Isolation and functional analysis of SrMYB1,a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 has rarely been explored.In this study,we identified a MYB transcription factor,SrMYB1,using a yeast one-hybrid screening assay.SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity.The transcript of SrMYB1 is predominantly accumulated in flowers,but is also present at a lower level in leaves.Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment(+50–(–141))of the SrUGT76G1 promoter.Furthermore,we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus.Taken together,our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S.rebaudiana.

Characterization of yogurt goat milk added Aloe vera and Stevia rebaudiana with functional potential

The search for functional foods with health benefits has increased,and yogurt represents one of these products due to their properties,such as,source of probiotics that favour the gastrointestinal system,contains vitamins,minerals and improves the action of enzymes.Present work investigated bromatological,microbiological,sensorial properties and shelf-life of different yogurt goat milk formulations with Aloe vera and natural sweeteners.The goat yogurt with 99.40%A.vera pulp and 0.60%S.rebaudiana presented the best nutritional composition,being suitable for human consumption representing an innovative product with a great functional potential.The synergy between the ingredients used in the yogurt processing resulted in an innovative product to the market.Goat milk has a lower cost per liter in comparison with cow milk,Aloe vera is accessible due to its fast-vegetative propagation,Stevia Rebaudiana has a great popularity for being a natural sweetener with no calories,and all these products are produced in México.

我国甜叶菊生产现状及未来展望

甜叶菊是一种重要的高价值药用植物和经济作物,其叶片富含的甜菊糖苷作为一类天然甜味剂被广泛应用。中国逐渐发展成为世界上甜叶菊种植面积最大、甜菊糖苷产量最多、加工最精细的国家,甜菊糖苷市场的增加有力推动甜叶菊种植产业的发展。本文以江苏、安徽、新疆等甜叶菊主产区为例,探讨甜叶菊在我国的生产现状,发现生产中存在的问题,并对未来提出展望。研究旨在为我国甜叶菊可持续生产提供理论参考。

不同添加水平甜菊糖苷对绵羊体外产气参数及瘤胃发酵的影响

为研究绵羊日粮添加不同水平甜菊糖苷对养分降解和绵羊瘤胃发酵的影响,共设8个处理,即绵羊日粮中甜菊糖苷添加水平分别为0(CK组)、0.01%、0.04%、0.07%、0.10%、0.15%、0.20%和0.30%(占日粮风干物质重),利用体外产气法评估不同添加水平甜菊糖苷对绵羊瘤胃发酵及48 h养分降解的影响。结果表明:各处理组产气量(GP)、干物质降解率(DMD)、挥发性脂肪酸(VFA)和甲烷(CH4)产量均随瘤胃降解时间的延长而增加,且在48 h时达到最大,pH随瘤胃降解时间的延长而降低。12 h后,0.10%组GP显著高于CK组(P<0.05)。24 h时,添加较低浓度甜菊糖苷的绵羊日粮DMD与CK组差异不显著(P>0.05),而较高浓度0.20%和0.30%组的日粮DMD则较CK组分别降低了9.71%和5.80%(P<0.05)。降解48 h时,各处理日粮的DMD和粗蛋白降解率(CPD)与CK组差异不显著(P>0.05)。随着发酵时间增加,各处理瘤胃液氨态氮(NH3-N)浓度介于7.05~13.97 mg·dL^(-1),在瘤胃微生物活动的适宜浓度范围内。0.07%组和0.10%组能够维持乙酸和总挥发性脂肪酸浓度于较高水平。综上,绵羊日粮中最适宜的甜菊糖苷浓度为0.07%~0.10%。

Influences of Na_2CO_3 Stress on Physiological Metabolisms of Different Alkali Tolerant Varieties of Stevia rebaudiana

