钙依赖蛋白激酶CPK14在花粉管生长中的功能分析  

Functional Analysis of Calcium-dependent Protein Kinase CPK14 in Pollen Tube Growth

作　　者：周利明 卢鑫蕊 马声葳 房玮 ZHOU Li-ming;LU Xin-rui;MA Sheng-wei;FANG Wei(College of Life Sciences,North China University of Science and Technology)

机构地区：[1]华北理工大学生命科学学院

出　　处：《生物技术通报》2019年第6期55-61,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31400168);河北省自然科学基金(C2014209216,C2018209112);国家重点实验室开放课题(SKLECRA2013OFP15)

摘　　要：旨在揭示钙依赖蛋白激酶CPK14 在花粉管极性生长中的生物学功能。利用基因芯片数据绘制花粉高量表达CPK 的表达图谱。去除CPK14 位于C 端的自抑制区及Ca2+结合区,保留完整的激酶区及N 端可变区,构建组成激活型(CA)的CPK14(CA-CPK14)。利用基因枪技术在烟草花粉中进行瞬时表达,比较分析全长CPK14、CA-CPK14 及GFP 对照之间的花粉管表型差异。通过CA-CPK14 与GFP-RIC4ΔC(活性ROP1 的标记物)共同表达试验,分析CA-CPK14 对ROP1 活性的影响。结果显示,CPK家族中存在8 个花粉高量表达成员(其中包括CPK14),它们在花粉中高量表达而在其他组织中表达较少。CPK14 过量表达可以造成花粉管顶端的膨大,而持续激活且不受Ca2+调控的CA-CPK14 则抑制花粉管萌发及生长。ROP1 是花粉管生长的调控关键因子,CA-CPK14 的表达可以抑制活性ROP1 的标记物(GFP-RIC4ΔC)在花粉管顶端的积累与分布,说明CPK14 在ROP1 所介导的花粉管生长调控机制中发挥一定的作用。The purpose of the study is to reveal the biological function of CPK14(calcium-dependent protein kinase 14)in pollen tube polar growth. Based on the available microarray database,the expression heat-map of pollen-specific CPK was depicted. The constitutively active CPK14(CA-CPK14)was constructed by removing the auto-inhibitor and Ca2+ binding domain at the C-terminal and retaining the complete kinase and N-terminal variable regions. The resulting construct(full-length CPK14,CA-CPK14 or GFP alone)was transiently expressed in tobacco pollen tubes via particle bombardment,and the overexpression phenotypes of transformed tubes were compared and analyzed. The effect of CA-CPK14 on the activity of ROP1 was analyzed by co-expression experiment of CA-CPK14 and GFP-RIC4ΔC(a marker of active ROP1). The results showed that eight pollen-specific CPKs(including CPK14)were identified,and they were highly expressed in mature pollen grains and less expressed in other tissues. CPK14 overexpression caused a swelling tip of pollen tubes,while CACPK14( continuous activation and lack of Ca2+ regulation)inhibited the germination and growth of pollen tube. ROP1 was a key regulator of pollen tube growth. The expression of CA-CPK14 inhibited the accumulation and distribution of active ROP1 maker(GFP-RIC4ΔC)at the top of pollen tube. These results suggest that CPK14 may play a role in the ROP1-mediated regulation mechanism of pollen tube growth.

关 键 词：花粉管 极性生长 钙依赖蛋白激酶 钙信号途径 

分 类 号：Q943.2[生物学—植物学]