【作者】 周颖；

【导师】 侯凯； 曾锐；

【作者基本信息】 四川农业大学 ， 农业硕士（专业学位）， 2023， 硕士

【摘要】 川芎是四川省的特色道地药材之一,年需求量超两万吨,农户种植川芎收益相当可观。但是川芎地上部分作为非药用部位被大量废弃,造成资源浪费。川芎地上部分的研究,有助于更好的利用川芎地上部分资源。氮是植物需要较多的一种元素,在植物的生长和物质的转换中扮演着重要的角色。拟在本草考证基础上结合营养成分分析等论证川芎地上部分可食性,且设定0.2 mmol/L、2.0 mmol/L、20 mmol/L三个氮水平,探究不同氮水平对川芎地上部分生长发育和次生代谢的影响。同时把缺氮(0.2 mmol/L)和氮过量(20 mmol/L)与正常氮水平(2.0 mmol/L)下材料的转录组和代谢组比较考察,探究氮素对川芎地上部分生长发育次生代谢的影响机理。筛选与川芎地上部分生长发育与品质形成相关的代谢产物和相关基因,为川芎地上部分优质高产生产提供科学支撑。主要研究结果如下:1、本草学考证及历史文献分析表明,川芎地上部分(靡芜)具有800多年的食用历史。川芎自汉代起就被收录在各种典籍中,其地上部分被当作蔬菜和茶叶已有相当长的历史,且在古诗歌中被大量提及。初步表明川芎非药用部位有广阔的研究和发展空间。与芹菜相比,川芎地上部分在膳食纤维和烟酸两个营养成分上有明显优势。本研究发现川芎地上部分膳食纤维含量5.9 g/100 g,是芹菜的4.2倍。同时,川芎地上部分具有较高的蛋白质和铁含量,而钠含量较低,是一个很有潜力的高纤维型新型保健蔬菜。2、通过对不同氮素水平处理川芎地上部分转录组分析发现,低氮处理差异基因主要集中在碳氮吸收利用方面,高氮处理差异基因主要集中在光合作用方面。初步揭示了川芎地上部分对试验浓度范围内不同氮素水平(0.2-20 mmol/L)均具有较好的耐受性:低氮浓度条件下,通过调整C/N平衡来消减低氮胁迫带来的影响;高氮水平下,通过提升光合作用通路相关基因表达来提升碳同化,以便更好适应体内高氮水平带来的压力。低氮处理差异基因富集在碳分解代谢调控转录(Carbon catabolite regulation of transcription)、细胞氮化合物分解代谢过程(Cellular nitrogen compound catabolic process)通路等,维持C-N同化平衡来抵御低氮水平带来的胁迫。高氮处理差异基因主要富集在色素绑定(Pigment binding)、叶绿素绑定(Chlorophyll binding)、光反应(Light reaction)、光收集(Light harvesting)、光系统I中的光收集(Light harvesting in photosystem I)、前体代谢产物和能量的产生(Generation of precursor metabolites and energy)等通路,促进光合色素、光反应等通路基因表达上调,对提升光合作用具有一定作用。高氮处理使得川芎地上部分大部分纤维素合成酶基因下调(T4813基因除外)。3、通过代谢组分析发现,低氮处理下,相对差异代谢物个数最多的通路是醛固酮-调节钠的再吸收(Aldosterone-regulated sodium reabsorption)。高氮处理下,相对差异代谢物个数较多的通路有生物合成(Benzoxazinoid biosynthesis)、II型聚酮产物的生物合成(Biosynthesis of type II polyketide products)、D-精氨酸和D-鸟氨酸代谢(D-Arginine and D-ornithine metabolism)、糖酵解/糖异生(Glycolysis/Gluc oneogenesis)。同时高氮水平下川芎地上部分烟酰胺(Nicotianamine)和5-甲基尿苷(5-Methyluridine)代谢物下调。不同水平氮处理对川芎地上部分代谢有明显影响,这些差异代谢都参与了川芎地上部分生长发育的调控,特别是对于水分、氮的吸收利用。高氮水平处理川芎地上部分对其次生代谢产物的形成和积累带来不利影响,特别是烟酰胺等健康成分减少尤为显著。转录组和代谢组关联分析结果表明,低氮水平下,川芎地上部分中异烟棒曲霉素C(Roquefortine C),醛固酮(Aldosterone)和延龄草苷(Diosgenin glucoside)三种差异代谢物与其差异基因的表达相关性较好。4、根据不同氮水平处理后川芎地上部分农艺性状的改变可以发现,氮肥显著提高了川芎地上部分根系生物量、株高、根长和根粗。川芎地上部分叶绿素含量和光合作用水平随着氮水平的增加而增加,高氮水平处理有利于川芎地上部分产量增加,但同时使川芎烟酸含量下降。通过不同氮水平处理发现,膳食纤维虽然没有发生明显变化,但是通过感官测试可以发现在口感上中氮处理材料纤维感更重。而且在转录组试验中可以发现高氮和低氮处理川芎地上部分中的纤维素合成酶基因有明显差异。说明虽然不同氮素处理下,膳食纤维总含量变化不大,但是膳食纤维的组成差异可能影响了川芎地上部分的口感。在高氮处理川芎地上部分材料中烟酸含量下降,同时在代谢组分析中也发现高氮处理时烟酰胺(Nicotianamine)含量下降。高氮处理下调控通路中的基因表达,使合成的维生素B6吡哆醇代谢物下调。总之,如果要使川芎地上部分中烟酸含量提高,应该控制氮水平。在高氮和低氮处理下川芎地上部分中的绿原酸含量下降。通过感官测试可以发现在口感上中氮处理时纤维感更重。综合考虑成本收益,为保证川芎地上部分品质和产量,中氮水平(2.0 mmol/L)更为合适。以上结果表明川芎地上部分具备高营养及可食性,且不同氮素水平处理影响川芎地上部分产量和品质,应当合理施用氮肥以更好保证优质川芎地上部分生产。更多还原

【Abstract】 Ligusticum Chuanxiong Hort.(CX)is one of the characteristic and authentic medicinal materials in Sichuan Province.The annual demand of CX exceeds 20,000 tons,and farmers can gain considerable profits from planting it.However,the above ground part of CX has been largely abandoned as non-medicinal parts,resulting in a waste of resources.The research of above ground part of CX is helpful to better use of above ground part resources.Nitrogen is an element that plants need more and plays an important role in plant growth and substance conversion.Proposed on the basis of literatures combining nutrition analysis demonstration from aerial parts of CX edibility,set and tendency for 0.2 L and 2.0 tendency for L,20 tendency/L three nitrogen levels,to explore the different nitrogen levels on the aerial parts of CX growth and the