含粘质沙雷氏菌几丁质酶SchiA基因的植物转化质粒pBG1112构建和水稻遗传转化

摘要:将一个2.8 kb的CaMV35S启动子/SchiA编码区/Nos终止区融合基因插入到植物转化载体pCAMBIA1301的多克隆酶切位点，得到1个14.6 kb的新植物转化质粒pBG1112,用花器介导法转化水稻（Oryza sativa ），经PCR检测，初步证实已将目的基因整合到受体植物的基因组中。一部分转基因T3代潮霉素抗性阳性植株对水稻纹枯病(Rhizoctonia solani )和稻瘟病（Pyricularia oryzae）的抗性较非转化对照增强。RT-PCR表明抗病性植株为阳性，而不抗病的植株为阴性。将RT-PCR产物测序后，用BLAST软件分析序列可知，该序列为细菌几丁质酶基因核苷酸序列而不是植物几丁质酶基因的核苷酸序列。T4代转基因水稻的几丁质酶活力高于对照未转基因植株，表明转入的外源几丁质酶基因能正常表达。     

PHAs合酶phaC2基因转化烟草叶绿体的研究

中长链羟基脂肪酸聚酯 (medium-chain-length-PHAs, mcl-PHAs) 属于微生物聚酯。Ⅱ型PHA合酶是mcl-PHAs生物合成途径中的关键酶。将编码Ⅱ型PHA合酶的基因phaC2与水稻(Oryza sativa )叶绿体psbA基因的启动子和终止子构建表达盒(RpsbA-pro-phaC2-RpsbA-ter)，连同壮观霉素抗性基因aadA表达盒(prrn-aadA-TpsbA-ter)一起克隆进烟草叶绿体基因组同源片段rbcL和accD中，构建了烟草叶绿体转化表达载体pTC2。基因枪法转化烟草(Nicotiana tobacum L.)叶片，获得具有壮观霉素抗性的转基因烟草。对T0代和T1代转基因植株的PCR和Southern blot鉴定结果表明, 外源基因确已整合进烟草叶绿体基因组中，T1代转基因植株已达同质化。RT-PCR分析结果证实phaC2基因已在转录水平上表达。转基因植株的正反交试验证明外源基因在转基因后代中遵循母性遗传规律，不存在转基因的花粉漂移现象。通过叶绿体遗传转化, 实现中长链羟基脂肪酸聚酯合成关键酶基因phaC2在烟草叶绿体基因组中的稳定转化。     

转基因水稻中ThEn-42基因的稳定遗传及其抗病性的提高*

用农杆菌介导法将CaMV35S启动子调控下的外源基因ThEn-42转入粳稻(Oryza sativa ssp．japonica)品种台北309中。对100多株再生植株进行了PCR鉴定，结果表明，90％以上的植株为阳性植株。Southern检测的结果进一步证实，外源基因已经整合到水稻基因组中。对T1代植株也进行了PCR鉴定和潮霉素抗性鉴定，结果表明，大约70％的T1代转基因株系符合3：1(阳性：阴性)的分离比，表明ThEn-42基因在这些株系中有单个插入位点；大约20％的T1代株系分离比符合15：1，表明这些株系含有2个整合位点。选择部分T1代潮霉素抗性植株进行Southern杂交鉴定，结果证实，外源基因已经稳定遗传给T1代。检测了T0代和T1代转基因植株对水稻纹枯病(Rhizoctonia solani)和两个分别属于A群和B群的稻瘟病(Magnapotthe grisea)小种的抗性，结果表明，30%株系表现了对这两种病害抗性水平不同程度的提高。有7个株系的抗性与对照相比有显著的提高，其中Tp64表现对两个稻瘟病小种完全免疫。这些结果表明内切几丁质酶基因ThEn-42已经在受体植物中准确表达，并且在提高水稻抗病性方面起了重要作用。     

