银杏小孢子母细胞响应秋水仙碱低频多倍化的细胞学和转录组分析

Analysis of Cytology and Transcriptome on Ginkgo biloba L.Microspore Mother Cells in Response to Low-Frequency Polyploidization Using Colchicine

银杏(Ginkgo biloba L.)三倍体具有很高的综合价值,但秋水仙碱诱导银杏有性多倍化仍较为困难,2n雄配子最高得率仅有7%。为初步探究秋水仙碱诱导银杏2n雄配子的形成机制,本研究使用0.6%的秋水仙碱对进入减数分裂粗线期的银杏小孢子母细胞进行避光瓶浸没处理,对不同处理时间下小孢子囊细胞中的微管骨架、生理生化变化以及转录本进行对比分析。结果显示,秋水仙碱处理的银杏小孢子母细胞游离脯氨酸、丙二醛含量及过氧化物酶活性在处理48 h时较高,可溶性蛋白含量和超氧化物酶活性低于对照组。与对照组相比,处理至24 h时,细胞微管蛋白开始减少,48 h呈弥散状,厚度变薄,荧光显微下几乎无微管蛋白显现。转录组分析共筛选到4690个差异表达基因,其中热激基因HSP11在48 h高差异表达。另外,在48 h鉴定到的41个差异转录因子中,有23个转录因子显著上调表达,其中最大的三个家族是AP2/ERF、MYB-related和NAC蛋白家族。上述结果表明,48 h是秋水仙碱诱导银杏小孢子母细胞加倍的关键时间点,HSP11基因、AP2/ERF、MYB-related和NAC转录因子家族成员在此过程中均起着关键的调控作用。本研究结果为探索与调控秋水仙碱诱导银杏2n雄配子形成有关的通路提供了参考依据,并为后续银杏有性多倍化分子机理研究奠定了理论基础。

Ginkgo biloba L.triploids has a high comprehensive utilization value,but the highest inducing rate of 2n male gametes was only 7%when treating G.biloba microspore mother cells with colchicine.In order to investigate the mechanism of colchicine induced 2n male gamete formation in Ginkgo,the microtubule skeleton,physiological and biochemical changes,and transcripts of ginkgo microspore mother cells entering the pachytene phase of meiosis treated by 0.6%colchicine with different time were studied.The results showed that the free proline(PRO)content,malonaldehyde(MDA)content,and peroxidase(POD)activity of ginkgo microspore mother cells treated with colchicine for 48 hours were relatively higher,while the soluble protein content and superoxidase(SOD)activity were lower than those of the control.Compared with the control group,when treated for 24 h,cell tubulin decreased gradually,and became diffused,thinned,and even disappeared under fluorescence microscopy when treated for 48 h.Totally 4690 differential expression genes were identified from transcriptome analysis,among which the heat shock protein HSP11 was higher expressed in group treated for 48 h than in control.Furthermore,41 transcription factors(TFs)were differentially expressed at 48 h,among which,23 TFs were significantly up-regulated.Among these 23 TFs,the richest three transcription factor families were the AP2/ERF,MYB-related,and NAC families.These results indicated that 48 h was the key time point fort responding to polyploidization of G.biloba microspore mother cells treated with colchicine.RNA-Seq analysis revealed that the HSP11 gene,AP2/ERF,MYB-related and NAC TFs may play key roles in polyploidization of G.biloba microspore mother cells.These results provide a reference basis for exploring genes or pathways related to the regulation of ginkgo 2n male gamete formation induced by colchicine,and lay a theoretical basis for the subsequent study on the molecular mechanism of ginkgo sexual multiplication.