等离子体预处理对穿心莲种子萌发过程基因表达及后期生长的影响

发布时间：2018-05-15 19:59

本文选题：穿心莲 + 等离子体处理　；参考：《广州中医药大学》2017年硕士论文

【摘要】：等离子体处理种子技术已广泛使用于多种作物,被证实可提高种子活力,促进植物生长,提高植物抗胁迫能力。等离子体处理种子的作用目前主要认为是通过刻蚀种皮来提高种皮通透性与放电过程产生的带电粒子对种子表面的消毒作用,与抗氧化酶活性提高有关,提高了种子的健壮度;在分子层面的探讨几乎空白,需要深层次挖掘分析。穿心莲来源于爵床科(Acanthaceae)植物穿心莲(Andrographis paniculata(Burm.f.)Nees)的地上部分,干燥后为临床常用中药材。研究报道等离子体处理穿心莲种子可提高的出苗率和整齐度,促进植物生长速率。本研究系统地考察了等离子体处理对穿心莲种子萌发、出苗、生长速率的影响,从分子层面考察了等离子体处理对种子萌发过程基因表达的影响,为揭示等离子体处理种子技术的作用机理提供理论基础。本论文的主要研究内容和结果如下:1.等离子体处理促进穿心莲种子萌发及幼苗生长采用大气压条件下低电压(30 V～50 V)高频交变电场激发常温空气等离子体处理穿心莲种子,考察了激发电压、处理时间、处理后存放时间等多个因素,筛选促进其萌发的最佳条件,探究了等离子体处理对穿心莲萌发、出苗、生长、等多方面的影响。用30 V激发的等离子体处理种子3 s后放置4～6 d再置种,发芽势显著提高;放置时间过长再置种,处理的效果下降或消失。等离子体处理穿心莲种子的效应主要表现在发芽势显著提高、出苗加快、出苗率升高、幼苗及植株生长量增加、生殖生长加快及抗逆性增强。2.等离子体处理后穿心莲种子萌发过程中基因表达概况本论文采用第二代高通量测序仪Illumina HiSeq 2500进行测序,共得到106.34 Gb 的 Clean Data,84749 条 Unigene,平均长度为 758.03 bp,注释到 Nr、KEGG、GO、Swiss-prot、COG等公共数据库的Unigene数共36567条,建立了穿心莲种子转录组平台。对对照组和处理组的吸胀期(分别为a,A)、萌动期(b,B)、萌发高峰期(c,C)三个阶段差异基因进行stem趋势聚类分析,发现对照组和处理组显著富集的趋势相同,表明等离子体处理并未改变种子萌发过程基因表达的主要表达趋势,整体遵循种子萌发基因表达特有的规律性变化。等离子体处理与未处理穿心莲种子相对应三个萌发过程时间点的转录组比较发现:在萌动期,处理组和对照组有125个差异表达基因,主要富集到物质代谢相关KEGG通路;差异表达基因主要GO富集到翻译、初级代谢过程、有机物代谢过程、代谢过程、氧化还原过程等,调控种子萌发物质代谢,与KEGG富集情况一致。分别对对照组和处理组中这125个基因间达显著相关的基因做基因相关性图,对照组和处理组这125个基因间的相关性关系存在巨大差别,体现了等离子体对基因表达调控关系的影响。本文对萌发过程中处理组与对照组相同阶段特有差异表达的基因进行了筛选,并进行富集分析,KEGG富集结果发现处理组从吸胀期到萌动期富集通路差异较大,处理组植物激素信号转导富集基因最多;在对照组则不在前10条富集通路,对照组中核糖体通路富集到基因数最多;从萌动期到萌发高峰期,两组基因富集情况类似,等离子体处理在种子萌发过程的调控主要表现在种子萌发的前期,即吸胀期到萌动期。对a_b(对照组从吸胀期到萌动期)、b_c(对照组从吸胀期到萌动期)、A_B(处理组从吸胀期到萌动期)、B_C(处理组从吸胀期到萌动期)这4个阶段特有的差异表达中物质代谢基因与该阶段特有的植物激素、转录因子、病程相关基因进行基因表达相关性分析,结果显示,对照组与处理组间基因调控网络具有显著差异性,等离子体处理改变了种子萌发过程中这些关键基因的表达调控模式。本实验选择了与种子萌发相关的6个基因进行qPCR,结果显示,这些基因的表达趋势模式与转录组测序结果大部分一致,说明测序可靠性较高。
[Abstract]:Plasma treatment seed technology has been widely used in a variety of crops, which have been proved to improve seed vigor, promote plant growth and improve plant resistance to stress. The main effect of plasma treatment of seeds is to improve the seed surface permeability and discharge through the etching seed skin to improve the disinfection of the seed surface. It is used to improve the activity of antioxidant enzymes and improve the robustness of the seeds; the discussion at the molecular level is almost blank and needs deep mining analysis. The Andrographis paniculata is derived from the upper part of the Acanthaceae (Andrographis paniculata (Burm.f.) Nees). After drying, it is used as a commonly used Chinese medicinal material. The effects of plasma treatment on the seed germination, emergence and growth rate of Andrographis paniculata were systematically investigated. The effects of plasma treatment on the expression of the seed germination process were investigated from the molecular level to reveal the plasma treatment seeds. The main contents and results of this paper are as follows: 1. plasma treatment promotes the seed germination and seedling growth of Andrographis paniculata by using low voltage (30 V ~ 50 V) high frequency alternating electric field under atmospheric pressure to excite the seed of Andrographis paniculata at normal temperature air plasma, and investigate the excitation voltage and treatment time. A number of factors such as storage time after treatment were used to screen the best conditions to promote the germination. The effects of plasma treatment on the germination, emergence and growth of Andrographis paniculata were investigated. The germination potential was significantly higher after 3 s of plasma treated with 30 V, and the germination potential was significantly higher. The effect of plasma treatment on the seed of Andrographis paniculata was mainly manifested in the remarkable improvement of the germination potential, the expedite emergence of the seedlings, the increase of