黄檗(Phellodendron amuranse)叶片总RNA提取方法研究

以黄檗(Phellodendron amuranseRupr.)叶片为材料,分别利用改进的盐酸胍法、Trizol法、CTAB法提取黄檗叶片总RNA,通过RNA产率、纯度、电泳图谱等分析,确立了1种从黄檗叶片中快速分离总RNA的方法。研究结果表明,改进盐酸胍法所提取的总RNA的A260/A280为1.928,28S和18S条带清晰谱图完整性好,而且具有产率高、时间短、成本低的特点,所提取的总RNA适用于mRNA分离、cDNA文库的建立、Northern杂交等分析。     

不同功能型植物叶氮含量与光合特性的关系研究

在山西南部的霍山七里峪林场,确定乔木、灌木和草本物种共26个,用Li-3000A叶面积测定仪测量了叶面积的大小、用Li-6400便携式光合作用测定系统测定了叶光饱和速率（A_area）,计算了比叶重（LMA）、单位重量的光饱和光合速率（A_mass）、单位面积叶氮含量(Narea)、单位重量叶氮含量(N_mass)及光合氮利用效率(PNUE),研究了它们之间的不同和相互作用关系。结果表明：不同功能型植物的N_mass、A_area、A_mass、N_area和PNUE差异显著(p<0.05),植物叶片氮含量与植物光合生理特性具有显著相关关系,N_mass与A_area、A_mass和PNUE呈线性显著的正相关(p<0.05);N_area与A_area、A_mass、PNUE之间呈极显著的负相关(p<0.01)。     

遮荫对虎耳草光合生理特性的影响

以盆栽1年的虎耳草为材料,研究遮荫条件下虎耳草叶片抗氧化酶体系活力、叶绿素含量、光合速率以及叶绿素荧光参数的变化,为虎耳草规模化栽培提供理论基础。结果表明：透光率50%条件下,叶绿素含量较高;虎耳草抗氧化酶活力最高;光饱和点（LSP）最大,最大光合速率（A_max）最高,光合速率日变化平均值最高,无“午休”;光系统Ⅱ的实际量子产量(Φ_PSⅡ)、PSⅡ最大量子产量(F_v/F_m)和光化学猝灭系数(qP)最高,非光化学猝灭系数(qN)最低。这表明,虎耳草在怀化地区的最适光照条件是50%左右的透光率。     

中国板栗自然居群微卫星（SSR）遗传多样性

采用8对微卫星分子标记对中国板栗(Castanea mollissima)的28个自然居群进行了遗传多样性与遗传结构分析。在849个个体上扩增得到128个等位基因, 每位点平均等位基因数(A)为16。中国板栗居群的平均预期杂合度(H_E)为0.678, 平均观察杂合度(H_O)为0.590。华中地区的中国板栗居群遗传多样性最高(A = 8.112, H_E = 0.705, H_O = 0.618), 其次为西北地区和华东地区, 而西南地区遗传多样性最低(A = 6.611, H_E = 0.640, H_O = 0.559)。基于无限等位基因模型(IAM)和基于逐步突变模型(SMM)的遗传分化系数分别为F_ST = 0.120和R_ST = 0.208。分子方差分析(AMOVA)结果表明中国板栗野生居群的遗传变异主要存在于居群内(87.16%)。Mantel检测揭示遗传距离与地理距离之间无显著相关性, 表明基因流不是主导中国板栗居群遗传结构的关键因素。华中地区(尤其是神农架及其周边地区)是中国板栗遗传多样性的现代分布中心, 因而应该得到优先保护, 同时该区域的野生板栗居群可优先作为栽培板栗遗传育种的材料和基因库。     

