响应地黄连作障碍LncRNA-RgAGT2的克隆及表达分析

Cloning and expression analysis of LncRNA-RgAGT2 responding to consecutive monoculture in Rehmannia glutinosa

连作障碍严重影响地黄的产量和品质,对其分子机制的探寻是地黄研究领域的重要问题之一。该研究基于前期数字表达谱(DGE)分析筛选到的特异响应连作障碍转录本Unigene29334_All,利用hi TAIL-PCR和RACE技术克隆到长度为1 573 bp的完整转录产物。借助ORF Finder预测发现,克隆产物所有ORF均小于300 nt,证实此转录产物为长链非编码RNA(long non-coding RNA,LncRNA)。与地黄转录组数据库比对发现,此LncRNA与丙氨酸-乙醛酸转氨酶2基因(Rg AGT2)部分区域同源,因此命名为LncRNA-RgATG2。为进一步研究LncRNA-RgAGT2的功能,该研究检测了LncRNA-RgAGT2与Rg AGT2基因在头茬和连作地黄生长发育5个关键时期(苗期,拉线期,膨大前期,膨大中期,膨大后期)的表达模式,结果暗示,LncRNA-Rg AGT2可能与地黄Rg AGT2基因存在调控关系。同时,连作地黄LncRNA-RgAGT2表达水平在各个时期均发生不同程度的变化,可以作为地黄连作障碍的"诊断标签"。该研究首次证实LncRNA-RgAGT2参与地黄连作障碍应答及调控过程,是对地黄连作障碍分子机制的有力补充。

The consecutive monoculture obstacle is a major problem in the field of Rehmannia glutiiwsa ( R. glutinosa), has severely declined the yield and quality of R. glutinosa. Here, using hiTAIL-PCR and RACE techniques, we have cloned the full-length transcript (1 573 bp) of Unigene 29334_A11 screened by DGE as a consecutive monoculture obstacle response gene of R. glutinosa. Based on ORF Finder prediction, all ORFs detected in the full-length transcript were less than 300 nt, which suggested that the above transcript was confirmed to be a long non-coding RNA ( LncRNA). With alignment in R. glutinosa transcriptome, this LncRNA was partially homologous to alanine glyoxylate transaminase 2 gene ( RgAGT2), which was named LncRNA-RgA TG2. To further explore the function of LncRNA-RgAGT2, we have examined expression patterns of LncRNA-RgAGT2 and RgAGT2 at five critical development stages (seedling, elongation, pre-expanding, mid-expanding, late-expanding) in the first and second year replanting of R. glutinosa, respectively. The results indicated that LrtcRNA-RgAGT2, as a potential regulator, is possible to play a vital role in RgAGT2 expression regulation. Meanwhile, LncRNA -RgAGT2 has presented significant variation in all development stages of R. glutinosa, which could be used as a " diagnostic label" to assess consecutive monoculture obstacle. This study, for the first time, showed that LncRNA was responsible for the response and regulation of consecutive monoculture obstacle, which would be a powerful supplement to reveal the molecular mechanisms of consecutive monoculture obstacle of R. glutinosa.