拟南芥SVL4和SVL5基因调控花粉管细胞壁合成的分子机制

Cell walls are essential for plants to grow and develop. The stucture and biosynthesis of cell wall in pollen tube, a tip-growth cell, have their own special characteristics. Although a few genes have been reported to involve in the biosynthesis of the pollen tube wall, the molecular mechanisms that control pollen tube wall biosynthesis remain largely unknown. SHV3 and its homolog SVL1 encode GPI-anchored proteins that are involved in the growth of root hairs and hypocotyl in Arabidopsis. The double mutant shv3 svl1 exhibit a marked cellulose deficiency. SHV3 and SVL1 may regulate the cellulose synthesis of cell wall by control the transports of sucrose. The cellulose is an important componant of the inner wall of pollen tubes. In previous work, we identitied that the SVL4 and SVL5, homologs of SHV3, are expressed specifically in the development of male gametophytes in Arabidopsis. The double mutant, svl4 svl5 has been achieved by the CRISPR-Cas9 assay, which shows short siliques and dramatically reduced fertility. Furthormore, the germination and growth of pollen tubes in svl4 svl5 are repressive. In this project, we mainly study the functional characterization of SVL4 and SVL5 genes. At first,their mutations phenotypes on the development of male gametophyte will be detailed observed. Then, the role of these SVL gens on the biosynthesis of pollent tube wall will be analyzed. Furthmore, the interacted protein of SVL will be identified or isolated. This project aims to understand new molecular mechanisms that regulate the biosynthesis of the pollen tube wall in order to provide the academic data for studies on the tip-growth of pollen tubes and the biosynthesis of plant cell walls.

细胞壁对于植物细胞的生长和发育非常重要，而花粉管这个顶端极性生长的细胞其壁的结构和合成又有其特殊性。虽然一些基因被报道参与花粉管细胞壁的合成，但我们对花粉管细胞壁合成的调控机制了解得仍有限。GPI锚定蛋白SHV3和同源蛋白SVL1调控拟南芥根毛和下胚轴的生长，两个基因突变后细胞壁纤维素的含量下降，可能通过影响蔗糖的运输调控细胞壁的纤维素合成。纤维素是花粉管内壁的重要成分，我们鉴定到SHV3的同源基因SVL4和SVL5在雄配子体中特异表达，通过CRISPR-cas9技术获得了svl4svl5双突变体：角果短小，育性下降，花粉管萌发和生长受到抑制。本项目主要研究SVL4和SVL5基因的功能，观察突变体的发育表型；分析它们在花粉管细胞壁合成中的作用；分离与SVL相互作用的蛋白。以期揭示SVL基因调控花粉管壁生物合成的分子机制，为研究花粉管的极性生长和植物细胞壁合成的调控机制提供理论资料。

雄配子体的正常发育是植物完成双受精和繁衍后代的前提, 雄配子体发育包括雄配子体的形成、花粉萌发和花粉管的生长, 而花粉管壁的完整性对花粉管的正常生长至关重要。SHAVEN3 (SHV3) 家族成员SHV3和SHV3-like 1 (SVL1) 参与细胞壁的合成, 该家族另外的两个成员SVL4和SVL5在花粉和花粉管中特异高表达, 本项目对SVL4和SVL5基因在花粉管生长中的功能进行了研究。SVL4和SVL5基因表达模式相似, 在花粉和花粉管中特异高表达。为了研究SVL4和SVL5基因的功能, 观察了svl4-1和svl5-1突变体的表型, 均未观察到明显的生长缺陷。由于SVL4和SVL5基因均位于5号染色体, 距离45 Kb, 很难通过杂交获得svl4 svl5双突变体。利用CRISPR/Cas9技术在svl4-1突变体背景下敲除SVL5基因, 获得了svl4 svl5双突变体。svl4 svl5双突变体角果结实率严重下降, 大量花粉刚萌发就破裂, 部分花粉能萌发出一定长度的花粉管但是花粉管形态异常, 花粉管萌发孔的位置出现裂口或是花粉管顶端破裂, 导致内容物流出。通过透射电子显微镜观察到svl4 svl5双突变体花粉管壁结构异常, 结构松散。染色和免疫荧光结果显示, 花粉管壁中胼胝质和纤维素含量增加而高度甲酯化果胶的含量减少。增加体外萌发培养基中硼酸和Ca2+浓度, 均不能恢复svl4 svl5双突变体花粉管生长缺陷的表型。SVL4蛋白定位在花粉和花粉管的细胞质, 以及花粉的营养核中。通过IP-MS实验筛选到一些可能与SVL4互作的候选蛋白, 包括与果胶甲酯化相关的蛋白以及与代谢相关的酶。这些结果表明SVL4和SVL5基因可能是花粉管生长所必需的。总之，SVL4和SVL5基因功能冗余地参与花粉管壁的合成与修饰，该研究结果不但揭示了SHV3家族蛋白的新功能, 还丰富了植物有性生殖的分子调控网络。