一种适用范围广的总RNA提取方法

介绍一种RNA提取方法,该方法以SDS、氯仿和Tris苯酚为主要提取试剂,以LiCl和乙醇为RNA沉淀试剂。分别以柽柳(木本植物)、星星草(草本植物)、天牛（昆虫）、酿酒酵母和白腐菌（真菌）为RNA提取材料,用该方法成功地提取出了它们的总RNA。获得的RNA条带清晰,A₂₆₀/A₂₈₀ 在1.8以上。通过对LiCl和乙醇沉淀RNA的效果分析表明,该方法可在10 min内完全沉淀RNA,同时也可以同时获得纯度较高的DNA。提取的RNA质量可满足cDNA文库构建,基因芯片探针标定和RT-PCR等对RNA质量要求较高的分子生物学操作,说明这是一种应用范围广的RNA提取方法。     

生物多样性的进化原理及其保护对策

本文论述了传统进化论学说对生物多样性解释的不足,探讨用生物适化学说解释生物多样性的形成,提出生物多样性产生的表达式： B_d=∫^T_Ｏ［(G_c-m+M)·E_c-(N_t+A_p+H_f)］dt, 并以此说明制订保育它们的原则对策。     

光强在低温弱光胁迫后番茄叶片光合作用恢复中的作用

为了研究光强在低温弱光胁迫后番茄叶片光合作用恢复中的作用,以番茄品种浙粉202为材料,研究了低温弱光后恢复期全光照与遮荫对光合作用和叶绿素荧光参数的影响。结果表明：低温弱光（8℃/12℃,PFD 80 μmol·m^-2·s^-1）导致番茄叶片P_n、Φ_PSⅡ、qP和F_v′/F_m′的下降,但诱导了NPQ的上升,未引起F_v/F_m的变化;全光照（100%光照）下恢复1 使得植株叶片P_n、F_v/F_m、Φ_PSⅡ、qP、NPQ和F_v′/F_m′均大幅下降,随后光合和荧光参数可缓慢恢复至对照水平;遮荫（40%光照）恢复植株F_v/F_m、Φ_PSⅡ和F_v′/F_m′仅在第一天稍有下降,而P_n和qP还略有上升,NPQ虽有所降低但仍显著高于对照水平,随后光合和荧光参数均可迅速恢复到对照水平。说明低温弱光虽抑制了光合作用的进行,但并未引起光抑制的发生;全光照恢复加剧了叶片光抑制的发生,而遮荫恢复可通过叶片PSⅡ光化学活性的快速恢复和天线色素热耗散能力的增强以保护光合机构免受伤害,有利于光合作用的迅速恢复。     

宝山堇菜（Viola baoshanensis）、紫花地丁(V. yedoensis)光合异质性比较

利用荧光成像技术,研究了特定和不同光合有效辐射下,宝山堇菜（Viola baoshanensis）、紫花地丁 (V. yedoensis)不同叶龄（幼叶和成熟叶片）叶片纵向（叶基、叶中部、叶尖）间的光合异质性特征。特定光化光照射下,两种堇菜不同叶龄的F_v/F_m、ΦpsⅡ、qP、PS/50和Abs在叶尖、叶中部、叶基间呈依次降低趋势,NPQ/4和qN变化趋势与之相反。F_v/F_m、ΦpsⅡ和Abs在两种堇菜不同叶龄的叶片纵向间均没有显著性差异,NPQ/4和qN均显示宝山堇菜不同叶龄的叶片纵向间存在显著差异;但NPQ/4和qN分别显示紫花地丁成熟叶和幼叶叶片的叶尖和叶基处差异显著。qP的显著差异只存在于宝山堇菜幼叶的叶尖和叶基处,PS/50在两种植物幼叶纵向间均有显著差异。快速光曲线的变化中,两种堇菜α由叶尖向叶基下降幅度不明显,幼叶纵向间P_m差异显著,成熟叶叶尖处P_m显著高于叶中部和叶基。两种堇菜成熟叶叶尖处I_k显著高于叶中部和叶基,宝山堇菜幼叶纵向间I_k差异显著,而紫花地丁幼叶纵向间I_k差异不显著。以上结果反映出两种堇菜叶片纵向间F_v/F_m和ΦpsⅡ具有较高的均质性,F_v/F_m和ΦpsⅡ的下降受到NPQ/4、qN、qP、PS/50和Abs的综合影响,但F_v/F_m和ΦpsⅡ的变化与NPQ/4、qN、qP、PS/50的显著变化并不一致。叶龄对两种堇菜叶片纵向间α影响不显著,对P_m影响显著,α的小幅下降反映两种堇菜叶片纵向间捕光能力基本相同。叶片纵向间I_k的显著差异受叶龄和植物种类的综合影响。     

