转基因及常规水稻幼苗对潮霉素敏感性的研究

研究了转基因及常规水稻幼苗叶面喷施潮霉素B溶液后的反应。结果发现,常规水稻吸收潮霉素B后,叶片会出现以褐斑为特征的中毒症状。在50～100mgL浓度范围内,褐斑的数目和面积随时间和浓度的增加而增加,溶液中滴加DMSO可提高潮霉素对稻苗的毒性。浓度在75mgL及以上时,喷后第7天,处理的所有幼苗都出现症状。而浓度在50mgL时,个别幼苗经处理后未出现中毒症状。在上述浓度范围内,带潮霉素磷酸转移酶基因(hpt)的Bt转基因水稻(克螟稻)幼苗均未出现中毒症状。苗期鉴定与抽穗前后成株期鉴定的结果相一致。讨论了叶面喷施潮霉素B溶液在转基因水稻育种和筛选潮霉素敏感转基因水稻突变体中的可能应用。     

Effect of temperature on growth,dry matter production and starch accumulation in ten mango (Mangifera indica L.) cultivars

Summary

Ten mango cultivars of tropical and subtropical origin (Carabao, Kensington, Nam Dok Mai, Alphonso, Dashehari, Florigon, Glenn, Irwin, Haden and Sensation) were grafted onto cv. Kensington seedling rootstock and held at four day/night temperatures for 20 weeks (15/10°C, 20/15°C, 25/20°C and 30/25°C). Vegetative growth increased with increasing temperatures. All grew vegetatively at 25/20°C and 30/25°C. Cultivars which did not grow at 20/15°C were Carabao, Kensington and Dashehari. Cultivars Kensington, Nam Dok Mai, Alphonso, Florigon, Glenn, Irwin, Haden and Sensation produced flower panicles at 15/10°C. The rise in temperature increased the average number of growth flushes (in responsive cultivars) from 0.48 at 15/10°C to 3.21 at 30/25°C, and the number of leaves per growth flush (1.22 at 15/10°C to 13.63 at 30/25°C). Distribution of dry matter from new growth was mostly to the roots at the lowest temperature (95% at 15/10°C) and to the leaves (58%) at 30/25°C. The mean daily temperature for zero vegetative growth was calculated to be 15°C. Temperature and related growth activity also affected the concentration of starch in the woody tissue of rootstock trunks at the end of 20 weeks (15.9% starch at 15/10°C v. 4.8% starch at 30/25°C). ‘Irwin’ had the highest starch concentration at the two higher temperatures (twice that of any other cultivar at 30/25°C) while ‘Kensington’ the lowest starch level at 25/20°C, ca. 50% of most other cultivars.     

一种不依赖于无菌培养的拟南芥活体转基因种子筛选方法

    拟南芥活体转基因种子的筛选仍主要采用无菌培养技术,存在操作麻烦,容易污染而造成转基因材料损失等问题.本研究在传统组织培养筛选技术的基础上,通过去除原培养基成分中对种子发芽和筛选不必需的蔗糖和有机成分,降低大量元素的浓度,缩短筛选时间,从而免除了传统转基因种子筛选中培养基和种子消毒的繁琐步骤,建立了一种不依赖于无菌培养的转基因种子筛选方法.与无菌培养筛选比较,筛选效果相同,但简化了试验步骤,提高了筛选操作的可靠性.     

小麦遗传率中潮霉素适宜筛选浓度的研究

研究了潮霉素不同质量浓度对西农 1376和京花 1号小麦愈伤组织及种子的筛选效果。结果表明 ,潮霉素对小麦愈伤组织的适宜筛选质量浓度为 110 mg/L,对转基因小麦后代种子筛选以 14 0 mg/L 较为适宜 ;不同基因型材料对潮霉素的敏感性存在一定差异     

拟南芥悬浮细胞及其愈伤组织对潮霉素的反应

研究了潮霉索对拟南芥悬浮细胞及其愈伤组织的作用效果和特性．结果表明：潮霉素对拟南芥悬浮细胞生长、愈伤组织诱导及其生长均有极明显的抑制作用。但作用效果和特性均有所不同；拟南芥悬浮细胞继代培养阶段、愈伤组织诱导阶段和愈伤组织增殖阶段的临界致死质量浓度分别为25、2．5、1．5mg／L，液体与固体培养条件下差异极大；潮毒素需要一定处理时间才能充分表现其抑制细胞生长或杀死细胞的效果，一般液体培养条件下3d以后、固体培养条件下5d以后效果趋于稳定，但不同的处理质量浓度有较大差异．还就液体与固体培养条件下作用效果差异的原因进行了讨论，并提出了对潮毒素抗性标记筛选的建议．     

