典型喀斯特石漠化生态系统土壤有机碳时空分布格局及其与环境的相关性

选取中国西南3个典型喀斯特石漠化生态系统(贵州毕节鸭池高原山地石漠化区、贵阳红枫湖高原盆地石漠化区和关岭花江高原峡谷石漠化区)为研究区,广泛建立野外样地,开展石漠化生态系统土壤有机碳分布及其与石漠化等级、地形地貌、植被、土壤性质等环境因子的相关性研究。结果表明:1)喀斯特石漠化生态系统土壤有机碳含量较低,毕节鸭池、贵阳红枫湖和关岭花江3个石漠化生态系统平均值分别为23.42、25.78、26.03 g/kg,且3个不同地貌类型石漠化土壤有机碳含量无明显差异。2)土地覆被变化明显影响了土壤有机碳含量,原生森林土壤有机碳平均含量31.32 g/kg,是所有类型中最高的。随着土地覆被由原生森林至石旮旯地退化不断增加的过程,土壤有机碳含量显示先降低后增加的变化趋势。3)土壤有机碳与土壤特性有明显的相关性,与土壤总氮、水解氮、速效钾、总孔隙度、自然含水量、毛管持水量、田间持水量和上层渗透性存在极显著地正相关,与总磷、下层渗透性存在显著地正相关,与容重存在极显著地负相关。4)植物多样性的丰富度指数(R)和多样性指数(H)与土壤有机碳含量有明显的相关性,达到了极显著的水平。5)不同石漠化等级土壤有机碳含量有显著差异,随着石漠化干扰程度的递增,土壤有机碳含量显示了先减小后增加的趋势。研究结果对中国西南喀斯特森林生态保护、石漠化生态系统恢复重建以及应对全球气候变化碳循环的减源增汇具有重要的理论意义和实践指导价值。     

中国南方喀斯特石漠化演替过程中土壤有机碳的响应及其影响因素分析

以中国南方典型喀斯特石漠化生态系统土壤为研究对象, 运用野外定点取样和实验室分析检测方法, 研究喀斯特石漠化生态系统土壤有机碳分布特征及其影响因素; 运用空间代替时间方法, 探讨石漠化演替过程中土壤有机碳的响应及其机制, 旨在为中国南方喀斯特森林生态保护和石漠化生态系统恢复重建提供理论支撑。结果表明: 1)喀斯特石漠化生态系统土壤有机碳含量较低, 三个研究区平均值分别为23.42、25.78 和26.03 g·kg–1; 2)不同等级石漠化环境土壤有机碳含量存在显著差异, 但土壤有机碳含量并不是随着石漠化程度增加而一直降低, 而是一个先降低后增加的趋势; 3)土壤有机碳与土壤特性有明显的相关性, 与土壤总氮、水解氮、速效钾、总孔隙度、自然含水量、毛管持水量、田间持水量和上层渗透性存在极显著地正相关, 与总磷、下层渗透性存在显著地正相关, 与容重存在极显著地负相关,而与pH、总钾、有效磷、土壤呼吸、毛管孔隙度、非毛管孔隙度无明显相关性; 4)土地覆被变化明显影响石漠化生态系统土壤有机碳含量, 而地貌的影响不明显。研究结果不仅对中国南方喀斯特森林生态保护和石漠化生态系统恢复重建具有重要的意义, 而且对应对全球变化碳循环的减源增汇同样具有重要的科学价值。     

西南喀斯特石漠化生态系统土壤有机碳分布特征及其影响因素

喀斯特石漠化已成为制约我国西南地区社会经济可持续发展最严重的生态地质环境问题,其恢复重建已成为我国社会经济建设中一项重要内容。土壤有机碳作为土壤质量评价的重要指标,可以综合反映土地生产力、环境健康功能,另一方面土壤有机碳也间接影响了陆地生物碳库,是陆地生态系统碳平衡的主要因子,它的转化和积累变化直接影响全球碳循环动态,已成为生态科学领域研究的热点之一。系统的总结了西南喀斯特石漠化地区不同土地覆被/土地利用、不同等级石漠化环境土壤有机碳的空间和季节分布特征。结合前人研究成果,进一步分析了影响喀斯特石漠化地区土壤有机碳分布的自然(气候、地形与土壤性质、植被等)和人为(土地覆被/土地利用变化、农业管理措施等)各因素,并提出增加喀斯特石漠化地区土壤有机碳含量的对策。研究结果为喀斯特石漠化退化生态系统恢复重建、石漠化地区土壤综合利用、增加碳截存应对全球碳循环减源增汇等提供了重要的科学参考。     

