橄榄(Canarium album)种子形态结构与数量性状分析

以福建主栽的橄榄(Canarium album (Lour.) Rauesch.)品种‘长营’种子为材料,观察其成熟种子及不同发育时期种子的形态结构,对数量性状进行测量与统计分析,并运用概率分级建立形态指标的正态分布分级标准。结果显示,橄榄种子表型性状和形态结构变异较大,各数量性状变异系数为6.77%~19.04%,具有丰富的遗传多样性;橄榄种子心室类型有12种,其中三心室占比最高(97.80%);胚数量0~4个不等;经K-S检验,各数量性状符合正态分布,依据各性状可划分为5级分级标准。本研究为橄榄种质的系统学研究提供了种子形态方面的新内容,可为橄榄资源描述的标准化及遗传育种研究提供一定的参考。

Tannins from Canarium album with potent antioxidant activity

The contents of total phenolics and extractable condensed tannins in the leaves, twigs and stem bark of Canarium album were determined. The structural heterogeneity of condensed tannins from stem bark was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS） and nuclear magnetic resonance （NMR） analyses The results show the predominance of signals representative of procyanidins and prodelphinidins. In addition, epicatechin and epigallocatechin polymers with galloylated procyanidin or prodelphinidin were also observed. The tannins were screened for their potential antioxidant activities using l,l-diphenyl-2-picrylhydrazyl （DPPH） and ferric reducing/antioxidant power （FRAP） model systems. Tannins extracted from leaves, twigs and stem bark all showed a very good DPPH radical scavenging activity and ferric reducing power.

Effect of Osmotic Stress with PEG6000 on Osmotic Adjustment Solute in Seedling Leaves of Chenopodium album L.

[ Objective] The purpose was to discuss drought resistance mechanism of Chenopodium album L. and supply theoretical basis and practical guidance for artificial cultivation and popularization of C. album. [ Method] C. album seedlings grown to 6th leaf stage were conducted osmotic stress treatment with PEG6000 osmotic whose concentration was set up as 0, 5%, 10% and 20% and the various physiological indices of the 3rd -5th function leaves in upper plant were determined after being treated for 0, 1,3, 5, 7 and 9 d. [ Result] Under osmotic stress with 5% PGE, the relative water content （RWC） of C. album reduced less. Under osmotic stress with 10%, the RWC in seedling leaves of C. album decreased to 62% on the fifth day and the leaves began to wither. Under osmotic stress with 20%, the RWC in seedling leaves of C. album decreased to 61.9% on the third day and the leaves appeared withering, and the RWC decreased to 48.6% on the 7th day and the leaves were dry and yellow. Proline contents in seedling leaves of C. album under osmotic stress with 5%, 10% and 20% PEG were 7.64, 10.9 and 29.4 times of CK on the 7th day. [ Conclusion] C. album hed some adaptability to moderate osmotic stress, but the PEG osmotic stress with high concentration and long time might lead to severe damage on C. album.

山东省长岛县南部四岛藜(Chenopodium album L.)天然种群遗传多样性的ISSR分析

采用ISSR分子标记对分布于山东省长岛县南部的北长山、南长山、大黑山和小黑山4个岛屿上的藜天然种群共81个个体进行了遗传多样性及遗传结构的研究。13个引物共检测到157个可重复的位点。遗传多样性研究结果表明:种内的多态位点比率为66.24%,具有较高的遗传多样性(Shannon(I)指数在物种水平上为0.332 0);种群间有一定的遗传分化,根据Gst值,种群间的遗传多样性占总群体的9.27%。遗传距离分析表明,XHS种群和NCS种群的遗传一致度最高,与地理距离无相关性。

大连地区岛屿与大陆藜(Chenopodium album L.)种群遗传多样性的ISSR分析

采用ISSR分子标记对分布于大连市周边的4个藜的天然种群100个个体进行了遗传多样性及遗传结构的研究。13个引物共检测了157个位点,多态位点比率为67.52%,Shannon信息指数在物种水平上为0.332 9。根据GST值,藜遗传变异有40.03%发生在种群间。遗传距离分析表明,DT种群与MLN种群遗传一致度最高,遗传距离与地理距离之间没有相关性。

