转基因作物对蜜蜂的影响

<正>转基因作物在世界范围内越来越多的被种植,但是有关转基因作物对蜜蜂以及其他传粉者的影响持续受到关注。转基因植物可能对蜜蜂产生直接或间接的影响。直接影响可能是指当蜜蜂摄入了转基因编码的蛋白质后所产生的影响。如果将转基因植入植物的过程导致植物表型发生不经意的变化,间接影响可能会发生。如果转基因产物(蛋白质)存在于转基因植物的花粉、花蜜或树脂中,对蜜蜂的直接影响可能会发生。     

利用egfp基因对中华蜜蜂卵进行显微注射的初步研究

利用显微注射法对中华蜜蜂0～12h卵注射绿色荧光蛋白基因,注射后的卵采取人工繁殖和蜂群自然培养两种方式饲养,结果蜜蜂卵注射后存活率低,绿色荧光蛋白基因表达不理想;实验探讨了中华蜜蜂卵显微注射转基因技术,为今后蜜蜂显微注射转基因研究和改进提供了一定参考价值。     

转基因植物对蜜蜂的安全性（下）

转基因植物已在全球范围内大面积推广应用 ,全球 5大转基因植物中的油菜、棉花、玉米均是主要的蜜粉源植物 ,对养蜂生产有重要意义 ,其对蜜蜂的安全性问题值得关注。 2种主要的抗虫 (Bt毒蛋白、蛋白酶抑制剂 )基因或抗真菌基因的产物只有在高浓度时才可能对工蜂的寿命、消化酶活性、行为等有不良影响。从目前的研究来看 ,转基因植物对蜜蜂是安全的     

禽传染性支气管炎病毒S1基因玉米表达载体的构建

禽传染性支气管炎病毒主要编码3种结构蛋白,其中S蛋白的S1蛋白是宿主的主要保护性抗原,能诱导机体产生中和抗体和血凝抑制抗体,并与病毒的组织嗜有关.本研究选取具有代表性的禽传染性支气管炎病毒分离株SAIB14和SAIB4,以其S1基因克隆质粒pUCSAIB14和pUCSAIB4为模板,用具有高保真度的pfu酶分别扩增,得到含先导序列的S1基因片段.把扩增产物分别通过ClaⅠ和BamHⅠ酶切纯化,替换中间载体pUGFPocs的GFPm1基因,构建中间表达载本pUSAIB14和pUSAIB4.用KpnⅠ和HindⅢ双酶切重组质粒pUSAIB14和pUSAIB4,回收Ubi-S1-Tocs片段,定向插入到pCAMBIA 300载体的多克隆位点.经酶切和PCR鉴定,证明获得了S1基因的玉米表达载体pCUSAIB14和pCUSAIB4.外源基因导入受体植物能否有效表达,表达载体构建是关键因素,构建时选用表达载体的类型、启动子和终止子、目的基因插入位点等都有影响.本研究用BarHⅠ和ClaⅠ双酶切载体pUGFPocs,用S1基因扩增片段替换GFPm1基因,将S1基因置于Ubi启动子和T-ocs终止子的控制之下,有助于S1蛋白的高效表达,最后将Ubi-S1-Tocs单元HindⅢ和KpnⅠ双酶切下来,插入到pCAMBIA1300的多克隆位点,得到可用于农杆菌介导的转化或直接转化的植物表达载体pCUSAIB4和pCUSAIB14. Ubi启动子来自玉米,属组成型启动子,内含有一个内含子和外显子,是已知的最强的单子叶植物启动子,外源基因在Ubi启动子的控制下,在转基因植物的所有部位和所有的发育阶段都会表达.pCAMBIA1300是中国农业科学院作物遗传育种中心构建的植物表达载体,可用于农杆菌介导的转化或直接转化,载体中T-DNA左右边界内带有多克隆位点和筛选标记Hyg基因,Hyg基因是玉米和水稻转化试验中广泛使用的标记基因,编码潮霉素磷酸转移酶,适于玉米和水稻转化后用化学试剂潮霉素进行筛选转化体.多克隆位点处可插入外源基因,在农杆菌介导的转化中,由于卸甲载体的作用,植物表达载体T-DNA左右边界内的序列,包括外源基因和筛选标记基因部分,发生转移并进入植物细胞内,用筛选试剂潮霉素可有效筛选出整合有外源基因的转化体.转基因植物疫苗生产技术是利用分子生物学技术把重组的疫苗基因导入植物,并使植物能够大量表达重组蛋白的一类生物技术.与常规疫苗及其它新技术疫苗相比,转基因植物疫苗具有生产简单、安全、廉价及保存和运输方便等优点,由于其应用前景可观,已引起免疫学家、分子生物学家和植物学家的极大兴趣与关注.利用该技术国外至今已获得成功的有乙型肝炎表面抗原、不耐热的肠毒素B亚单位(LT-B)、诺沃克病毒外壳蛋白、流感病毒血凝素和艾滋病病毒抗原、口蹄疫病毒抗原和鼻病毒抗原、痢疾抗原等10多种疫苗,国内在利用转基因植物生产疫苗的研究方面还远远滞后于其他国家.本研究利用含先导序列的禽传染性支气管炎病毒S1基因,构建了玉米表达载体系统,为进一步在玉米中表达IBV S1基因提供了基础材料.     

