表达豇豆胰蛋白酶抑制剂转基因杨树的虫试试验

将转豇豆胰蛋白酶抑制剂基因的三倍体毛白杨回交杂种 [(毛新杨×毛白杨 )×毛白杨 ]]于春季萌发前截干 .用萌条上的树叶对 3种主要杨树害虫 :天幕毛虫 (Malacosomadisstria)、舞毒蛾 (Lymant riadisparL .)和杨雪毒蛾 (StilpnotiacandidaStaudinger)进行离体虫试试验 ,年底测量各转基因株系萌条树高和地径 ,以衡量其生长性状 .结果发现 ,与对照相比 ,转基因株系的树高和地径没有显著降低 ,说明外源基因的导入没有影响转基因杨树的速生特性 ,相反 ,TG0 4的树高和地径较对照有明显的提高 ,这可能是外源导入后促进转基因杨树生长的结果 ;转基因植株均能显著提高害虫的死亡率 ,降低幼虫的排粪量、蛹重、体重增加量和成虫的产卵量 ;其中 ,TG0 7、TG0 8和TG713个转基因株系的抗虫特性较为突出 ,表明CpTI基因在它们的树体内表达较为活跃和稳定     

双抗虫基因对三倍体毛白杨的转化和抗虫性表达

建立三倍体毛白杨叶片再生体系,用部分改造BtCry1Ac基因与慈菇蛋白酶抑制剂(API)基因构建的双抗虫基因表达载体,通过农杆菌介导法转化三倍体毛白杨无性系,在含卡那霉素50 mg·L-1的分化培养基上诱导产生不定芽的叶片占18%,在生根培养基上对转基因植株做进一步筛选,获得50个转化再生株系.PCR检测表明:80%的植株呈现阳性反应,部分株系进行Southern Blot检测,证明外源基因已经插入杨树基因组中.用Bt毒蛋白抗血清进行ELSA检测,结果表明:7个转基因株系都有Bt杀虫蛋白表达,表达量最高的株系约占叶总可溶性蛋白的0.016 1%.用经分子生物学检测的28个转基因株系叶片进行舞毒蛾和杨扇舟蛾幼虫饲虫试验,结果表明:不同株系幼虫杀死率明显不同,有39.3%的株系对舞毒蛾和杨扇舟蛾幼虫的致死率在80%以上;25.0%的株系对两种害虫的幼虫死亡率均在60%～80%之间;另有35.7%的株系幼虫死亡率在50%以下,有些株系基本未表现出抗虫性.同时具高抗虫性的转基因株系能够明显抑制存活幼虫的生长和发育.     

通过RNAi技术抑制杨树c3h基因表达提高糖转化效率

利用克隆得到的毛白杨c3h1基因构建其RNAi抑制表达载体,通过根癌农杆菌介导的叶盘法转化银腺杨无性系84 K,Realtime PCR检测表明其转基因株系323、325和322中c3h1基因表达量较野生型植株分别下调89.04%、82.22%和68.38%;茎横切片组化染色和显微结构观察表明转基因植株木质部发育和木质素沉积方式发生了改变;木质素、纤维素含量测定及苯酚—硫酸法总糖含量与HPLC法可溶性总糖和单糖含量检测结果表明:转基因植株木质素含量平均降低23.00%,最高可达39.71%;酸前处理效率最高提高了41.39%;未经酸处理直接酶解的糖化效率是对照植株的2.34～2.72倍,322株系和323株系比对照植株经酸前处理后再酶解的糖化效率高出81.18%和375.53%。     

干旱胁迫对转SacB基因、转BADH基因的美丽胡枝子的影响

以同时培育的转SacB基因、转BADH基因及未转基因的美丽胡枝子盆栽苗为材料,研究干旱条件下3种试材的脯氨酸、甜菜碱、可溶性糖、过氧化物歧化酶、丙二醛、叶绿素含量的变化.结果表明:在正常土壤水分状态下,3种试材的这几项指标含量没有明显差异,但随着干旱胁迫强度的增加,2类转基因的美丽胡枝子所积累的脯氨酸、可溶性总糖量明显高于非转基因植株,在甜菜碱积累方面.转入BADH基因的美丽胡枝子要强于非转基因植株及转入SacB基因的植株.尽管在干旱状态下,3种试材的SOD活性增强,但2种转基因植株的SOD活性并没有明显大于非转基因植株.不论是转入SacB基因,还是转入BADH基因,转基因植株明显可抑制丙二醛在植物体内的快速积累和叶绿素的降解.田间观测的结果也进一步表明:转基因植株的耐旱性明显强于非转基因植株.     