Comparison of the Evolvement Structure of Chenopodium album L.under the Stress of Different Saline Environments

[Objective] The aim of this study was to reveal the evolvement structures,especially the crystal characteristics of Chenopodium album L.under saline stress,so as to providing the first-hand data for utilizing biological techniques to control saline environment.[Method] Employing high definition display method of plant crystal structure and paraffin-section method,we performed a comparative study on the evolvement structures of C.album growing in high salinity areas in the coast of Egyptian Red Sea and common salinity areas in the grasslands in Changling County of Jilin Province.[Result] The regionally distributed crystal and the developed assimilating tissue of C.album are the key structural characteristics to antagonize the saline stress during the evolving process.Stem cortex of C.album growing in both the high salinity areas in coast of Egyptian Red Sea and common salinity areas in the grasslands in Changling County of Jilin Province has similar discontinuous crystal rings.Assimilating tissue in C.album growing in high salinity areas is highly developed than that in common salinity environment.Comparative analysis indicates that the developed stratum corneum and marrow is also the key structural characteristics to antagonize the saline stress.[Conclusion] Our results provide a valuable approach to study the salt-tolerance mechanism of plant using structural botanical techniques,i.e.,crystal may become the identification characteristics of salt tolerant plant.

基于SLAF-Seq技术的橄榄种质资源SNP标记开发与遗传关系鉴定

【目的】开发橄榄SNP标记并分析其种质资源遗传多样性,为橄榄种质资源保护和利用提供依据。【方法】基于SLAF-Seq技术进行橄榄SNP标记开发,同时采用系统进化分析、群体聚类分析和主成分分析等研究了橄榄种质资源遗传结构和遗传多样性。【结果】基于SLAF-Seq技术共挖掘到506 701个SLAF标签,其中多态性SLAF标签27 108个,开发获得361 386个群体SNP标记;基于SNP标记,利用系统进化树和群体聚类分析可分别将橄榄种质资源分为3和6个类群,整体Nei多样性指数和Shanon-Wiener指数分别为0.321和0.472。两种分类方法分析结果均发现,不同地区之间的橄榄种质资源并未严格按照地域分布归类。【结论】橄榄种质资源遗传多样性相对丰富,且不同地域间存在种质资源交流,而采用SNP标记可有效鉴定橄榄种质资源。

Effects of different culture media on the growth of Indian sandalwood(Santalum album L.) seedlings in Zhanjiang,Guangdong,southern China

We studied the effects of different culture media on the growth of India sandalwood （Santalum album L.） seedlings in Zhanjiang, Guangdong Province in southern China. Five different growth substrates, lateritic subsoil, burnt soil, agricultural soil, peaty soil and coconut dust, were used as the basic culture materials and seven different treatments of composition were used as potting media. Kuhnia rosmarinifolia Vent. was used as a primary host plant for all treatments. Statistically significant differences were found between treatments in respects of survival rate （p 〈 0.001）, height （p 〈 0.001）, ground diameter （p 〈 0.001） and biomass （p = 0.002）, as well as for quality index （p 〈 0.001） of S. album seedlings after 6-month growth in containers with different culture media. Among all treatments, the treatment combining burnt soil, peat and coconut dust in a weight ratio of 1：1：1 plus 2% calcium super-phosphate as basal manure achieved the best performance for most of the seedling growth parameters, including survival rate （98%）, height （35.81 cm）, ground diameter （0.56 cm）, biomass （4.46 g） and quality index （0.65）, followed by the treatment using only burnt soil plus 2% calcium super-phosphate as the culture medium （survival rate 86%, height 29.23 cm, ground diameter 0.48 cm, biomass 3.36 g and quality index 0.52）, while the treatment using only lateritic subsoil plus the basal manure as the medium obtained the poorest results in survival rate （38%）, height （12.04 cm）, ground diameter （0.19 cm）, biomass （0.26 g） and quality index （0.043）. Increasing the proportion of lateritic subsoil in the medium when mixed with peat and coconut dust did not show statistically significant differences in survival, height, ground diameter, biomass nor in quality index. In consideration of cost, using burnt soil plus 2% calcium super-phosphate as basal manure may be the optimum culture medium for large-scale production of Indian sandal- wood seedlings in Guangdong, southern China.