转基因植物对蜜蜂的安全性（上）

转基因植物已在全球范围内大面积推广应用 ,全球 5大转基因植物中的油菜、棉花、玉米均是主要的蜜粉源植物 ,对养蜂生产有重要意义 ,其对蜜蜂的安全性问题值得关注。 2种主要的抗虫 (Bt毒蛋白、蛋白酶抑制剂 )基因或抗真菌基因的产物只有在高浓度时才可能对工蜂的寿命、消化酶活性、行为等有不良影响。从目前的研究来看 ,转基因植物对蜜蜂是安全的。     

唐古特白刺类黄酮-3-O-葡萄糖基转移酶基因(NtUFGT)的克隆与功能分析

花青素是植物体内重要的黄酮类次生代谢产物,其强大的抗氧化能力对植物抵抗由各种非生物胁迫带来的氧化损伤发挥着重要的作用。本研究基于转录组数据,从唐古特白刺cDNA中克隆得到一个类黄酮3-O-葡萄糖基转移酶基因,将其命名为NtUFGT。该基因开放阅读框长度为1407 bp,编码468个氨基酸,预测该基因编码的蛋白质相对分子质量为51.37 kDa。多重序列比对分析结果显示,其编码蛋白属于UDP-glycosyltransferases蛋白家族。实时荧光定量PCR(qRT-PCR)分析了该基因在唐古特白刺中的表达模式,发现该基因的表达具有组织特异性,由高到低依次为花果实茎叶根;同时该基因能被聚乙二醇(PEG)和脱落酸(ABA)等非生物胁迫强烈诱导表达。构建该基因的真核表达载体pPZP221:35S:NtUFGT,使用花序浸染法转化拟南芥并筛选至T_3代,RT-PCR验证表明,NtUFGT基因在转基因拟南芥3个株系中均明显表达。测定干旱胁迫条件下野生型(WT)和转基因拟南芥(OE株系)生长状况和抗逆相关生理生化指标,结果发现转基因拟南芥生长状况明显优于野生型,根长更长,鲜重和叶绿素含量更高,同时OE株系积累了更多的花青素和总黄酮。与表型一致,相较于WT,OE株系具有更高的抗氧化酶活性[超氧化物歧化酶(SOD);过氧化物酶(POD);过氧化氢酶(CAT)],积累了更多的还原性谷胱甘肽(GSH)和脯氨酸,同时其丙二醛(MDA)、过氧化氢(H_2O_2)含量显著低于WT;基因定量分析结果显示,OE株系拟南芥中抗逆相关基因AtCAT1、AtPOD1、AtRD29A及脯氨酸合成基因AtP5CS的表达量明显高于WT。以上结果说明,NtUFGT能有效提高转基因拟南芥中花青素和总黄酮含量,赋予植物更强的活性氧清除能力和渗透调节能力,从而增强了植物对干旱胁迫的耐受性。     

