| Plants are inevitably subjected to a variety of biological and abiotic stresses in the process of life due to their growth patterns fixed on the ground.Copper is one of the heavy metal elements that affect the growth and development of the plant.As the influence of human activity leads to the accumulation of copper in the soil,the threat of the plant to the copper stress is often a long-term and multi-generation.Many studies have shown that stress can affect the subsequent growth of plants and the response of their sexual or asexual offspring to this stress.It is called "stressful memories" of plants.When the progeny with stress memory is subjected to the same stress again,it can respond quickly and positively to stress and improve the tolerance of plant to stress.The phenomenon of plant stressful memories is of great significance in species distribution,population evolution and induction of plant pharmacodynamic components.It can not only induce resistant breeding,breeding excellent varieties,but also be related to the formation of traditional Chinese medicinal materials.Continuous multiple generations of identical stress on plants is one of the main ways to study the phenomenon of plant stressful memories.1.ObjectiveIn this paper,the effects of multi-generation copper stress on the growth and physiological changes of the in vitro roots of Aquilaria sinensis were explored.In combination with the data analysis of the transcriptome,the response of the physiological and genetic level of the in vitro roots of Aquilaria sinensis to multi-generation heavy metal stress was explored,Which provide a reference for the study of stressful memories phenomenon in Aquilaria sinensis.2.Methods2.1 Culture of the in vitro roots and the screening of the concentration of copper stressThe aseptic seedlings were cultured from the seeds of Aquilaria sinensis.The isolated roots were induced by nutrient solution culture.The secondary rooting rate was used as the index to screen the suitable concentration of copper stress.2.2 Acquisition and Detection of Multi-generation Copper-stressed in Vitro Root MaterialsMulti-generation culture and stress were carried out to obtain the in vitro root materials which had experienced multi-generation copper stress.The contents of proline and malondialdehyde(MDA)were measured before and after stress in each generation,and the variation of proline and MDA was analyzed before and after four generations of stress to judge whether the materials had the effect of stress memory.2.3 Analysis of Transcriptional sets of the in vitro Roots of Aquilaria sinensis under multiple generations of Copper stressThe transcript group data of Control group C(without stress,both contemporary and parental)and Treatment group TTTC(Contemporary parents are subjected to three generations of stress without being subjected to stress)were obtained by means of transcript sequencing technique.The differential gene expression among groups was analyzed,and the genes related to proline biosynthesis pathway,peroxisome biogenesis pathway,copper transporter,cell wall membrane related gene and transcription factor were excavated.3.Results3.1 Screening of copper stress concentration and rooting of the next generation Copper stress could affect the growth of in vitro roots and the rooting rate of the next generation.With the increase of stress concentration,in vitro root wilt was yellow and root tip turned yellow or brown.The root rooting of the next generation after stress subculture was significantly inhibited.The 5 m·L-1 Cu2+ concentration had no significant effect on the rooting rate of the next generation of Aquilaria sinensisJStarting with the concentration of 10 mg·L-1、Cu2+,Gthe rooting rate of the next generation of Aquilaria sinensis was reduced to less than 50%.The rooting rate of the next generation was close to 0 under the concentration of 25 mg · L-1 Cu2+.3.2 Acquisition and Detection of Multi-generation Copper stressed in Vitro Root MaterialsThe in vitro root samples of Aquilaria sinensis before and after four generations of stress were obtained.The results showed that multiple generations of copper stress had the effect of accumulation of proline content at background level.With the increase of copper stress generation of their parents,the proline background value of the isolated roots of Aquilaria sinensis increased when they were not subjected to stress at present.3.3 Analysis of Transcriptional Group data on the in vitro Roots of Aquilaria sinensis under multiple generations of Copper stressA total of 56467 unigene,were obtained from the group C and group TTTC’s transcriptome data,with the content of GC is 42.4179%.All genes were compared to four databases and annotated to 29037 unigenes,comments with a rate of 51.423%.Using FDR<0.05 and | log2 FC |>1 as the screening conditions,a total of 18704 differentially expressed genes were obtained,of which 7794 were up-regulated and 10910 were down-regulated.KEGG enrichment analysis of differentially expressed genes showed that 2186 differentially expressed genes were annotated to 132 KEGG pathways.On the Arginine and Proline pathway,the bifunctional enzyme P5CS was up-regulated,the P5CR was down-regulated,and the proline dehydrogenase PRODH was up-regulated on the glutamic acid pathway of proline biosynthesis.The expression of COX17 mediated by copper transport was up-regulated and the expression of V-type ATPase was down-regulated in Oxidative Phosphorylation pathway.77.8%of DEG was down-regulated in Flavonoids biosynthesis pathway.The expression of Peroxisome biogenic protein PEX16,PEK 1,PEX 2,PEX 7,and two PXMP2,three MPV17 on the metabolic pathway of reactive oxygen species ROS in PMP 34,were down-regulated by Peroxisome biogenic pathway.Three CAT differentially expressed genes and four sod differentially expressed genes were up-regulated in the hydrogen peroxide metabolic pathway of antioxidant system.The differentially expressed transcription factors included 47 TFs families and 579 transcription factors,of which 220 were up-regulated and 359 were down-regulated.The 10 families with the largest number of differentially expressed tfs are ERF、NAC、bHLH、MYB、C2H2、GRAS、MYB related、WRKY、B3 and bZIP respectively.Most of the TFs family genes were down-regulated,while the WRKY family mainly up-regulated the expression,4.ConclusionIn this study,under the nutrient solution culture system established by our research group,four generations of copper stress were used to obtain the materials which showed multi-generation stress memory effect,and the transcript set sequencing technique was used to analyze the effects of transcription group sequencing on the effects of multiple generations of stress memory on the in vitro roots of of Aquilaria sinensis.The genes related to proline synthesis were screened from the data by transcript sequencing technique,and other differentially expressed genes involved in oxidative phosphorylation,peroxisome pathway and transcription factors were analyzed.It laid a foundation for exploring the mechanism of multi-generation stress memory. |
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