西南喀斯特长期植被修复对土壤有机碳组分的影响

揭示西南喀斯特土壤有机碳分布积累及其组分构成对长期植被修复的响应规律和内在机理,可为喀斯特石漠化科学治理和阐明喀斯特植被修复的土壤碳汇效应提供科学依据。以西南典型喀斯特石漠化植被恢复区实施了28-31年的4种植被修复工程内的7种典型修复措施(人造乔木林:柏木和柚木种植;人造灌木林:花椒和火龙果种植;人造藤林:金银花种植;人工草地:砂仁和皇竹草种植)为研究对象,系统分析了土壤总有机碳、活性有机碳、缓效性有机碳和惰性有机碳分布积累对长期植被修复的响应。结果表明:(1)西南喀斯特长期植被修复显著改变了土壤有机碳及其组分的分布积累。人造乔木和藤本显著提升土壤有机碳及其各组分的分布积累,但人工种草不仅不能提高土壤有机碳的累积,反而在多数情况下降低了土壤总有机碳含量和储量以及土壤有机碳各组分含量。(2)西南喀斯特长期植被修复明显影响着土壤有机碳库组分结构。除人工种草外,植被修复显著提升了土壤有机碳库中缓效性有机碳的占比。人造花椒明显降低了土壤有机碳库中活性有机碳的占比。柏木种植显著增加了土壤有机碳碳库中的惰性有机碳的比例,而火龙果和砂仁种植明显降低了土壤有机碳碳库中的惰性有机碳的比例。(3)土壤总氮、总磷和容重与土壤有机碳及其各组分的分布积累具有极显著正/负相关,是长期植被修复背景下西南喀斯特土壤有机碳及其组分分布积累的主要影响因子。研究结果为西南喀斯特脆弱生态系统科学植被恢复,以及基于植被修复的土壤碳循环调控助力碳中和提供了科学理论依据。     

西南喀斯特典型石漠化生态系统土壤养分生态化学计量特征及其影响因素

喀斯特石漠化生态系统土壤养分元素生态化学计量特征及其对环境变异的生态响应是喀斯特退化森林生态系统恢复重建必需明确的关键科学问题。为探明喀斯特石漠化土壤C、N、P、K养分元素生态化学计量特征,探讨其对环境因子的响应,对西南喀斯特3个典型石漠化调查点(贵州毕节鸭池、清镇红枫湖和关岭-贞丰花江) 90个样方土壤及环境因子调查取样,研究了其土壤有机碳(C)、全氮(N)、全磷(P)及全钾(K)的化学计量特征及其影响因素。结果表明:西南喀斯特典型石漠化生态系统土壤C、N、P、K平均含量分别为45.61、2.54、0.79 g/kg和3.33 g/kg,计量比C∶N、C∶P、C∶K、N∶P、N∶K、P∶K平均值分别为19.56、65.07、23.65、3.45、1.32和0.39。4个土壤养分元素中,K元素表现明显高于其他元素的波动性。土壤养分含量及化学计量比在不同调查点、石漠化等级及植被覆盖率环境均有显著差异。无石漠化环境土壤养分C、N、P含量显著大于潜在、轻度、中度和强度石漠化,而强度石漠化环境土壤养分K含量却显著高于其他等级石漠化。土壤养分含量之间及其与化学计量比之间多具有显著的非线性相关关系。降水、温度、岩石裸露率和土地覆被是西南喀斯特石漠化生态系统土壤养分及其化学计量比最主要的影响因素。研究结果对丰富土壤生态化学计量学科学理论和我国西南喀斯特石漠化退化植被科学恢复具有重要意义。     