Study on the Microscopic Identification of Chenopodium album L.

[Objectives]This study was conducted to identify the microscopic characteristics of Chenopodium album L.[Methods]The microscopic identification method was adopted.[Results]The xylem vessels and fiber bundles of the roots are arranged into 3-4 intermittent ring belts in a concave-convex pattern,alternately with the parenchymal cell ring belts;and the xylem rays vary in width.The cross section of the stems is polygonal;there are parenchyma cells at the corners of the cortex;there are many vascular bundles of varying sizes;and calcium oxalate clusters are common in the medulla and cortex.There are 2 to 4 vascular bundles in the main leaf vein,peltate;and fiber bundles exist below the phloem.The powder is characterized by numerous clusters of calcium oxalate,which are uniform in size,sharp at edges and corners.[Conclusions]This study can provide reference for the identification and quality standard of the crude drug.

The Effect of Air Pollution on Chenopodium album L. Pollen Structure

Air pollution is harm and discomfort to human or other living organisms, it also causes damage to the environment. The aim of this project was to study the effect of air pollutions on structure and pollen grains development in Chenopodium album. Anthers of Chenopodium album L. were collected at different stages of development from control （less polluted） and polluted areas （mainly SO2, NO2, CO and APM）. Structure and development of pollen grains were studied and compared. The effects of pollution on pollen structure was investigated under Light and Scanning electron microscopy and the results showed that when pollen grains were exposed to polluted air they became abnormality in form and covered with large amounts of pollutants compared to control ones. Pollen abnormalities were seen as irregularity, shrinkage, thinning and breakage of the exine. Cellular material release was induced also. The data presented suggest that prolonged exposures of plants to air pollution may cause different biological effects at the cellular tissue and organ levels.

Folin-Ciocalteu比色法测定橄榄中多酚含量的研究

采用Folin-Ciocalteu比色法研究了橄榄多酚的定量测定方法.结果表明:橄榄多酚提取液在加入Folin-Ciocalteu试剂1.5 mL、10%Na2CO3 6 mL、反应时间2 h、温度30 ℃的条件下进行显色,并于765 nm处测定其吸光值,多酚质量浓度在7～70 mg/L范围内与吸光值有良好的线性关系,根据此线性回归方程进行橄榄多酚的定量测定,该方法简单方便、稳定性好、准确度高.

橄榄基因组AFLP扩增体系的优化

为建立适于橄榄研究用的AFLP技术体系,以10个橄榄品种为材料,对其基因组DNA从酶切、连接、预扩增和选择性扩增等方面进行条件优化。研究结果表明,DNA模板用量为500 ng,酶切体系中限制性内切酶EcoRⅠ和MseⅠ各加入3 U,37℃水浴4 h;连接体系中T4DNA连接酶加入1.5 U,16℃连接6 h;并利用优化后的预扩增体系和选择性扩增体系筛选出6对适合于橄榄AFLP分析的引物组合:E/AAC-M/CAT、E/AAC-M/CTT、E/ACA-M/CAA、E/AGG-M/CAA、E/ACC-M/CAA、E/ACT-M/CAT。采用该体系得到的电泳条带清晰可靠、多态性好,可用于对橄榄遗传多样性的研究。