the seedling rate, the increase of seedling and plant growth, the accelerated growth of the reproductive growth and the increase of the resistance to stress in the seed germination of the seed of Andrographis paniculata after.2. plasma treatment. The second generation high throughput sequencing instrument Ill was used in this paper. Umina HiSeq 2500 was sequenced. A total of 106.34 Gb Clean Data, 84749 Unigene, and an average length of 758.03 BP were obtained. A total of 36567 public databases such as Nr, KEGG, GO, Swiss-prot, COG and other public databases were set up. The three phase difference gene of C (C) was analyzed by stem trend cluster analysis. It was found that the trend of significant enrichment in the control group and the treatment group was the same, which showed that the plasma treatment did not change the main expression trend of gene expression in the seed germination process. Compared with the transcriptional group at the time point of three germination process, it was found that there were 125 differentially expressed genes in the treatment group and the control group at the germination stage, which mainly enriched the KEGG pathway related to the material metabolism, and the main GO of the differential expression genes was enriched in the translation, the primary metabolic process, the organic metabolism process, the metabolic process, and the redox process. And so on, the regulation of the metabolism of seed germination material was consistent with the enrichment of KEGG. The correlation between the 125 genes in the control group and the treatment group was significantly related to the genes. The correlation of the 125 genes between the control group and the treatment group had a huge difference, which reflected the influence of the plasma on the regulation of gene expression. The genes of specific differential expression in the same stage of the treatment group and the control group were screened, and the enrichment analysis was carried out. The enrichment results of KEGG enrichment found that the enrichment pathway of the treatment group from the bloating period to the germinating stage was different, and the plant hormone signal transduction was the most abundant in the treatment group; in the control group, the first 10 enrichment pathways and the control groups were not in the control group. The number of genes in the middle nuclear sugar pathway is the most. From the germinating period to the peak period, the two sets of gene enrichment are similar. The regulation of the plasma treatment in the seed germination process is mainly in the early stage of seed germination, that is, the bloating period to the germinating period, and the a_b (the control group from the bloating period to the germinating period), and the b_c (the control group from the bloating period to the germinating period), A_B (treatment group from suction period to germinal period), B_C (treatment group from suction period to germination stage) of the 4 stages of the specific differential expression of material metabolism gene and the specific plant hormones, transcription factors, disease related genes gene expression correlation analysis, the results show that the control group and the treatment group gene regulation network has a significant difference. The expression regulation mode of these key genes during seed germination was changed by plasma treatment. 6 genes related to seed germination were selected for qPCR. The results showed that the trend patterns of these genes were most consistent with that of the transcriptional group, and that the reliability of the sequencing was higher.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.219;Q943.2

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