草莓乙烯受体FaEtr1和FaErs1基因的克隆及其反义表达载体的构建

在已报道的FaEtr1和FaErs1基因序列的基础上,设计特异引物,分别克隆草莓(Fragaria ananassa)乙烯受体FaEtr1基因580bp部分特异序列和FaErs1基因445bp部分特异序列,将上述2个片段分别反向插入植物表达载体pBI121的CaMV35S启动子和Nos终止子之间,构建了反义表达载体pBI121Etr1和pBI121Ers1。将带有完整启动子和终止子的FaEtr1和FaErs1基因引入pCAMBIA1301中,最终获得FaEtr1和FaErs1的双价植物表达载体pFRS。将这3个重组表达质粒pBI121Etr1、pBI121Ers1和pFRS导入根癌农杆菌(Agrobactrium tumefaciens)EHA105中,PCR及限制性内切酶酶切确定质粒已被导入。     

pBI121-Lyz-GFP表达载体的构建及其对和田苜蓿愈伤组织的转化

利用分子克隆技术,将GFP基因片段插入到pBI121-Lyz多克隆区,构建了重组表达载体pBI121-Lyz-GFP。经SmaI与BamHI双酶切和PCR验证重组质粒,均能得到约750bp的目的片段,通过进一步测序证明：GFP基因已成功连接到pBI121-Lyz中,且重组质粒连接方向正确。利用冻融法将重组质粒导入农杆菌菌株LBA4404,用叶盘法转化和田苜蓿,筛选出具有卡那霉素抗性的愈伤组织,经PCR扩增和荧光显微镜检测证明重组基因已成功转化到和田苜蓿愈伤组织中。     

Expression Pattern of Ａ Recombinant Phloem-specific Promoter from Pumpkin in Transgenic Potato Plants

以本实验室构建的重组南瓜(Cucurbita moschata)韧皮部特异启动子dENP构建了植物表达载体pBdENP。利用根癌农杆菌(Agrobacterium tumefaciens)LBA44O4介导转化马铃薯(Solanum tuberosum)品种Favorita，经过抗生素筛选，共获得转pBdENP和对照pBI121的抗卡那霉素马铃薯再生植株106株。通过PCR初步筛查，筛选出65株为转基因阳性植株。通过Southern blot对部分植株进一步分析,确证外源gus基因已经插入到转基因马铃薯植株的基因组中，插入拷贝数在1个或2个以上。对这些转基因马铃薯植株进行GUS染色结果表明, dENP和CaMV35S启动子一样均能驱动gus基因的表达，前者仅在马铃薯的韧皮部内特异表达，而CaMV35S启动子驱动的gus基因为组成型性表达。GUS酶活力测定结果进一步表明dENP和CaMV35S启动子驱动gus基因表达水平没有明显区别。以上结果证明dENP启动子驱动的外源基因在马铃薯中也具有韧皮部特异而高效表达的特征，从而可用于马铃薯抗病、抗蚜虫转基因研究。     

Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.     

转基因抗虫棉花重组DNA在土壤中分布的实时定量PCR分析

根据转基因抗虫棉花35S启动子与Cry1A（c）基因以及35S启动子与nptⅡ基因之间的构建特异性序列分别设计引物和探针,建立荧光定量PCR检测方法,并对采集于3个生长时期（播种后40、50d和60d）的不同根区（根表、根际和非根际）土壤中35S-Cry1A和35S-nptⅡ片段进行定量分析。结果表明,所建立的荧光定量PCR方法最低能够检测到10个拷贝数的外源重组DNA片段,定量标准曲线相关系数均达到0.998以上,具有很好的重复性。实时定量PCR分析表明,同一生长时期不同根区土壤中35S-Cry1A和35S-nptⅡ片段拷贝数变化情况均为根表土〉根际土〉非根际土,即转基因抗虫棉花重组DNA片段主要分布于根表土壤中,其次为根际土壤和非根际土壤。在60d生长期内,土壤中35S-Cry1A和35S-nptⅡ片段的拷贝数随生长时期的推进均呈现上升趋势,其分布范围逐渐扩大。土壤中35S-nptⅡ片段拷贝数均高于同一生长时期相应根区中35S-Cry1A片段的拷贝数。转基因抗虫棉花对环境可能存在潜在影响。     

