青花菜花粉外壁蛋白基因的克隆与表达特征分析

Cloning and Expression Analysis of a Pollen Coat Protein Gene from Broccoli

花粉外壁蛋白参与多种植物生理进程,在花粉发育过程中起着重要的作用。本试验以青花菜保持系‘WN12-95B’为材料,利用RT-PCR技术克隆得到青花菜花粉外壁蛋白（PCP）基因。结果显示,该基因全长578 bp其中开放阅读框长度为561 bp共编码186个氨基酸。该蛋白为疏水性蛋白,在第1~24位有一个信号肽序列,预测相对分子量为18.82 kD等电点为10.91。青花菜PCP蛋白的二级结构主要由a-螺旋和不规则卷曲构成,不含β-转角。系统进化分析表明青花菜花粉外壁蛋白与同属植物的进化关系相近,其中与甘蓝进化关系最近。采用qRT-PCR技术分析青花菜PCP基因的表达特性,结果显示该基因仅在花蕾中表达,具有明显的组织特异性。青花菜Ogu不育系及其保持系不同发育阶段花蕾中PCP基因差异明显,表明其在花粉发育中具有重要的作用。本研究结果为进一步研究PCP基因在青花菜花粉发育过程中的作用奠定基础。

The pollen coat protein （PCP） involved in a variety of plant physiological processes, playing an important role in pollen development. A PCP gene was cloned using RT-PCR technology from a broccoli advanced inbred line ‘WN12-95B＇ in this study. The results revealed that the full length ofPCP gene was 578 bp, containing a 561 bp open reading frame to encode a total of 186 amino acid residues. Meanwhile, the PCP protein from broccoli was hy- drophobic and had a signal peptide sequence from 1 to 24. Prediction of the relative molecular mass of PCP was 18.82 kD with the theoretical isoelectric point of 10.91. The two-dimension structure of PCP protein was mainly consisted of α-helixs and random coils, and this protein does not contain the β-sheet. Homology analysis showed that the homologous species should be mainly belonged to the Cruciferae, the PCP from broccoli exists a highly i- dentity with that from Brassica oleracea. The characteristic ofgene expression was analyzed through qRT-PCR tech- nology. The results indicated that this gene should express only in the bud of broccoli and has distinct tissue speci- ficity. Among the different development stage of bud in Ogu CMS and its maintenance line, the PCP gene expres- sion had great differences, indicating that this gene should play an important role in pollen development of broccoli. The results could facilitate to further research the role ofPCP gene during pollen development in broccoli.