Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures

The activities of the biosynthetic enzmes phenylalanine ammonia lyase (PAL) and 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) were measured in cells transferred from growth to production medium in a two-stage batch culture. It was found that both these enzymes showed transient increases, PAL (three- to fourfold) and HMGR (two- to four-fold), at or near the point of exhaustion of nitrogen source (NO(3)). Production of shikonin derivatives also started at this time. The addition of excess nitrate to the medium shortly before nitrate exhaustion (days 6-8) markedly reduced the final product yield (by 70-80%) while addition of excess nitrate in the later stationary growth phase (days 14-16) had no significant effect. When the production rate of shikonin derivatives was correlated with PAL activity, it was observed that production rate is very low (less than 1 mg/L . day) at low levels of PAL activity (below 0.1 unit/mg protein). Once a threshold level of PAL activity (about 0.15 unit/mg protein) is reached, the biosynthetic rate of shikonin derivatives increases. Such a relationship could not be deduced for HMGR activity. It was concluded that the production of shikonin derivatives may be limited at the phenylalanine deaminating step at low levels of PAL activity.     

Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

Exploring the evolutionary process of alkannin/shikonin O-acyltransferases by a reliable Lithospermum erythrorhizon genome

Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be ∼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contig_N50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon’s roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT’s functional differentiation in evolution.     

Enhancement of shikonin production in single- and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound

This work demonstrates the use of low-energy ultrasound (US) to enhance secondary metabolite production in plant cell cultures. Suspension culture of Lithospermum erythrorhizon cells was exposed to low-power US (power density < or = 113.9 mW/cm(3)) for short periods (1-8 min). The US exposure significantly stimulated the shikonin biosynthesis of the cells, and at certain US doses, increased the volumetric shikonin yield by about 60%-70%. Meanwhile, the shikonin excreted from the cells was increased from 20% to 65%-70%, due partially to an increase in the cell membrane permeability by sonication. With combined use of US treatment and in situ product extraction by an organic solvent, or the two-phase culture, the volumetric shikonin yield was increased more than two- to threefold. Increasing in the number of US exposures during the culture process usually resulted in negative effects on shikonin yield but slight stimulation of shikonin excretion. US at relatively high energy levels caused slight cell growth depression (maximum 9% decrease in dry cell weight). Two key enzymes for the secondary metabolite biosynthesis of cells, phenylalanine ammonia lyase and p-hydroxybenzoic acid geranyltransferase, were found to be stimulated by the US. The US stimulation of secondary metabolite biosynthesis was attributed to the metabolic activity of cells activated by US, and more specifically, the defense responses of plant cells to the mechanical stress of US irradiation.     

硬紫草细胞悬浮培养和紫草宁及其衍生物的形成

硬紫草细胞悬浮培养形成紫草宁及其衍生物时．细胞生长曲线呈扁平的s形。细胞停止生长后,紫草宁及其衍生物大量形成,二者的动态变化呈负相关。测定丁此过程中培养液的无机元素和可溶性糖含量、溶氧、pH值以及细胞形态的变化情况,可作为硬紫草细胞大规模培养的参考。     

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation.The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.     

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics. Received: 2 March 1998 / Revision received: 25 May 1998 / Accepted: 8 July 1998     

固定化培养和产物释放促进剂对硬紫草细胞代谢的影响

固定化培养的硬紫草细胞生长缓慢,仅包埋球外层的细胞生长明显。其蛋白质合成的量也低。培养３０ｄ的细胞色素产量达到４．２ｍｇ／ｇＦＷ,相对色素分泌量达到７０％,而色素的组成成分及各组分的比例也与悬浮细胞的不同。以正十六烷处理固定化细胞可促进产物释放,其不同的处理时间对细胞没有显著影响。连续培养的固定细胞保持其色素形成能力达８０ｄ之久,色素总产量达２０ｍｇ／ｇＦＷ。     

Regulation of shikonin production by glutamine in Lithospermum erythrorhizon cell cultures

Amino acid analysis has shown that Lithosperum erythrorhizon cell suspension cultures which are unable to produce shikonin derivatives in LS medium containing ammonium accumulate a large quantity of glutamine, as compared with shikonin-producing cells cultured in the production medium M9 containing nitrate as the sole nitrogen source. The addition of glutamine to M9 medium proved to be strongly inhibitory to shikonin production. Furthermore, culture experiments using an inhibitor of glutaminase suggested that shikonin synthesis is not inhibited by ammonium released from glutamine but by glutamine itself. These findings indicate that the repression of shikonin synthesis occurs in close association with an accumulation of glutamine in cultured cells grown in a medium containing ammonium.     