水稻Rubisco小亚基启动子的克隆及光诱导表达载体的构建

高效特异表达的启动子往往是转基因研究的关键因素。Rubisco小亚基启动子具有光诱导性、组织特异性和高表达的特性,可利用该启动子为转基因研究服务。本文以水稻（Oryza sativa）中花11叶片为材料,根据GenBank所报道的水稻Rubisco小亚基启动子序列,设计特异引物从水稻基因组DNA中扩增得到长度约1600bp的DNA片段,将该片段连接至T载体pMD18-TSimple,测序结果表明该片段序列与GenBank报道序列一致为100%。Plant CARE序列分析表明,该启动子具有12个与光诱导表达相关的元件。为构建光诱导表达载体,将该启动子和植物表达载体pCactF分别以KpnⅠ和XbaⅠ酶切后连接。光诱导表达载体的构建为进一步研究基因功能及利用光诱导表达载体改良作物品质奠定基础。     

Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.     

蓝猪耳胚珠磷脂酶C基因TfPLC1启动子的克隆及载体构建

应用衔接头PCR技术，以蓝猪耳全基因组DNA分别经DraI、EcoRV、PvuII、SmaI消化后与衔接头连接产物为模板，用衔接头引物和TfPLC1基因的特异引物经过多轮的巢式PCR，先后克隆到两个大小为798bp、813bp的TfPLC1基因上游序列；经测序、blastn比较分析和拼接得到一个蓝猪耳TfPLC1基因的启动子序列，共1432bp。序列分析表明它含有类似于TATA box和CAAT box的元件，在其远端上游区域还有多个AT富含区，而且还含有多个胚乳特异表达启动子的元件。将该启动子全序列和5’端缺失的700bp序列与PBI121分别构建了植物表达载体PPP1326和PPP700，用于转化蓝猪耳，以验证该启动子的功能。该启动子的克隆对于研究蓝猪耳胚珠TfPLC1基因的表达调控及功能具有重要意义。     

Degradation of endogenous and exogenous genes of roundup-ready soybean during food processing

Roundup-Ready soybeans have been genetically modified to resist the effects of the herbicidal glyphosate and have become the most prevalent transgenic crop in the world. In this work, Roundup-Ready soybeans were used as raw material to study the effects of critical processing procedures such as grinding, cooking, blending, homogenization, sterilization, and spray-drying on the length of DNA fragments of an endogenous gene (lectin) and an exogenous gene (epsps) examined in material from three soybean foods of bean curd, soy milk, and soy powder and from samples taken during their processing. The results showed that various processing procedures caused degradations of both the endogenous and exogenous genes to different degrees. In the grinding procedure, endogenous gene DNA was degraded from 1883 to approximately 836 bp, and exogenous gene DNA was degraded from 1512 to approximately 408 bp. In the blending and squeeze-molding procedures, exogenous gene DNA was also degraded from about 408 to 190 bp, but there was no obvious action on the endogenous gene. After the endogenous and exogenous genes had been degraded to some degree, such as 836 and 408 bp, respectively, they were not evidently affected by cooking procedure at 100 degrees C for 15 min. However, the endogenous gene was further considerably degraded from around 836 to 162 bp in the sterilization procedure at 121 degrees C for 30 s. The effect of the homogenization step on endogenous and exogenous genes was similar to that of the cooking procedure. The coagulation procedure, principally a biochemical reaction, did not greatly affect the exogenous gene but did affect endogenous gene, reducing DNA size from about 836 to 407 bp. Furthermore, the spray-drying procedure, a process of physical shearing, high temperature, and sudden high pressure, distinctly caused degradation of both the lectin and epsps genes, rapidly decreasing the sizes from about 836 to 162 bp for the endogenous gene and from about 408 to 190 bp for the exogenous gene.     