转AmGS基因红叶石楠的分子检测及抗寒性分析

对转AmGS基因(从沙冬青中克隆的肌醇半乳糖苷合成酶基因)的红叶石楠植株进行多项分子检测(PCR,Southern,RT-PCR),结果表明AmGS基因已经整合到转基因株系R6和R7的基因组DNA中,并检测到转录水平上的表达。随后,经对R6和R7两个转基因株系进行连续6代芽切扩繁继代株系的PCR检测,发现导入的AmGS基因传递到所有芽切扩繁后代植株中。植株抗寒能力试验结果表明,在相同低温处理条件下,转基因株系均比未转基因植株的存活率要高。相对电导率测定结果表明,随着处理温度的降低,2个转基因株系相对电导率升高的程度明显低于未转基因的对照植株。低温半致死温度(LT50)测定结果表明,2个转基因株系(R6和R7)的LT50明显低于未转基因的对照植株。上述结果说明导入的AmGS基因提高了转基因株系的抗寒性。     

一个毛白杨MADS盒基因的克隆与功能分析

利用PCR技术,从毛白杨花序材料中分离出一个MADS盒基因PtSEP3.蛋白序列比较和同源树分析表明,该基因与拟南芥SEP3同源.表达分析显示PtSEP3不仅在成年植株雌蕊和雄蕊中表达,也在幼茎、幼叶和顶芽中表达.在35S启动子驱动下,PtSEP3超表达的毛白杨转基因植株发生了明显的形态变化,表现为叶序不规则,在同一节间常数枚集生;并且叶片基部形态与野生型的心形不同,大部分为楔形.在离体条件下,在芽分化培养基上,PtSEP3转基因株系的根和叶外植体可以诱导发生带有花柱和子房室的子房状结构.试验结果说明PtSEP3参与了毛白杨花器官的发生,并可能在维持茎端分生组织分化出正常形态的叶片中也起着作用.     

过量表达LAR3和Bbchit1以提高毛白杨对叶枯病的抗性

利用转基因技术将多种抗病基因共同转入毛白杨中以提高其抗性,从而获得毛白杨抗病新品种是目前解决杨树真菌病害的主要研究方向之一。本研究通过根癌农杆菌介导的二次遗传转化,将来源于球孢白僵菌几丁质酶基因Bbchit1转入过量表达无色花色素还原酶基因LAR3转基因毛白杨中,实时定量PCR显示Bbchit1与LAR3均能有效表达,离体抗病试验显示Bbchit1+LAR3共表达转基因毛白杨细胞粗提液对杨树叶枯病菌具有明显抑制作用,进一步将叶枯病菌接种在转基因和非转基因毛白杨叶片上培养30天,转基因植株的感病面积均低于非转基因植株且Bbchit1+LAR3共表达转基因株系抗病效果更明显。上述抗病试验结果表明:LAR3和Bbchit1在杨树中共表达可提高其对叶枯病的抗性。     

利用转基因番茄生产葡激酶的研究

利用农杆菌介导的方法,将葡激酶(Staphylokinase,SAK)基因导入番茄中。经PCR、Southern杂交和Northern杂交检测,葡激酶基因已整合到再生番茄植株基因组中,共获得8个转基因株系。经ELISA检测,转基因番茄的果实和叶片均能表达SAK蛋白,SAK蛋白在果实和叶片可溶性蛋白中的比例最高分别为3.42%和2.47%。转基因番茄中的SAK蛋白具有一定的溶栓活性,溶栓比活力为3 866 AU·mg-1。     

转基因杨树对美国白蛾幼虫中肠保护酶系统的影响(英文)