橄榄果实转录组SSR和SNP/InDel位点特征

简单重复序列标记(simple sequence repeats,SSR)和单核苷酸多态性标记(single nucleotide polymorphism,SNP)具有高灵敏度和高特异性特征,挖掘不同类型橄榄果实性状相关分子标记可为其分子辅助育种提供参考。本研究以充分成熟的长营和惠圆橄榄果实为材料,经总RNA提取和cDNA文库构建后采用Illumina Novaseq平台进行转录组测序,并采用MISA 1.0和GATK3软件分析转录组的SSR、SNP和插入缺失标记(Indel)位点特征。结果在橄榄果实转录组的10124条unigenes上鉴定到13935个SSR位点,发生频率为22.98%,平均每1 kb序列出现0.25个SSR位点,平均长度为14.34 bp;其中,单碱基重复基元类型的SSR位点数量最多(占比66.80%),长度为10~64 bp,平均长度为12.85 bp,重复基元的重复次数集中在9~12次,频率最高的基元为A/T(占比66.67%);六碱基重复基元类型的SSR位点数量最少(占比0.47%),长度为30~54 bp,平均长度31.76 bp,基元重复次数集中在5~8次,频率最高的基元为AGATGG/ATCTCC(占0.04%)。在橄榄果实转录组中共检测到284992个SNP位点,平均每1 kb序列含有5.21个;其中,转换类型的SNP位点数量为166162,包括C/T和A/G两种;颠换类型的SNP位点数量为118830,包括A/T、A/C、T/G和C/G四种。此外,橄榄果实转录组中共挖掘到18548个InDel位点,平均每1 kb序列存在2.95个,其中含有1个InDel位点的unigenes数量最多(7853条);通过比对预测发现,含有最多InDel位点的unigene可能是胼胝质合成酶基因。研究结果表明,通过转录组测序可有效开发橄榄SSR和SNP/InDel标记,不同性状橄榄果实中存在丰富多样的SSR位点且广泛分布SNP/InDel位点。该研究结果为橄榄果实性状鉴定标记的开发提供数据基础。

NaCl胁迫对橄榄种子萌发的影响

用不同浓度的Na Cl溶液对橄榄种子进行胁迫萌发实验,观察其发芽率和发芽后种苗的生长情况,并测定淀粉酶活性。结果表明,供试4个品种橄榄种子对盐胁迫敏感程度。当Na Cl溶液浓度≤0.8%时,4个品种橄榄种子发芽率均在90%以上;但随着Na Cl溶液浓度的增加,种子萌发吸水受到抑制,淀粉酶活性降低,发芽率、发芽指数和活力指数均呈逐渐下降趋势。但4个品种表现不一致。当Na Cl溶液浓度≤0.4%时,4个品种橄榄种子发出的幼苗基本没有影响;但当Na Cl溶液浓度增加时,4个品种橄榄胚根生长停止的临界指数:惠圆Na Cl盐度指数为1.0%;茶橄榄Na Cl盐度指数为1.2%;三方Na Cl盐度指数为1.4%;油橄榄Na Cl盐度指数为1.6%。4个品种盐胁迫敏感度:檀香﹥茶橄榄﹥三方﹥油橄榄。

NaCl和Na2CO3处理对土荆芥和藜的生长及抗氧化酶活性的比较研究

采用5个浓度水平的NaCl和Na2CO3溶液,分别模拟盐胁迫和碱胁迫对入侵植物土荆芥及其近缘植物藜进行浇灌处理,比较二者在2种盐胁迫下的生长以及抗氧化酶活性的变化。结果表明:土荆芥根重比和根冠比在盐胁迫下显著增加,在碱胁迫条件下则极显著降低;在各浓度梯度下,盐胁迫诱导的SOD和CAT活性均高于碱胁迫,而POD的活性仅在高浓度盐胁迫时才高于碱胁迫。表明Na2CO3胁迫对土荆芥造成的危害更大,土荆芥耐受NaCl胁迫能力强于Na2CO3胁迫。另外,近缘植物藜的根重比、根冠比在盐、碱胁迫时均呈下降趋势,抗氧化酶活性的变化也低于土荆芥,说明土荆芥比藜更能耐受盐、碱胁迫。

NaCl胁迫对藜幼苗叶片渗透调节物质和保护酶的影响

[目的]研究不同浓度NaCl胁迫对藜叶片中渗透调节物质和保护酶活性的影响,以探讨藜的耐盐机理,为藜的人工栽培及推广提供理论依据。[方法]待藜幼苗长到4叶期时,用0%、0.6%、1.2%、1.8%NaCl溶液进行根部胁迫处理,分别于处理0、1、3、5、7 d后从藜幼苗上部取第3～5片功能叶片进行各项生理指标的测定。[结果]在0.6%NaCl胁迫下,渗透调节物质(可溶糖、脯氨酸和可溶性蛋白质)含量和保护酶(SOD、POD和CAT)活性都是缓慢增加的,丙二醛含量增加不大,膜脂过氧化水平低。在1.2%、1.8%NaCl胁迫下,渗透调节物质(可溶性糖、脯氨酸和可溶性蛋白质)含量随胁迫时间的延长而增加,SOD、POD、CAT活性随胁迫时间的延长先升高后降低,丙二醛含量显著提高,膜脂过氧化水平增大。[结论]藜可以通过渗透调节物质增加和保护酶活性增大来对适度的盐胁迫有一定的适应性,但高浓度、长时间的盐胁迫会对藜造成严重损伤。