转柽柳金属硫蛋白基因（MT1）烟草的获得及对重金属镉的抗性分析

将柽柳（Tamarix. sp）金属硫蛋白基因（MT1）插入到植物表达载体pBI121，利用农杆菌（Agrobactrium tumefaciens）介导法将MT1导入烟草基因组。对转基因T1植株的卡那霉素抗性分析表明，多数转基因植株后代表现为3:1的分离比例，只有少数转基因植株的抗性分离表现为15:1或不符合孟德尔遗传的分离比例。说明整合到烟草基因组的外源基因多为单拷贝基因，也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR-Southern检测和Northern杂交分析表明，外源MT1基因已整合到烟草基因组，并且得到了正确表达。转基因植株对重金属镉的抗性比对照显著提高，表现为转基因植株的株高和鲜重均明显优于非转基因株系。     

香蕉乙烯受体基因RNA干扰表达载体的构建

设汁两对引物，PCR扩增香蕉乙烯受体基因cDNA序列自AUG启始密码子起长538bp区段的正向(记为5‘I)、反向(记为AI’)DNA区段。构建含该正、反向互补重复序列DNA片段的中间载体，经测序证实连接正确后。克隆到植物表达载体pCAMBIA-1301中的GUs基因位置并经双酶切证实。在所得表达载体pCAMBIA-1301-S5’I-AI’中，该插入正反互补重复片段处于35S启动子之后，由此转录的mRNA因两端序列反向互补而形成发夹式RNA，因此可应用于RNA干扰研究。同时，文中对构建含正、反向重复序列插入片段植物表达载体过程中出现的插入片段链内退火引起连接困难等问题进行了分析讨论。     

油菜毛状体发育相关基因GLABRA2启动子的克隆与功能验证

GLABRA2(GL2)基因在拟南芥毛状体发育中具有重要作用.实验利用基因组步移巢式PCR的方法,通过两次步移克隆得到一个油菜(Brassica napus)GL2基因启动子序列,序列分析发现它与拟南芥GL2启动子同源性较差,但有一些共同的顺式作用元件如MYB结合位点、G box等,也有油菜GL2基因所特有的应答元件如E-box.将其中具有启动子的基本特征的序列GP1与GUS报告基因融合构建重组载体pBI121-GP1,转化拟南芥(Arabidopsis).用卡那霉素筛选得到10株阳性苗.在T2代转基因株系中有6个株系的绿苗与黄花苗的比例都接近3:1,推定T-DNA是以单拷贝的形式插入拟南芥基因组DNA.GUS组织化学染色表明,GP1调控下报告基因主要在子叶、真叶的表皮毛以及根部的幼嫩组织中表达,与拟南芥GL2基因启动子表达模式基本一致,但也有明显不同:油菜GL2启动子GP1只在表皮毛发育早期强烈表达,而拟南芥GL2启动子调控表皮毛发育的整个过程.     

转CBF1基因烟草植株的花器官变异及其插入区侧翼序列的分析

在转CBF1基因烟草的后代中发现了花器官变异的植株，主要有花瓣裂瓣、花型变小、花丝变短花粉萌发率降低和育性降低等表型变化。采用TAIL－PCR方法，从花器官变异明显和不明显的植株中分别克隆到了各自的T－DNA插入区域的侧翼序列。将这些侧翼序列与GenBank database及pBI121质粒进行比对。结果表明表型差异明显的2个植株的T－DNA插入位点相同，属于一个转基因株系；且T－DNA插入区位于烟草基因组的非编码区。说明这些花型的变异并非由T－DNA在受体基因组中插入位点不同造成的，也不是由外源转基因对受体功能基因的破坏引起的。     

Influence of phosphate and copper on reductive dechlorination of thiobencarb in California rice field soils