中国南方喀斯特石漠化演替过程中土壤理化性质的响应

以中国西南典型喀斯特石漠化生态系统土壤为研究对象,运用野外定点取样和实验室分析检测方法,研究不同等级石漠化环境土壤理化性质特征;运用空间代替时间方法,探讨石漠化演替过程中土壤理化性质的响应及其机制,旨在为中国西南喀斯特森林生态保护和石漠化生态系统恢复重建提供理论支撑。结果表明:1)不同等级石漠化环境土壤理化性质存在显著差异,土壤容重、毛管孔隙度、总孔隙度、田间含水量、毛管含水量、pH值、有机质、水解氮、有效磷和全钾均在不同等级石漠化环境间具有显著差异。但这些指标并不是随着石漠化程度增加而一直退化,而是一个先退化后改善的趋势;2)土壤有机质、氮素、毛管持水量、容重和孔隙度与土壤其它绝大多数理化因子具有明显的相关性,是土壤理化性质的关键因子,在改善土壤理化性质和促进养分循环方面起着关键作用;3)主成分分析也表明,土壤有机质、氮素、钾素、容重、持水状况、孔隙度等是基于土壤理化性质评价石漠化程度的关键指标。作者提出了强度石漠化环境裸岩对土壤养分的聚集效应学说和喀斯特石漠化演替过程中土壤理化性质的响应及其机制。研究结果对中国西南喀斯特森林生态保护和石漠化生态系统恢复重建具有重要的理论意义和实践指导价值。     

西南喀斯特石漠化环境下土地利用变化对土壤有机碳及其组分的影响

土壤有机碳是喀斯特生态系统中碳转移的动力学媒介和碳流通的主要途径,土壤有机碳及其组分是土壤碳循环的重要组成部分,然而目前缺乏关于喀斯特地区土壤有机碳及其组分的研究。本研究以西南典型喀斯特石漠化区——贵州关岭花江6种典型土地利用方式下(花椒林、火龙果林、花椒火龙果混交林、圆柏林、圆柏女贞混交林和坡耕地)的土壤为研究对象,分析土地利用方式变化对土壤有机碳含量(SOC)和储量(SOCS)、土壤水溶性有机碳(WSOC)、易氧化有机碳(EOC)、颗粒有机碳(POC)、轻组有机碳(LFOC)及重组有机碳(HFOC)含量及其分配比例的影响。结果表明: 6种土地利用方式下SOC和SOCS均表现为圆柏林、圆柏女贞混交林和花椒林显著大于火龙果林、花椒火龙果混交林和坡耕地;在0～20 cm土层,土壤SOCS平均值为花椒林＞圆柏林＞圆柏女贞混交林＞坡耕地＞花椒火龙果混交林＞火龙果林。土壤WSOC、EOC、POC、LFOC和HFOC含量均表现为圆柏林、圆柏女贞混交林和花椒林大于其他3种土地类型。土壤SOC与其各组分(WSOC、EOC、POC、LFOC和HFOC)均呈显著正相关,且各组分两两之间也呈显著正相关。花椒林可作为中国西南喀斯特石漠化生态恢复和山地农业发展优先考虑的经济物种。土壤WSOC、EOC、POC、LFOC和HFOC可作为反映土壤有机碳库的有效指标。     

喀斯特小流域土壤有机碳空间异质性及储量估算方法

为了准确估算土壤有机碳储量,利用网格法采集2755个土壤剖面,共计23536个土壤样品,研究了喀斯特小流域土壤有机碳含量分布特征,并以"土壤类型法"为基准,对土壤分布面积、石砾含量、岩石裸露率、土层厚度等指标进行修正,合理的优化了土壤有机碳储量计算公式,探索出一种专属于喀斯特地区土壤有机碳储量的估算方法,结果表明:不同土层深度和土壤类型下土壤有机碳含量存在明显差异,土壤有机碳含量随着土层深度的增加而逐渐减小,不同土属的有机碳含量减小的幅度有所差异,不同坡位和坡向的有机碳含量大小为:阳坡阴坡,坡中上部坡顶坡中坡中下坡坡底,不同土地利用方式下土壤有机碳含量大小顺序为:林地灌草地旱地水田;土壤有机碳含量与坡度、海拔、岩石裸露率均呈极显著正相关关系,与土层厚度、土壤容重呈显著负相关;喀斯特地区土壤异质性较大,不同修正指标对土壤有机碳储量估算的影响程度为:土壤厚度岩石裸露率石砾含量土壤有机碳含量土壤容重;通过修正后的计算公式估算出普定后寨河小流域表层20 cm土壤有机碳密度区间为3.53—5.44 kg/m~2,平均值为:1.24 kg/m~2,100 cm土壤有机碳密度区间为4.44—14.50 kg/m~2,平均值为12.12 kg/m~2,土壤有机碳储量为5.39×10~5t。     

