甜叶菊微卫星富集文库的构建与多态性标记的筛选

甜叶菊是我国一种重要特种经济作物, 其分子标记相关遗传背景研究甚少。本研究基于生物素与链霉亲和素的强亲和性原理, 用链霉亲和素顺磁颗粒捕捉人工合成的标记有生物素的寡核苷酸探针(AG)₁₅, 间接筛选出含有甜叶菊基因组微卫星序列的DNA酶切片段, 将筛选得到的片段连接到pUC-T载体中, 构建甜叶菊微卫星序列的富集文库。挑取354个克隆进行菌落PCR检验, 从中筛选出158个阳性克隆进行测序。结果表明, 134个(84.81%)克隆中含有微卫星序列, 其中完美型85个(63.43%)、非完美型15个(11.19%)、复合型34个(25.38%)。根据微卫星序列共设计出71对微卫星引物, 其中62对能扩增出稳定的条带。利用24个甜叶菊品系对这62对引物的遗传多样性的分析表明, 有16个位点表现出多态性, 等位基因数为2~8个, 平均每个位点扩增得到4.5个等位基因, 多态性信息含量在0.3163~0.7595之间, 观测杂合度(H_o)与期望杂合度(H_e)的范围分别为0.2174~0.9167与0.3555~0.8076。通过聚类分析, 将甜叶菊分为大小叶两大类。本研究开发出的微卫星标记可为甜叶菊的分子遗传育种提供有效的遗传标记。     

甜叶菊中莱苞迪苷D、莱苞迪苷A含量测定方法的优化及应用

为比较不同品系甜叶菊中甜味品质较好的莱苞迪苷D(RD)、莱苞迪苷A(RA)含量组成,在传统C18、HSS T3、Amide色谱柱中选择适宜固定相建立高效液相色谱(HPLC)方法进行测定与分析。结果表明,HSS T3柱对甜菊糖苷选择性较好,可同时分离RA、甜菜苷(ST)、莱苞迪苷F(RF)、莱苞迪苷C(RC)、甜茶苷(RBS)、莱苞迪苷B(RB)、甜菊双糖苷(SB),其HPLC分析参数为:流动相32%乙腈和68%磷酸水(0.01%),等度洗脱,柱温40℃,波长210 nm,进样量10μL,流速1.0 mL·min^-1;Amide柱对RD分离能力最佳,其HPLC分析参数为:流动相76%乙腈和24%水,等度洗脱,柱温40℃,波长210 nm,进样量10μL,流速0.8 mL·min^-1。分析比较12个扦插培育的甜叶菊品系,以编号2甜叶菊中RD含量及其占比最高,提示以其为原材料可生产含RD较高的甜菊糖苷;编号3、5、7、11甜叶菊中具较有高含量的甜菊糖苷(主要为RA),提示这些品种富含RA且甜菊糖苷产量较高;编号1、8甜叶菊中RA+RD占比较高,提示...     

干旱胁迫对甜叶菊生理指标及单株干叶产量的影响

为了给干旱胁迫条件下的甜叶菊栽培管理提供理论依据,利用温室盆栽法研究了干旱胁迫对不同种质材料甜叶菊生理指标及干叶产量的影响。结果表明：正常水分处理下5份甜叶菊种质材料的可溶性蛋白质含量、相对电导率、丙二醛（MDA）含量、超氧化物歧化酶（SOD）、过氧化物酶（POD）及甜叶菊单株干叶产量不同。随着干旱时间的持续,5份甜叶菊种质材料的可溶性蛋白质含量均表现为下降趋势,仅IS-1在T3（停止灌溉15天）略有上升;5份甜叶菊种质材料相对电导率均出现较平稳的显著增加;IS-1和AL-4叶片MDA的含量随着干旱胁迫时间的持续先降低后升高,而QF-1、‘青甜杂4’及JD-1叶片MDA的含量持续升高;SOD与POD的活性均表现出先升高后降低再升高的变化趋势;5份甜叶菊种质材料的单株干叶产量均出现不同程度的下降趋势。相同处理中不同材料的相同指标变化程度不同,说明其抗旱能力不同：其中IS-1最强,QF-1最弱,其他3份材料的抗旱性顺序分别为：AL-4>‘青甜杂4’>JD-1。     

The effect of different nitrogen levels on yield and quality of stevia (Stevia rebaudiana bert.)

In the present work, the efficiency of different nitrogen doses (0, 50, 100, 150, and 200 kg ha^?1) on growth, yield, and quality of stevia (Stevia rebaudiana Bert.) was investigated in 2011–2013. The study was conducted in Antalya located in the Mediterranean Region of Turkey. Terra rossa type soil (LVx, FAO) characteristics of the experimental field were clay loam, with high amounts of lime (33,9%) and slightly alkaline (pH 7.7). The experiment was carried out in randomized block design with four replications. All the results were summarized as mean of three years. The highest fresh and dry biomass yields (26.75 t ha^?1 and 7.5 ha^?1, respectively) were obtained from 150 kg ha^?1 N dose and followed by 100 kg ha^?1 N dose (26.29 t ha^?1 and 7.24 ha^?1, respectively). Whereas the highest fresh and dry leaf yields (13.27 t ha^?1 and 3.82 t ha^?1, respectively) were realized in 100 kg ha^?1 N dose. Actually, all nitrogen doses gave higher biomass and leaf yields compared to the control. On the hand, major steviol glycosides (stevioside and rebaudioside A) in the leaf were not influenced by nitrogen levels. In conclusion, 100 kg ha^?1 N dose was found to be suitable for cultivation of stevia under field conditions.     