The potential for reductive dechlorination of the herbicide thiobencarb (TB) by microbes and its prevention in saturated anaerobic rice field soils was examined in laboratory microcosms. TB is effective in controlling both annual grasses and broadleaf weeds. In anoxic microcosms, TB was effectively degraded within 30 days to its dechlorinated product, deschlorothiobencarb (DTB), in two Sacramento Valley rice field soils. TB dechlorination, and subsequent degradation, followed pseudo-zero- (lag phase) and first-order (degradation phase) kinetics. Logistic regression analysis (r2 > 0.841) produced a half-life (t(1/2)) in nonsterile soils ranging from 10 to 15 days, which was also observed when microcosms were amended with low concentrations (<3 mg L(-1)) of copper (Cu2+; as the fungicides Cu(OH)2 and CuSO4.5H2O). High Cu2+ concentrations (>40 mg L(-1)) were added to the microcosms to determine if copper toxicity to dechlorinating microbes is concentration dependent within the range used. After 30 days, the low-copper-amended soils closely resembled the nonsterile experiments to which no Cu2+ was added while the high-copper-amended microcosms were similar to the sterile experiment. Microcosms were also separately amended with 5.7 g L(-1) phosphate (PO4(2-); as KH2PO4), a nutrient regularly applied to rice fields. Phosphate-amended experiments also showed TB degradation, but no DTB formation, indicating the phosphate played a role, possibly as a microbial inhibitor or an alternative electron acceptor, in limiting the dechlorination of TB. In summary, TB dechlorination was inhibited at high Cu(OH)2, CuSO4.5H2O, and KH2PO4 concentrations.     

植酸酶根特异表达载体的构建及大豆转化

应用PCR方法分别从胡萝卜基因组中扩增出96bp的外展蛋白信号肽编码序列片段,从拟南芥基因组中扩增出1454bp的pyk10启动子片段,用RT-PCR方法从无花果曲霉（Aspergillus ficuum3.4322）中扩增出phyA基因,长1347bp。然后,分别克隆到pMD18-T载体。应用已设计的限制酶切位点,通...     

Detection of transgenic and endogenous plant DNA in digesta and tissues of sheep and pigs fed Roundup Ready canola meal

The persistence of plant-derived recombinant DNA in sheep and pigs fed genetically modified (Roundup Ready) canola was assessed by PCR and Southern hybridization analysis of DNA extracted from digesta, gastrointestinal (GI) tract tissues, and visceral organs. Sheep (n = 11) and pigs (n = 36) were fed to slaughter on diets containing 6.5 or 15% Roundup Ready canola. Native plant DNA (high- and low-copy-number gene fragments) and the cp4 epsps transgene that encodes 5-enolpyruvyl shikimate-3-phosphate synthase were tracked in ruminal, abomasal, and large intestinal digesta and in tissue from the esophagus, rumen, abomasum, small and large intestine, liver, and kidney of sheep and in cecal content and tissue from the duodenum, cecum, liver, spleen, and kidney of pigs. High-copy chloroplast-specific DNA (a 520-bp fragment) was detected in all digesta samples, the majority (89-100%) of intestinal tissues, and at least one of each visceral organ sample (frequencies of 3-27%) from sheep and swine. Low-copy rubisco fragments (186- and 540-bp sequences from the small subunit) were present at slightly lower, variable frequencies in digesta (18-82%) and intestinal tissues (9-27% of ovine and 17-25% of porcine samples) and infrequently in visceral organs (1 of 88 ovine samples; 3 of 216 porcine samples). Each of the five cp4 epsps transgene fragments (179-527 bp) surveyed was present in at least 27% of ovine large intestinal content samples (maximum = 64%) and at least 33% of porcine cecal content samples (maximum = 75%). In sheep, transgene fragments were more common in intestinal digesta than in ruminal or abomasal content. Transgene fragments were detected in 0 (esophagus) to 3 (large intestine) GI tract tissues from the 11 sheep and in 0-10 of the duodenal and cecal tissues collected from 36 pigs. The feed-ingested recombinant DNA was not detected in visceral tissues (liver, kidney) of lambs or in the spleen from pigs. Of note, however, one liver and one kidney sample from the pigs (different animals) were positive for a 278-bp fragment of the transgenic cp4 epsps (denoted F3). Examination of genomic libraries from these tissues yielded no conclusive information regarding integration of the fragment into porcine DNA. This study confirms that feed-ingested DNA fragments (endogenous and transgenic) do survive to the terminal GI tract and that uptake into gut epithelial tissues does occur. A very low frequency of transmittance to visceral tissue was confirmed in pigs, but not in sheep. It is recognized that the low copy number of transgenes in GM feeds is a challenge to their detection in tissues, but there was no evidence to suggest that recombinant DNA would be processed in the gut in any manner different from endogenous feed-ingested genetic material.     