贵州喀斯特石漠化地区植物多样性与土壤理化性质

以贵州典型喀斯特石漠化生态系统环境为研究对象,运用野外取样调查和实验室检测分析方法,研究不同等级石漠化环境植物多样性和土壤理化性质特征及其相关性;运用空间替代时间方法,探讨石漠化演替过程中植物多样性和土壤理化性质的响应,旨在为贵州乃至整个中国西南喀斯特森林生态保护和石漠化生态系统恢复重建提供理论支撑。结果表明:1)石漠化环境植物群路组成简单,物种丰富度也很低,且随着石漠化程度增加,植被物种组成呈递减趋势;不同等级石漠化环境植物多样性具有显著差异,均匀度指数变化与石漠化等级演替明显耦合,显示了随石漠化程度增加而减小的变化趋势。2)不同等级石漠化环境土壤理化性质存在显著差异,随着石漠化程度增加,土壤理化性质显示了先退化后改善的响应过程。土壤有机质、氮素、毛管持水量、容重和孔隙度与植物多样性具有明显的相关性,在改善土壤理化性质和促进植物多样性恢复方面起着关键作用。3)主成分分析表明,土壤有机质、氮素、钾素、持水状况、孔隙度和植物多样性均匀度指数等是基于土壤理化性质和植物多样性评价石漠化程度的关键指标。基于上述结果,进一步阐述了石漠化演替过程中植物多样性和土壤理化性质的变化规律和响应机制。研究结果对我国西南喀斯特森林生态保护和石漠化生态系统恢复重建具有一定的理论意义和实践指导价值。     

喀斯特不同土地利用类型裂隙土壤有机碳及磷素赋存特征

以喀斯特石漠化区不同土地利用类型裂隙土壤为研究对象，运用野外调查和实验室内分析相结合方法，探索其有机碳和磷素含量变化及其赋存特征，以期为喀斯特地区开展石漠化治理和植被恢复提供理论依据。结果表明：4种土地利用类型裂隙土层土壤有机碳赋存含量变化范围为16.067-39.436 g/kg，总体呈现出随土层深度增加而降低的变化趋势；土壤全磷、有效磷赋存含量变化范围分别为0.093-0.274 g/kg、3.836-8.025 mg/kg，整体上在裂隙表层显著高于其他土层，具有上层高下层低的特点；同时，土壤有机碳和磷素总体上属于中度变异。乔木林地和灌丛地的C/P总体上表现出随土层的加深而减小的趋势，而草地和撂荒地先减小后增加，土壤C/P在各土地利用类型裂隙土层变化范围为86.499-268.343，磷的有效性较低；随土层深度的增加，各土地利用类型裂隙土壤有机碳、全磷和有效磷含量逐渐在减少，有机碳对土壤碳磷比、有效磷含量变化有一定影响。     

叶绿体转化三角褐指藻表达外源蛋白

为建立三角褐指藻(Phaeodacty lum tricornutum)叶绿体表达体系,从其叶绿体基因组中克隆了rnS-trnI、trnAml序列作为遗传转化同源重组序列,并以氯霉素抗性基因(CAT)表达盒作为筛选标记,以及绿色荧光蛋白基因(GFP)表达盒作为报告基因.以TA克隆载体pMD19-T为基础,将CAT表达盒...     

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha-T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcp A promoter. pPha-T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat-1 genes confer resistance to the antibiotic nourseothricin, and npt II confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uid A and gfp were also introduced into P. tricornutum using pPha-T1. Gus expression in some transformants reached 15 μg·μg⁻¹ of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum , enhancing the potential of this organism for exploring basic biological questions and industrial applications.     

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.)

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.     