基因枪转化MAR序列介导水稻bar基因的表达分析

以水稻（Oryza sativa L.) 成熟胚愈伤组织作为水稻遗传转化受体，采用带有MAR（matrix attachment region)和不带MAR的两种植物表达质粒进行基因枪轰击转化bar基因，获得抗basta转基因水稻。分析了MAR序列介导在转基因水稻中对转基因表达的影响。研究结果表明，利用MAR序列介导bar基因表达，获得的转基因系的数量比对照提高66．7％，单块抗性愈伤组织再生植株数比对照提高27．3％，MAR序列介导bar基因表达的转基因系中，bar基因的表达水平比对照高，并且不同转基因系间bar基因表达差异比对照小。     

CHI-PAT双价基因遗传转化贵州禾来拢

以贵州禾来拢幼胚为转化受体,用农杆菌介导法将几丁质酶和抗除草剂抗性双价基因（CHI-PAT）导入来拢幼胚,筛选出抗性愈伤组织并获得抗性植株.抗性植株经GUS组织化学及PCR检测呈阳性,转基因植株对50 mg/L的Basta溶液有抗性.初步证明CHI和PAT基因已整合进了水稻基因组中.     

甘蔗尿苷二磷酸葡萄糖焦磷酸化酶基因(UGPase)转化拟南芥及转基因植株的生理特性分析

尿苷二磷酸葡萄糖焦磷酸化酶(UDP-glucose pyrophosphorylase,UGPase)是植物糖代谢的主要参与酶之一,在植物的生长发育过程中起着重要作用。本研究将甘蔗(Saccharum officinarum)UGPase基因cDNA片段连接至载体pBI121,通过BamHⅠ和SacⅠ酶切鉴定及测序验证,结果表明,植物表达载体成功构建;通过农杆菌(Agrobacterium tumefaciens)的介导,采用浸花法转化拟南芥(Arabidopsis thaliana)。结合卡那霉素抗性筛选和PCR检测,获得了5株T0代转基因植株。对T1代转基因植株进行PCR及Southern blot分析,结果表明,目的基因已成功转入拟南芥中,并且不同的转化植株含有目的基因的拷贝数不同。对T2代转基因植株进行PCR和RT-PCR检测,结果表明,目的基因不仅能在自交系后代中稳定遗传,而且在RNA水平也有表达。同时,对T2代转基因植株的可溶性总糖、蔗糖及淀粉含量进行测定,结果表明,与野生型相比,转基因植株中可溶性总糖含量没有明显的变化,但蔗糖含量有所提高,并且差异明显,比野生型植株提高了50.85%~96.99%,而淀粉含量都较野生型植株的低,降低了9.69%~36.76%。说明UGPase在蔗糖与淀粉的转换过程中起着较为重要的作用,其催化的反应方向影响着组织中这两种产物(蔗糖和淀粉)的分配。     

水稻钙依赖型蛋白激酶0sCPK9基因RNAi表达载体的构建及遗传转化

水稻钙依赖型蛋白激酶（CDPKs）是一个响应逆境胁迫并在植物发育过程中起重要作用的蛋白激酶。我们采用RT—PCR从水稻品种日本晴（Oryza sativa L．CV．Nipponbare）中克隆了OsCPK9基因靠近翻译起始位点下游的一段280bp的特异基因片段。将该片段以正、反向分别连接到来源于小麦TAK14基因的548bp内含子片段（NCBIaccessionnumber：AF325198）两侧,从而获得pSK．OsCPK9-RNAi的中间表达载体。进而将其克隆到植物双元表达载体pCAMBIA1301中,构建shRNAi表达载体pCAMBIA．05CPK9一RNAi。经酶切和测序鉴定正确后,利用农杆菌介导转化水稻。通过抗性筛选和标记基因hyg和gus进行PCR鉴定,筛选到20株转基因水稻植株,实时荧光定量PCR分析显示,部分转基因植株OsCPK9的表达受到显著抑制。     

