玉米根部酵母双杂交cDNA文库的构建及评价

从玉米各生长时期的根部中提取总RNA后,再利用磁珠法从中分离纯化出mRNA,然后以mRNA为模板,反转录合成cDNA第一链,再在DNA聚合酶作用下,通过长距离PCR,扩增双链cDNA(ds cDNA).利用SMART技术,通过同源重组的方法,在酵母菌株AH109中构建了玉米根部全长cDNA文库.经检测,文库的转化率为1.76×107转化子/3μgpGADT7-Rec,文库滴度为1.17×106pfu/mL,重组率为95%.     

Proteomics Identification of Differentially Expressed Proteins Relevant for Nicotine Synthesis in Flue-Cured Tobacco Roots Before and After Decapitation

Nicotine is a secondary substance synthesized in tobacco roots. In flue-cured tobacco planting, tobacco decapitation is an effective practice to promote nicotine biosynthesis by regulation of the redistribution of total nitrogen amounts. However, proteins relevant to nicotine synthesis in tobacco roots has not been identified and characterized yet. It is important to explore the regulation of nicotine biosynthesis in tobacco roots. To identify the proteins relevant to nicotine synthesis, the protein patterns in roots of flue-cured tobacco （cv. K326） before and after decapitation were analyzed. In the present study, the protein patterns in roots of flue-cured tobacco were analyzed by two-dimensional electrophoresis （2-DE）, and the differentially-expressed spots were identified by matrix assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS）. Paired comparison of 2-DE maps revealed 26 spots of differentially-expressed proteins in roots before and after decapitation. Furthermore, nine differentially-expressed spots were identified. There were four proteins which were enzymes possibly involved in nicotine biosynthesis. In addition, the roles of the four enzymes in nicotine biosynthesis were discussed in a putative network. Our results would contribute to the understanding of the regulation pathway of nicotine biosynthesis and further to the molecular manipulation on the nicotine contents in flue-cured tobacco.     

胡杨根、茎、叶、愈伤组织总RNA提取的研究

胡杨根、茎、叶、愈伤组织等器官和组织的总RNA高质量提取是进行以胡杨RNA为基础的分子生物学研究的基础。以2 a生胡杨实生苗根系为材料,用CTAB-PVP提取缓冲液裂解细胞并变性蛋白,经氯仿/异戊醇(24∶1,v/v)抽提后,用3种不同的沉淀方法获得胡杨根系总RNA,电泳和定量测试等分析提取质量。结果表明,LiCl沉淀法结合70%乙醇洗涤沉淀两次获得的的胡杨根系总RNA,条带清晰、亮度高,且没有明显的DNA污染;actin基因片段设计引物对胡杨根系总RNA进行RT-PCR扩增,扩增片段与预期大小一致,表明该RNA可用于逆转录合成cDNA,提取物RNA中的Li 残留对逆转录酶活性的抑制作用不明显;该方法用以提取2 a生胡杨实生苗茎、叶及愈伤组织总RNA,均获得高质量总RNA。CTAB-PVP-LiCl(附70%乙醇洗涤RNA沉淀2次)的方法可用于胡杨根、茎、叶和愈伤组织总RNA的提取。     

Isolation and Analysis of Drought-Induced Genes in Maize Roots

Maize roots are important component for plant adaptation to soil water deficits because they are supposed to take up water and necessary solutes from the soil. In the present study, the drought-induced genes were isolated in maize roots. A suppression subtractive hybridization protocol was applied to construct a forward subtractive cDNA library from CN165 for drought-stressed maize roots and a number of drought-induced genes were isolated. Totally, 126 uniESTs （containing 82 singlets and 44 contigs） were obtained from 503 available ESTs sequences after macroarray hybridization. UniESTs were analyzed using BLASTN and BLASTX and the results showed that 92% of the uniESTs had homolgous sequences in maize nr database by BLASTN. About 89% of uniESTs appeared the homlogous amino acid sequences in rice protein database but not in maize protein database by BLASTX, implying that those genes are likely new functional genes in maize. Function analysis showed that those genes were involved in a broad spectrum of biological pathways, mainly in signaling and regulatory pathways related to stress tolerance.     

Regrowth and dry matter allocation in Quercus robur (L.) seedlings root pruned prior to transplanting

Two-year seedlings of Quercus robur (pedunculateoak) were root-pruned before transplanting in order toevaluate the importance of coarse versus fine roots onregrowth. Root systems were pruned by leaving c. 19,13 or 7 cm root from the root collar. Alternatively,coarse roots (>2 mm in diameter) were removed, leaving only the taproot and the fine roots (<2 mm)attached, or fine roots were removed from coarse rootsand taproot. Growth of shoots and roots after onegrowing season was compared to an unpruned controlunder standard nursery conditions. Seedlings rootpruned to 19, 13 or 7 cm were further tested undercompetition achieved by transplanting into a mixtureof clover and grass. Pruning of the root systemsignificantly reduced regrowth in terms of total plantDW in accordance with the severity of pruning, shootDW being more affected than root DW. Removal of coarseroots depressed final root DW whereas removal of fineroots reduced shoot DW and hence root:shoot ratioincreased. The study suggests that fine and coarseroots have different roles in root:shoot allocation.The competition test increased root:shoot ratioindicating that competition induced seedlings toallocate more of their resources into growth of theroot system.     

