Abstract：
Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson\'s correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.
摘要：
茉莉酸（JAs）是主要的植物激素之一，调节植物生长和发育，应激反应，和次生代谢。作为JA信号通路的主要调节因子,MYC2在植物次生代谢产物的合成和积累中也起着重要作用。在这项研究中,我们对在不同处理时间接受茉莉酸甲酯（MeJA）的石蒜幼苗进行了比较转录组分析。通过RNA测序鉴定了总共31,193个差异表达基因(DEGs)。其中,对包含51个TF家族的732个差异表达的转录因子(TF)进行了表征。最丰富的TF家族是WRKY蛋白（80），其次是AP2/ERF-EFR(67)，MYB（59），bHLH(52),和NAC蛋白(49)家族。随后，通过计算TFDEGs表达水平与石蒜碱含量之间的Pearson相关系数(PCC)，鉴定出41个参与石蒜碱积累的潜在TF基因(|PCC|>0.8)，包括36个正调节器和5个负调节器。此外，在转录组测序的基础上克隆MeJA诱导的MYC2基因(即LaMYC2)。生物信息学分析表明，LaMYC2蛋白含有bHLH-MYC_N结构域和bHLH-AtAIB_样基序。LaMYC2蛋白定位于细胞核,并能部分拯救拟南芥中的MYC2突变体。LaMYC2蛋白可以与先前鉴定的大多数LaJAZ（尤其是LaJAZ3和LaJAZ4）相互作用。LaMYC2的瞬时过表达增加了L.aurea花瓣中的石蒜碱含量，这可能与酪氨酸脱羧酶（TYDC）和苯丙氨酸解氨酶（PAL）基因转录水平的激活有关。通过分离LaTYDC起始密码子（ATG）上游的887bp长度的启动子片段，我们在LaTYDC的启动子中发现了几种不同类型的E-box基序(CANNTG)。进一步的研究表明，LaMYC2确实能够结合存在于LaTYDC启动子中的E-box(CACATG)，验证了参与石蒜碱生物合成的通路基因可以被LaMYC2调节，并且LaMYC2在石蒜碱生物合成的调节中具有积极作用。这些发现表明，LaMYC2是石蒜碱生物合成的正调节因子，可能有助于LaMYC2基因的进一步功能研究。特别是其在金黄色葡萄球菌中的石豆科生物碱积累中的潜在调节作用。