唐古特白刺类黄酮-3-O-葡萄糖基转移酶基因(NtUFGT)的克隆与功能分析

花青素是植物体内重要的黄酮类次生代谢产物,其强大的抗氧化能力对植物抵抗由各种非生物胁迫带来的氧化损伤发挥着重要的作用。本研究基于转录组数据,从唐古特白刺cDNA中克隆得到一个类黄酮3-O-葡萄糖基转移酶基因,将其命名为NtUFGT。该基因开放阅读框长度为1407 bp,编码468个氨基酸,预测该基因编码的蛋白质相对分子质量为51.37 kDa。多重序列比对分析结果显示,其编码蛋白属于UDP-glycosyltransferases蛋白家族。实时荧光定量PCR(qRT-PCR)分析了该基因在唐古特白刺中的表达模式,发现该基因的表达具有组织特异性,由高到低依次为花>果实>茎>叶>根;同时该基因能被聚乙二醇(PEG)和脱落酸(ABA)等非生物胁迫强烈诱导表达。构建该基因的真核表达载体pPZP221:35S:NtUFGT,使用花序浸染法转化拟南芥并筛选至T₃代,RT-PCR验证表明,NtUFGT基因在转基因拟南芥3个株系中均明显表达。测定干旱胁迫条件下野生型(WT)和转基因拟南芥(OE株系)生长状况和抗逆相关生理生化指标,结果发现转基因拟南芥生长状况明显优于野生型,根长更长,鲜重和叶绿素含量更高,同时OE株系积累了更多的花青素和总黄酮。与表型一致,相较于WT,OE株系具有更高的抗氧化酶活性[超氧化物歧化酶(SOD);过氧化物酶(POD);过氧化氢酶(CAT)],积累了更多的还原性谷胱甘肽(GSH)和脯氨酸,同时其丙二醛(MDA)、过氧化氢(H₂O₂)含量显著低于WT;基因定量分析结果显示,OE株系拟南芥中抗逆相关基因AtCAT1、AtPOD1、AtRD29A及脯氨酸合成基因AtP5CS的表达量明显高于WT。以上结果说明,NtUFGT能有效提高转基因拟南芥中花青素和总黄酮含量,赋予植物更强的活性氧清除能力和渗透调节能力,从而增强了植物对干旱胁迫的耐受性。     

病程相关蛋白与植物抗病性关系的研究及其在草坪草抗病育种中的应用

植物被各类病原物侵染后,发生一系列的生理生化变化,包括细胞壁变厚,产生植保素以及防御相关蛋白。本研究从病程相关蛋白编码基因的诱导和表达、在植物组织细胞中的分布和运输、生物学功能与抗病性的关系、转基因植物的研究等方面依次进行总结,并对草坪病害的研究进展以及病程相关蛋白在草坪草等植物抗病育种工作中的作用与前景进行了讨论。关键词：病程相关蛋白;植物抗病性;病害;抗病育种;草坪草     

瘤胃厌氧真菌纤维素酶的研究与开发进展

瘤胃厌氧真菌被归为壶菌纲,在超显微结构和生物化学性质上与需氧真菌有严格的区分.瘤胃厌氧真菌能够分泌活性很高的纤维素酶,甚至是酶活最高的超级酶,这些酶是降解植物细胞壁纤维素所必需的.本文介绍了主要瘤胃厌氧真菌所产纤维素酶的研究现状,对瘤胃厌氧真菌纤维素酶开发利用的各种途径进行了综述,重点是瘤胃厌氧真菌纤维素酶基因的克隆表达、转基因动植物的研究以及瘤胃厌氧真菌纤维素酶的分子改造.     