[Objective] The research aimed to reveal physiological mechanisms of alkali tolerances of different Stevia rebaudiana varieties under alkali stress.[Method] By using matrix culture method,the influences of Na2CO3 on chlorophyll content,malondialdehyde（MDA）,superoxide dismutase（SOD）,peroxidase（POD） and Proline（Pro） content of leaves from different alkali tolerance varieties of S.rebaudiana [No.2 Shoutian（relative alkali tolerance variety） and No.4 Zhongshan（alkali sensitivity variety）] were studied.[Result] 1.2 g/L of Na2CO3 stress made that the chlorophyll contents of leaves from No.2 Shoutian and No.4 Zhongshan seedlings both decreased in different degrees.Moreover,MDA content of No.4 Zhongshan was higher than control during the whole stress period,and the largest increase amplitude was 43.2%.MDA content of No.2 Shoutian was lower than control in early and latter periods of stress,and increased the maximum on the 14th day of alkali stress,which was 24.4% higher than control.SOD activities of No.2 Shoutian and No.4 Zhongshan both showed a trend of first increasing and declining then in the alkali stress period,but the increasing extent of SOD activity in No.2 Shoutian was higher than that in No.4 Zhongshan.In latter period of Na2CO3 stress,SOD activity of No.2 Shoutian declined,but POD activity was higher than that of No.4 Zhongshan.It illustrated that POD had stronger scavenging capability of active oxygen.Pro contents of No.2 Shoutian and No.4 Zhongshan were higher than control in the stress period.It showed that the osmoregulation of Pro might not be key regulatory factor of alkali tolerance difference of the two S.rebaudiana varieties.[Conclusion] The research not only provided theoretical basis for further breeding new salt tolerance variety of S.rebaudiana,but also had important significance for improving utilized ratio of kaline soil and growing environment for mudflat in China.

电感耦合等离子体质谱法测定甜叶菊中25种元素

建立电感耦合等离子体质谱(ICP-MS)法测定甜叶菊中25种元素(Cd、Pb、As、Hg、Co、V、Ni、Cu、Li、Sb、Ba、Mo、Sn、Cr、Na、Mg、Al、Ca、Ti、Mn、Fe、Zn、Ga、Se、Sr、Tl)含量的分析方法。称取甜叶菊样品粉末约0.5 g(过3#筛),置于消解罐中,加入5 mL硝酸,3 mL过氧化氢,充分进行混合,摇匀,预消解后,经微波消解处理样品,利用ICPMS法测定各元素含量。25种元素质量浓度在不同范围内与质谱响应值线性良好,标准曲线相关系数均大于0.999,各元素的检出限为0.0001~0.5 mg/kg,样品加标回收率为92.4%~102.4%,相对标准偏差为1.09%~4.11%(n=6)。该方法简便快速,线性范围宽,灵敏度高,可用于甜叶菊中多种元素的测定。

甜菊(Stevia rebaudiana)糖基转移酶基因的克隆及序列分析

利用与植物次生代谢产物糖基化相关的UDPG糖基转移酶的特征性保守区域 ,设计了同源简并引物 ,以甜菊基因组DNA为模板 ,PCR扩增获得甜菊糖基转移酶基因片段 .根据获得的基因片段设计引物GSTr和GSTf,分别与cDNA文库的T3 和T7通用引物扩增获得全长核苷酸序列的甜菊糖基转移酶基因 ,定名为sudpt 2 ,该基因全长 16 6 2bp ,包括poly(A)和一个 136 2bp的开放阅读框 ,编码 45 4个氨基酸 .与常见的糖基转移酶基因的相似性达 44 %～ 77% ,且具有UDPG糖基转移酶的特征性保守序列 .分子发育进化分析表明 。

TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in <i>Stevia rebaudiana</i>Bertoni: An Important Medicinal Plant and a Natural Sweetener

An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.

NMR Spectral Analysis and Hydrolysis Studies of Rebaudioside N, a Minor Steviol Glycoside of <i>Stevia rebaudiana</i>Bertoni

The complete proton and carbon NMR spectral assignments of a diterpene glycoside isolated from the commercial extract of the leaves of Stevia rebaudiana Bertoni, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid-[(2-O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1);also known as rebaudioside N, was achieved by the extensive 1D and 2D NMR (1H and 13C, COSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside N using acid and enzymatic studies to identify aglycone and sugar residues in its structure.

Reversed-Phase HPLC Analysis of Steviol Glycosides Isolated from Stevia rebaudiana Bertoni

High Performance Liquid Chromatography (HPLC) experiments have been performed on nine steviol glycosides namely rebaudioside A, steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A isolated from the leaves of Stevia rebaudiana using Reversed-Phase (RP) column. Using RP-HPLC method, the individual retention times for nine naturally occurring ent-kaurane diterpene glycosides of S. rebaudiana reported in JECFA have been determined at four different temperatures: 20℃, 40℃, 60℃, and 79℃. Also, calculated the relative retention times of the eight steviol glycosides steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A against the major steviol glycoside rebaudioside A. HPLC results suggested that temperatures 40℃ and 60℃ would be ideal conditions for better separation of steviol glycosides.