转5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)基因抗草甘膦烟草和棉花的获得

以从抗草甘膦的荧光假单胞菌(Pseudomonas fluorescens)G2中克隆的、并按双子叶植物偏爱密码子改造的5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)基因aroAG2M为目的基因,用菊花Rubisco小亚基的启动子驱动,在基因的5'端加叶绿体定位信号肽,构建植物表达载体。用根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘法转化烟草(Nicotiana tabacum)获得转基因植株,PCR检测表明,外源基因已整合到烟草基因组中。转基因和非转基因植株6～8叶龄苗的叶片涂抹不同浓度的草甘膦异丙胺盐,表明转基因植株可抗4‰浓度的草甘膦,而非转基因对照植株则在2‰草甘膦时即死亡。花粉管通道法转化棉花(Gossypium hirsutum),得到3株具有草甘膦抗性的转基因植株,PCR和Southern检测显示,外源基因已整合到棉花基因组中,田间喷洒草甘膦异丙胺盐水剂,表明T1代转基因植株具有草甘膦抗性。     

抗草甘膦EPSPS转基因棉花的研究

林敏教授课题组从具有草甘膦抗性的荧光假单胞菌G2中克隆了EPSP合酶基因aroAG2，根据aroAG2的氨基酸序列，按照双子叶植物偏爱密码子人工合成aroAG2M基因。我们利用aroAG2M基因和Impactvector1.4，构建了以菊花Rubisco启动子和叶绿体信号肽驱动aroAG2M基因高效表达的植物表达载体Pim1.4-epsps-plus，以农杆菌介导法转化烟草，转基因烟草经PCR和Southern检测，证明外源基因已整合到基因组中，转基因烟草具有草甘膦抗性。以花粉管通道法转化棉花，经草甘膦抗性筛选得到T1代转基因棉花3株，转基因棉花的PCR和Southern检测结果均为阳性，说明目的基因已通过花粉管通道法导入棉花。     

甘露糖阳性选择系统的建立及在番茄转化中的应用

利用PCR克隆了大肠杆菌(Escherichia coli)6-磷酸甘露糖异构酶基因（pmi)并进行了序列测定。将该基因替换植物表达载体pAMBIA2301中的卡那霉素抗性基因，构建了以pmi为选择标记基因的植物表达载体pPMI，并导入根癌农杆菌(Agrobacterium tumefaciens)EHA105中。研究了甘露糖对番茄(Lycopersicum esculentum)生长和生根的影响及叶盘对甘露糖的敏感性。通过根癌农杆菌介导对番茄进行遗传转化，获得转化番茄再生植株，并对转化植株进行了PCR扩增检测。研究表明以甘露糖阳性选择系统在番茄遗传转化中应用是可行的。     

盆栽蔬菜土壤中汞的形态变化

以四九黄菜芯作为供试蔬菜,采用盆栽试验和连续化学萃取法-氢化物原子吸收光谱法,研究了种植蔬菜前后土壤Hg形态分布特点及蔬菜生长对Hg形态变化的影响.结果表明,外源Hg进入土壤后,在种植蔬菜前后的土壤中均主要以残渣态形式存在,蔬菜的生长明显影响土壤中的Hg存在形态.种植蔬菜后的土壤Hg形态变化表现为水溶态和腐植酸络合态明显减少,而强有机质结合态显著增加,随着外源Hg量的增加,土壤Hg形态由残渣态、强有机质结合态向交换态和碳酸盐铁锰氧化态转化,Hg的生物活性增强,蔬菜的生物量下降.除强有机质结合态和残渣态外,土壤中各形态Hg含量和总Hg量与蔬菜根、茎叶中Hg含量呈显著正相关.     

Transformation of Applied Phosphorus in a Calcareous Fluvisol

A new inorganic phosphorus (IP) fractionation scheme developed by Jiang and Gu was used in an incubation experiment to investigate the transformation of applied P in a calcareous fluvisol. The results show that after addition of common superphosphate (CSP), the Ca2-P in the soil decreased gradually and transformed largely to the less available Fe-P, Al-P and Ca8-P, rather than to the unavailable forms of Ca10-P and O-P. The different IP fractions ranked in the following order with respect to the increment by addition of CSP after 120 days of incubation: Fe-P> Al-P>Ca8-P>Ca2-P. After addition of pig manure, the content of Ca2-P in the soil increased rapidly at first and then decreased slowly, and the amount of different IP fractions accumulated after 120 days of incubation ranked in the following order: Ca2-P > Fe-P > Ca8-P > Al-P.     

