耐除草剂转基因大豆对花粉生活力的影响

转基因作物的环境安全评价是其商业化推广前的必要环节,其基因漂移风险及其可能引起的生态环境效应是安全性评价的重要内容。为了明确转EPSPS、PAT基因抗草甘膦大豆S4003. 12和S4003. 14外源基因的逃逸风险,于大豆盛花期选取温室种植的转基因大豆、其受体品种及常规栽培大豆材料花粉,使用液体培养基培养,检测其萌发率,并于带标尺显微镜下观察其花粉粒直径。结果表明:大豆花粉刚离体后萌发活力最强,培养2 h后花粉萌发率基本达到最大值约88. 2%,随离体时间延长萌发活力随之下降,室温放置3 h后,花粉几近完全失活;与非转基因对照相比,转EPSPS、PAT基因耐除草剂大豆S4003. 12和S4003. 14的花粉离体后的生存能力无显著差异,对花粉粒直径大小无显著差异。研究表明耐除草剂转基因大豆花粉传播能力与非转基因大豆间无显著差异,通过设置隔离区可有效规避外源基因逃逸风险。     

甘蔗锌指蛋白ShZP基因正义反义植物表达载体的构建及转化烟草

根据本实验室克隆的甘蔗锌指蛋白ShZP基因cDNA序列,在开放阅读框两侧设计特异引物,通过PCR扩增引入相应的酶切位点,把甘蔗ShZP基因的编码区(大小为725bp)cDNA片段,分别以正向和反向插入到中间载体pCRBI的Prd29A启动子和NOS终止子之间,构建了其正义植物表达载体pCRBIShZP和反义表达载体pCRBIantiShZP.采用冻融法分别将pCRBIShZP和pCRBIantiShZP导入根瘤农杆菌菌株EHA105中,通过农杆菌介导法对烟草进行转化,经过12mg/L的PPT连续抗性筛选,共获得23株抗性植株,对其中的5株转正义基因烟草植株和9株转反义基因烟草植株进行PCR检测,分别获得3株转正义基因的阳性植株和4株转反义基因的阳性植株.PCR检测结果初步证明外源甘蔗锌指蛋白ShZP基因已成功整合到烟草基因组中,这一研究为植物抗逆基因工程研究打下了一定的基础.     

转Bar基因抗除草剂两系杂交早稻恢复系Bar 68-1的培育研究

通过基因枪转化方法将抗除草剂基因Bar转入早籼稻两系恢复系D68中,育成了转基因抗除草剂恢复系Bar 68-1和杂交早稻组合香125S/Bar 68-1;Southern杂交检测和除草剂抗性鉴定结果显示,它们具有抗除草剂特性并能稳定遗传。转基因抗除草剂杂交稻组合香125S/Bar 68-1和对照香两优68(香125S/D68)相比,株高显著降低,直链淀粉含量极显著降低,其它农艺、品质和产量性状没有明显变化。     

农杆菌介导大豆子叶节转化cry1Iem基因

利用农杆菌介导法,以东北地区种植的9个大豆基因型和国外品种Williams82的子叶节为外植体,将抗虫基因cry1Iem转入栽培大豆品种中。共转化外植体1 459个,获得84棵再生植株。采用除草剂叶片涂抹法、PCR及Bar蛋白试纸条检测法对得到大豆再生植株进行鉴定,转cry1Iem基因大豆T_0阳性植株为61株。对部分T_1转基因植株的遗传分析表明,外源基因能够稳定遗传到下一代。通过对部分T_1阳性转基因植株进行Southern blot分析,证明目的基因片段均已整合到受体大豆的基因组DNA中,单拷贝率为22%左右。对获得的部分T_2材料进行了室内抗大豆食心虫鉴定,有2份转基因材料抗性较对照显著提高。     