强光胁迫对濒危植物金花茶幼苗生长和叶绿素荧光参数的影响

以当年生盆栽金花茶实生苗为材料,研究不同程度的强光胁迫（25％、50％和100％自然光强,以8%自然光强为对照）对其生长、生物量、叶片光合色素含量、叶绿素荧光参数的影响。结果表明：在不同程度的强光胁迫下,金花茶幼苗的生长均受到抑制,随着胁迫程度的增强,金花茶叶片颜色由深绿变为浅绿、黄绿色,叶片灼伤愈来愈严重;植株抽稍时间推迟,抽稍后长出的新叶长势较差;幼苗死亡率越来越高。幼苗根生物量、茎生物量、叶生物量和总生物量均随胁迫程度的升高而显著降低,强光胁迫对叶生物量的影响最大,根生物量次之,对茎生物量的影响最小。随着胁迫程度的增强,叶片叶绿素总量（Chl）、叶绿素a（Chla）、叶绿素b（Chlb）含量均显著降低,Chla/Chlb和Car/Chl显著升高。叶绿素荧光参数F_o、F_m、F_v、F_v/F_m、F_v/F_o均随胁迫程度的升高降低,强光胁迫使PSⅡ受到了伤害,光合作用原初反应过程受抑制,光合电子传递受到影响,从而抑制植株的正常生长。     

外源NO对盐胁迫下黑麦草幼苗活性氧代谢、多胺含量和光合作用的影响

采用溶液培养法研究了外源一氧化氮(nitric oxide,NO)供体硝普钠(sodium nitroprusside,SNP)对100 mmol·L^-1 NaCl胁迫下黑麦草幼苗生长、活性氧代谢、多胺含量和光合作用的影响。结果表明,50 μmol·L^-1 SNP显著提高盐胁迫下黑麦草幼苗叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性及谷胱甘肽(GSH)、精胺(Spm)、亚精胺(Spd)含量和(Spm+Spd)/Put比值,降低了腐胺(Put)、超氧阴离子(O^—·₂)、H₂O₂和丙二醛(MDA)含量,使幼苗叶片叶绿素和类胡萝卜素含量、净光合速率(P_n)和气孔导度(G_s)升高,胞间CO₂浓度(C_i)下降,相对生长量增加。叶绿素荧光动力学资料显示,SNP处理降低盐胁迫下黑麦草叶片的初始荧光(F₀),表明它对光合膜系统具有保护效应。SNP处理不仅提高了盐胁迫下叶片的最大荧光(F_m)、PSⅡ潜在光化学效率(F_v/F₀)和PSⅡ最大光化学效率(F_v/F_m),而且提高了PSⅡ实际光化学效率(Φ_PSⅡ)、光化学荧光猝灭系数(q_P)、表观光合电子传递速率(ETR)和光化学速率(PCR),降低了非光化学荧光猝灭系数(NPQ)和天线热耗散(D),而1 mmol·L^-1 NO清除剂PTIO和0.5 μmol·L^-1 NaNO₂处理(对照)则无此效应。由此表明,外源NO可能通过提高植株的抗氧化能力及光能的捕获与转换而增强盐胁迫下黑麦草叶片的光合能力,从而缓解盐胁迫对幼苗生长的抑制作用。     

条斑紫菜丝状体总RNA提取方法比较

目的:为了获得质量较高的条斑紫菜丝状体总RNA,对几种常用提取方法进行研究。方法:以条斑紫菜自由丝状体为材料,比较了用异硫氰酸胍法、CTAB法、SDS/酚法、TRIzol法、RNAplant法提取的RNA的质量和纯度。结果:异硫氰酸胍法提取RNA的成本低,但纯度不高;CTAB法产率较小,且不能完全去除多糖或蛋白质;SDS/酚法未能获得完整的RNA;TRIzol法未能见到5SrRNA条带,且带有杂带;而RNAplant法提取RNA的质量好、纯度高、提取效率高,其D260nm/D280nm值为1.836,经逆转录得到的双链cDNA扩增产物长度在200bp以上。结论:实验结果表明RNAplant法更适于条斑紫菜丝状体总RNA的提取。     