Sodium Application Enhances DDT Transformation in a Long-Term Contaminated Soil

Bioremediation is an economically attractive option to remediate soil contaminated with DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and other organochlorine pesticides. However, lack of DDT bioavailability in soil presents one major obstacle to this technology particularly in soils that have been contaminated for long periods. In this work, sodium ion (Na⁺) was applied to a long-term DDT contaminated soil as Na⁺ is known to disperse clays, which would potentially release and/or expose physically protected DDT thereby enhancing DDT bioavailability. Sodium ion addition significantly increased dissolved organic carbon (DOC) levels, anaerobic bacterial numbers and the amount of DDT residues measured in soil solution. DDT transformation ranged from 95% (30—80 mg Na⁺ kg^-1 soil) to 72% (no Na⁺ added) with the optimum level of DDT transformation occurring at 30 mg Na⁺ kg^-1 soil. Higher Na⁺ levels repressed DDT transformation and this appeared to be related to lower DOC levels and flocculation of soils. The anaerobic incubation conditions employed (high water content) prevented DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] production and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane] was the major breakdown product formed. Overall it appeared that Na⁺ has potential as a cheap and safe alternative to surfactants as a method for increasing DDT transformation in contaminated soil.     

外源硫在草甸草原黑钙土中的转化

  目的  探讨元素硫添加后在草甸草原黑钙土中的氧化速率,明确土壤无机S组分的变化特征。  方法  依托内蒙古额尔古纳草甸草原硫磺添加试验平台,测定了元素S添加量（0、1、2、4、6、8、10 t hm?2）1年后的表观氧化速率及表层土壤（0 ~ 10 cm）各无机硫组分的变化。  结果  研究表明,对照中的土壤全硫含量为627.5 mg kg?1,其中无机硫和有机硫分别占2.4%和97.6%。硫添加处理的表土全硫含量分别比对照增加646.5 ~ 5182.5 mg kg?1,总无机硫分别比对照增加169.4 ~ 887.9 mg kg?1;硫添加处理中,表土中未氧化的元素硫分别为473.9 ~ 4264.0 mg kg?1,元素硫的表观氧化速率分别为173.5 ~ 271.3 g kg?1 a?1。在增加的表土总无机硫中,85.0% ~ 91.9%为（CaCl₂浸提）,7.8% ~ 17.1%为吸附性硫（ Ca(H₂PO₄)₂浸提）,0.8% ~ 1.6%为难溶性硫（HCl浸提）。土壤无机硫组分与土壤pH呈显著负相关,与土壤电导率呈显著正相关。  结论  外源添加的元素硫主要氧化为植物可利用态的易溶性硫 和吸附性硫,而氧化为难溶性硫的比例很低。     

Transformation of fertilizer P in a Vertisol amended with farmyard manure

Availability, fixation, and transformation of added P were studied in a 16-week incubation experiment with a Vertisol amended with farmyard manure in pots with 500 g soil each. P availability, as measured by Olsen P, decreased for up to 8 weeks with various rates of added P, when no manure was applied. In the presence of farmyard manure, P availability decreased during the first 6 weeks and then showed a considerable increase from the 8th week onwards. P fixation increased for up to 8 weeks with the rates of P in the absence of manure. With manure application, P fixation increased only during the first 6 weeks and thereafter decreased continuously. Thus the presence of farmyard manure shortened the period of P fixation and promoted its availability. After 16 weeks of incubation, when manure and fertilizer P were applied together, P was transformed into labile organic (NaHCO₃–P), moderately labile organic P (NaOH-P), and calcium-bound inorganic P (HCl-P). When manure was not applied. P accumulated predominantly as labile inorganic (NaHCO₃–P), moderately labile inorganic (NaOH-P), and inorganic HCl-P. The application of farmyard manure enriched long-term P fertility through NaHCO₃–P and NaOH–P and a shortterm P supply as HCl-P. All fractions except inorganic NaOH-P showed good relationships with Olsen P.     