杂草环境下转EPSPS基因大豆NZL06-698的生态适应性研究

为研究转EPSPS基因大豆NZL06-698在杂草环境中的生态适应性,试验设置杂草处理模拟野外环境,研究转基因大豆NZL06-698(GT698)的生存竞争能力以及外源EPSPS蛋白表达情况。结果表明:在相同处理下转基因大豆GT698的株高(R8期)、百粒重显著高于亲本大豆蒙豆12(MD12),但单株产量、单株籽粒数、结实率显著低于亲本大豆,说明外源基因的存在并未增强转基因大豆的生存竞争力,且转基因大豆在野外的生存竞争能力与亲本大豆相比较弱。试验注意到,杂草环境对转EPSPS基因大豆的生长有明显的限制作用,杂草处理下转基因大豆的单株产量、单株籽粒数、百粒重、结实率等指标均显著低于对照处理。ELISA检测结果表明,转基因大豆叶片及籽粒中外源EPSPS蛋白在杂草处理及对照中均正常表达,且两种处理下的EPSPS蛋白表达量无显著差异。以上结果表明杂草环境中转基因大豆的EPSPS蛋白正常表达,正常提供抗草甘膦性状,但是外源基因的存在并没有增加转基因大豆的生存竞争能力,且转基因大豆的生长受到杂草环境的显著抑制,由此得出结论:与亲本大豆相比,转EPSPS基因大豆NZL06-698在野外环境中生态适应能力较弱。     

转Bt基因抗虫棉毒蛋白检测方法及其表达规律研究进展

研究转Bt基因抗虫棉毒蛋白的表达规律及其对标靶害虫毒杀性,对评价Bt基因成功转化、转Bt基因抗虫棉选育和推广、目标害虫抗性预测以及生物安全评价管理都具有重要意义。概述了国内外转Bt基因棉毒蛋白检测方法及其表达变化规律,并提出引起Bt毒蛋白表达变化的原因及改进对策。     

转Bt基因棉的抗虫性遗传及其生化基础研究

本文从卡那霉素抗性鉴定、抗虫性鉴定Bt毒蛋白含量测定等三个方面对转Bt基因棉的抗虫性遗传及其生化基础进行了研究。结果表明，转Bt基因棉美棉33B和GK-12对棉铃虫具有显著的抗性。盛蕾期饲喂美棉33B、GK-12棉抹顶端叶片72h后，初孵棉铃虫幼虫死亡率分别为86．8％、75．1％，对照TM-1、泗棉33号、苏棉12号三个常规品种(系)初孵幼虫死亡率分别为10.9％、13．9％、9．2％。美棉33B、GK-12盛蕾期功能叶片Bt毒蛋白含量分别为每克鲜重836.68纳克、682．56纳克。饲喂美棉33B、GK-12与常规品种(系)杂种一代棉抹顶端叶片72h后。初孵棉铃虫幼虫平均死亡率分别为84．1％、77．2％，两个转Bt基因棉品种与常规棉品种(系)杂种一代功能叶片的Bt毒蛋白含量平均值分别为每克鲜重820．58纳克、683．77纳克。转Bt基因棉与常规棉杂种一代的抗虫性表现及其生化基础与转Bt基因棉非常接近，杂种二代群体抗、感虫植抹的分离比例符合3：1，回交世代BC1群体抗、感虫植株的分离比例符合1：1，转Bt基因抗虫性状的遗传是受一对完全显性基因控制的。Bt基因与NPTⅡ基因是紧密连馈或完全连锁的。段毒蛋白表达量按照一对显性基因的表达方式在转Bt基因棉杂交后代中进行传递。不存在基因的剂量效应。三种鉴定方法都能对玫基因进行有效的追踪检测，且三者之间具有高度的一致性。并对国产转Bt基因棉GK-12的抗虫性不及美棉33B的原因进行了探讨。     

抗除草剂两系杂交水稻香125S/Bar68-1栽培试验初报

2002-2003年对转bar基因抗除草剂两系杂交水稻香125S/Bar68-1进行了栽培应用初步试验,结果表明,香125S/Bar68-1去杂提纯靠专用除草剂,苗期喷施1次Basta对保证其纯度和提高产量是至关重要的;各种育秧方式上均可喷施,喷药质量与环境条件是关键,旱育软盘秧有利于喷施Basta操作和秧苗均匀受药,其喷施效果最好.此外,对转bar基因两系法杂交水稻应进一步研究的问题进行了讨论.     