A quick method to isolate RNA from wheat and other carbohydrate-rich seeds

No reports on isolating RNA from carbohydrate-rich wheat seeds have been published. Because of the presence of carbohydrates, published protocols yield small amounts of poor quality RNA. Extracting seeds in a buffer (pH 9, 150 mM NaCl, 1% sarcosyl) ensured maximum RNA solubility and the removal of most interfering substances. Extracted RNA was purified using a guanidine hydrochloride-based buffer system. This protocol yields up to 148 μg of RNA from 100 mg of tissue in 3.5 h. An A₂₆₀/A₂₈₀ ratio of 1.85 indicates RNA purity. Isolated RNA was amenable to downstream applications such as differential display. The developed method was extended to other carbohydrate-rich seeds, such as barley and maize, with success.     

DNA as a Flocculation Factor in Pseudomonas sp.

A component responsible for flocculation was extracted from Pseudomonas strain C-120 by treating the cells with 3 M guanidine hydrochloride. The guanidine hydrochloride-extracted cells were reflocculated, not only with the guanidine hydrochloride extract but with DNA prepared from various bacteria. The reconstituted flocs were deflocculated by deoxyribonuclease or guanidine hydrochloride which indicated that the reconstituted flocs closely resembled natural flocs. In reconstitution experiments using Escherichia coli DNA at different molecular weights, it was found that DNA with a molecular weight higher than about 6 × 10⁶ was required to flocculate the guanidine hydrochloride-extracted cells. Heat-denatured DNA did not flocculate the guanidine hydrochloride-extracted cells. DNA with a high molecular weight was detected in the guanidine hydrochloride extract. It was concluded that the component involved in flocculation of this organism was highly polymerized double stranded DNA.     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

An improved method for high-quality RNA isolation from needles of adult maritime pine trees

When conventional RNA isolation methods optimized for pine seedlings are applied to needles of adult pine trees, poor-quality RNA results. Here we describe a modified procedure to isolate high-quality RNA from needles of 30-year-old maritime pines, exhibiting high levels of phenolics, polysaccharides, and RNases. Major changes are the inclusion of proteinase K in the extraction medium followed by incubation at 42°C. Integrity and purity were evaluated by using denaturing gel electrophoresis and spectrophotometry (A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀). The total RNA could be successfully used for poly(A)⁺-RNA isolation and cDNA library construction.     

从少量培养细胞中同时提取微量蛋白和RNA的方法探讨

为建立一项从少量培养细胞中同时提取RNA和蛋白质的技术 ,向 2～ 3× 10 5细胞中加入 1ml自制RNA提取试剂 ,RNA抽提后剩下的中下两相 ,用异丙醇、盐酸胍和无水乙醇抽提蛋白质 .同时用进口Tripure试剂、经典的异硫氰酸胍 苯酚 氯仿一步抽提RNA法和分子克隆实验手册裂解液制备蛋白质的方法 ,作为对照 .自制试剂提取的总RNA ,18S、2 8S清晰可见 ,2 8S比 18S带亮度强 2～ 3倍 ,带与带之间无拖尾现象 ,5S隐约可见 ,而且成功地进行了Northern印迹、RT PCR分析 ,与经典方法差异不大 ;用此法所提蛋白质 ,经SDS PAGE检测 ,蛋白分离效果很好 ,无杂质 ,且Western印迹检测Giα蛋白 ,可见一条清晰的特异带 ,与常规提取蛋白质 ,结果相似 .从微量细胞中同时提取的RNA和蛋白质 ,得率高、纯度好 ,具有化学完整性和生物学性质     

A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10⁸ spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.