植物基因工程疫苗的研究进展

植物基因工程疫苗是利用植物系统表达病原菌抗原蛋白一个或几个亚单位的疫苗，它没有病原菌完整的侵染能力，却可以使机体对特异的病毒或细菌产生免疫应答反应。由于转基因植物产生的疫苗对动物实施免疫只是简单地摄取含这种疫苗的植物组织或种子，所以这种疫苗不会被酶类所破坏，可通过机体肠道黏膜作用激发特异性免疫应答，使动物获得持久性疾病防御能力，且没有或很少出现病原体的过敏反应。     

AMV和WCMV在转基因红三叶中的基因累集

随着转基因技术的发展及其在植物育种中的应用，出现了一大批表现优良、经济效益高的转基因植物，但生产实践要求一种植物同时具有多个优良的转基因性状。目前解决这一问题的方法主要是基因累集(Gene pyramiding)，就是将多个基因集中到同一植株，可以将已经转入1个基因的植株做原材料，用农杆菌介导或基因枪等方法将另一基因转进去。也可以采用人工杂交，在不同转基因植株之间进行人工授粉，从后代中筛选出所需要的基因型。本研究对抗苜蓿花叶病毒(AMV)的转基因红三叶和抗白三叶花叶病毒(WCMV)的转基因红三叶进行了人工杂交，以期获得兼有2个基因、同时抵抗2种病毒的新植株，为下一步的育种打好基础。几乎所有的杂交组合都结了种子，杂交一代的发芽率随杂交亲本转基因拷贝数的不同而有所变化，只有1个转基因拷贝的亲本其杂种发芽率最高。对所有幼苗先用PCR进行检测，从中挑出较理想的基因型作进一步的分子生物学分析。Southern和Northern印迹分析结果表明，有些杂交一代的转入基因多于1个拷贝，有一部分同时兼有2种抗病基因，并且得到了表达。为进一步检测其抗病性，对它们进行了温室接种和田间评估，结果显示，尽管有的个体Northern分析呈阳性，但在田间仍然对病毒敏感，不过其感病程度都比对照有显著降低。大多数兼有2种转入基因的植株都能同时抵抗2种病毒，可以作为免疫个体供下一步育种之用。与遗传转化相比，人工杂交以其操作简单、省时省力、预见性强和准确率高而具有明显优势，是常规育种方法在分子育种中的具体体现。     

Design of vectors for transgene expression: The use of genomic comparative approaches

The design of transgenes has always been limited by the extent of available information on the endogenous locus whose expression pattern had to be replicated. Those genes whose expression domain had not been entirely documented resulted, usually, in transgenes with an unpredictable expression patterns and suboptimal performance in transgenic animals. The use of genomic comparative approaches, highlighting evolutionary conserved homologous DNA sequences, helps to identify crucial regulatory elements that are associated to a given expression domain. The inclusion of these conserved regulatory sequences in transgenic constructs would normally result in optimal expression levels of transgenes in recipient animals. The use of artificial chromosome-type transgenes usually ensures the inclusion of these preserved regulatory elements that are required for the faithful expression of the gene. These constructs could also contain insulators, a subset of regulatory sequences whose application is being addressed in transgenesis. Therefore, the generation of transgenic animals with genomic-type constructs is the recommended approach to achieve optimal transgene expression, according to the expected pattern of the corresponding endogenous locus.     