influence of secondary metabolism.At the same time,the transcriptomes and metabolites of materials with nitrogen deficiency(0.2 mmol/L)and nitrogen excess(20 mmol/L)were compared with those with normal nitrogen level(2.0mmol/L)to explore the effect mechanism of nitrogen on the growth and development of above ground parts of CX.To screen metabolites and genes related to the growth,development and quality formation of above ground part of CX,and provide scientific support for high quality and high yield production of above ground part of CX.The main research results are as follows:1.The herbological research and historical literature analysis show that the above ground part of CX has a history of more than 800 years.CX has been included in various classics since the Han Dynasty.The above ground part of CX has a long history as a vegetable and tea leaf,and it is mentioned a lot in ancient poetry.The results showed that the non-medicinal parts of CX had broad space for research and development.Compared with celery,the above ground part of CX has obvious advantages in dietary fiber and niacin.The study found that the dietary fiber content of the above ground part of CX was 5.9 g/100g,4.2 times that of celery.At the same time,the above ground part of CX has high protein and iron content,but low sodium content,so it is a potential high fiber type of new health care vegetable.2.Through transcriptomic analysis of above ground part of CX treated with different nitrogen levels,it was found that the differential genes in low nitrogen treatment were mainly concentrated in carbon and nitrogen absorption and utilization,while those in high nitrogen treatment were mainly concentrated in photosynthesis.It was preliminarily revealed that the above ground part of CX had good tolerance to different nitrogen levels(0.2-20 mmol/L)within the experimental concentration range.Under the condition of low nitrogen concentration,the influence of low nitrogen stress was reduced by adjusting the C/N balance.Under high nitrogen levels,carbon assimilation was improved by increasing the expression of genes related to photosynthetic pathways,so as to better adapt to the pressure caused by high nitrogen levels in the body.Differential genes of low nitrogen treatment were enriched in Carbon catabolite regulation of transcription and Cellular nitrogen compound catabolic process,maintain C-N assimilation balance to resist stress caused by low nitrogen levels.Different genes of high nitrogen treatment are mainly enriched in Pigment binding,Chlorophyll binding,Light reaction,Light harvesting,and Light collection in photosystem I harvesting photosystem I,Generation of precursor metabolites and energy and other pathways promote up-regulated gene expression of photosynthetic pigment,photoreaction and other pathways.It has certain effect on the promotion of photosynthesis.Most cellulose synthetase genes of CX were downregulated by high nitrogen treatment(except T4813 gene).3.The metabolome analysis showed that the pathway with the highest number of relative differentiated metabolites under low nitrogen treatment was Aldosterone-regulated sodium reabsorption.Under high nitrogen treatment,the pathways with relatively large numbers of different metabolites include Benzoxazinoid