Potassium deficiency inhibits steviol glycosides synthesis by limiting leaf sugar metabolism in stevia(Stevia rebaudiana Bertoni)plants

The steviol glycosides(SGs)in stevia(Stevia rebaudiana Bertoni)leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivation.Optimizing fertilization management can effectively increase SGs productivity,but knowledge on the relationship between potassium(K)fertilization and SGs production is still lacking.In this study,pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves,as well as the underlying mechanisms.Our results showed that when compared with standard K fertilization,K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents.However,K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes.The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition.Moreover,spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency.Our results also revealed the significant positive correlations between sucrose,glucose and SGs contents.Overall,our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves,and this effect may be mediated by the leaf sugar metabolism.Our findings provide new insights into the improvement of SGs production potential.

Isolation and Structural Characterization of a New Minor Penta β-D-Glucopyranosyl Diterpene from Stevia rebaudiana Bertoni

From the commercial extract of the leaves of Stevia rebaudiana Bertoni, a new minor ent-kaurane diterpene glycoside having five β-D-glucopyranosyl units has been isolated. The chemical structure of the new compound was characterized as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester (1) on the basis of extensive 1D (1H & 13C) and 2D NMR (TOCSY, HMQC, and HMBC), and High Resolution (HR) mass spectroscopic data as well as hydrolysis studies.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Rapid HPLC Method for Determination of Rebaudioside D in Leaves of Stevia rebaudiana Bertoni Grown in the Southeast of Mexico

Stevia leaves contain glycosides on which biological activity and sweetening capacity has been reported. Besides the main glycosides—stevioside and rebaudioside A—there are minor glycosides that may contribute to the activity and thus it is important to quantify them. Rebaudioside D is one of the minor glycoside present in S. rebaudiana leaves and there are no reports of a validated method to quantify it. Therefore a simple and sensitive high performance liquid chromatography (HPLC) method was validated for the determination of rebaudioside D in leaves of Stevia rebaudiana B. grown in the southeast of México. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 ml/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The retention time of rebaudioside D was found to be 3.47 min ± 0.04 (S.D.). The calibration curves were linear over the working range (25 - 150 μg/ml), with correlation coefficient ≥0.99 and determination coefficient ≥0.98. The calculated limit of detection (LOD) was 8.53 μg/ml, while the limit of quantitation (LOQ) was 25.85 μg/ml. The percent recoveries of fortified samples were 100% ± 10% and precision relative standard deviation was ≤2.79%. The criteria of validation showed accuracy, linearity, and precision;therefore the method is suitable for quantitative analysis of rebaudioside D in Stevia rebaudiana leaves. Rebaudioside D content (g/100g) in Morita II and Criolla varieties grown in the southeast of Mexico were 0.43 and 0.46, respectively with no significant differences (p > 0.05) between them.

Influence of Additives on Enhanced in Vitro Shoot Multiplication of <i>Stevia rebaudiana</i>(Bert.)—An Important Anti Diabetic Medicinal Plant

The present study was designed to develop an efficient protocol for micro propagation of S. rebaudiana from nodal explants and study the influence of additives on enhancement of shoot proliferation. A two-step protocol has been standardized in which, first step comprising growth hormones concentration is optimized and it was found that MS medium supplemented with 2.0 mg/l BAP + 0.5 mg/l Kin + 0.1 mg/l NAA turned out to be the best treatment for shoot induction. In the second step, the best treatment for shoot induction was fortified with different growth additives for further shoot proliferation. Among the different types of additives used, casein hydrolysate at 0.05% (w/v) was found to be most effective, resulted with maximum of 15.0 shoots. 90% regeneration frequency and shoot length of 6.0 cm were recorded per explant. Thus, the procedure described is a quick and reliable method which could be applied for efficient large scale propagation, genetic transformation assays and secondary metabolite production of Stevia.

Molecular Analysis of Genetic Fidelity in Micropropagated Plants of <i>Stevia rebaudiana</i>Bert. Using ISSR Marker

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg·L-1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L-1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of? fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.

