A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization,heterologous expression in transgenic plants and transactivation in yeast cells

The coordinate expression of anthocyanin biosynthetic genes in leaves and stems of a red forma of Perilla frutescens is presumably controlled by regulatory gene(s). A Myc-like gene (Myc-rp) was isolated from a cDNA library prepared from the leaves of red P. frutescens, and its deduced amino acid sequence shows 64% identity with that of delila from snapdragon. The Myc-rp gene was expressed in leaves and roots of both red and green P. frutescens equally. Comparison of deduced amino acid sequence of Myc-rp with that of Myc-gp, the second allele isolated from a green forma of P. frutescens, indicates that the 132nd amino acid, alanine, existing in MYC-RP was changed to serine in MYC-GP. The heterologous expression of these two alleles of Myc-like gene in tobacco and tomato resulted in an increase of the anthocyanin contents in flowers of tobacco and vegetative tissues and flowers of tomato. However, the flowers of transgenic tobacco expressing the fragment with a partial deletion (encoding 1–115 amino acids deleted) of Myc-gp gave no change in anthocyanin accumulation, but some morphological changes of the flower were observed. In yeast, the MYC-RP/GP and Delila protein exhibited transactivation activity on the GAL-1 promoter from yeast and the promoter of dihydroflavonol 4-reductase (DFR) gene from P. frutescens. A transactivation domain of MYC-RP/GP and Delila could be located in the region between the 193rd and the 420th amino acid of MYC-RP/GP proteins. Our data indicate that this Myc-like gene presumably functions in the regulation of anthocyanin biosynthesis similarly in different tissues of dicot plants.     

Effect of light irradiation on anthocyanin production by suspended culture of Perilla frutescens

After a series of experiments on photoperiodicity and light intensity under daylight supplied by an ordinary fluorescent lamp in cultivations using a flask and a roux bottle, it was found that irradiation at 27.2 W/m(2) for the whole period was effective for anthocyanin production by a suspended culture of Perilla frutescens (shiso). A high amount of anthocyanin pigments, 3.0 g/L, was obtained in a bubble column bioreactor after 10 days of cultivation at an aeration rate of 0.1 vvm with light irradiation at 27.2 W/m(2), while 2 g/L was obtained at 13.6 W/m(2) and very little at 54.4 W/m(2). A high amount of anthocyanin pigments, 2.9 g/L, was also produced using an aerated and agitated bioreactor at an agitation speed of 130 rpm, an aeration rate of 0.1 vvm and light irradiation intensity of 27.2 W/m(2). The amount of anthocyanin produced was more than twice that without light irradiation, Keeping the other cultivation conditions the same. The results obtained also showed that the amount of anthocyanin pigment accumulated in a shake flask could be rather well reproduced in bioreactors for both aerated culture, and aerated and agitated culture, by improving the conditions of light irradiation, which conspicuously affects metabolite formation.     

Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydoxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.     

HPLC法测定紫苏不同来源不同部位中迷迭香酸的含量

采用 HP LC 法测定14种不同来源紫苏不同部位的迷迭香酸的含量,并进行统计与聚类分析.结果表明：14种来源紫苏间叶、果穗、茎及根中迷迭香酸含量均有显著差异(P ＜0．05),同一来源不同部位间迷迭香酸含量也存在显著差异(P ＜0．05).各种来源紫苏叶中的迷迭香酸含量均最高;大部分来源紫苏果穗中迷迭香酸的含量较紫苏茎高.14份材料经聚类分析可分成4个类群.研究结果表明紫苏果穗可能是潜在的新药源,来源于长江中下游紫苏不同部位的迷迭香酸含量要比西南地区的高.     

Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla frutescens

We have investigated metabolite profiles and gene expression in two chemo-varietal forms, red and green forms, of Perilla frutescens var. crispa. Striking difference in anthocyanin content was observed between the red and green forms. Anthocyanin, mainly malonylshisonin, was highly accumulated in the leaves of the red form but not in the green form. Less obvious differences were also observed in the stems. However, there was no remarkable difference in the contents and patterns of flavones and primary metabolites such as inorganic anions, organic anions and amino acids. These results suggest that only the regulation of anthocyanin production, but not that of other metabolites, differs in red and green forms. Microscopic observation and immunohistochemical studies indicated that the epidermal cells of leaves and stems are the sites of accumulation of anthocyanins and localization of anthocyanidin synthase protein. By differential display of mRNA from the leaves of red and green forms, we could identify several genes encoding anthocyanin-biosynthetic enzymes and presumptive regulatory proteins. The possible regulatory network leading to differential anthocyanin accumulation in a form-specific manner is discussed.     

Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation

Anthocyanins are a class of products of plant secondary metabolism and are responsible for tubers color in potato.The biosynthesis of anthocyanins is a complex Researchbiological process,in which multiple genes are involved including structural genes and regulatory genes.In this study,StAN11,a WD40-repeat gene,was cloned from potato cultivar Chieftain(Solanum tuberosum L.).StAN11(HQ599506)contained no intron and its open reading frame(ORF)was 1,029 bp long,encoding a putative protein of 342 amino acids.In order to verify its role in anthocyanin biosynthesis,StAN11 was inserted behind the CaMV-35S promoter of pCMBIA1304 and the recombination vector was introduced into the potato cultivar Désirée plants by Agrobacterium-mediated transformation.The color of transgenic tuber skin was significantly deepened,compared to the wild-type control,which was highly consistent with the accumulation of anthocyanin and expression of StAN11 in transgenic lines tuber skin.Further analysis on the expression of Flavonone-3-hydroxylase(F3H),Dihydroflavonol reductase(DFR),Anthocyanidin synthase(ANS),and Flavonoid 3-O-glucosyl transferase(3GT)in transgenic plants revealed that only DFR was upregulated.This result suggested that StAN11 regulated anthocyanin biosynthesis in potato by controlling DFR expression and accumulation of anthocyanin could be increased through overexpression of StAN11 in the tubers with the genetic background of anthocyanin biosynthesis.     

A quantitative analysis of shear effects on cell suspension and cell culture of perilla frutescens in bioreactors

The short-time effects of shear on suspended cells of Perilla frutescens were quantitatively analyzed by exposing the cells to a well-defined flow field in a rotating drum reactor. It was found that both shear rate and shearing time significantly affected cell viability. The quantitative effects of shear on cell growth and the production of anthocyanin, a secondary metabolite, by the cell cultures were further investigated in a series of batch cultivations using a 5-L plant cell bioreactor with a marine impeller. The results indicated that there was an optimum range of shear rate; i.e., an average shear rate of 20 to 30 s(-1) or an impeller tip speed of 5 to 8 dm/s, which maximized all the values of the following parameters: the specific growth rate, the maximum cell concentration, the (specific) production and productivity of anthocyanin, and the cell and anthocyanin yields. (c) 1994 John Wiley & Sons, Inc.     

Cloning of thyroid hormone receptor genes expressed in metamorphosing flounder

Two distinct cDNAs encoding thyroid hormone receptors (THRs) were cloned from a λ gtl0 library prepared from the whole bodies of metamorphosing flounder larvae (Paralichfhys olivaceus). Deduced amino acid sequences of the two isolated cDNAs shared 96% and 92% homologies in their DNA- and hormone-binding domains, respectively. These were highly conserved when compared to THRs for other vertebrates: 88–96% in the DNA-binding domain and 84–94% in the hormone-binding domain. Other receptors in the nuclear receptor family showed lower homologies than those of THRs. Both THRs for the flounder had higher homologies with the α-type THRs of other vertebrates than with the β-type. Thus, the two THRs for flounder were designated as fTHRαA and fTHRαB. © 1994 Wiiey-Liss, Inc.     

Expression of anthocyanin biosynthesis pathway genes in red and white grapes

The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.     

Molecular Cloning and Expression Analysis of 3-Ketoacyl-ACP Synthases in the Immature Seeds of Perilla frutescens

We report the isolation and expression analysis of two cDNAs encoding 3-ketoacyl-acyl carrier protein synthases (KAS) that are involved in the de novo synthesis of fatty acids in plastids of perilla (Perilla frutescens L.). The cDNAs, designated PfFAB1 and PfFAB24, encoded polypeptides with high sequence identities to those of KAS I and KAS II/IV, respectively, of various plants. Genomic Southern blots revealed that there was a single PfFAB1 gene but two PfFAB24 genes in the perilla genome. Of interest is that the expression of both genes was developmentally regulated in seeds. Their mRNA expression patterns in seeds were also discussed in comparison with the profile of fatty acid accumulation.     

家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析

吡哆醛激酶（pyridoxal kinase,PLK, EC2.7.1.35）是维生素B₆关键代谢酶,其cDNA的克隆在昆虫类还未见报道。利用生物信息学原理和使用PCR方法,克隆出编码家蚕Bombyx mori吡哆醛激酶的cDNA (GenBank登录号DQ452397),体外原核表达成功,并对表达粗提产物进行了酶活检测。克隆到的cDNA含有一894 bp的完整可读框,编码一条分子量为33.1 kD,含298个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类吡哆醛激酶具有52.84%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但比哺乳动物和植物克隆到的吡哆醛激酶均少10多个氨基酸残基,几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换。依据家蚕基因组数据库信息和PLK的cDNA,家蚕PLK基因包含5个外显子和4个内含子,跨越10 kb DNA序列,所有外显子/内含子交接点都遵从gt/ag剪接规则,基因的5′端启动子调控区发现有TATA-box和CAAT-box保守基序。     