潮霉素对紫花苜蓿愈伤组织诱导和分化的影响

潮霉素是当今基因工程中的一种转化选择剂。通过对苜蓿叶片诱导阶段、分化阶段施加不同浓度的潮霉素,确定潮霉素对紫花苜蓿叶片愈伤组织形成及其分化的影响。研究表明,潮霉素对紫花苜蓿叶片愈伤组织诱导与分化有显著的抑制作用。当潮霉素浓度达到15mg/L时苜蓿叶片完全无法诱导愈伤;愈伤和分化阶段都添加10mg/L潮霉素时,虽然可以诱导出愈伤组织但无法分化。因此建议在遗传转化时诱导愈伤阶段及分化阶段都添加10mg/L潮霉素。     

转Cry1A(b)基因抗虫水稻的获得及鉴定

利用农杆菌混合液介导的共转化法,研究不同浓度的农杆菌混合液浸染5种水稻品种的愈伤组织,进而获得共转化再生植株。对转基因植株进行叶片潮霉素抗性检测和螟虫田间抗性鉴定的结果表明,农杆菌混合液浓度OD600为0.6左右时,其浸染能力较强;5个品种中以浙粳27的再生效率最高。转基因植株叶片经潮霉素抗性检测,潮霉素阳性率为78.13%,经PCR检测转基因植株Cry1A(b)基因阳性率为15.11%。自然抗性鉴定结果表明:Cry1A(b)基因阳性的转基因植株对稻纵卷叶螟具有很好的抗性。基因分离的鉴定结果表明:HPT基因与Cry1A(b)基因在共转化转基因植株的后代中发生了分离。     

The mechanism of resistance to sterol 14α demethylation inhibitors in a mutant (Erg 40) of Ustilago maydis

Resistance to DMI fungicides is a problem in both agriculture and medicine. Several mechanisms of resistance exist, but, as yet, few have been characterised in field resistant strains of plant pathogens. One approach to evaluating the role of mutations in the sterol 14α demethylase (14DM) target site requires cloning this gene and confirming its identity by complementation in an appropriate mutant. The azole‐resistant mutant, Erg 40, of Ustilago maydis which is totally blocked at the 14α demethylation step in sterol biosynthesis seems to be suitable for such expression studies. Transformation of Erg 40 with a plasmid containing the yeast 14α demethylase (CYP51A1) gene removed the block in sterol biosynthesis and generated azole‐sensitive transformants. Detailed analysis of these transformants failed to detect the presence of the yeast gene and suggested, instead, that changes in sterol biosynthesis resulted simply from the transformation protocol and not from the incorporation of extracellular DNA. Subsequent sequence analysis has revealed a mutation in the 14α demethylase gene of Erg 40. The results suggest that azole resistance in Erg 40 is not simply controlled by this mutation but involves some additional regulatory function, and consequently Erg 40 is not suitable for complementation studies with CYP51A1 genes. © 2000 Society of Chemical Industry     

黄瓜遗传转化体系优化及表皮毛基因Gl的转化

选用黄瓜无毛突变体gl的子叶节为材料,对芽诱导阶段及根诱导阶段潮霉素的浓度进行筛选,分别确定2和3mg/L为其最适浓度;以pCAMBIA2301-gusA为载体,农杆菌GV3101介导,筛选出Silwet L-77(表面活性剂)的最佳体积分数为0.04%,侵染液浓度以原菌液(OD600=0.6)等倍稀释最佳;另外,对快速提取黄瓜DNA方法进行了优化。结果表明,添加500μL 0.5mol/L NaOH与200μL 100mmol/L Tris-HCl的组合最好。用优化的体系对表皮毛基因Gl进行遗传转化,获得7株阳性苗,为研究Gl基因的调控机制奠定了基础。     

一种适合水稻农杆菌转化的RNAi载体的构建和潮霉素对水稻转化的影响

以感染水稻条纹病毒(RSV)水稻叶片的总RNA为模板,利用RT-PCR扩增出了RSV的CP基因。分别将正反向的CP基因连接到载体pMCG161,再将此载体和pCAMBIA1301载体同时双酶切,产物回收后连接,成功构建了适合农杆菌介导的水稻转化RNAi载体。在农杆菌水稻转化过程中,比较了分化阶段是否加潮霉素对愈伤组织分化的影响,结果表明,分化阶段去除潮霉素的筛选可以提高愈伤组织的分化程度但同时降低获得水稻植株的阳性率。