MYB转录因子是植物最大的转录因子家族之一,广泛参与植物的生长发育、逆境胁迫和次生代谢产物积累.该研究通过同源比对和功能注释,在地黄(Rehmannia glutinosa)转录组中筛选出MYB的转录本,设计特异性引物对MYB基因的cDNA序列进行PCR扩增,用水杨酸(SA)、Ag+、茉莉酸甲酯(MeJA)和腐胺(Put)这4种诱导子处理地黄毛状根,并通过实时荧光定量PCR(qRT-PCR)检测候选MYB基因的表达.结果显示:(1)成功克隆到1个地黄MYB基因,命名为RgMYB10;该基因编码247个氨基酸残基,蛋白质相对分子质量28.48 kD,等电点为5.14,属于R2R3-MYB转录因子.(2)qRT-PCR结果显示,RgMYB10在须根中表达量最高,其次为茎,块根中的表达量最低.(3)RgMYB10在MeJA处理后的毛状根中显著上调表达,为特异响应MeJA诱导的基因,推测Rg-MYB10基因可能是响应MeJA参与地黄毛蕊花糖苷生物合成的关键转录因子.研究表明,地黄MYB10基因可能参与地黄毛蕊花糖苷的生物合成,为进一步研究MYB10基因在地黄毛蕊花糖苷合成中的功能奠定了基础.

Rehmannia glutinosa is a common bulk medicinal material that has been widely used in China due to its active ingredients. Acteoside, one of the ingredients, has antioxidant, antinephritic, anti-inflammatory, hepatoprotective, immunomodulatory, and neuroprotective effects, is usually selected as a quality-control component for R. glutinosa herb in the Chinese Pharmacopeia. The acteoside biosynthesis pathway in R. glutinosa has not yet been clearly established. Herein, we describe the establishment of a genetic transformation system for R. glutinosa mediated by Agrobacterium rhizogenes. We screened the optimal elicitors that markedly increased acteoside accumulation in R. glutinosa hairy roots. We found that acteoside accumulation dramatically increased with the addition of salicylic acid (SA); the optimal SA dose was 25 μmol/L for hairy roots. RNA-seq was applied to analyze the transcriptomic changes in hairy roots treated with SA for 24 h in comparison with an untreated control. A total of 3,716, 4,018, and 2,715 differentially expressed transcripts (DETs) were identified in 0 h-vs.-12 h, 0 h-vs.-24 h, and 12 h-vs.-24 h libraries, respectively. KEGG pathway-based analysis revealed that 127 DETs were enriched in “phenylpropanoid biosynthesis.” Of 219 putative unigenes involved in acteoside biosynthesis, 54 were found to be up-regulated at at least one of the time points after SA treatment. Selected candidate genes were analyzed by quantitative real-time PCR (qRT-PCR) in hairy roots with SA, methyl jasmonate (MeJA), AgNO3 (Ag+), and putrescine (Put) treatment. All genes investigated were up-regulated by SA treatment, and most candidate genes were weakly increased by MeJA to some degree. Furthermore, transcription abundance of eight candidate genes in tuberous roots of the high-acteoside-content (HA) cultivar QH were higher than those of the low-acteoside-content (LA) cultivar Wen 85-5. These results will pave the way for understanding the molecular basis of acteoside biosynthesis in R. glutinosa, and can serve as a basis for future validation studies.

为研究地黄根系分泌物的化感作用,探索其中的化感物质种类和分布,以70％甲醇提取地黄茬后根际土壤,利用石油醚、氯仿、乙酸乙酯、正丁醇分步萃取得到不同极性的部位.通过种子发芽测试和田间盆栽试验来检测和验证各部位的化感强度,利用HPLC检测各部位中的阿魏酸、香豆酸、香草酸、对羟基苯甲酸、丁香酸五种酚酸的含量.结果表明,正丁醇部和水部能抑制种子的萌发和幼根的伸长生长,化感作用效应指数分别达到-0.22和-0.43;正丁醇部对盆栽地黄块根的生长抑制作用最强,超过水部,其酚酸类总含量高达2.094 μg· mL-1,主要含有香豆酸、对羟基苯甲酸和香草酸;地黄根系分泌物对种子和地黄块根的影响是不同的,需要在种子发芽试验的基础上进一步进行盆栽块根繁殖试验,才能确定某些化感物质是否是导致地黄连作障碍的化感物质.

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of phenylethanoid glycosides. Based on the transcriptome data, we cloned the full-length cDNA (GenBank accession NO. KU640395) of RgTyDC gene from Rehmannia glutinosa, and then performed bioinformatic analysis of the sequence. Further, we detected the expression pattern in different organs and hair roots treated with four elicitors by qRT-PCR. The results showed that the full length of RgTyDC cDNA was 1 530 bp encoding 509 amino acids. The molecular weight of the putative RgTyDC protein was about 56.6 kDa and the theoretical isoelectric point was 6.25. The RgTyDC indicated the highest homology with Sesamum indicum SiTyDC and Erythranthe guttata EgTyDC, both of them were reached 88%. RgTyDC highly expressed in R. glutinosa leaf, especially in senescing leaf, and rarely expressed in tuberous root. After the treatment of SA and MeJA, the relative expression level of RgTyDC mRNA was substantially increased. The results provide a foundation for exploring the molecular function of RgTyDC involved in phenylethanoid glycosides biosynthesis.

