. These results suggest that the Cc expansin1 involves in stipe elongation growth. In this research project, researchers will clone and heterologously express the recombinant Cc expansin1 protein in yeast cells to exclude interference with wall extension assay by contamination of minor endogenous proteins to determine whether Cc expansin1 possesses hydrolytic activity, and which one of breaking hydrogen-bonds or hydrolyzing covalent-bond corresponds to stipe wall extension; researchers will study effects of knockdown of Cc expansin genen by dsRNA-inducing gene silencing or overexpressing of Cc expansin1 gene on growth and development of fruiting foodies, analyze expression level of Cc expansin1 in Cc expansin overexpression strain and suppression strain, identify the spatial and temporal expression pattern of Cc exansin1 to elucidate the physiological role of Cc expansin1 in vivo in gene level; researchers will perform a multiple sequence alignment to examine amino acid sequence similarity between 16 members of Cc expansin family and to identify conserved motifs, inspect where each CC expansin is expressed and how the expression of each Cc expansin is associated with a specific developmental stage or a specific region to examine whether each member of the Cc expansin family has a distinct role during C. cinerea growth and development. This project aims at elucidating the mechanism and physiological function of expansion-mediated wall extension in Coprinopsis cinerea.

研究子实体生长发育对食用菌生产具有重要理论和实践意义。本研究前期从灰盖鬼伞菌细胞壁分离到扩张蛋白，可诱导菌柄细胞壁伸长，其功能未见报道，基因组中共有16个同源基因蛋白。采用RNA干扰基因沉默法使扩张蛋白基因“knockdown”, 子实体原基形成和子实体伸长生长受到抑制，说明扩张蛋白特异性作用菌柄伸长生长。本课题异源重组表达纯化扩张蛋白，排除分离纯化内源扩张蛋白可能存在的微量杂蛋白干扰，分析扩张蛋白通过水解酶作用还是打破氢键作用诱导菌柄细胞壁伸长；研究RNAi介导扩张蛋白基因“knockdown”和扩张蛋白基因过表达对子实体生长发育影响，分析扩张蛋白基因表达时间和空间特异性及与子实体生长发育关系，在体内基因水平上阐明扩张蛋白生理功能；分析扩张蛋白家族16个不同成员蛋白质同源性和亲缘关系，以及它们在子实体生长发育不同阶段和不同部位表达差异，阐明不同扩张蛋白在子实体生长发育不同生理过程中作用。

食用菌菌柄生长表现为特征性的伸长生长，主要取决于细胞壁扩张作用，但菌柄细胞壁扩张的分子机制尚不清楚。本课题发现酸诱导扩张菌柄细胞壁释放葡萄糖、N-乙酰氨基葡萄糖和N-乙酰氨基葡萄二糖，并在部分纯化的有扩张活性菌柄细胞壁蛋白组分中测到几定质酶活和葡聚糖酶活，因此分离纯化或异源重组表达并鉴定了灰盖鬼伞8个几丁质酶、5个beta-1,3-葡聚糖酶和3个几丁质脱乙酰基酶。研究结果揭示几丁质酶是菌柄细胞壁扩张蛋白，在菌柄伸长生长中起关键作用：1，外切几丁质酶ChiE1和内切几丁质酶ChiⅢ能重组热灭活菌柄细胞壁持续而稳定地扩张，释放N-乙酰氨基葡萄糖和N-乙酰氨基葡萄二糖，2，ChiE1或ChiⅢ重组热灭活菌柄细胞壁扩张像天然菌柄扩张一样具有酸性pH依赖性；3，ChiE1或ChiⅢ重组热灭活菌柄细胞壁扩张活性与菌柄伸长生长区域一致；4，酸诱导扩张的天然菌柄细胞壁和几丁质重组诱导扩张的热灭活菌柄细胞壁内表面几丁质微丝间出现很多相互分离裂隙，而对照未扩张的菌柄没有这些裂隙；5，免疫胶体金标记显示ChiE1和ChiIII作用部位在菌柄细胞壁上；6，ChiE1/ChiⅢ-RNAi双干扰菌株子实体伸长生长受到阻遏，酸诱导天然菌柄细胞壁扩张活性下降。研究结果还揭示了葡聚糖酶协同几丁质酶参与菌柄细胞壁扩张作用：1，内切beta-1,3-葡聚糖酶单独可诱导菌柄细胞壁扩张，外切beta-1,3-葡聚糖酶或beta-葡萄糖苷酶单独不能诱导菌柄细胞壁扩张，但可协同内切beta-1,3-葡聚糖酶以低浓度诱导菌柄细胞壁扩张,释放葡萄糖；2，内切beta-1,3-葡聚糖酶重组热灭活菌柄细胞壁扩张显示酸性pH依赖性和不同菌柄区域依赖性；3，beta-1,3-葡聚糖酶诱导菌柄细胞壁扩张起始速率最高，然后快速下降至零；4，内切beta-1,3-葡聚糖酶重组诱导扩张热失活菌柄细胞壁内壁几丁质微丝没有相互分离裂隙；5，低浓度内切beta-1,3-葡聚糖酶和几丁质酶单独没有伸长活性，但混合作用时诱导菌柄细胞壁扩张。这样，项目揭示了几丁质酶和葡聚糖酶介导菌柄细胞壁扩张的分子机制。项目发表SCI收录研究论文17篇，其中SCI 一区、二区top 论文12篇。