根癌农杆菌转化紫草的研究

紫草 (ＬｉｔｈｏｓｐｅｒｍｕｍｅｒｙｔｈｒｏｒｈｉｚｏｎＳｉｅｂ .ｅｔＺｕｃｃ)是传统中药。其根部含有萘醌类化合物—紫草素及其衍生物 ,具有显著的抗菌、抗炎、抗癌以及促进伤口愈合等生理活性。紫草素同时也是一种名贵化妆品染料。科学家对紫草的研究兴趣是基于其资源的缺乏及紫草植物本身所具有的一些特点 ;如 :紫草素及其衍生物的颜色特性可凭借肉眼观察 ,紫草素及其衍生物只在紫草的根部积累 ,紫草素合成的次生代谢途径受多种酶和外界条件 (光照 ,营养等 )的调节等。紫草细胞培养 (Ｆｕｊｉｔａ等 ,1983;叶和春等 ,1991)可以产…     

Induction of shikonin biosynthesis by endogenous polysaccharides in Lithospermum erythrorhizon cell suspension cultures

Endogenous polysaccharides capable of inducing shikonin biosynthesis in a growth medium were isolated from shikonin-producing Lithospermum cells cultured in a production medium. Chemical analysis showed that these active polysaccharides consist of galacturonic acid, galactose, arabinose and glucose. Their activity, however, was lost by a treatment with pectinlyase. Addition of a small amount of pectinase to cell cultures in the growth medium induced shikonin formation. This is the first report that endogenous polysaccharides participate in inducing normal secondary metabolism of higher plants.     

Involvement of LeMRP,an ATP‐binding cassette transporter,in shikonin transport and biosynthesis in Lithospermum erythrorhizon

• Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown.
• In this study, we isolated a cDNA encoding LeMRP, an ATP‐binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real‐time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes.
• Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real‐time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up‐regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down‐regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots.
• Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.

     

藻类活性物质对紫草细胞生长和色素形成的影响

本文研究了螺旋藻、栅列藻和织线藻的水提物对新疆紫草和硬紫草细胞生长和色素形成的作用。结果表明不同种类的藻的提取物对不同紫草细胞作用呈现差异。在生长阶段,对新疆紫草,上述3种藻的低浓度提取物促进生长但作用不大,高浓度的螺旋藻和栅列藻提取物强烈抑制生长;对硬紫草,各种藻提取液的所有浓度处理均有促生长作用。在色素形成阶段,连续用高浓度织线藻提取物处理可以加速新疆紫草色素形成,同时提高色素含量。低浓度的织线藻提取液处理能提高硬紫草色素含量。栅列藻的水提物对两种紫草的色素形成均起抑制作用。螺旋藻水提物的适当浓度可加速两种紫草的色素形成。B_5培养基中加高浓度织线藻水提物,可抑制新疆紫草生长阶段的色素形成。     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of shikonin derivatives of cultured cells and ko-shikon

A method for quantitative analysis of shikonin derivatives using high pressure liquid chromatography (HPLC) was established. With this method the composition of shikonin derivatives in cultured cells and roots of Lithospermum erythrorhizon (ko-shikon) was compared. The composition of shikonin derivatives produced by cell suspension cultures was similar to that of the ko-shikon, and the composition in cultured cells was found to fluctuate less than that of the ko-shikon.     

不同林型地表甲虫食性及营养级关系：基于碳氮稳定同位素分析

土壤动物联结着生态系统地上与地下部分的物质循环和能量流动,对生态系统的结构、功能及过程起着重要的调控作用。地表甲虫作为典型的大型土壤动物,在食物网中占有重要的位置,因此对不同林型地表甲虫的δ¹³C、δ¹⁵N同位素特征及营养关系研究对了解森林土壤动物的食性特征进而保护森林生物多样性是十分必要的。采集了小兴安岭凉水自然保护区6种不同林型的地表甲虫共10科31种,利用稳定同位素技术测定了甲虫中的δ¹³C、δ¹⁵N含量,并分析不同林型内地表甲虫的δ¹³C、δ¹⁵N值及营养级差异。结果表明6、7月份不同林型地表甲虫的δ¹³C、δ¹⁵N值差异显著(P<0.05),其中δ¹³C值在原始阔叶红松林和次生白桦林显著高于落叶松人工林和阔叶红松择伐林。δ¹⁵N值在阔叶红松择伐林显著高于其他5种林型。不同林型地表甲虫的营养级差异显著(P<0.05),林型内各物种营养级差异不显著(P...     

