月季RhPR10.2基因克隆及生物学功能分析

为解析月季PR-10家族基因RhPR10.2在月季花瓣衰老中的生物学功能,采用Real-Time PCR、RACE PCR、异源过表达、VIGS(病毒诱导的基因沉默)等生物技术方法,分析了月季RhPR10.2基因在月季不同开放阶段花瓣中的表达谱,克隆了RhPR10.2基因全长,构建了pSuper1300-RhPR10.2异源过表达载体以及pTRV2-RhPR10.2VIGS载体.结果表明,RhPR10.2基因的表达受月季花瓣衰老显著诱导,其开放阅读框为483bp,编码160个氨基酸,含有PR-10家族特有的P-loop基序;蛋白分子式为C796H1250N204O239S2,分子量为17 566.02,等电点(pI)为6.07;RhPR10.2蛋白与葡萄VvPR10.1亲缘关系最近;与拟南芥野生型植株(WT)相比,异源过表达RhR10.2基因的拟南芥T2代纯合子植株表现出显著延迟叶片衰老的表型,伴随着更高的叶绿素含量以及更低的离子渗透率;此外,与TRV2对照相比,沉默RhPR10.2基因的月季花瓣表现出加速衰老的表型,伴随着更高的离子渗透率以及衰老marker基因RhSAG12的表达.

Cloning and Biology Functional Characteristic of RhPR10.2 in Rose (Rosa hybrida)

In order to understand the role of PR-10 family gene RhPR10.2 in the regulation of rose petal senescence, real time PCR was used to explore the expression of RhPR10.2 in rose petals of different opening stages, RACE PCR was used for cloning the full length of RhPR10.2. In addition, pSuper1300-RhPR10.2 and pTRV2-RhPR10.2 vectors were constructed for the analysis of the biological function of RhPR10.2. The results showed that the expression of RhPR10.2 was significantly induced by rose petal senescence. Its open reading frame was 483 bp, encoding a protein of 160 amino acids. RhPR10.2 protein had a typical P-loop motif which was conserved in PR-10 protein family. The molecular formula of this protein was C₇₉₆H₁₂₅₀N₂₀₄O₂₃₉S₂, with a molecular weight of 17 566.02, and a theoretical pI of 6.07. Phylogenetic tree analysis suggested that RhPR10.2 had the highest similarity with Vitisvinifera VvPR10.1. To evaluate the biological role of RhPR10.2, transgenic arabidopsis (Arabidopsis thaliana) was observed. Compared with wild type (WT), RhPR10.2 transgenic T2 plants showed that the senescence of leaves was significantly delayed, accompanying with marked higher content of chlorophyll and significant lower ion leakage. On the contrary, compared with TRV2 control, RhPR10.2 gene silence markedly promoted rose petal senescence, accompaniying with significant higher ion leakage and senescence marker gene RhSAG12 expression.