Toward molecular trait‐based ecology through integration of biogeochemical,geographical and metagenomic data

Using metagenomic ‘parts lists’ to infer global patterns on microbial ecology remains a significant challenge. To deduce important ecological indicators such as environmental adaptation, molecular trait dispersal, diversity variation and primary production from the gene pool of an ecosystem, we integrated 25 ocean metagenomes with geographical, meteorological and geophysicochemical data. We find that climatic factors (temperature, sunlight) are the major determinants of the biomolecular repertoire of each sample and the main limiting factor on functional trait dispersal (absence of biogeographic provincialism). Molecular functional richness and diversity show a distinct latitudinal gradient peaking at 20°N and correlate with primary production. The latter can also be predicted from the molecular functional composition of an environmental sample. Together, our results show that the functional community composition derived from metagenomes is an important quantitative readout for molecular trait‐based biogeography and ecology.     

转Cry1Ah基因抗虫玉米HGK60对田间节肢动物及杂草多样性的影响

近年来,随着转基因技术快速发展,转基因作物环境释放风险成为需要重点关注的问题,而对生物多样性的影响研究是客观评价其风险的重要手段。本研究以2018年海南春、冬两季转Cry1Ah基因抗虫玉米‘HGK60'及其对照常规玉米‘郑58'为研究对象,重点探讨了其对田间节肢动物、虫害及杂草多样性的影响。 结果表明: 田间调查共记录节肢动物43753头,隶属7目19科69种,转基因抗虫玉米HGK60与对照常规玉米郑58的节肢动物群落组成、结构及各生态指标均无显著差异;与郑58玉米相比,转基因抗虫玉米HGK60对钻蛀类害虫具有明显的抗性;田间杂草隶属8科16种,转基因抗虫玉米HGK60与郑58玉米田间杂草在密度及各生态指标上差异均不显著,且二者在整个生育期的变化趋势基本一致。说明转Cry1Ah基因抗虫玉米HGK60的种植对田间节肢动物及杂草多样性无显著的影响。该结果为转基因作物的环境风险研究提供了一定的数据支持。     

Rootstock‐to‐scion transfer of transgene‐derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus‐resistant transgenic rootstocks developed through siRNA‐mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock‐to‐scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV‐hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV‐hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long‐distance (1.2 m) transfer of PNRSV‐hpRNA‐derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for ‘using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.     

转cry1Ac／cpti基因水稻对土壤酶活性和养分有效性的影响

【背景】转基因作物可能通过根系分泌物和植株残体组成的改变对土壤酶活性和养分转化产生影响,转基因作物对土壤质量的影响是其环境安全性评价的重要方面。【方法】本研究通过田间定位试验分析了连续3a种植2种转crylAc／cpti双价抗虫基因水稻后,土壤酶活性和养分有效性等土壤质量性状的变化。【结果】在水稻各生育期内,除齐穗期转基因稻科丰8号（GM1）田的土壤酸性磷酸酶活性显著（P〈0．05）高于其受体非转基因稻明恢86（CK1）外,转基因稻GM1、GM2（Ⅱ优科丰8号）的土壤酸性磷酸酶、碱性磷酸酶和脲酶活性与对应非转基因稻CK1、CK2（Ⅱ优明恢86）间均无显著差异。同时,在水稻生长过程中,土壤pH、有机质、有效氮、有效磷和速效钾等土壤理化指标在GM1和CK1或GM2和CK2间也均无显著差异。【结论与意义】连续38种植转crylAc／cpti双价抗虫基因水稻并未改变稻田土壤中主要营养元素的有效性及其相关土壤酶活性,即短期内种植转crylAc／cpti双价抗虫基因水稻不会影响土壤酶活性和养分状况。该结果为进一步评价转基因水稻的生态风险提供了一定的依据。     

Enhancing the stability and ecological safety of mass‐reared transgenic strains for field release by redundant conditional lethality systems

The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically‐based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline‐suppressible (Tet‐off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline‐containing diet, but female‐lethal and male sterile in tetracycline‐free conditions. This would allow their most efficacious use in a unified system for sterile male‐only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet‐off lethal survival determinations, development of large‐scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet‐off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosβ2¹ mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host‐specific cognates to be created for a wide range of insect species.     

Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice

Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage in fact being inferior to free water for short-term survival of nonfeeding first-instar larvae. These results support the view that OCI, an effective inhibitor of a substantial fraction of digestive enzymatic potential in P. bioculatus, should not interfere with its predation potential when expressed in potato plants fed to its prey at a maximum level of approximately 0.8% of total soluble proteins in mature foliage.     

