梨花粉管质膜谷氨酸类受体通道PbrGLR3.3介导钙离子内流的分子机制

Fertilization with calcium ion (Ca2+) in flowering period is an important agronomic measures in pear production. Main reason of this is that the Ca2+ is one of the main factors for controlling pollen germination and pollen tube growth. Exogenous Ca2+ enter in the cytosolic of pollen is mainly through the Ca2+ channel that localized in the plasma membrane. Recent years, we have detected several types of Ca2+ channels in the pollen of pear, however, the encoding genes of these Ca2+ channels are still unknown. The glutamate receptor-like (GLR) channel extensively exists in the plants. Based on analysis of pear whole genome sequence, we found that the pear GLR gene family have 34 members. The expression of PbrGLR3.3 in pollen tube showed after rising to decline phenomenon during pollen tube growth, but the specific function of PbrGLR3.3 need to be further exploration. In this study, we intends to comprehensive utilization of multiple techniques, such as patch clamp, laser confocal microscope, antisense oligonucleotide gene expression suppression, to clone and functional analysis the PbrGLR3.3, identification the ligand of PbrGLR3.3, characterization of calcium current mediated by PbrGLR3.3, and identification the PbrGLR3.3 interaction protein in the intracellular of pollen. Finally, we want to clarify the signal transduction mechanisms of PbrGLR3.3 mediation Ca2+ influx in the pear pollen tube and controlling their growth, and provide the theoretical basis for the agronomic measures of fertilization with Ca2+ in flowering period.

“花期施钙”是梨生产的一项重要农艺措施，因为花粉胞质钙离子是其萌发和生长的决定性因子。外源的钙离子主要通过质膜钙通道进入胞内。近年来，申请人已在梨花粉质膜上检测到多种钙通道，但其编码基因尚不清楚。谷氨酸类受体通道是一类重要的钙通道，在植物中广泛存在。基于梨全基因组序列分析，申请人发现梨谷氨酸类受体通道基因家族共有34个成员，其中PbrGLR3.3表达量随着花粉管的生长，呈现先上升后下降的现象。本研究中，申请人拟综合利用膜片钳、激光共聚焦显微镜、反义寡核苷酸基因抑制表达等前沿技术方法，开展PbrGLR3.3的克隆与功能分析，PbrGLR3.3配体的鉴定及其介导的钙电流特性解析，以及PbrGLR3.3的花粉胞内互作蛋白鉴定及其调控分析等方面的研究，最终阐明梨花粉管质膜谷氨酸类受体通道PbrGLR3.3介导钙离子内流、调控花粉管生长的信号转导机制，从而为梨“花期施钙”的农艺措施提供理论依据。

本研究为阐明梨花粉管质膜谷氨酸类受体通道PbrGLR3.3介导钙离子内流，调控花粉管生长的机制，综合运用生物信息学、生理学、细胞生物学、分子生物学和生物化学等研究方法，对调控花粉管生长的信号转导路径开展了研究取得了如下进展：本研究探明了蔷薇科植物谷氨酸受体家族的进化过程；发现蔷薇科植物GLR基因主要分为四个亚家族（GLR1，GLR2，GLR3和GLR4）。对这些基因进行进化、共线性和扩张模式分析，发现GLR1和GLR2亚家族在木本植物中大量扩张。组织特异性表达模式研究发现GLR1和GLR2家族基因优先在木本植物形成层和顶端分生组织中表达。此研究为谷氨酸类受体基因在木本植物生长过程中所起的作用提供了生物信息学证据。梨GLR家族中GLR3亚家族具有较高保守性，其中PbrGLR3.3在多种组织中表达量均较高。对花粉萌发和花粉管生长8个阶段的基因表达研究发现，PbrGLR3.3表达量随着花粉管发育逐渐上升，使用ODN技术抑制PbrGLR3.3表达可导致花粉管钙离子浓度对D-丝氨酸的敏感性下降，并抑制花粉管生长。本研究证明了PbrGLR3.3通过调控花粉管尖端钙离子的流入进而调控花粉管生长。研究发现PbrRALF2是调控花粉管生长的关键因子。PbrRALF2作为细胞外信号分子与细胞膜受体激酶PbrCrRLK1L13的胞外域相互作用，并激活细胞内激酶活性，进而通过PbrMPK18诱导花粉管尖端活性氧产生，最终造成花粉管生长抑制。此外还发现了PbrOSCA2.6、PbrPRK6、PbrTTS1和PME等多个基因可能作为参与PbrGLR3.3调控花粉管生长的潜在因子。