Transformation of a nonnormal distribution of quantitative traits for breeding purposes

Journal of Agricultural, Biological and Environmental Statistics - Some continuous quantitative traits such as yield are not always normally distributed. This article proposes an underlying normal...     

Transformation of catechol in the presence of a laccase and birnessite

The transformation of naturally occurring phenols to humic polymers through oxidative coupling reactions may involve oxidoreductive enzymes and soil minerals as catalysts. There is limited information on the possible inhibitory or synergistic interactions between oxidoreductases and mineral catalysts as they participate in oxidative coupling of phenolic substrates. In this study, a ternary system was investigated, in which a fungal enzyme (Trametes villosa laccase), birnessite (δ-MnO₂), and a naturally occurring phenolic compound (catechol) were reacted together to model soil processes. Binary systems (catechol/laccase and catechol/birnessite) were included for comparison. In the absence of the mineral, T. villosa laccase (950 katal ml⁻¹) transformed 31% of catechol, whereas birnessite (1 mg ml⁻¹) in the absence of the enzyme showed a 24% catechol transformation. The percentages of catechol transformation in the binary systems did not accumulate in the ternary system; instead, birnessite and laccase tested together transformed only 36% of catechol. This suggested that birnessite had an inhibitory effect on substrate transformation by laccase catalysis. Enzyme assays indicated that inhibition was a result of enzyme deactivation by humic-like polymers produced by birnessite, and by Mn²⁺ ions released from the mineral. These observations underscore the importance of considering enzyme-soil mineral-organic matter interactions in studies of humus formation and contaminant removal.     

大豆叶绿体转化载体pJY系列的构建及其转化研究

杂交大豆是我国具自主知识产权的科研成果，具有广阔的应用前景。为解决杂交大豆制种过程中不育系易与保持系混杂的问题，我们开展了大豆叶绿体转基因的研究，目的是通过转基因的方法在不育系的细胞质中，植入筛选标记基因，以区分不育系和保持系。根据已发表的大豆叶绿体序列（GenBank X07675）设计引物，用PCR方法获得两段大豆叶绿体同源重组序列LHRR和RHRR，克隆到质粒pUC19中，并将来源于质粒pTF102的壮观霉素抗性基因aadA克隆到两段同源重组序列之间，以构建大豆叶绿体转化表达载体pJY01。 aadA基因的启动子Prrn和终止子psbA基因3’序列，分别从烟草叶绿体基因组中扩增而来。在此基础上，我们还将草丁膦抗性基因或绿色荧光蛋白与GUS的融合蛋白基因克隆到该载体上，得到pJY03与pJY04，并初步应用于烟草的叶绿体转化，获得了具壮观霉素抗性的绿色愈伤和幼苗，PCR扩增出aadA基因的条带，并在激光共聚焦扫描下检测到GFP的表达，由此证实了这一实验思路的可行性，为后期大豆叶绿体转化工作的顺利进行打下了良好的基础。     

大豆叶绿体转化载体pJY系列的构建及其在烟草上的转化研究

为解决杂交大豆(Glycinemax)制种过程中不育系易与保持系混杂的问题,开展了大豆叶绿体转基因的研究。通过转基因方法在不育系的细胞质中植入筛选标记基因,以区分不育系和保持系。根据已发表的大豆叶绿体序列(GenBankX07675)设计引物,用PCR方法获得两段大豆叶绿体同源重组序列LHRR和RHRR,克隆到质粒pUC19中,并将来源于质粒pTF102的壮观霉素抗性基因aadA克隆到两段同源重组序列之间,以构建大豆叶绿体转化表达载体pJY01。aadA基因的启动子Prrn和终止子psbA基因3′序列,分别从烟草叶绿体基因组中扩增而来。在此基础上,将草丁膦抗性基因或绿色荧光蛋白与GUS的融合蛋白基因克隆到该载体上,得到pJY03与pJY04,并初步应用于烟草(Nicotianatobacum)的叶绿体转化,获得了具壮观霉素抗性的绿色愈伤和幼苗,PCR扩增出aadA基因的条带,并在激光共聚焦扫描下检测到GFP的表达。