Isolation of pea matrix attachment region and study on its function in transgenic tobaccos

A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and downstream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which β-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and     

Effects of seed mixture sowing with transgenic Bt rice and its parental line on the population dynamics of target stemborers and leafrollers,and non‐target planthoppers

The widespread planting of insect‐resistant crops has caused a dramatic shift in agricultural landscapes, thus raising concerns about the potential impacts on both target and non‐target pests. In this study, we examined the potential effects of intra‐specific seed mixture sowing with transgenic Bt rice (Bt) and its parental non‐transgenic line (Nt) (100% Bt rice [Bt₁₀₀], 5% Nt+95% Bt [Nt₀₅Bt₉₅], 10% Nt+90% Bt [Nt₁₀Bt₉₀], 20% Nt+80% Bt [Nt₂₀Bt₈₀], 40% Nt+60% Bt [Nt₄₀Bt₆₀] and 100% Nt rice [Nt₁₀₀]) on target and non‐target pests in a 2‐year field trial in southern China. The occurrence of target pests, Sesamia inferens, Chilo suppressalis and Cnaphalocrocis medinalis, decreased with the increased ratio of Bt rice, and the mixture ratios with more than 90% Bt rice (Bt₁₀₀ and Nt₀₅Bt₉₅) significantly increased the pest suppression efficiency, with the lowest occurrences of non‐target planthoppers, Nilaparvata lugens and Sogatella furcifera in Nt₁₀₀ and Nt₀₅Bt₉₅. Furthermore, there were no significant differences in 1000‐grain dry weight and grain dry weight per 100 plants between Bt₁₀₀ and Nt₀₅Bt₉₅. Seed mixture sowing of Bt rice with ≤10% (especially 5%) of its parent line was sufficient to overcome potential compliance issues that exist with the use of block or structured refuge to provide most effective control of both target and non‐target pests without compromising the grain yield. It is also expected that the strategy of seed mixture sowing with transgenic Bt rice and the non‐transgenic parental line would provide rice yield stability while decreasing the insecticide use frequency in rice production.     

转Bt水稻对土壤可溶性有机碳氮及微生物学性质的影响

研究了3种转Bt水稻［克螟稻（KMD）、华恢1号（HH1）和Bt汕优63（BtSY63）］及对应亲本在2年大田条件下对土壤可溶性有机碳（DOC）和可溶性有机氮（DON）及微生物学性质的影响.结果表明：测定指标均随采样时间发生显著变化.与对应亲本相比,转Bt水稻对土壤DOC、DON和微生物生物量氮（MBN）的影响不显著,而对土壤微生物生物量碳（MBC）、基础呼吸（BR）和微生物代谢熵（qCO₂）的影响在大田种植第1年某些时段达到显著水平（P<0.05）,但这种影响没有持续到第2年;3种亲本水稻土壤DOC、DON及微生物学性质差异均不显著,但相应转Bt水稻土壤MBC、BR、qCO₂差异显著,BtSY63土壤MBC和BR显著高于KMD及HH1,而qCO₂显著低于KMD及HH1.转Bt水稻2年的大田种植对土壤DOC和DON及微生物学性质的影响较小,但3种转Bt水稻之间微生物学性质的差异比亲本之间的差异大,表明长期监测可能有助于发现转Bt水稻对土壤生态系统结构和功能的影响.     

Communities of ground‐dwelling arthropods in conventional and transgenic maize: background data for the post‐market environmental monitoring

To verify the validity of concerns about environmental safety of maize expressing insecticidal Cry toxins (referred to as Bt maize), we compared communities of ground beetles (Carabidae), rove beetles (Staphylinidae) and spiders (Araneae) in plots planted either with Bt maize cultivar YieldGard^® or with the non‐transgenic parental cultivar Monumental. Each cultivar was grown on 5 plots of 0.5 ha for three consecutive years. To increase the field load of Cry toxin, the fully grown maize of the first study year was shredded to small pieces that were ploughed into the soil. Arthropods were collected in pitfall traps and determined to the species level. The abundance and species richness of all studied groups greatly varied over the season and between the seasons but without statistically significant differences between the Bt and non‐Bt plots. A single spider species and three ground beetle species dominated in the catches every year, whereas a set of 1–4 most abundant rove beetle species changed every year. Frequently occurring species were typical for most of Europe. The total counts of ground beetles, rove beetles and spiders collected once or twice per season are proposed to serve as bioindicators in the post‐market environmental monitoring (PMEM).     

Production of a non‐triple helical collagen α chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture

We generated transgenic silkworms that synthesized human type I collagen α1 chain [α1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant α1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans‐activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the α1(I) chain content to 8.0% (4.24 mg per cocoon). The α1(I) chain was extracted and purified from the cocoons using a very simple method. The α1(I) chain contained no hydroxyprolines due to the absence of prolyl‐hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the α1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the α1(I) chain‐coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the α1(I) chain‐coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human α1(I) chain is a promising candidate biomaterial as a high‐quality and safe gelatin substitute for cell culture. Biotechnol. Bioeng. 2010;106: 860–870. © 2010 Wiley Periodicals, Inc.     

Highly interactive nature of flower‐specific enhancers and promoters,and its potential impact on tissue‐specific expression and engineering of multiple genes or agronomic traits

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co‐existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer–promoter and promoter–promoter interactions in transgenic plants and demonstrated that three of four flower‐specific enhancer/promoters were capable of distantly activating a pollen‐ and stigma‐specific Pps promoter (fused to the cytotoxic DT‐A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen‐ and carpel‐specific DT‐A expression, thus resulting in tissue ablation in an orientation‐independent manner; this activation was completely abolished by the insertion of an enhancer‐blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue‐specific DT‐A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant‐derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue‐specific engineering of multiple traits using a single‐vector stacking approach. Therefore, our work highlights the importance of adopting enhancer‐blocking insulators in transformation vectors to minimize promoter–promoter interactions. The practical and fundamental significance of these findings will be discussed.     