Crop Productivity,Steviol Glycoside Yield,Nutrient Concentration and Uptake of Stevia rebaudiana Bert. under Mediterranean Field Conditions

Stevia rebaudiana Bertoni (Asteraceae Family), characterized by a high content of steviol glycosides (SG) in its leaves, represents an interesting source of non-sucrose and no-calorie sweeteners. Stevia, which is native to Paraguay, is a relatively new crop for the Mediterranean region, where it is grown as an annual or poly-annual crop. In order to assess the crop’s environmental requirements, productive performance and nutrient uptakes, a two-year field experiment was carried out in north-eastern Italy, where the species had never previously been cultivated. The aim was to evaluate the effects of important pre-harvest factors, such as cultivation site, harvest time and their reciprocal interaction, on productivity, leaf SG yield and nutrient requirements. Results showed that the timing of the harvest and the pedo-climatic conditions of the cultivation sites had a significant effect on the main biometric traits, total and leaf dry yield, SG production, macronutrient concentration and uptake.     

Effect of Different Nitrogen Doses on Dry Matter Ratio,Chlorophyll and Macro/Micro Nutrient Content in Sweet Herb (Stevia rebaudiana Bertoni)

With this study, the effect of different nitrogen (N) doses applied to sweet herb (0, 50, 100, 150 and 200 N kg ha^?1) on the macro/micro nutrient element content, dry matter ratio and on the chlorophyll amount in the plant leaves is examined. The research was carried out in the research and trial field of Faculty of Agriculture Agronomy department, Akdeniz University, during the 2012–2013 growing season under field conditions for 2 years with four replications according to the randomized block trial design. Chlorophyll amounts were determined and the average was obtained with three readings from each leaf by a chlorophyll meter when the plants were in the beginning of flowering. Post-harvest macro and micro nutrient elements of the sweet herb were also investigated. According to the obtained data, while the highest chlorophyll and N amounts in the leaf for both the first and second years were determined as 46.97 SPAD (Minolta Corp, NJ, USA) and 1.34 N% 44.9 SPAD, 1.42 N%, respectively, under 200 kg ha^?1 N application, the lowest chlorophyll and N amounts were determined in the control group. In addition, N application in different doses for both years increased the phosphorus (P) and potassium (K) contents and increased the microelement contents, especially in the second year. At the same time, a correlation between the chlorophyll content in the plant leaves and the N amount was determined and a linear increase was observed in the chlorophyll amount with the increased green part.     

甜菊醇糖苷生物合成途径关键酶基因KA13H的克隆及序列分析

本研究利用RT-PCR方法从甜叶菊叶片中分离了一个与甜菊醇糖苷生物合成密切相关的基因KA13H,对该基因的ORF、编码产物结构、同源性比对及次级结构等进行了生物信息学预测分析,同时初步分析该基因的组织表达和原核表达。经DNAMAN6.0软件分析,该基因编码一条分子量为54.476kD的由476个氨基酸残基组成的多肽,含有典型的细胞色素P450的血红素结合位点FXXGXXXCXG,TMPRED程序预测其C-端含有一个明显的跨膜区MIQVLTPILLFLIFFVFWKVY,是一个典型的细胞色素P450基因。推断的KA13H编码产物与其它生物合成相关细胞色素P450同源比对和系统发生分析表明,该蛋白与苜蓿(Medicago truncatula)CYP716A12和云杉(Sitka spruce)CYP720B1的一致性较高,分别为51%和46%,推测KA13H可能与CYP716A12和CYP720B1具有类似的功能。KA13H次级结构分析结果表明,KA13H与CYP74A2的一致性仅为15%,但是它们却有着类似的次级结构,都含有6个基质识别位点(SRSs)。半定量RT-PCR分析表明:KA13H在根、茎、叶和花中...     

甜菊不同层次叶片叶绿体超微结构观察及其糖苷含量变化

甜菊Stevia rebaudiana Bertoni不同层次叶片叶绿体超微结构的观察及其糖苷含量的测定表明:在上层幼嫩叶片中,叶绿体小,类囊体膜较简单,基粒片层和基质片层数量少,糖苷含量居中。在中层成叶的叶片中,叶绿体大,类囊体膜复杂,基粒片层多,基质片层密集,在基质中出现“淀粉区”结构,糖苷含量最高。在下层衰老的叶片中,叶绿体趋于解体,类囊体膜退化,糖苷含量最低。上述结构与功能密切相关,说明甜菊在现蕾期,中层叶最多,且糖苷含量也最高,是收获的最适时期。     

安甜菊2号选育·特征特性·高产栽培技术

[目的]介绍高产、高糖苷含量的甜叶菊(Stevia rebaudiana Bertoni)新品种安甜菊2号。[方法]对高糖苷含量甜叶菊新品种安甜菊2号的选育过程、特征特性、产量表现及栽培技术要点进行综述。[结果]2012年通过安徽省非主要农作物品种鉴定登记委员会审定,命名为安甜菊2号。在安徽明光、霍邱等地种植,平均产量约为3 127.5 kg/hm2,比守田2号(CK)增产11.54%。总甜菊糖苷含量占干叶重12%以上,RA3含量超过65%。[结论]该研究可为安甜菊2号的推广应用提供理论论据。     

甜叶菊残渣在蔬菜育苗上的应用效果

用甜叶菊残渣与基础基质（土、蛭石、锯末）配制不同配比的蔬菜育苗土。结果表明,含甜叶菊残渣的育苗土对黄瓜、青椒、番茄幼苗有促进生长的作用。甜叶菊与基础基质的最佳配比为黄瓜１∶３,青椒１∶４,番茄１∶４。