以转Bt基因欧洲黑杨(P.nigra L.)和转CpTI基因毛白杨(Populus tomentosa)叶片饲喂4-5龄美国白蛾（Hyphantria cunea Drury）幼虫，对其体内保护酶系统活性进行测定。结果表明，饲喂两种转基因杨树叶片的幼虫中肠保护酶表现出相似的变化规律，SOD、CAT和POD三种酶的活性在饲喂后数小时内逐渐增加，某一时刻达到最高值，此后突然下降。饲喂转Bt基因杨树叶片的幼虫，其中肠SOD、CAT活性峰值出现在饲喂后的24小时，POD活性峰值出现在饲喂后的12小时；饲喂转CpTI基因杨树叶片的幼虫，其中肠三种保护酶活性高峰出现时间均较前者滞后12小时。本文还比较了饲喂两种转基因叶片不同中毒程度的美国白蛾幼虫体内保护酶活性，发现不论饲喂那种转基因叶片，中毒较轻者其体内保护酶活性显著高于中毒较重者，这种差异在饲喂CpTI叶片的处理株表现尤为明显。     

多拷贝rol基因对转基因杨树生长及内源激素的影响

对转入多拷贝rolB、rolC基因的三倍体毛白杨进行高生长量、生根率和内源激素(IAA、ABA)含量等指标的测定,以研究多拷贝rol基因在转基因植物中的表达。结果显示:转Ri质粒三倍体毛白杨再次分别转入rolB、rolC基因后,部分株系试管苗的生长受到了抑制,但各株系生根率均有不同程度提高;rolB基因转化植株的内源IAA平均含量高于rolC基因转化植株,其IAA/ABA值亦高于rolC基因转化植株。试验结果表明rol基因的多拷贝促使转化植株快速大量生成毛状根。     

Identification of CpTI Gene Integration for 2-year-old Transgenic Poplars at DNA Level

The putative transgenic hybrid triploid poplars [(P. tomentosa P. bolleana) P. tomentosa] with CpTI gene have been outplanted in test field for 2 years. Although the authors previous studies have proved that they are highly resistant to 3 species of poplar-threatening insect pests and contain high content of CpTI protein in foliage, incorporation status of foreign CpTI gene in poplar genome is uncertain. In this present study, the incorporation of foreign CpTI gene in genome of 5 transgenic poplars was confirmed by PCR and Southern blotting analysis. DNA amplification showed that there were clear DNA bands of about 450bp specific to CpTI gene in transgenic lanes, while no corresponding band in non-transgenic lane was observed. Correspondingly, clear DNA hybridization signals and no signal were exhibited on film for DNA Southern blotting analysis in transgenic lanes and non-transgenic lane, respectively, which further confirmed the stable integration of foreign CpTI gene in genome of 2-year-old transgenic poplar.     

Assessment of Rhizospheric Microorganisms of Transgenic Populus tomentosa with Cowpea Trypsin Inhibitor (CpTI) Gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for microorganism culture, counting assay and PCR analysis to assess the poten-tial impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1:10 000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem.     

Identification of <Emphasis Type="Italic">CpTI</Emphasis> gene integration for 2-year-old transgenic poplars at DNA level

The putative transgenic hybrid triploid poplars [(P. tomentosa × P. bolleana) × P. tomentosa] with CpTI gene have been outplanted in test field for 2 years. Although the authors’ previous studies have proved that they are highly resistant to 3 species of poplar-threatening insect pests and contain high content of CpTI protein in foliage, incorporation status of foreign CpTI gene in poplar genome is uncertain. In this present study, the incorporation of foreign CpTI gene in genome of 5 transgenic poplars was confirmed by PCR and Southern blotting analysis. DNA amplification showed that there were clear DNA bands of about 450bp specific to CpTI gene in transgenic lanes, while no corresponding band in non-transgenic lane was observed. Correspondingly, clear DNA hybridization signals and no signal were exhibited on film for DNA Southern blotting analysis in transgenic lanes and non-transgenic lane, respectively, which further confirmed the stable integration of foreign CpTI gene in genome of 2-year-old transgenic poplar.     

