耐旱苔藓植物DNA提取及优化RAPD、ISSR反应体系的建立

耐旱苔藓植物常常单个个体矮小、生物量低,如何从细小的单个个体中有效提取总DNA是进一步开展居群遗传多样性研究的关键。本研究以古尔班通古特沙漠广泛分布的刺叶墙藓(Tortula desertorum)为对象,使用快速提取法、2×CTAB法及DNeasy plant mini kit试剂盒提取法等3种方法对刺叶墙藓单个个体的总DNA进行提取。结果表明,2×CTAB法提取的DNA纯度高,凝胶电泳显示无明显降解现象,适宜作为PCR扩增的模板。利用所提取的单个个体DNA为模板,建立了优化的RAPDI、SSR反应体系。

顺磁共振和傅里叶变换红外光谱法研究高温对荒漠苔藓质膜结构影响

运用顺磁共振波谱和原位傅立叶变换显微红外光谱研究了高温对荒漠植物刺叶墙藓不同叶龄水合组织质膜透性和膜蛋白二级结构的影响。自旋标记法研究质膜透性结果表明,处理前野生叶质膜透性普遍高于室内培养获得的原丝体和次生叶,处理后次生叶膜透性变化最大,比处理前增加4倍,其次为原丝体。质膜透性随叶龄增加,变化幅度逐渐降低。红外光谱的二阶求导、傅立叶自解卷积及拟合分峰等结果显示,各叶龄间蛋白质二级结构含量差异较大,表明各龄组织蛋白成分不完全相同;高温处理后次生叶和原丝体α螺旋含量分别比对照增加40%和16%;其它叶龄组织二级结构含量变化范围小,表明热胁迫下老龄组织蛋白质二级结构稳定。膜透性和膜蛋白二级结构分析证明,膜透性和蛋白质稳定性呈正相关,指示蛋白质组成和含量不同是造成各龄组织不同耐热性的主要原因之一。

Electron paramagnetic resonance study of amphiphiles partitioning behavior in desiccation-tolerant moss during dehydration

Desiccation tolerance is a crucial characteristic for desert moss surviving in arid regions. Desiccation procedure always induces amphiphiles transferring from the polar cytoplasm into lipid bodies. The behavior of amphiphiles transferring can contribute to the enhancement of desiccation tolerance and the reduction of plasma membrane integrity simultaneously. The effects of amphiphiles partitioning into the lipid phase during water loss has been studied for pollen and seeds using electron paramagnetic resonance (EPR) spectroscopy. However, desiccation-tolerant high plants occur among mosses, several angiosperms and higher plants seeds or pollens. They have different strategies for survival in dehydration and rehydration. A desiccation-tolerant moss Tortula desertorum was used to investigate the behaviors of amphiphilic molecules during drying by spin label technology. There are small amount of amphiphilic probes partitioning into membrane during moss leaves dehydration, comparing with that in higher plants. Cytoplasm viscosity changed from 1.14 into glass state only dehydration less than 60 min. Moss leaves lost plasma membrane integrity slightly, from 0.115 to 0.237, occurred simultaneously with amphiphiles partition. The results showed the more advantages of mosses than higher plants in adapting fast dehydration. We propose that EPR spin label is feasible for studying the amphiphiles partitioning mechanisms in membrane protection and damage for desiccation-tolerant mosses.

不同沙埋深度对刺叶墙藓植株碎片生长的影响

采用撒茎法对构成古尔班通古特沙漠苔藓结皮的优势种刺叶墙藓(Tortula desertorumBroth.)进行室内沙培实验,探讨不同沙埋深度对刺叶墙藓无性繁殖的影响。结果表明:(1)在不添加任何营养元素或激素的原沙基质培养条件下,刺叶墙藓的植株碎片仍然能够生长并发育成完整的植物体。(2)在所设置的不同沙埋厚度条件下,刺叶墙藓的植株碎片在2 mm沙埋厚度时生长较好,生长趋势表现为:前12 d内生长较快,之后生长缓慢。第22 d以后,未见新生植株,苔藓植株数趋于稳定。大于2 mm的沙埋厚度则抑制其生长。同时指出,沙埋也是影响古尔班通古特沙漠苔藓植物生长和分布的因素之一。

荒漠刺叶墙藓DNA的提取及PCR扩增反应条件

采用改进CTAB法、NaOH法和直接PCR法（direct PCR amplification）提取藓类生物结皮中刺叶墙藓（Tortula desertorum Broth．）基因组DNA，比较其分离效果以及ISSR扩增效果。结果表明：改进CTAB法提取的基因组DNA质量好、纯度高，ISSR扩增反应效果好。直接PCR法一旦体系建立，则是最简易快速、经济高效的模板制备方法，适于植株矮小、生物量很小的荒漠藓类植物个体模板DNA的制备。利用改进CTAB法获得的基因组DNA作为模板。对ISSR反应组分浓度及反应程序中的一些重要参数进行摸索和优化实验，建立了刺叶墙藓最优ISSR反应体系。反应总体积为20μL，各反应组分终浓度为：2μL 10×Buffer，2mM Mg^2＋，0．1mM dNTPs，0．2μM引物，10-40ng模板DNA，1U Taq酶。扩增程序为：94℃ 4min，1个循环；94℃45s，退火温度（Tm）45s，72℃1min，35个循环；72℃7min，1个循环；4℃保存。刺叶墙藓是组成古尔班通古特沙漠藓类生物结皮的优势种，研究结果为进一步开展刺叶墙藓遗传多样性的研究奠定了基础。