转基因741杨节肢动物群落主要害虫及天敌的动态变化

在时间序列过程中,转基因741杨对目标害虫鳞翅目食叶害虫表现出持续的抗性,其数量明显减少,高抗和中抗741杨之间差异不大.研究表明,转基因741杨对目标昆虫和非目的植食性害虫存在负效应,而对天敌和中性节肢动物组成和发生无明显负作用.因而在制定害虫综合治理策略和途径上宜采取与对照741杨不同的措施,抗性株系应以生态调控为主.     

不同基因型转Bt水稻种植对土壤螨类群落的影响

随着转基因技术的飞速发展,越来越多的转基因作物新品种被培育成功并得以推广应用,但转基因作物对非靶标生物的生态安全性问题日益引起人们的广泛关注。为加强转基因作物的生态风险评估,以Cry1Ab纯合基因型转Bt（Bacillus thuringiensis）水稻"克螟稻"和Cry1Ab/Ac融合基因型转Bt水稻"华恢1号"及其对照亲本水稻稻田土壤螨类为研究对象,系统调查研究了纯和基因型和融合基因型转Bt水稻种植下土壤螨类的群落组成、数量动态及其群落多样性的变化。研究结果显示,转Bt水稻对土壤螨类的群落组成无负面影响,仅一些稀有类群（<1%）和常见类群（介于1%和10%）消失或出现。且与对照亲本相比,纯和基因型转Bt水稻中仅上罗甲螨科（Epilohmanniidae）上罗甲螨属（Epilohmannia）的百分比含量显著增加了525%。此外,转Bt水稻与其对照亲本稻田土壤螨类的数量动态、群落多样性、群落均匀度和科属丰富度之间均差异不显著（P > 0.05）。而与纯合基因型转Bt水稻相比,融合基因型转Bt水稻可显著提高大田土壤螨类的数量和科属丰富度（P < 0.05）。可见,融合基因型转Bt水稻种植比纯合基因型转Bt水稻更有利于土壤螨类等非靶标生物的发生及其生物多样性保护。     

Effect of species‐counting protocols and the spatial distribution of effort on rarefaction curves in relation to decision making in environmental‐impact assessments

Rarefaction Curves are frequently used in Environmental Impact Assessments to evaluate sampling sufficiency, but without clear guidelines of how to ensure that the assumptions of the methods are met. Infrastructure projects in the Brazilian Amazon and elsewhere often occupy extensive areas in remote locations with difficult access, and random sampling under such conditions is impractical. We tested the influence of sampling unit (sample or individual), and geographic distance between samples on rarefaction curve s, and evaluated the magnitude of errors resulting from the misuse of rarefaction curve in decision making, using frogs from four Amazonian sampling sites. Individual‐based rarefaction curve were steeper than those generated by sample‐based rarefaction curve. Geographic distance influenced the number of exclusive species in a predictable fashion only in one area, and not in the Environmental Impact Assessment site. Misuse of rarefaction curve generated large errors in the identification of vulnerable taxa. Because the rarefaction curve model is sensitive to the assumption of randomness and geographic distance can influence it unpredictably, we suggest that rarefaction curve should generally not be used to estimate sample completeness when making management decisions for environmental licensing purposes.     

转GAFP基因棉花生态适合度及其对棉田昆虫群落的影响

新型转基因棉花生态适合度及其对棉田昆虫群落的影响是转基因棉花环境安全评价的重点内容.本文选用新型转GAFP(Gastrodia anti-fungal protein)基因棉花及其非转基因亲本棉花为试验材料,于2013和2014年在河南安阳研究了田间棉花生态适合度及其对昆虫群落的影响.结果表明: 转基因棉蕾期、花铃期和铃期叶片干物质量与其亲本无显著差异;苗期比叶面积显著低于亲本,蕾期、花铃期和铃期叶面积显著大于亲本;苗期株高显著低于亲本,蕾期、花铃期和铃期株高与亲本无显著差异;棉花生长的4个关键时期(苗期、蕾期、花铃期和铃期),转基因棉单株果枝数、蕾数和脱落数均与亲本无显著差异,而铃期单株大铃数显著低于亲本.棉铃虫发生的3个高峰期,转基因棉对棉铃虫和甜菜夜蛾无明显控制作用,且对棉田昆虫群落、害虫亚群落和天敌亚群落的昆虫个体总数、昆虫群落结构与组成、均匀性指数、多样性指数和优势集中性指数均无显著影响.表明外源基因GAFP导入后,新型转基因棉花营养生长增强,产量构成性状整体无显著变化,未引起棉田昆虫群落变化.研究初步明确了新型转基因棉花生存竞争势态和棉田昆虫群落发生规律与动态,可为新型转基因棉花环境安全评价提供理论依据,同时为转基因棉花环境安全评价积累科学数据.     

In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.