To study the effect of continuous cropping obstacles on the growth of different germplasm resources,and screen germplasm of R. glutinosa resistant to continuous cropping,using 18 R. glutinosa germplasm resources as plant materials,the differences in crown width,leaf length,leaf width,leaf number,stem diameter,tuberous root number,tuberous root length,tuberous root diameter and fresh weight of tuberous root per plant between first-year plants and second-year plants were researched. And phenotypic differences among different germplasm resources were compared by principal component analysis,cluster analysis and correlation analysis based on the tolerance index of continuous cropping disease( TCC). The results showed that there were notable differences among 18 R. glutinosa germplasm resources in response to continuous cropping. All 14 cultivars showed various degrees of replant problems,but the four wild germplasms showed good tolerance to replant problems. The performance of replant problems in R. glutinosa cultivars were as follows: size of leaf and number of leaf decreased,diameter of tuberous root reduced,number of fibrous root increased,and fresh weight of tuberous root per plant declined. Jinzhuangyuan showed the smallest continuous cropping effect in tuberous weight with the largest,whereas Beijing No. 3showed the lightest replant problems in aboveground parts of plants. The first four principal components which accounted for 82. 489% of total variance were extracted from the principal component analysis. Leaf length,stem diameter and number of tuberous root were significantly or high significantly correlated with tuberous root weight per plant. The 18 germplasm resources could be divided into two clusters,which belonged to cultivars and wild germplasms,respectively. The results showed that there were ample morphological diversities in TTC of nine traits of 18 R. glutinosa germplasms.

The mediator complex, a conserved assembly of multi-functional proteins, is required for the transcription of almost all protein-encoding genes in eukaryotes. In this report, 36 mediator complex subunit transcripts in Rehmannia glutinosa were identified from a leaf and tuberous root EST database. Nine of these transcripts had integrated open-reading frames (ORFs). A comparison of predicted amino acid sequences with putative mediator complex subunit homologues from another six flowering plants revealed high identities and several conserved domains. Phylogenetic analysis of the nine mediator complex genes in seven flowering plants revealed a closer evolutionary relationship between R. glutinosa and Solanum lycopersicum. The relative abundances of the 36 transcripts were analysed in the leaves and tuberous roots of R. glutinosa. qRT-PCR analysis revealed that the transcripts of approximately one-third of the mediator complex genes investigated in this study were expressed at similar levels. This result suggested that these transcripts might contribute to the formation, maturation or functions of the analysed tissues. The effects of consecutive monoculture on the transcription of R. glutinosa mediator complex genes were also analysed by qRT-PCR. This analysis revealed that one-third of the mediator complex genes presented differential expression patterns in the leaves of second-year plants, whereas over half of the mediator genes were differentially expressed in the tuberous roots of second-year plants. These patterns suggest that one of the subunits of the mediator complex regulates the consecutive monoculture problem in R. glutinosa.

The v-myb avian myeloblastosis viral oncogene homolog (MYB) superfamily constitutes one of the most abundant groups of transcription factors (TFs) described in plants. To date, little is known about the MYB genes in Rehmannia glutinosa. Forty unique MYB genes with full-length cDNA sequences were isolated. These 40 genes were grouped into five categories, one R1R2R3-MYB, four TRFL MYBs, four SMH MYBs, 25 R2R3-MYBs, and six MYB-related members. The MYB DNA-binding domain (DBD) sequence composition was conserved among proteins of the same subgroup. As expected, most of the closely related members in the phylogenetic tree exhibited common motifs. Additionally, the gene structure and motifs of the R. glutinosa MYB genes were analyzed. MYB gene expression was analyzed in the leaf and the tuberous root under two abiotic stress conditions. Expression profiles showed that most R. glutinosa MYB genes were expressed in the leaf and the tuberous root, suggesting that MYB genes are involved in various physiological and developmental processes in R. glutinosa. Seven MYB genes were up-regulated in response to shading in at least one tissue. Two MYB genes showed increased expression and 13 MYB genes showed decreased expression in the tuberous root under continuous cropping. This investigation is the first comprehensive study of the MYB gene family in R. glutinosa.

The v-myb avian myeloblastosis viral oncogene homolog (MYB) superfamily constitutes one of the most abundant groups of transcription factors (TFs) described in plants. To date, little is known about the MYB genes in Rehmannia glutinosa. Forty unique MYB genes with full-length cDNA sequences were isolated. These 40 genes were grouped into five categories, one R1R2R3-MYB, four TRFL MYBs, four SMH MYBs, 25 R2R3-MYBs, and six MYB-related members. The MYB DNA-binding domain (DBD) sequence composition was conserved among proteins of the same subgroup. As expected, most of the closely related members in the phylogenetic tree exhibited common motifs. Additionally, the gene structure and motifs of the R. glutinosa MYB genes were analyzed. MYB gene expression was analyzed in the leaf and the tuberous root under two abiotic OPEN ACCESS Int. 15010 stress conditions. Expression profiles showed that most R. glutinosa MYB genes were expressed in the leaf and the tuberous root, suggesting that MYB genes are involved in various physiological and developmental processes in R. glutinosa. Seven MYB genes were up-regulated in response to shading in at least one tissue. Two MYB genes showed increased expression and 13 MYB genes showed decreased expression in the tuberous root under continuous cropping. This investigation is the first comprehensive study of the MYB gene family in R. glutinosa.