Assessment of rhizospheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibitor ( CpTI ) gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1:10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem. [Supported by the National Project in Transgenic Plant and Application (Grant No. J2002-2003)]     

转基因杨树对杨小舟蛾幼虫解毒酶活性的影响

用转Bt单基因和转Bt CpTI双价基因杨树叶片饲喂杨小舟蛾,幼虫中肠酯酶的活力在饲喂初期显著升高,但在饲喂一定时间后受到抑制:转Bt单基因杨树在饲喂24 h开始受到抑制,48 h受到明显抑制,较对照下降了15.8%;而转双价基因杨树在饲喂12 h受到明显抑制,较对照下降了38.1%.转双价基因杨树对中肠酯酶的抑制作用大于转Bt单基因杨树.转Bt单基因杨树对幼虫中肠羧酸酯酶的抑制能力不强,而转双价基因杨树对中肠羧酸酯酶的抑制作用大于转Bt单基因杨树,饲喂12 h后活力开始受到抑制,饲喂24、36、48 h的活力分别较对照下降了33.4%、22.5%、29.6%,与对照均有显著差异.转基因杨树主要通过抑制幼虫中肠酯酶和羧酸酯酶这2种解毒酶活性而干扰昆虫正常的生理代谢,从而起到毒杀害虫的作用.     

转Bt基因南林895杨抗虫性及对土壤微生物影响分析

以转Bt基因南林895杨株系B1、B4、B17、B21扦插苗为试验材料,分析其在室内和野外自然条件下对靶标害虫杨小舟蛾幼虫的抗虫性。结果表明:转Bt基因杨树4个株系均有一定的杀虫活性,其中株系B21对杨小舟蛾1龄幼虫12 d校正死亡率高达95.3%;虫试表明转Bt基因杨树各株系扦植苗在野外自然条件下的12 d幼虫校正死亡率8月份为35.0%88.8%,9月份为40.5%95.3%。用转Bt基因杨树叶片饲养杨小舟蛾,对照植株杨小舟蛾幼虫化蛹率为83.3%96.0%,而转Bt基因株系幼虫化蛹率为8.0%76.7%,二者有显著差异。转Bt基因杨树对杨小舟蛾幼虫的生长发育有抑制作用,饲喂8 d后害虫取食量、体质量增长速率显著低于对照。此外,为了解转Bt基因杨树对土壤微生物的影响,对转Bt基因杨树株系及对照进行了根际土壤微生物可培养类群的分析,初步结果显示:转Bt基因杨树根际土壤微生物中细菌、真菌、放线菌的数量与对照比较无显著差异。     

两种杨树对铅和铅-镉复合胁迫的生理响应

不同品种的杨树对重金属污染土壤的修复和耐受性存在显著差异,为了解不同品种的杨树对铅(Pb)和镉(Cd)的耐抗特性,以南林95杨和南林895杨为研究对象,研究其在不同浓度Pb胁迫和PbCd复合胁迫下的生理响应,对其根系活力、过氧化氢酶(CAT)活性、可溶性蛋白质和叶绿素含量等指标进行对比分析,并采用主成分分析和隶属函数法综合评价2种杨树对Pb和Cd胁迫的耐受性。结果表明,南林95杨和南林895杨在Pb胁迫和Pb-Cd复合胁迫下,各生理指标的变化趋势大致相同,均表现出较强的耐受性;在Pb胁迫和Pb-Cd复合胁迫下,南林895杨的根系活力和CAT活性均强于南林95杨,但其叶绿素含量少于南林95杨。Pb胁迫下,南林95杨的可溶性蛋白质含量略高于南林895杨,在Pb-Cd复合胁迫下则相反。综合评价结果表明,南林895杨对重金属Pb和Cd的耐受性强于南林95杨,具有更好的修复前景。     

Laboratory and field evaluation of the transgenic Populus alba × Populus glandulosa expressing double coleopteran-resistance genes

Expression of the two coleopteran-resistant proteins (Bt-Cry3A and oryzacystatin I) was detected in the leaves of field-grown transgenic poplar (BOGA-5) in two or three subsequent years. The BOGA-5 contained ～10 μg g(-1) of Cry3A over the individual years with no detection in the control, and protein extracts from BOGA-5 displayed a higher reduction in papain activity (～42%) compared with ～21% in the control. Laboratory feeding experiments showed that the total mortality of the target pest Plagiodera versicolora (Coleoptera, Chrysomelida) larvae fed with BOGA-5 leaves was 76.7%, significantly higher than that of the control (P相似文献