The Study Of NaCl Stress Treatment And Transcriptome In Three Consecutive Generations Of Aquilaria Sinensis Vitro Roots

It shows that the stimulating memory experienced by plants under stress can be inherited across generations.The contemporary stress of plants can influence the response of offspring or itself to stress.Stress memory has great significance in the study of plant stress mechanisms and the breeding of new stress-tolerant varieties.Stress memory can also provide references for secondary metabolite accumulation in herbs,efficient cultivation of medicinal plants,and traditional of Chinese medicine.We used the in vitro root of Aquilaria sinensis as a material to establish a salt-stress memory system,explore the biomarker compounds under the salt stress of Aquilaria sinensis.We used transcriptome technology to search for its biomarker genes in order to study changes in stress memory system.objectiveA salt stress memory system was established for isolated roots of Aquilaria sinensis.We explored the change rules of soluble sugar and antioxidant enzymes and signature compounds in the system.And using transcriptome technology to find biomarker genes.Methods 1.Salt Stress ScreeningIn this experiment,the in vitro roots of Aquilaria sinensis were used as experimental materials and added 0,150,300,450 m mol/L of salt stress with 1,3,5 days,respectively.The rooting rate,lateral root growth,and proline content were selected as indicators.Ssalt stress conditions with little change in regeneration and significant proline content were selected as suitable salt stress.2.The Establishment of a Salt Stress Memory SystemThree generations of in vitro roots of Aquilaria sinensis were treated with selected salt stress concentration to establish a salt-stress memory system.The growth and proline content of lateral roots under three consecutive salt stresses were tested to verify whether the series of materials had a “memory effect”.Subsequently,the changes of antioxidant enzyme activity,soluble sugar content and chemical composition under salt stress memory systems were measured.3.Transcriptome Data Analysis of Salt Stress Memory SystemRNA-seq technology was used to extract total RNA and perform transcriptome sequencing.Differential gene expression in salt-stress memory systems was analyzed from the transcriptome.Simultaneously,genes associated with changes in proline,soluble sugar content,secondary metabolism were screened to find biomarker genes of salt-stress memory system.Results 1.Salt Stress ScreeningAfter treatment with in vitro roots of 0,150,300,450 m mol/L NaCl with 1,3,5 days,the growth of the second generation roots showed a trend of decreased root length and lateral root number,and increased lateral root diameter with increasing salt concentration.The rooting rate and lateral root number at low concentration were similar to those of the control group,and the root length increased,suggesting that low concentrations(150 mmol/L)can promote lateral root growth.After treatment with salt,the proline content of the explant roots of Aquilaria sinensis was significantly increased.With the increase of time,the proline content in the same concentration increased first and then decreased,showing the adaptation process to salt stress.The results showed that the salt stress at 150 mmol/L with 3 days had no significant effect on the growth of vitro roots and the content of proline was increased.We used this treatment to establish salt stress memory system.2.The Establishment of a Salt Stress Memory SystemThe salt stress memory system was established with 150 mmol/L with 3 days.The results showed that the rooting rate of explants in vitro root was maintained at 100%,while the content of proline in three generations was 95.37,218.91,1224.77 ?g/g,respectively.It shows that there is a faster and stronger response under continuous stress which a stress memory effect.We found that changes in soluble sugar content showed a memory effect,which increased with salt stress treatment.Comparing chemical components in stress memory system with control,we found that eight new compounds were produced after stress,of which 7,6,5 compounds were added to N1,N2,N3,respectively.However,there was no significant change in antioxidant enzyme content.3.Transcriptome Data Analysis of Salt Stress Memory SystemBy processing the obtained transcriptome data,a total of 48,286 Unigenes were obtained with a GC percentage of 42.341%.Through comparison with the four major databases,25 575 unigenes were annotated and the annotation rate was 52.966%.The Unigene and KEGG databases were blasted and the annotation results showed that there were 5,202 unigene annotations in 132 KEGG pathways.There were no significant changes in the number of differentially expressed genes in the in vitro roots of three generations of Aquilaria sinensis.The number of up-regulated differentially expressed genes was decreased,and the number of down-regulated differentially expressed genes was increased.The KEGG annotation results showed that there are 42 pathways with significant changes.We focused on pathways related to proline biosynthesis,soluble sugar biosynthesis and degradation and secondary metabolite synthesis.The final screened genes were: P5 CSB,TPRP-F1,SPE2,PIP,ASP1 genes related to proline synthesis,PFK3,UGP2,SDH related to carbohydrate synthesis and degradation,C12RT1,FLS related to flavonoid synthesis,CYP98A2,CYP73A11,CHS genes,and PMA4,AVP1 genes associated with salt stress.We verified the expression trend of some high expression genes by Quantitative real-time PCR.At the same time,it was found that the expression of SPE2 and P5 CSB genes in the salt stress memory system continued to increase for three consecutive generations.They showed a significant positive correlation with proline.The expression of TPRP-F1 in the salt stress memory system was significantly negatively correlated with proline.We speculated that these three genes can be used as molecular biomarker genes related to proline in the salt stress memory system.ConclusionIn this experiment,the salt stress memory system of in vitro root was established using the proline content as an indicator.Finally,three genes that were significantly associated with proline biosynthesis were selected as their molecular level biomarker genes.