转基因动物研究进展

本文从转基因动物的产生、转基因细胞载体的运用、基因的转移、转基因的命运、转基因的载体、基因功能的研究,生物医学模型、制备重组蛋白、改善乳成分等方面综述了转基因动物的最新研究成果。     

Effect of dietary supplementation of recombinant Cry and Cp4 epsps proteins on haematological indices of growing rabbits

Genetically modified (GM) crops expressing insect resistance and herbicide tolerance provide a novel approach for improved crop production but their advent at the same time presents serious challenges in terms of food safety. Although prevailing scientific proof has suggested that transgenic crops are analogous to their conventional counterparts, their use in human and animal diet gave rise to emotional public discussion. A number of studies had been conducted to evaluate the potential unintended effects of transgenic crops expressing single transgene, but very few studies for those with multiple transgenes. As the crops with single and multiple transgenes could impart different effects on non‐target organisms, thus, risk evaluation of transgenic crops expressing more than one transgene is required to declare their biosafety. The present study was therefore designed to assess the effects of different levels of dietary transgenic cottonseed expressing recombinants proteins produced by Cry1Ac, Cry2A and Cp4epsps genes on haematological indices of growing rabbits. A total of 48 rabbits were assigned to four dietary treatments containing different levels of transgenic cottonseeds (i.e., 0% w/w, 20% w/w, 30% w/w and 40% w/w) with 0% w/w serving as control. Haematological parameters were measured at periodic intervals (0, 45, 90, 135 and 180) days. No significant (p > 0.05) dose‐dependent effects were observed in most of the haematological parameters evaluated. Though, significant differences (p < 0.05) were recorded in the level of MCHC, MCH and HCT in some of experimental male and female rabbits, yet, they were not biologically significant, as all the differences were within the normal reference values. Our study suggested that feeding transgenic cottonseed of up to 40% could not adversely affect rabbit's haematological profile. However, further study needs to be conducted with different cotton genotypes expressing both single and polygenic traits before recommending the utilization of transgenic cottonseed in routine livestock feeding.     

The absence of detectable fetal microchimerism in nontransgenic goats (Capra aegagrus hircus) bearing transgenic offspring

Regulations for the disposal of genetically engineered animals are strict due to concern for their inappropriate introduction into the food chain, and of the possible public health and environmental impacts of these organisms. Nontransgenic animals that give birth to transgenic offspring are treated as if they are transgenic due to concern of fetal cells crossing the placental barrier and residing in the mother (fetal-maternal microchimerism). Determining whether or not fetal-fetal or fetal-maternal transfer of DNA or cells occurs during caprine gestation is critical to effectively protect the public without culling animals that pose no risk. Additionally, fetal-maternal transfer, should it exist in the goat, could contraindicate the rebreeding of nontransgenic dams due to the possible transfer of fetal cells from 1 pregnancy to the fetus of subsequent pregnancies. Fetal-maternal transfer in Capra hircus has not been reported in the literature, although it has been reported in another ruminant, Bos taurus. We examined blood from nontransgenic dams that carried transgenic offspring using a PCR method sensitive enough to detect the presence of a spider silk transgene to a 1:100,000 dilution. At this sensitivity, we did not detect the occurrence of fetal-maternal transfer in 5 nontransgenic dams. Likewise, fetal-fetal transfer was not observed from a transgenic to a nontransgenic twin in utero. To test tissue-specific expression of the silk transgene, proteins purified from standard necropsy tissue from a lactating transgenic dam were examined by Western blot analysis. Silk protein expression was only observed in mammary tissue consistent with the tissue specificity of the β-casein promoter used in the transgenic construct. We report evidence collected from a limited caprine breeding pool against transfer of transgenes in utero from fetus to dam and fetus to fetus. In addition, we show evidence that the β-casein promoter in our expression construct is not expressed ectopically as previously suggested. These results suggest that transgene transfer in utero does not occur, but further study is warranted with a larger sample group to confirm these results.     

