植物非编码RNA（ncRNA，non-coding RNA）是一类重要的生物分子，在植物生长发育以及代谢反应等多方面都发挥着不可替代的作用。按照序列的长短，非编码RNA能被划分为lncRNA（long non-coding RNA）与RNA（small RNA）两大种类，而后者还能进一步细化成多种形式。长非编码反义转录RNA（lncNAT）在lncRNA中发挥着独特的作用，而非编码小RNA以微小RNA（miRNA，microRNA）研究最广泛。

北马兜铃（Aristolochia contorta Bunge）作为一种备受争议的药用植物，有着独特的药用价值，古时被用于多种治疗，现代因其马兜铃酸肾毒性从药典中摘除。探究北马兜铃体内次生代谢通路尤其是毒性生物碱的合成，减小有害成分保留药用成分对于马兜铃及马兜铃酸类药材的发展有重要意义。而非编码RNA等生物分子在植物生长代谢过程中发挥着不可替代的作用。因此本文探究北马兜铃植物中的非编码RNA及其调控作用，以期找到参与次生代谢尤其是生物碱代谢的相关分子。

本研究获得的主要结果如下：（1）对北马兜铃根茎花叶四个组织植物样进行RNA-seq测序，得到35.94Gb Clean Data。对测序得到的数据进行lncRNA和lncNAT分析。得到718个lncRNA，其中443个在四个组织中差异表达。得到441个lncNAT，包含437个顺式lncNAT和4个反式lncNAT。并得到560个lncNAT和其正义链组成的SAT对。441个lncNAT中有133个差异表达lncNAT。对顺式lncNAT的正义链进行了功能注释。

（2）通过对北马兜铃四个组织进行小RNA组测序，分别得到20,509,711、19、120,948、27、955、448和22,405,111条clean reads。四个组织进行混合降解组测序，总共得到26,317,348条clean reads。这些序列筛选后用于miRNA靶基因的预测分析。对测序序列进行分析后得到223个miRNA，包括51个保守的已知miRNA和172个新miRNA。共得到125个miRNA家族，其中20个为保守miRNA家族，105个为新miRNA家族。对miRNA表达进行分析，有120个差异表达的miRNA。MiRNA的靶基因预测分析结果显示：96个miRNA有363个靶向mRNA；39个miRNA靶向63个lncRNA，这些lncRNA中有12个顺式lncNAT。通过对miRNA靶基因的注释分析，发现一些参与北马兜铃次生代谢的miRNA。

丹参（Salvia miltiorrhiza）是传统中药材，现临床上主要用于心血管疾病，主要药用成分是丹酚酸类和丹参酮类化合物。Smi-miR12112是从丹参中鉴定出的靶向多酚氧化酶（PPO）的microRNA，而PPO在丹参体内可能调控丹酚酸类化学物的代谢合成。因此构建丹参smi-miR12112过表达材料，对于探究其对丹参药用成分的调控具有重要作用。

（3）从丹参中扩增得到了smi-miR12112的基因序列MIR12112，并将其构建到改造的pCAMBIA1391双元载体上。接着将构建好的载体转化到GV3101根癌农杆菌和ACCC10060发根农杆菌中，利用丹参的遗传转化体系，得到丹参转基因材料，接着对转基因材料进行DNA水平的PCR验证和RNA水平的qRT-PCR验证，成功获得丹参的毛状根和植株过表达smi-miR12112材料。对过表达转基因毛状根使用UPLC进行丹酚酸类化合物检测，发现丹酚酸B以及迷迭香酸含量与野生型相比有所下降。

综上所述，本文对北马兜铃的lncRNA、lncNAT和miRNA进行了预测与分析，简单构建了北马兜铃lncRNA-miRNA-mRNA调控网络。意在为后续北马兜铃次生代谢等分子生物研究提供理论基础。获得丹参smi-miR12112过表达材料，对于后续更深入的smi-miR12112及其靶基因过氧化物酶在丹参的体内研究及功能鉴定立下了基础。

Plant non-coding RNA (ncRNA) is an important class of biomolecules, which plays an irreplaceable role in plant growth and development and metabolic response. NcRNA can be divided by the length of the sequence into long non-coding RNA (lncRNA, long non-coding RNA) and non-coding small RNA (small RNA), then lncRNA and miRNA can be divided into many types. Long non-coding antisense transcription RNA (lncNAT) is an antisense transcript relative to a strand mRNA. LncNAT plays a unique role in lncRNA, while non-coding small RNA has been the most widely studied as microRNA (miRNA).

