外源茉莉酸甲酯对UV-B胁迫下颠茄生物碱积累及TAs代谢途径调控的机制探究

In order to preliminarily explore the mechanism of TAs metabolic pathway in A. belladonna treated with exogenous methyl jasmone under UV-B stress, the effects on the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, signal molecule and the expression level of key enzyme genes of secondary metabolism were studied under different concentrations of exogenous MeJA and different treatment time under using A. belladonna as materials. UV-B stress treatment significantly reduced the contents of hyoscyamine and scopolamine, inhibited the accumulation of precursors in the TAs synthesis pathway, which harmful to TAs synthesis. The content of TAs in A. belladonna increased to some extent, the contents of precursor amino acids (ornithine, arginine), polyamine content and key enzymes activities in the synthesis of putrescine in the secondary metabolic pathway all increased to some extent after treatment with the appropriate concentration of MeJA. The concentrations of signal molecule NO firstly increased and then decreased with the rising MeJA concentration, and reached the highest when MeJA concentration was 250 μmol L ^-1. The expression of key enzyme genes in TAs synthesis pathway showed that exogenous MeJA could increase the relative gene expression levels of TR I, PPAR, H6H to some extent. Those indicated that exogenous MeJA could induce the increase in the contents of upstream products in TAs synthesis by stimulating the burst of NO resulted in more precursor materials for the TAs synthesis pathway and affect the high expression of TR I, PPAR and H6H. It alleviated the inhibiton of UV-B stress on TAs of A. belladonna and increased the contents of hyoscyamine and scopolamine effectively. The results provided a theoretical basis for further studying the mechanism of exogenous elicitors to regulate the TAs secondary metabolic pathway of A. belladonn under stress, and effectively improved the stress resistance of A. belladonn and the accumulation of medicinal ingredients in actual production.     

基于颠茄发根的外源基因表达系统的建立

 用携带植物高效表达载体pCAMBIA1304 ⁺ “解除武装”的重组C58C1工程菌转化颠茄无菌苗真叶, 发根诱导率达100%。PCR检测证实发根型质粒pRiA4和表达型质粒pCAMBIA1304 ⁺均能整合到颠茄发根基因组内, 共转化率达30%。建立了基于颠茄发根的高效外源基因表达系统, 为将托品烷类生物碱东莨菪碱的生物合成关键酶基因导入颠茄, 实现其次生代谢工程及开展分子育种奠定了基础。     

外源性5氨基乙酰丙酸对盐胁迫下颠茄生理特性及次生代谢产物含量的影响

通过对颠茄幼苗生理特性及生物碱含量的研究,寻找提高颠茄在盐胁迫条件下的抗性能力以及提高生物碱含量的途径.采用100 mmol/L NaCl溶液和不同质量浓度5-氨基乙酰丙酸(5-ALA)溶液处理颠茄幼苗,对颠茄叶片的叶绿素含量、叶绿素荧光参数、可溶性蛋白含量、可溶性糖含量、游离脯氨酸含量、丙二醛(MDA)含量、抗氧化酶(SOD,POD)活性及莨菪碱、东莨菪碱含量进行测定.结果表明:叶面喷施5-ALA可显著(p0.05)提高抗氧化酶活性及叶绿素含量、渗透调剂物质含量,降低盐胁迫对细胞质膜过氧化程度和PSⅡ的损伤程度,同时可提高盐胁迫下颠茄中东莨菪碱的产量,但是对莨菪碱产量的作用不明显.外源5-ALA可有效缓解盐胁迫对颠茄生长的抑制,提高其抗盐能力.     