Transgenic fish resistant to infectious diseases, their risk and prevention of escape into the environment and future candidate genes for disease transgene manipulation

Transgenic fish have been produced that have improved growth, disease resistance, survival in cold and body composition, have altered color, that can act as bioindicators for estrogenic pollutants and that can produce pharmaceutical proteins. The largest amount of transgenic research has focused on growth hormone transfer. A relatively small amount of research has focused on enhancing disease resistance, but significant enhancement has been accomplished. Pleiotropic effects from the transfer of other transgenes, particularly growth hormone gene can alter disease resistance in both positive and negative ways. Most negative effects for all transgenes appear to lower fitness traits, which is positive for biological containment. Transgenic fish appear to pose little environmental risk, but this research is not fully conclusive. To expedite commercialization and minimize environmental risk, transgenic sterilization research is underway. A large amount of functional genomics research has resulted in a much better understanding of gene expression when fish are experiencing disease epizootics. This information may allow the future design of more effective transgenic approaches to address disease resistance.     

Obtaining raw material: plants as tool sources for nigerian chimpanzees

We investigated the acquisition of plant materials from which Nigerian chimpanzees manufacture wooden tools to harvest insects and honey from nests of army ants, honey bees and stingless bees. Slender trunks of juvenile trees and branches are most commonly used, and bendable vines rarely, probably reflecting the need to work with relatively sturdy tools to extract resources. While several tools are sometimes sourced from the same plant, there is also evidence for a depletion effect, as multiple tool sources at the same site are often spaced several metres apart. Identified tool sources belong to 27 species of at least 13 families. Honey-gathering implements are often chewed upon by chimpanzees. Interestingly, twigs of the most commonly used honey-gathering species possess antibacterial propensities and are favoured by Nigerians as chewing sticks. This suggests that extractive tools might possess associated medicinal or stimulatory properties. We do not know if chimpanzees actively select specific plant parts or species as we cannot compare observed with expected frequencies. Nevertheless, about three quarters of tools are picked from plants more than 6 m away from the extraction site, potentially indicating some degree of forward planning.     

CRISPR/Cas9 knock-in介导的输卵管特异性表达转基因鸡研究进展

利用动物生物反应器生产重组蛋白是一种具有应用前景的生物技术。鸡输卵管生物反应器是理想的动物生物反应器之一,其优点在于表达的外源蛋白能够分泌到蛋清中,可避免蛋白提取过程中对鸡本身造成伤害,同时蛋清成分简单,便于后期的纯化。目前利用慢病毒结合原始生殖细胞(PGCs)制备转基因鸡被认为是最可行的方法,但因外源基因随机整合且生殖系传递效率较低,使转基因鸡研究受到技术上的限制。而2013年问世的CRISPR/Cas9基因敲入(CRISPR/Cas9 knock-in)技术能够使外源基因精准定向插入基因组特异性位点,这对生产输卵管特异性转基因鸡具有重大意义。文章综述了鸡输卵管反应器的研究进展、CRISPR/Cas9 knock-in技术在输卵管特异性表达转基因鸡研究和鸡育种领域的应用现状,并指出了目前存在的问题和相应的解决办法。     

在工蜂产卵超过23天的中蜂群中介绍蜂王

在中蜂的饲养管理中,蜂群失王且工蜂产卵后,一般处理方法都是并入它群;实验是在不得已的情况下,将3群失王且工蜂产卵超过23 d的中蜂群合并的同时,介绍蜂王获得成功.     

推进蜜蜂授粉与高效生态农业互促发展的对策研究——以浙江为例

蜜蜂授粉在促进现代农业发展中的生态价值、经济价值正在被越来越多的人所认识到。以蜜蜂授粉发展较快的浙江省为例,系统分析了蜜蜂授粉与高效生态农业互促发展的现状,对二者发展推进中存在的认识不到位、种养主体衔接不紧密、养殖户经济效益分享有偏、优质农产品市场机制不完善等问题进行分析。在此基础上,较为深入地剖析了蜜蜂授粉与高效生态农业互促共进的机理,最后从加大宣传示范力度、加快先进适用授粉技术的研发、促进配套的服务体系建设以及完善蜜粉源植物保护、推进授粉农产品市场认证机制等方面提出了加强蜜蜂授粉业与高效生态农业互促发展的政策建议。