硒是一种重要的微量元素，存在于动物、植物和微生物中并且是动物和人体中谷胱甘肽过氧化物酶等的活性成分，具有抗氧化、抗衰老，提高人体免疫力等作用。不过食用硒也要适量，因为过量摄入会导致中毒，出现脱发、脱甲等现象。目前对硒元素的研究，主要集中在资源分布、富硒栽培、在植物中存在的形式以及其对植物生理生化的影响等方面，而在植物富硒分子机理方面研究较少。野葛（pueraria lobata （willd.） ohwi），为豆科葛属多年生植物，是一种大宗重要中药材，于2002年被国家卫生部认定为药食两用植物，也是一种富硒植物。野葛以其干燥的根入药，称为葛根，具有提高大脑记忆能力、改善和治疗心脑血管疾病等功效，其主要生物活性成分为异黄酮类化合物。本研究首先通过使用不同浓度亚硒酸钠处理野葛幼苗，接着测定植株中硒含量和部分生理指标，筛选出最适的亚硒酸钠浓度。然后借助转录组技术研究亚硒酸钠作用下野葛中蛋白质编码基因的差异表达情况，明确差异表达基因所在的信号通路及其表达模式，根据注释信息找到硒代谢和异黄酮代谢相关的编码基因。最后利用小rna测序技术揭示了亚硒酸钠作用下野葛microrna（mirna）及其靶基因的差异表达情况，从而掲示葛幼根响应硒刺激的分子机制，为深入研究富硒野葛提供理论依据。本研究主要结果如下：

1. 用不同浓度的亚硒酸钠处理野葛幼苗，然后测定植株中的硒含量，初步明确了随着亚硒酸钠浓度的升高，葛根幼苗叶和茎中硒的含量表现为先增高后降低的模式，并在25 mg/l的浓度时野葛中硒的含量达到最大值。

2. 根据上述结果，使用6种不同浓度的亚硒酸钠处理野葛幼苗，然后测量根长、可溶性糖和丙二醛含量，超氧化物歧化酶的活性，综合以上结果，明确了适宜的亚硒酸钠处理浓度为25 mg/l。

3. 用25 mg/l亚硒酸钠处理野葛幼苗，设置三次生物学重复，分别于处理后第0、1、3、5、7、9天采集野葛幼根，使用改良的trizol法提取rna，建立转录组文库，再利用illumina hiseq x ten平台进行测序，获得了18个碱基数目分别大于6,200,000,000 nt的文件。使用trimmomatic软件质控后，再经过trinity软件拼接共得到323,717个转录本，聚类去冗余后保留最长的序列，得到150,567条平均长度为624.31 bp的unigene，其n50为1,135 bp，平均gc含量大于46%。接着对测序组装的unigene序列进行功能注释，发现有59,266、64,759、5,416、58,101、33,401、31,796个基因分别在nr、go、kegg 、swissprot、kog和pfam等数据库得到了注释。在kegg数据库中，共有9,746个基因在23个二级目录下的215个途径中得到了识别，其中包括29个硒代谢相关基因和41个异黄酮及其上游合成途径基因。以0天作为对照共获得了4,246个差异表达编码基因，硒代谢途径中有19个基因差异表达，而在异黄酮及其上游途径中则有9个基因差异表达，并且这9个基因均为上调表达。本研究将转录因子与硒代谢途径和异黄酮代谢途径基因进行共表达分析，以|R|> 0.75为阈值，共发现了301个转录因子与硒代谢基因相关，341个转录因子与异黄酮代谢基因相关。

4．使用illumina hiseq x ten平台进行小rna测序。再使用psrobot的软件共鉴定出88个保守mirnas，它们属于26个mirna家族；以及166个非保守mirnas，它们属于118个mirna家族。对这些mirnas进行靶基因预测，以期望值小于3.0为阈值，共预测出2541个靶基因。对差异表达mirna的靶基因进行功能富集后发现：go富集分析中前五位分别为“integral component of membrane”、“nucleus”、“atp binding”、 “tranion, dna-templated”和“plasma membrane”；kegg富集分析显示125个差异表达mirnas的靶基因富集到了74条代谢途径上，其中有69、23、20、13个靶基因分别富集到了“metabolism”、“genetic information processing”、“environmental information processing”和“cellular processes”四个大类中，表明了野葛在亚硒酸钠处理后，幼根内差异表达mirna靶基因应答具有复杂性。