Aristolochia contorta Bunge as a controversial medicinal plant, was removed from the pharmacopoeia due to its aristolochic acid nephrotoxicity. It is of great significance to explore the secondary metabolic pathway of aristolochic, especially the synthesis of toxic alkaloids, and reduce the harmful components for the development of aristolochic and aristolochic acid. Therefore, we explored the non-coding RNA and its regulatory role in Aristolochic plants in order to find related molecules involved in secondary metabolism, especially alkaloid metabolism. Salvia miltiorrhiza is a traditional Chinese medicine, which is mainly used for cardiovascular diseases in clinical practice. The main medicinal ingredients are dananolic acids and tanshinones. Smi-miR12112 is the microRNA targeting polyphenol oxidase (PPO) identified from Salvia miltiorrhiza, and PPO may regulate the metabolic synthesis of polyphenolic acid chemicals in Salvia. Therefore, the construction of miltiorrhiza Smi-miR12112 overexpression materials plays an important role in exploring the regulation of its medicinal components.

The main results are divided into two parts, one was non coding RNA analysis of Aristolochia contorta Bunge, the other one was overexpressed MIR12112 in Salvia miltiorrhiza.  

In Aristolochia contorta Bunge, RNA-seq sequencing of four tissues: root, stem, flowers and leaves were used. Totally obtained 35.94Gb clean data. The data obtained from sequencing were analyzed by lncRNAs and lncNATs. We obtained 718 lncRNAs, of which 443 were differentially expressed in the four tissues. 441 lncNATs containing 437 cis-lncNATs and 4 trans-lncNATs. And obtained 560 SAT pairs consisting of lncNATs and its sense gene. 133 of 441 lncNATs were differentially expressed lncNATs. Functional annotation of the sense transcripts of cis-lncNAT yielded three lncNAT potentially involved in secondary metabolism of Aristolochia contorta Bunge.

Small RNA sequencing of four tissues were used to predicted miRNA. Totally obtained 20,509,711,191,120,948,27,955,448 and 22,405,111 clean reads, respectively. Four tissues were subjected to mixed degradome sequencing, resulting in a total of 26,317,348 clean reads. These sequences were screened for prediction analysis of miRNA target genes. Analysis of the sequencing sequences yielded 223 miRNAs, including 51 conserved miRNAs and 172 novel miRNAs. In total, 125 miRNA families were obtained, of which 20 were conserved miRNA families and 105 were novel miRNA families. Analysis of miRNA expression, with 120 differentially expressed miRNAs. The target gene prediction analysis of miRNA showed 363 targeted mRNAs. 39 miRNAs targeting 63 lncRNAs, and 12 cis-lncNATs in these lncRNAs. The annotation analysis of miRNA target genes revealed some miRNAs involved in the secondary metabolism of Aristolochia contorta Bunge.

MIR12112 was amplified from Salvia miltiorrhiza and constructed onto pCAMBIA1391 binary vector. Then the constructed vectors were transformed into Agrobacterium GV3101 and ACCC10060, and the genetic transformation system of Salvia miltiorrhiza was used to obtain the overexpressed smi-miR12112 material. The resulting transgenic material was verified by PCR at the DNA level and qRT-PCR at the RNA level, and successfully obtained the Salvia miltiorrhiza overexpressed MIR12112 material. Tanphenolic acid compounds using UPLC for overexpressing transgenic hairy roots revealed a decrease in salvianolic acid B, and rosmarinic acid compared with wild type.

In conclusion, this paper predicted and analyzed the non-coding RNA: lncRNA, lncNAT and miRNA of Aristolochia contorta Bunge, and simply constructed the aristoliochic lncRNA-miRNA-mRNA network. Its intended to provide the theoretical basis for the subsequent molecular biological research such as secondary metabolism. The overexpression material of Salvia miltiorrhiza smi-miR12112 was obtained, which laid the basis for the in vivo study and functional identification of miRNA.