甜菊自交不亲和及自交亲和无性系自花授粉柱头的荧光显微观察和转录组分析

为了探究甜菊(Stevia rebaudiana Bertoni)自交不亲和的生理和分子机制,以甜菊自交不亲和无性系‘B4’及自交亲和无性系‘Z8-42’为研究对象,对2个无性系的自花授粉柱头进行荧光显微观察和转录组分析。荧光显微观察结果显示:‘B4’自花授粉柱头无花粉附着,而‘Z8-42’自花授粉柱头有花粉附着。2个无性系自花授粉柱头转录组整体测序质量较高,共获得43.84 Gb clean data。在组装的51819个unigene中,表达unigene有49214个;注释到NR数据库的表达unigene最多(29523);并且,在NR数据库中,注释到向日葵(Helianthus annuus Linn.)同源序列的unigene最多(20117)。在2个无性系中共筛选到7560个差异表达unigene;以‘B4’为对照,‘Z8-42’自花授粉柱头中表达量上调的差异表达unigene有3122个,表达量下调的差异表达unigene有4438个。GO功能注释和富集分析结果表明:甜菊差异表达unigene的功能包括生物过程、细胞组分和分子功能3个大类47个小类,其中,生物过程大类中富集到信号转导的差异表达unigene最多(225),细胞组分大类中富集到膜部分、膜固有成分和膜组成成分的差异表达unigene非常多(分别为1700、1665和1664),分子功能大类中富集到离子结合的差异表达unigene最多(1650)。共有1605个差异表达unigene被注释到KEGG代谢通路的6个大类中,其中,注释到代谢大类的差异表达unigene最多(882),占比为55.0%,分布在10个二级分类的91条代谢通路中。单个代谢通路中,植物-病原体互作通路的差异表达unigene最多(81)。综合上述研究结果,初步判定甜菊自交不亲和可能发生在柱头与花粉互作的起始阶段,如识别、黏附过程;差异表达unigene引起的柱头细胞信号转导、膜成分、离子结合等功能和结构改变可能是导致供试的2个无性系自交亲和性不同的原因,并且植物-病原体互作通路可能参与柱头和花粉的识别过程,与自交不亲和性密切相关。

Enzymatic Synthesis and Structural Characterization of Rebaudioside D3, a Minor Steviol Glycoside of <i>Stevia rebaudiana</i>Bertoni

Rebaudioside D3, a novel steviol glycoside, is produced by specific UDP-glycosyltransferase of rebaudioside E, a minor steviol glycoside of Stevia rebaudiana Bertoni. The complete proton and carbon NMR spectral assignments of rebaudioside D3, 13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester, was achieved by the extensive 1D and 2D NMR (1H and 13C, TOCSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside D3 using acid and enzymatic studies to identify aglycone and sugar residues in its structure. Rebaudioside D3 is detected in the commercial extract of the leaves of Stevia rebaudiana by LC-MS analysis, suggesting rebaudioside D3 is a natural steviol glycoside.

甜叶菊主要功能性成分研究进展

甜叶菊是一种重要的中草药、新型糖料作物和具有极高价值的经济作物。其富含糖苷类化合物、黄酮类化合物、绿原酸类化合物和其他多种功能性成分,具有抗氧化、抗菌、抗癌症、降血压、降血脂、降血糖、防龋齿等多种药理活性,日益受到人们的关注,目前在全球范围内广泛应用于食品、药品、保健品和化妆品等多个领域,具有极高的经济价值和广阔的市场前景。本文对甜叶菊主要功能性成分糖苷类化合物、黄酮类化合物和绿原酸类化合物的组成、检测、提取、分离纯化、功能活性、开发利用等方面进行了综述,并对其目前存在的问题和发展前景进行了展望,以期为我国甜叶菊资源的进一步开发、研究和利用提供一定的参考和借鉴。

IR Spectral Analysis of Diterpene Glycosides Isolated from <i>Stevia rebaudiana</i>

The objective is to identify the Infra-Red (IR) spectral analysis of the diterpene glycosides present in the commercial extracts of Stevia rebaudiana was achieved by PerkinElmer Spectrum 400 Fourier Transform (FT) spectrometer employing a PerkinElmer Universal Attenuated Total Reflection (ATR) accessory. Using this technique the IR spectral pattern of 15 steviol glycosides which belongs to three different classes of ent-kaurane diterpene glycosides namely ent-13-hydroxykaur-16-en-19-oic acid, ent-13-hydroxykaur-15-en-19-oic acid, and 13-methyl-16-oxo-17-norent- kauran-19-oic acid were identified. From the wave numbers found for all 15 steviol glycosides, it was observed that that though there are differences in the number of sugar units, nature of sugar units, and their attachments;there are not any notable differences in the IR values.