The synthetic growth hormone2,4-dichlorophenoxyacetic acid (2,4-D) significantly inhibits shikonin and its derivatives biosynthesis in the medicinal plant Lithospermum erythrorhizon. However, the molecular mechanism of this regulation remains unclear.In this study, we attempt to address this issue by comparing the transcriptome of 2,4-D treated cell cultures with that of the control (CK). A total of 216 up-regulated and 269 down-regulated genes by 2,4-D were discovered. Gene ontology (GO) enrichment analysis revealed that differentially expressed genes (DEGs) were statistically significantly related to the metabolic process. Pathway classification enrichment analysis further confirmed that the DEGs were mainly related to the secondary metabolism. More importantly, 2,4-D has no effect on the two key enzyme genes ACS and ACO for ethylene biosynthesis. However,2,4-D significantly down-regulated the genes directly involved in ethylene signal transduction pathway (ESTP), including IllvL45, EIN3, and ORCA3. 2,4-D also down-regulated two indirect genes, LOX and GA2ox, thereby conferring the jasmonate (JA) and GA biosynthesis, which regulates ESTP by targeting EIN3. By blocking ESTP, 2,4-D fmally down regulated the phenylpropanoid formation-related genes, thereby conferring shikonin and its derivatives biosynthesis. Our fmdings provide new hints for the deep understanding of the molecular mechanism of shikonin formation.

BACKGROUND:Shikonin is a naphthoquinone secondary metabolite with important medicinal value and is found in Lithospermum erythrorhizon. Considering the limited knowledge on the membrane transport mechanism of shikonin, this study investigated such molecular mechanism.RESULTS:We successfully isolated an ATP-binding cassette protein gene, LeMDR, from L. erythrorhizon. LeMDR is predominantly expressed in L. erythrorhizon roots, where shikonin accumulated. Functional analysis of LeMDR by using the yeast cell expression system revealed that LeMDR is possibly involved in the shikonin efflux transport. The accumulation of shikonin is lower in yeast cells transformed with LeMDR-overexpressing vector than that with empty vector. The transgenic hairy roots of L. erythrorhizon overexpressing LeMDR (MDRO) significantly enhanced shikonin production, whereas the RNA interference of LeMDR (MDRi) displayed a reverse trend. Moreover, the mRNA expression level of LeMDR was up-regulated by treatment with shikonin and shikonin-positive regulators, methyl jasmonate and indole-3-acetic acid. There might be a relationship of mutual regulation between the expression level of LeMDR and shikonin biosynthesis.CONCLUSIONS:Our findings demonstrated the important role of LeMDR in transmembrane transport and biosynthesis of shikonin.

The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.

BACKGROUND:The phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation.RESULTS:The mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production.CONCLUSIONS:The results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.

Novel shikonin derivatives were synthesised and probed as anticancer agents. Compound 40 showed the best anticancer activity with an IC50 of 1.26 μM, could induce apoptosis and cause cell cycle arrest at the G2/M phase via the P21 p-CDC2 (Tyr15) pathway independent of P53.

植物次生代谢产物是人类重要的药物及化工原料来源,其产生与植物正常的生长发育及对环境的适应密切相关,并受到多种因素的调控.乙烯作为一种植物内源激素,广泛参与植物的生长、发育、抗逆和次生代谢产物合成等重要生理过程的调控.该文综述了乙烯的信号转导机制及其调控作用;重点归纳了乙烯对植物次生代谢产物形成所表现出的双重调控效应,即在一定浓度范围内,乙烯对植物次生代谢产物的合成起促进作用,低于或超过该浓度范围则起抑制作用;并对今后该领域的研究方向进行了展望.

Cellulose is the main component of plant cell walls,and the cellulose synthase encoded by CesA gene is the most important enzyme involved in cellulose biosynthesis.In this study,genome sequence of three model plant species(Arabidopsis thaliana,Oryza sativa,Populus trichocarpa) had been utilized to investigate the phylogenetic relationship of the CesA gene family.The results showed that CesA genes had already existed before species differentiation and that gene expansion occurred after species differentiation.There was a variation in the degree of gene expansion among different species and the entire gene family showed negative selection pressure.To further demonstrate the expression regulation patterns of CesA genes,the promoters of OsCesAs were analyzed and the results suggest that OsCesA genes might be regulated by various stresses and hormones.RT-PCR results indicated that some OsCesAs were down-regulated by abscisic acid.

利用随机扩增多态性DNA分子标记技术,对江苏太湖和安徽升金湖共4个区域的黄颡鱼种群中共192条个体进行遗传多样性的比较,并分析太湖蓝藻水华产生的藻毒素及其衍生污染物的复合污染对种群遗传结构的影响.在试验的引物中筛选出6条在PCR扩增时能得到清晰、稳定性和重复性好条带的引物,各引物扩增得到的总位点数为8～18个,多态位点比率为50.00％ ～ 100.00％,其中胥口湾黄颡鱼的多态位点比率呈现最高(86.46％),而梅梁湾黄颡鱼最低(67.71％).4个黄颡鱼种群的Shannon指数和Nei基因多样性指数大小都表现出和多态位点比率一致的趋势,即胥口湾黄颡鱼＞升金湖黄颡鱼＞东太湖黄颡鱼＞梅梁湾黄颡鱼.由此可见,即使实际距离相隔不远,不同水质环境下黄颡鱼种群的多样性已经表现出一定程度的差异,并且随着水环境污染的严重,黄颡鱼的多样性也随之降低.在水质最佳的胥口湾区域,多样性呈现最高,作为对照区域的升金湖区域,其种群多样性同样也高于污染严重区域的梅梁湾地区.总体来看,近年来太湖蓝藻水华产生的环境污染在一定程度上影响着生物多样性,因此改善环境,保护生物多样性刻不容缓.