The effects of developmental stage and source leaf position on integration and sectorial patterns of carbohydrate movement in an annual plant, Perilla frutescens (Lamiaceae)

A well-integrated plant shows extensive carbohydrate translocation through the plant body. Even in highly integrated plants, however, translocation patterns will be sectorial if vascular tissue restricts carbon movement to sectors along stems. Both integration and sectorial translocation patterns are sensitive to plant architecture and thus may change as a plant develops. These patterns should vary also with the position of the source leaf because leaves at each node are unique in age and vascular relationship to the rest of the plant. I measured the effects of developmental stage and location of the source leaf on integration and sectoriality in an annual plant, Perilla frutescens, by labeling plants with C at one of three leaves and four developmental stages. Stage and source leaf affected both integration and sectoriality. Most notably, integration declined and sectoriality increased during seed fill, when resource demand at each node was high. Furthermore, translocation was least extensive from the leaf supporting the largest number of seeds on its axillary branch. These results suggest that plants are not homogeneous collections of subunits; rather, the role of each leaf in a plant's carbon budget is a function of its age and location on the plant.     

志贺毒素保护性抗原的基因克隆与序列分析

根据GenBank报道的志贺毒素A、B亚单位基因序列设计合成两对引物,以I型痢疾志贺茵的基因组为模板经PCR扩增获得3条DNA序列,将其分别插入pMD18—T载体,测序证明所获得的3条序列为志贺毒素A、B亚单位基因及其串联片段,与GenBank中相应序列比较,同源性分别为99．5％、100％和99．8％。通过计算机软件对所推测的氨基酸序列进行了蛋白质参数分析。志贺毒素保护性抗原基因的获得为志贺毒素的免疫学研究准备了必要的材料。     

紫苏硬脂酰-ACP脱氢酶基因家族鉴定及表达分析北大核心CSCD

硬脂酰-ACP脱氢酶(SAD)催化硬脂酸脱氢生成油酸,是形成不饱和脂肪酸的关键酶。该研究从紫苏转录组数据库中筛选鉴定紫苏硬脂酰-ACP脱氢酶(PfSAD)家族基因,并进行生物信息学分析及保守功能域分析,用qRT-PCR技术检测PfSADs各成员在不同组织中的表达特性,以探讨PfSAD家族基因在调控种子脂肪酸组分中的作用,为紫苏脂肪酸组分的遗传改良提供基因元件。结果显示:(1)从该课题组前期自测的紫苏转录组数据库中共检测出6个PfSAD家族基因,其编码蛋白的氨基酸长度介于367~396 aa之间,均具有SAD的保守结构域和二铁中心,预测其基因编码蛋白均定位于叶绿体。(2)多序列比对结果显示,紫苏PfSADs蛋白序列与拟南芥、蓖麻及可可等植物的SAD蛋白序列相似性均在50%以上;系统进化分析显示,6个紫苏SAD蛋白被分为3个亚组,其中第一个亚组包含PfSAD1,第二亚组包含PfSAD2、PfSAD3,第三亚组包含PfSAD4、PfSAD5和PfSAD6。(3)实时荧光定量PCR分析发现,PfSADs各成员在‘晋紫苏1号’不同组织中的表达量差异显著,其中PfSAD1主要在叶中表达,PfSAD2、PfSAD3、PfSAD4和PfSAD5在种子中表达量较高,PfSAD6在花中具有显著表达优势。研究表明,PfSADs具有典型的保守基序及催化SAD的活性中心,其各成员在不同的组织中高表达,推测这6个基因均参与了硬脂酰ACP(C18∶0-ACP)脱氢生成油酰基ACP(Δ9C18∶1-ACP)的过程,在紫苏油脂合成代谢过程中发挥重要作用。     

海洋破囊壶菌Thraustochytrium sp. FJN-10 DHA生物合成 途径相关延长酶的克隆与表达

二十二碳六烯酸(Docosahexaenoic acid,DHA)是具有各种重要生理功能的高度不饱和脂肪酸.以海洋真菌Thraustochytrium sp.FJN-10为研究对象,利用RT-PCR结合RACE,获得了两个碳链延长酶(TFD6和TFD5)的完整基因,其中TFD6 cDNA全长816 bp,编码271个氨基酸;TFD5 cDNA全长831 bp,编码276个氨基酸.将TFD6、TFD5酶切后分别连接到HindⅢ/Xba Ⅰ处理过的pYES2载体,醋酸锂法转化酿酒酵母感受态细胞,成功构建了延长酶酵母表达系统.气相色谱分析表明TFD6可延长C18:3n-6至C20:3n-6,TFD5可延长C20:5n-3至C22:5n-3.