selenium is an important trace element, which is present in animals, plants and microorganisms and is an active ingredient of glutathione peroxidase in animals and humans, and has anti-oxidation, anti-aging effects and enhances human immunity. however, selenium supplementation should not be excessive, because excessive intake of selenium can lead to poisoning, hair loss, dislocation and other phenomena. at present, the research on selenium mainly focuses on the distribution of selenium, selenium-enriched cultivation, the form of selenium in plants, and the effects of selenium on plant physiology and biochemistry. however, there are few studies on the molecular mechanism of selenium in plants. pueraria lobata (willd.) ohwi, a perennial woody vine of the genus leguminosae, is a large and important chinese medicinal material. it was recognized as a dual-use plant by the ministry of health in 2002, also a selenium-enriched plant. p.lobata is used as a dry root to be used as a genus of puerarin. it has the functions of improving brain memory, improving and treating cardiovascular and cerebrovascular diseases, and its main biological active ingredient is isoflavones. in this study, the seedlings of p.lobata were treated by using different concentrations of sodium selenite, and then the selenium content, and some physiological indexes of the plants were determined, and the optimum concentration of sodium selenite was screened. then, the tranome was used to study the differential expression of the geranium functional gene under the action of sodium selenite. the signaling pathway and expression regulation mode of the differentially expressed genes were identified, and the functional genes related to selenium metabolism and isoflavone metabolism were found according to the annotation information. finally, small rna sequencing technology was used to reveal the differential expression of genomic dna and its target genes under the action of sodium selenite, revealing the complexity of differentially expressed microrna(mirna) target gene responses in radicle, and providing insight into the mirnas that respond to selenium stimulation in gesong root. thus, the molecular mechanism of ge's root response to selenium stimulation was revealed, which provided a theoretical basis for the in-depth study of selenium-rich kudzu.  theoretical basis. the main results are as follows:

1. the seedlings of p.lobata were treated with  different concentrations of sodium selenite, and then the selenium content in p.lobata was determined. the content of selenium in leaves and stems of p.lobata seedlings was determined with the increase of sodium selenite treatment concentration. it is expressed as a mode of increasing first and then decreasing, and the content of selenium in p.lobata reaches a maximum at a concentration of 25 mg/l.

2. according to the above results, six different concentrations of sodium selenite were used to treat the p.lobata seedlings, and then the root length, soluble sugar and malondialdehyde content, superoxide dismutase, and the above results were measured. it is clear that 25 mg/l is a suitable treatment concentration for sodium selenite.

3. treat the p.lobata seedlings with 25 mg/l sodium selenite, set up three biological replicates, collect the geranium roots on days 0, 1, 3, 5, 7, and 9 after treatment, and extract rna using the modified trizol method. the tranome sequencing of the above samples was performed using the second-generation sequencing illumina hiseq x ten platform, and 18 files with a number of bases greater than 6,200,000,000 were obtained. after trimmomatic software quality control, 323,717 trans were obtained by trinity software splicing. after clustering and redundancy, the longest sequence was retained, and 150,567 unigenes with an average length of 624.31 were obtained. the n50 was 1,135 bp and the average gc content was more than 46%. . functional annotation of the sequenced assembled unigene sequence revealed that 59, 266, 64, 759, 5, 416, 58, 101, 33, 401, 31, 796 genes were respectively nr, go, kegg, swissprot, kog, and pfam were annotated. in the kegg database, a total of 9,746 genes were identified in 215 pathways in 23 secondary catalogues, including 29 selenium metabolism-related genes and 41 isoflavones and their upstream synthetic pathway genes. a total of 4,246 differentially expressed genes were obtained using 0 days as a control group. among these genes, we found that 19 genes were differentially expressed in the selenium metabolic pathway, while 9 genes were differentially expressed in the isoflavones and their upstream pathways, and these 9 genes were up-regulated. in this study, many tranion factors were found to be co-expressed with selenium metabolic pathways and isoflavone metabolic pathway genes with |r|> 0.75. we fuond 301 tranion factors were associated with selenium metabolism genes, and 341 tranion factors were related to isoflavone metabolism genes.

4. small rna sequencing was performed using illumina hiseq x ten platform. in total, 88 conserved mirnas were identified using psrobot software which  belong to 26 mirna families and 166 non-conserved mirnas were obtained, which could be classfied to 118 mirna families. a total of 2541 target genes were predicted for these mirnas with a predicted value of less than 3.0. functional enrichment of target genes for differentially expressing mirna revealed that the top five in go terms were “integral component of membrane”, “nucleus”, “atp binding”, “tranion”, “dna-templated”, and “plasma”. the kegg enrichment analysis showed that the target genes of 125 differentially expressed mirnas were enriched into 74 metabolic pathways, of which 69, 23, 20, and 13 target genes were enriched in “metabolism”, “genetic information processing”, “ environmental information processing”, and “cellular processes” , respectively. it indicated that the differential expression of mirna target gene response in juvenile roots was complicated after treatment with sodium selenite.