玉米叶绿体表达载体的构建及AsRED基因原核表达

从玉米幼嫩叶片中提取玉米叶绿体基因DNA,通过PCR克隆出叶绿体同源重组片段trnA和trnI、叶绿体特异性启动子Prrn以及终止子psbA.构建玉米叶绿体表达载体pBAIRTARED,含有一个人工操纵子,其中,筛选标记基因aadA和红色荧光蛋白报告基因AsRED处于Prrn启动子和psbA终止子控制.将构建的载体转化大肠杆菌BL21（DE3）,观测到重组细胞呈现红色,表明构建的载体可以用于玉米叶绿体转化以及表达报告基因.     

三角褐指藻(Phaeodactylum tricornutum)通用转化载体的构建

三角褐指藻(Phaeodactylum tricornutum)是开展微藻生物柴油研究的理想材料。克隆了内源fcp基因簇的多个调控序列(启动子、终止子),构建了包括fcpB启动子-bar基因-fcpA终止子、以及fcpA启动子-多克隆位点(MCS)-fcpA终止子两个表达盒的通用转化载体pfcpA-MCS/fcpB-Bar,其特征是以bar基因作为选择标记,MCS区方便插入一至多个目的基因。新载体可用于三角褐指藻的重组蛋白表达、或油脂代谢相关基因的功能验证和代谢调控研究。     

烟草质体多顺反子定点整合表达载体的构建和转化

构建了烟草质体多顺反子定点整合表达载体pLM4(-psaA-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3'-psbC-).用基因枪将该载体轰击烟草叶片5次,用添加了壮观霉素的选择分化培养基筛选,获得质体转基因烟草6株.用PCR、激光扫描、Western blot和RFLP等方法检测都证实多顺反子表达盒中的3个基因甘露聚糖酶基因(man)、绿荧光蛋白基因(gfp)、氨基糖苷3'-腺苷酰基转移酶基因(aadA)已整合到烟草质体基因组中,且均得到表达.     

A retained selection cassette increases reporter gene expression without affecting tissue distribution in SPI3 knockout/GFP knock-in mice

The human serpin, proteinase inhibitor 6 (PI-6/SERPINB6), is a protease inhibitor expressed in many tissues. It inhibits a large number of proteases, including cathepsin G in granulocytes and monocytes. To determine the temporal and spatial distribution of PI-6, mice were generated in which exon 2 of the PI-6 ortholog SPI3 (Serpinb6) was replaced with a green fluorescent protein (GFP) reporter gene. This placed GFP under the control of the regulatory elements and initiation codon of the SPI3 gene. The neomycin selection cassette was flanked by loxP sites to allow excision from the targeted allele. GFP expression in heterozygous and SPI3-deficient mice accurately reflected the tissue distribution of SPI3 in all organs tested and allowed precise comparisons of expression levels. Interestingly, retention of the neomycin cassette in targeted mice resulted in 2-10-fold increases of GFP in leukocytes, but without affecting tissue-specific expression patterns. This is the first example of selection cassette retention specifically increasing reporter gene expression in targeted mice and reinforces the view that selection cassettes must be removed to avoid confounding effects on reporter gene expression patterns.     

Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression

Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.     

Beta-lactamase reporter system for selecting high-producing yeast clones

In modern production of protein biopharmaceuticals, a good screening and selection method of high-producing clones can dramatically influence the whole production process and lead to lower production costs. We have created a rapid, simple, and inexpensive method for selecting high-producing clones in the yeast Pichia pastoris that is based on the beta-lactamase reporter system. By integrating the reporter gene and the gene of interest into the same genome locus, it was possible to use beta-lactamase activity as a measure of the expression level of the protein of interest. A novel expression vector with two independent expression cassettes was designed and tested using green fluorescent protein (GFP) as a model. The first cassette contained the GFP gene under the control of a strong, inducible AOX1 promoter, while the second cassette consisted of the beta-lactamase reporter gene under the control of a weak constitutive YPT1 promotor. High-producing GFP clones were selected directly on the plates based on the color change after hydrolysis of the beta-lactamase substrate added to the medium. beta-lactamase activity was found to positively correlate with GFP fluorescence. The reporter system described is widely applicable-it can be easily applied to other, also pharmaceutically relevant proteins and to other yeast expression systems, such as Saccharomyces cerevisiae and Hansenula polymorpha.