一种不依赖酶切位点的分子克隆方法

本研究介绍一种分子克隆的新方法。该方法要求在PCR引物设计时,目的片段上游引物的5＇端和下游引物的5＇端各设计约25 bp与目标载体末端同源的序列,或目的载体反向游引物的5＇端和正向引物的5＇端各设计约25 bp与目标片段同源的序列,以便进行融合PCR,PCR反应扩增目的片段后,与载体进行融合PCR。用DpnⅠ消化原始甲基化模板,然后进行转化和重组子鉴定。结果表明利用该方法成功将目的片段插入载体。证明这是一种简便、通用、高效并值得推广的分子克隆的新方法。该方法不需要骨架载体上的特异性酶切位点,特别适用于插入片段中无限制性酶切位点的载体改造;该方法还可以引入定点突变,可便捷构建分析启动子功能等需引入定点突变的载体;该分子克隆方法还可以实现基因的无缝克隆。     

Soil metabolism of a new herbicide, [14C]Pyribenzoxim, under flooded conditions

To elucidate the fate of a new pyrimidinyloxybenzoic herbicide, pyribenzoxim, a soil metabolism study was carried out with [14C]pyribenzoxim applied to a sandy loam soil under flooded conditions. The material balance of applied radioactivity ranged from 96.4 to 104.4% and from 96.1 to 101.9% for nonsterile and sterile soils, respectively. The half-life of [14C]pyribenzoxim was calculated to be approximately 1.3 and 9.4 days for nonsterile and sterile soils, respectively. The metabolites identified during the study were 2,6-bis(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid (M1) and 2-hydroxy-6-(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid (M2), resulting from the cleavage of the ester bond and subsequent hydrolysis. The nonextractable radioactivity levels increased to 37.8% for nonsterile conditions at 50 days after treatment and to 38.2% for sterile conditions at 60 days after treatment. Fractionation of the nonextractable soil residues indicated that bound radioactivity was associated mainly with humin fraction. No significant volatile products or [14C]carbon dioxide was observed during the study. On the basis of these results, pyribenzoxim is considered to undergo rapid degradation in soil by microbial and chemical reactions, mainly hydrolysis, which limits its transfer to and accumulation in lower soil layers and groundwater. Therefore, the possibility of environmental contamination from the use of pyribenzoxim is expected to be very low.     

金柑LEAFY同源基因克隆与全序列分析

通过PCR扩增,从融安金柑基因组DNA中克隆出一条2 361 bp的DNA片段,该DNA克隆含有1个2 081 bp的LEAFY同源基因全长序列(FcLFY)。金柑LEAFY同源基因包含2个内含子和3个外显子,编码398个氨基酸。比对结果表明,金柑LEAFY同源基因与甜橙、枳的LEAFY同源基因的核苷酸和氨基酸序列的同源性均为98%。该研究为今后从分子水平上研究金柑开花调控机理打下了坚实的基础。     

Use of the gef gene in a biological containment system in soil microcosms

Summary Data on the use of a biological containment system in microcosms are presented. The system is based on a transposon Tn5-cassette containing a fusion of the lac promoter and gef controlled by lacI^Q. The suicide function was inserted into different bacterial hosts and its induction by isopropyl--d-thiogalactoside (IPTG) was tested in sterile and non-sterile soil microcosms. The results suggest that the killing effect of this gef-based suicide mechanism is insufficient under soil conditions.