碳氮源对转基因鱼腥藻Anabaena sp.PCC7120培养的影响

对碳源、氮源种类和用量对转rhTNF-α基因鱼腥藻7120（Anabaena sp.PCC7120）培养的影响进行了研究,发现最适碳源为蔗糖,最适氮源为NaNO3,最佳用量分别为9g/L和2．25g/L,此时生物量远高于自养方式,达2．52g/L,比相同条件下在BG－11培养基培养高71．66％,ATNF－α表达量为16％－22％,生物活性为10^5U/mg。     

晋西北黄土高原丘陵区不同土地利用方式下土壤碳氮储量

对晋西北黄土高原丘陵区杨树-小叶锦鸡儿人工林、小叶锦鸡儿人工灌丛、杨树人工林、撂荒地和农田5种土地利用方式下土壤碳氮储量进行研究.结果表明: 不同土地利用方式下土壤碳氮含量、碳氮密度和碳氮储量存在显著差异.5种土地利用方式0～20 cm表层土壤碳氮含量和碳氮密度均显著大于20～40 cm和40～60 cm土层.5种土地利用方式同一土层碳氮含量和碳氮密度大小为: 杨树-小叶锦鸡儿人工林>小叶锦鸡儿人工灌丛>杨树人工林>撂荒地>农田;0～60 cm土层土壤有机碳储量大小为:杨树-小-叶锦鸡儿人工林(30.09 t·hm^-2)>小叶锦鸡儿人工灌丛(24.78 t·hm^-2)>杨树人工林(24.14 t·hm^-2)>撂荒地(22.06 t·hm^-2)>农田(17.59 t·hm^-2);土壤氮储量与有机碳储量变化规律相似,杨树-小叶锦鸡儿人工林0～60 cm土层土壤氮储量(4.94 t·hm^-2)最高,其次是小叶锦鸡儿人工灌丛(3.53 t·hm^-2)、杨树人工林(3.51 t·hm^-2)和撂荒地(3.40 t·hm^-2),农田土壤氮储量(2.71 t·hm^-2)最低.杨树-小叶锦鸡儿人工林和小叶锦鸡儿人工灌丛是晋西北黄土高原丘陵区植被建设和生态恢复过程中较好的两种土地利用方式.     

氮素形态对小白菜生长和碳氮积累的影响

水培条件下,研究不同氮素形态(硝态氮、铵态氮、甘氨酸、谷氨酰胺、丙氨酸、牛血清蛋白,以及甘氨酸与硝态氮、牛血清蛋白与硝态氮的混合氮源)对小白菜生长和碳氮积累的影响.结果表明:不同氮素形态对小白菜质量、碳氮积累量、可溶性蛋白质含量、可溶性糖含量和游离氨基酸含量的影响不同;硝态氮处理下小白菜地上部分和根的干质量与鲜质量均最大;甘氨酸对小白菜根系的生长及碳氮积累具有明显的促进作用;在3种氨基酸中,谷氨酰胺更有利于小白菜地上部分的生长和氮积累.聚类分析表明,9种氮素形态处理按营养效应大小分为:硝态氮、谷氨酰胺＞甘氨酸与硝态氮混合氮源、牛血清蛋白与硝态氮混合氮源、甘氨酸、铵态氮＞丙氨酸、牛血清蛋白、对照.有机氮源可以作为小白菜生长的氮源,不同的氮素形态对植物产生的生理效应不同.     

10种常见甲藻细胞体积与细胞碳、氮含量的关系

选择10种常见甲藻,通过构建相应细胞几何模拟图形从而计算了每种甲藻细胞的体积,利用元素分析仪测定了每种甲藻的单个细胞碳、氮含量,并分析了细胞体积与细胞碳、氮含量之间的关系。结果表明,10种常见甲藻的细胞体积差异显著,最小仅为2.97×10² μm³(卡特双甲藻),最大可达到4.50×10⁴ μm³(红色赤潮藻),相差2个数量级;单个细胞碳、氮含量变化范围分别为54.50-2238.00 pg/个和11.42-482.28 pg/个,均相差40多倍。细胞体积与单个细胞碳、氮含量存在极显著的正相关线性关系(P<0.0001)。