外源SA对盐胁迫下颠茄生理生化、氮代谢及次生代谢的影响

采用无土栽培的方式,研究0.75 mmol/L 水杨酸(SA)对100 mmol/L NaCl胁迫下颠茄幼苗生理生化、氮代谢与次生代谢的影响。结果表明,外源SA处理后:颠茄叶片叶绿素含量增多,最大光化学效率(F_v/F_m)、PSⅡ的潜在活性(F_v/F_o)、PSⅡ激发能捕获效率(F_v'/F_m')、表观光合电子传递速率(ETR)、PSⅡ实际光化学效率(ΦPSⅡ)、光化学猝灭系数(qP)显著升高,初始荧光(F_o)、非光化学猝灭系数(qN)明显降低;超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性明显增强,丙二醛(MDA)含量明显降低;可溶性蛋白含量显著升高,脯氨酸和可溶性糖含量降低;NO3-含量增加,NH4+含量减少,谷氨酰胺合成酶(GS)和谷氨酸合成酶(GOGAT)活性提高,谷氨酸脱氢酶(GDH)活性降低;次生代谢产物莨菪碱和东莨菪碱含量显著提升。说明0.75 mmol/L外源SA能显著改善100 mmol/L NaCl胁迫下颠茄幼苗的光合性能,调节有机渗透物质含量,提高抗氧化酶活性,减小膜脂过氧化程度,以缓解盐胁迫造成的伤害。并使氮代谢相关酶活性恢复或增强,为生物碱的合成提供更多的前体物质和能量,从而直接或间接地促进了次生代谢产物的合成与积累。     

不同基质配方对颠茄育苗的影响

为了解各种基质对颠茄育苗的影响,采用壮苗1号、蛭石、珍珠岩、药渣、腐叶糠为材料,进行颠茄育苗试验。结果表明:以药渣、珍珠岩、园土的混合基质与以壮苗一号为基质的差异性不显著,出苗率、茎高、主根、侧根数、移栽成活率等农艺性状,均优于其他配比基质。     

Induction and development of adventitious shoots of Atropa baetica as a means of propagation

In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l^-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l^-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l^-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l^-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l^-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced. This revised version was published online in July 2006 with corrections to the Cover Date.     

颠茄H6H基因的克隆及高效植物表达载体的构建（摘要）

[目的]克隆颠茄（Atropa belladonna）H6H基因并构建高效植物表达载体。[方法]采用RT-PCR方法从颠茄（Atropa belladonna）中克隆莨菪碱-6β-羟化酶和1,4-丁二胺-氮-甲基转移酶基因编码区,插入经改造后获得双元三价植物高效表达载体p2301-gus,构建植物表达载体p2301-H6H,并将该表达载体导入根癌农杆菌LBA4404和发根农杆菌C58C1。[结果]获得了可直接用于遗传改良的颠茄工程菌p2301-H6H-LBA4404和p2301-H6H-C58C1。     

颠茄高频再生体系的建立及卡那霉素抗性筛选

[目的]建立颠茄高频再生体系及筛选卡那霉素（Kan）抗性。[方法]以颠茄叶片及腋芽为外植体,研究了培养基中6-BA和NAA不同配比对其不定芽分化的影响以及叶片不定芽对Kan的敏感性。[结果]MS＋4．5m#L6-BA＋0．2mg／LNAA为叶片不定芽分化的最佳培养基,不定芽分化率达100％,在1cm×1cm叶块上的不定芽分化数平均达5．85;MS＋3．0mg／L6-BA＋0．1mg／LNAA为腋芽不定芽分化的最适培养基,不定芽分化率达100％,每个腋芽不定芽分化平均数为4～8个;400．0m#LKan为颠茄叶片遗传转化的最佳筛选浓度。[结论]为颠茄无菌苗的快速繁殖以及基于叶盘法的遗传转化奠定了基础。     

颠茄最佳采收期的研究

通过研究颠茄干物质、生物碱含量积累动态来确定最佳采收期,为颠茄规范化种植提供理论依据.结果表明:在整个生育期内,颠茄的干重呈不断增加的趋势,于9月下旬达到最大值,单株干重可达152.48 g；生物碱的含量花果期积累最快,9月中旬达到最大值,可达4.86％,而后则出现下降的趋势.综合分析产量与质量因素,9月中旬颠茄经济产量最高,为最佳采收时间.     

颠茄草害虫瘤缘蝽的初步研究

[目的]为科学防治颠茄草害虫瘤缘蝽提供依据。[方法]在颠茄草生长季节定期、定点调查瘤缘蝽的发生规律,喷施无公害药剂观察防治效果。[结果]瘤缘蝽在信阳一年发生2代,4月中旬至11月中旬成虫和若虫均可为害颠茄草,人工捕杀、摘除卵块或喷施3%农家盼可以达到较理想的防治效果。[结论]3%农家盼是一种较理想的瘤缘蝽杀虫剂。