檀香叶中的γ-氨基丁酸的HPLC测定研究

建立了檀香叶中γ-氨基丁酸(GABA)含量的高效液相色谱检测方法。以乙醚为溶剂,采用索氏提取除去色素,水提醇沉去除糖类和蛋白质的净化方法,以丹酰氯为衍生剂,高效液相色谱法分离,荧光检测器测定,外标法定量。峰面积与浓度之间线性关系良好,回归方程为y=475 211 x+69 037,R2=0.999 8,加标回收率为112%～120%,检测限为7μg/g。该方法操作步骤简单,灵敏度高,用于檀香叶中GABA的检测,方法简便、准确、高效。

檀香醇提物的化学成分研究

目的对檀香Santalum album L.中的化学成分进行研究。方法采用ODS凝胶柱层析方法以及HPLC等色谱技术进行分离纯化,结合UV、MS和NMR等波谱数据进行化合物的结构鉴定。结果从檀香乙醇提取物中分离得到9个化合物,分别鉴定为原儿茶酸(1)、原儿茶酸甲酯(2)、香草酸(3)、7-甲基-10-羟基喜树碱(4)、阿魏酸(5)、肉桂酸乙酯(6)、松柏醛(7)、(7R,8S)-3,5'-dimethoxy-4',7-epoxy-8,3'-neolignane-5,9,9'-triol(8)、(3S,4S)-3-methyl-4-{[6-O-(3,4,5-trihydroxybenzoyl)-β-D-glucopyranosyl]oxy}octanoic acid(9)。结论化合物2、4、8、9为首次从檀香属植物中分离得到。

聚类分析和主成分分析法研究檀香HPLC特征图谱

目的:建立檀香药材甲醇提取部位的HPLC特征图谱,为合理地评价檀香药材质量提供方法与依据。方法:采用Waters Symmetry C18色谱柱,以乙腈-0.2%磷酸溶液为流动相,梯度洗脱;检测波长为205 nm,柱温为30℃,流速为1.0 mL/min,建立13批檀香样品的HPLC特征图谱,并进行相似度评价、聚类分析和主成分分析;同时测定样品中挥发油含量。结果:建立了13批檀香样品的特征图谱,共标记8个共有峰,相似度为0.369~0.993,差异较大;聚类分析和主成分分析结果一致;13批檀香样品挥发油含量为0.58%~5.52%,其中S3、S4号样品挥发油含量偏低,与相似度评价结果一致。结论:本研究建立的檀香药材HPLC特征图谱检测方法简便快速,专属性强,可对不同来源檀香的质量进行评价,为其质量控制提供依据。

橄榄黑斑病病原菌的分离鉴定及防治药剂筛选

为明确引起潮州市橄榄黑斑病的病原菌种类,筛选有效防治药剂,对发病果实采用组织分离法进行了病原菌的分离与纯化,并结合形态学和分子生物学技术对病原菌进行鉴定。鉴定结果表明,引起橄榄黑斑病的病原菌为链格孢菌(Alternaria alternata)。通过菌丝生长速率法测定了14种杀菌剂对病原菌的抑菌效果,结果表明14种杀菌剂对链格孢菌均有抑制作用,其中3%中生菌素可湿性粉剂、80%克菌丹水分散粒剂、8%氟硅唑微乳剂和40%苯甲·嘧菌酯悬浮剂的抑菌效果较好,EC_(50)分别为1.267、1.207、1.100、0.944 mg·L^(-1)。田间防效测定结果表明,40%苯甲·嘧菌酯悬浮剂、8%氟硅唑微乳剂的防治效果显著,防效为68.58%、65.44%;而30%王铜、2%春雷霉素的防效较差,仅为10%左右。综上,推荐将8%氟硅唑微乳剂和40%苯甲·嘧菌酯悬浮剂2种药剂作为防控橄榄黑斑病的首选药剂。本研究可为橄榄黑斑病高效杀菌剂的筛选提供数据参考,并为其防控工作提供科学依据。