biosynthesis and Biosynthesis of type II polyketide Products,D-Arginine and D-ornithine metabolism,Glycolysis/sugar dysplasia.At the same time,some Nicotianamine and 5-Methyluridine metabolites in CX were downregulated under high nitrogen levels.Different levels of nitrogen treatment have significant effects on the abovement-part metabolism of CX.These differences in metabolism are involved in the regulation of the growth and development of abovement-part of CX,especially the absorption and utilization of water and nitrogen.The aboveground part of CX treated with high nitrogen level had adverse effects on the formation and accumulation of secondary metabolites,especially the reduction of health components such as niacinamide.Transcriptomic and metabolite association analysis showed that three different metabolites of Roquefortine C,Aldosterone and Diosgenin glucoside in the ground part of CX have good correlation with their differential genes expression at low nitrogen level.4.According to the changes of agronomic characters of the above ground part of CX treated with different nitrogen levels,it can be found that nitrogen fertilizer significantly increased the root biomass,plant height,root length and root diameter of the above ground part of CX.The chlorophyll content and photosynthesis level of above ground part of CX increased with the increase of nitrogen level.High nitrogen level treatment was beneficial to increase the yield of above ground part of CX,but at the same time,the content of nicotinic acid of CX decreased.Although dietary fiber did not change significantly through different nitrogen levels of treatment,sensory tests showed that medium nitrogen treatment materials had a heavier sense of fiber in taste.In addition,it was found that there were significant differences between cellulose synthetase genes in the above ground part of CX treated with high nitrogen and low nitrogen.These results indicated that although the total dietary fiber content changed little under different nitrogen treatments,the difference in dietary fiber composition may affect the taste of the above ground part of CX.The content of niacin in the above ground part of CX treated with high nitrogen decreased,and the content of Nicotianamine was also found to decrease after high nitrogen treatment in metabolome analysis.The gene expression in the regulatory pathway under high nitrogen treatment down-regulated the synthesis of vitamin B6 pyridoxine metabolites.In short,if the content of niacin in the above ground part of CX is to be increased,the nitrogen level should be controlled.The content of chlorogenic acid in the above ground part of CX decreased under the treatment of high nitrogen and low nitrogen.Sensory tests show that the texture is more fibrous when treated with medium nitrogen.In order to ensure the quality and yield of the above ground part of CX,the nitrogen level(2.0 mmol/L)is more appropriate.The above results showed that the above ground part of CX had high nutritional value and edible quality,and different nitrogen application levels had effects on the yield and quality of the above ground part of CX.Therefore,nitrogen application should be reasonable to better ensure the high quality production of the above ground part of CX.更多还原