Amounts of carbon mineralised and leached as DOC during decomposition of Norway spruce needles and fine roots

Changes in climate or forest management practices leading to increased litter production will most likely cause increased leaching rates of dissolved organic carbon (DOC) from the O horizon. The rhizosphere is often assumed to have a large carbon flux associated with root turnover and exudation. However, little has been done to quantify the amount of DOC originating from root litter. We studied decomposition of fine root and needle litter of Norway spruce (Picea abies) through a combined incubation and leaching experiment in the laboratory using five different litter types: fresh needle litter, aged needles from the litter layer, fresh and dead roots from mineral soil samples, and seven-year-old roots from a previous litterbag study. After respiration measurements, the samples were percolated with artificial throughfall water and DOC and UV absorbance were measured in the leachate. Mineralisation of dissolved organic matter in the leachate and sorption of DOC to ferrihydrite were determined as a measure of DOC ability to be stabilised by iron (hydr)oxide surfaces.The mineralisation rate and DOC production rate of root samples were always lower than that of needle samples. However, root and needle derived dissolved organic matter (DOM) were similar in terms of aromaticity, as indicated by their specific UV absorbance, and ability to be sorbed by ferrihydrite. For seven-year-old roots, a significantly higher fraction of carbon was lost as DOC (30%) than for younger roots (20%). Furthermore, DOM from old roots bound more strongly to ferrihydrite and is mineralised at a lower rate than DOC from younger roots, suggesting that roots at late stages of decomposition, although a small fraction of total litter, significantly contribute to carbon build-up in mineral soils. The slower decomposition rate of roots compared with needles must be taken into account when modelling litter decomposition.     

从黄芩毛状根中提取、纯化黄芩苷的研究

本试验以黄芩苷质量浓度为指标,考察了影响大孔吸附树脂对黄芩毛状根中黄芩苷富集效果的相关因素,并利用硅胶柱层析对其进行纯化。结果显示,LX-68型大孔吸附树脂的富集效果最好,较优工艺条件为:上样浓度为8.69 g/L,吸附流速为6 BV/h,上样体积为16 BV,树脂床径高比为1:14;洗脱剂为70%乙醇,洗脱流速为6 BV/h,用量为10 BV;硅胶柱层析洗脱剂条件为氯仿:乙酸乙酯:甲醇:甲酸(7:3:1.5:0.5)。在所确定的工艺条件下,该树脂可有效地提取黄芩毛状根中的黄芩苷,黄芩苷吸附量达131.2 mg/g,黄芩苷的回收率超过69.1%,经纯化后黄芩苷纯度为98.45%。     

基于支持向量机理论的植物根系图像边缘检测方法

由于传统边缘检测方法中存在噪声大、粗糙边缘和不准确边缘等缺点,因此在植物根系的研究中,采用传统的图像边缘检测方法检测出来的边缘信息都无法达到令人满意的效果.为此,基于支持向量机方法给出了一种改善的边缘检测算法.同时,提出了边缘检测算法流程,然后使用支持向量机分类方法对图像进行边缘检测;用所得到的边缘检测算法与Canny算法和Prewitt算法的性能进行了比较.仿真结果表明,给出的算法与Canny算法和Prewitt算相比,不仅边缘检测性能得到提高,而且可以一定程度地克服噪声干扰.     

枣棉间作系统枣树根系空间分布特征研究

[目的]对环塔里木盆地中心地带——阿克苏市的枣-棉花间作系统枣树吸收根根系空间分布特征进行研究,以期为枣-棉花间作系统枣树根系吸水运移模型的建立、枣树保护带宽度的确定及田间灌水、施肥技术的改进提供基础资料.[方法]采用根系分层挖掘法,挑拣每一个土层样方内的活根,清洁、阴干、分类、扫描,并用专业软件对根系长度等指标进行测定分析.[结果](1)垂直方向上,枣树吸收根根量主要集中在0～ 120 cm土层内,约占总量87.09;;(2)水平方向上,主要分布在距枣树树干0～150 cm范围内,占总量的83.67;;(3)根据棉花根系的分布,得到枣树与棉花根系分布密集区在距枣树100～150 cm、土层深度0～80cm的范围内.[结论]保护带的设置对于枣树根系的生长、缓解枣树与棉花间水分、养分的竞争有极其重要的作用,而保护带宽度的设定值得探讨,需要根据枣树的年龄、冠幅等进行综合判断.就此试验地的情况来看,在不改变该试验地枣树保护带宽度的条件下,枣树的施肥区域应在距枣树树干100～ 150 cm.     

基于根肿菌早期侵染量的白菜抗性分析

为探究不同抗性白菜品种对根肿菌侵染量的影响,通过水培法观测4个白菜品种接种根肿菌后其在寄主根内的发展动态,实时荧光定量PCR检测技术对同期根内含菌量进行分析,并结合温室盆栽试验获得的各品种发病数据进行相关性分析。结果表明,早熟长江5号根肿病的发病率和病情指数分别为82.5%和36.2,华良早五号为66.7%和37.5,CR-春美为68.9%和36.0,可归为感病品种,而CR-英雄为52.5%和16.5,可归为抗病品种;水培法接种观测和PCR定量结果显示,根毛侵染率、皮层侵染数以及根内含菌量均表现为CR-英雄最低,显著低于其它3个品种。根毛侵染率、皮层侵染数、根内含菌量、发病率四者间呈显著相关,其R2最大值变幅为0.84~0.98,且根毛侵染率与根内含菌量、发病率与根内含菌量的最大相关系数均出现在第6天,说明根肿菌早期侵染量直接影响白菜根肿病的发生程度。表明根内早期侵染是根肿菌致病的关键环节,通过水培法观测并结合实时荧光定量PCR检测可以评价品种的抗性,可作为根肿病抗性评价的一种方法。