Effects Of Plasma Pretreatment On Gene Expression In Andrographis Paniculata Seed Germination Stage And On Its Late Growth

The technology that employing plasma pretreatment on seed has been widely used in a variety of plants,which has been confirmed that can improve seed vigor,promote plant growth,and enhance plant stress resistance.Its effective mechanism was mainly considered to be the change of the seed coat permeability and seed photosensitivity by etching seed coat,strengthening seed due to disinfection effect on the surface of seeds and activating antioxidase system by the gas mixture produced during plasma discharge process.However,the research at the molecular level is almost blank,and need to be further studied.Andrographis herba is the dried aboveground parts of Andrographis paniculata(Burm.f.)Nees belongs to Acanthaceae family.It is widely used in the clinic.It has been reported that the plasma treatment of Andrographis paniculata seeds can improve seed emergence rate and growth uniformity,promote plant growth rate.In this paper,the effects of plasma on the seed germination,emergence,growth rate and resistance to low temperature stress were studied systematically.The effect of plasma treatment on the gene expression during seed germination was investigated at molecular level.It will also provide a theoretical basis for mechanism of action of plasma treatment on seed.The main contents and results of this thesis are as follows:1.Effects of plasma treatment on seed germination and growth of A.paniculataHigh frequency alternating electric field under low voltage(30 V?50 V)was used to stimulate the seeds of A paniculata seeds at atmospheric temperature.The factors such as excitation voltage,treatment time and storage time were investigated to optimize the plasma treatment condition.The effects of plasma treatment on the germination,emergence,growth and biochemical indices of A.paniculata were studied.It showed that the optimum air plasma seed treatment condition for A.paniculata were as follows:a voltage of 30 V,with a duration of 3 seconds,and stored for 4?6 days before sowed.The effect of plasma treatment on the seed of A.paniculata mainly resulted in the increase of germination potential,seedling emergence in germination experiment;the increase of seedling and plant growth,the increase of reproductive growth and increased resistance to stress in pot experiment.2.Transcriptomic analysis of plasma pretreatment A.paniculata seedThe global transcriptome of plasma pretreatment on A.paniculata seed was sequenced using Illumina 2500 high-throughput sequencing technology.A total of 2.58 Gb of Clean Data and 84,749 Unigenes,with a mean length of 1,045 bp were generated.After bioinformatics annotation,36567 Unigenes were mapped to Nr,COG,GO and KEGG database,and a transcriptome platform of A.paniculata seed was established.Using the STEM(Short Times-series Expression Miner)software,expression trends of differential expressed genes(DEGs)in the three stages of seed germination process in both control and treatment group were obtained.The results showed that the trend of the enrichment of the control and the treatment group were almost the same,which indicated that plasma pretreatment did not change the main expression trend of gene expression,and the gene expression rules of seed germination process were followed.Comparison was done on transcriptome of plasma pretreat and control group in the three stages of seed germination process.There are 125 DEGs between the two group at seed germinating stage.Most of these 125 DEGs'enriched on KEGG and GO database are related to substance metabolism.The gene correlation diagram of these 125 genes of two groups was generated based on Pearson Correlation Coefficient(r?0.8),respectively,and results showed that the regulation relationship between genes was different between these two groups,which demonstrated the effect of plasma pretreatment on gene expression at seeds germinating stage.In this paper,the unique genes differentially expressed of the treatment group and the control group at the same stages were filtered for gene expression enrichment analysis.The DEGs' KEGG enrichment results from seed imbibition stage to seed germinating stage exhibits difference between the treatment group and control group,In the treatment group,the plant hormone signal transduction pathway enriched the most genes,but not in the top 10 enrichment pathway in the control group;the DEGs' KEGG enrichment results from seed germinating stage to seed germinating peak stage are similar.This results suggest that the regulation of the plasma pretreat in the seed germination process mainly focus on the early stage of seed germination,that is,the period from seed imbibition stage to seed germinating stage.The unique differentially expressed genes related to energy metabolism,plant hormones,transcription factor,pathogenesis-related 'genes at a_b?b_c?A_B?B_C period have been filtered to generate the gene correlation diagram for each period respectively.The results showed the regulation relationship between these genes was different between control and treatment group,which shows the plasma pretreatment changed the gene expression mode during seed germination of A.paniculata.Six genes related to seed germination process were selected for real-time quantitative PCR,most of their expression mode were consistent with the results of the transcrip tome,which validated reliability of the transcriptome analysis.