茶树无色花色素还原酶基因克隆及表达分析

采用EST测序技术和3′RACE技术,获得了茶树儿茶素代谢途径中的一个重要酶—无色花色素还原酶的全长基因,在GenBank的登录号为EF205148,序列全长1 301 bp,其中开放阅读框长1 029 bp,编码342个氨基酸,3′端有一个明显的多聚腺苷酸加尾信号,推测的蛋白分子量约为37.5 kD,理论等电点为5.81。将该基因重组到表达载体pET-32a(+)中进行原核表达,经IPTG诱导、SDS-PAGE检测,结果表明茶树无色花色素还原酶基因能在大肠杆菌BL21中表达,电泳检测到一条大约60 kD的外源蛋白,与预测的融合蛋白分子量相符。同源性分析表明茶树无色花色素还原酶基因编码的氨基酸序列与其他植物具有较高的相似性,例如与亚洲棉、草莓和葡萄的相似性分别为70%、68%、71%。利用半定量PCR技术检测总儿茶素含量不同的4个茶树品种中与类黄酮合成相关的黄酮醇合成酶(FLS)、二氢黄酮醇-4-还原酶(DFR)、无色花色素还原酶(LAR)、花色素合成酶(ANS)等7个基因的表达情况,结果表明DFR和LAR基因的表达量与茶树中总儿茶素含量呈一定的相关性,而其他基因则与其相关性不大。     

转Bt基因玉米的生殖毒性评价

将昆明种小鼠按照体重随机分为5组(分别为高、中、低剂量组、阳性对照组和正常对照组),进行显性致死试验和精子畸形试验,评价转Bt基因玉米的生殖毒性。结果显示,饲喂转基因玉米的3组小鼠各项指标间的差异不显著(P0.05),与正常对照组相比亦无显著差别(P0.05),与阳性对照组差别显著(P0.05)。说明使用添加不同剂量转Bt基因玉米的饲料饲喂小鼠不会造成对雄性小鼠的生殖毒性。     

Bar基因及转Bar基因水稻的研究和利用

Ｂａｒ基因是广泛应用于基因工程育种中的除草剂抗性基因,也是遗传转化中的标记基因。综述了Ｂａｒ基因的作用机制、Ｂａｒ基因在转基因植物中的表达和遗传及其对转基因水稻农艺性状的影响,讨论了转Ｂａｒ基因水稻在杂种优势利用中的途径和前景     

一种新的水稻香味基因功能标记的开发与应用

香味是影响稻米品质的一个重要性状,Badh2基因突变是水稻产生香味的主要原因,水稻香味产生主要有Badh2基因第2、4、5、7、8外显子突变类型,其中研究最多的是第7外显子突变类型.为了提高水稻香味性状检测的准确性、安全性和效率,本研究根据水稻Badh2基因突变(第7外显子8 bp缺失、3 bp突变)的序列设计开发了3...     

Recent Advances in Development of Herbicide Resistant Transgenic Hybrid Rice in China

In addition to weed control in direct seeding field of hybrid rice, herbicide resistance genes were used by Chinese scientists to increase and identify the purity of hybrid seeds, and to realize the mechanization of hybrid seed production. The elite restorer lines, such as Minghui 63, R752, T461, R402, D68 and E32 were transformed directly with herbicide resistance genes, in which D68 and E32 are restorer lines of two-line system and the others are of three-line system. Because almost all of important restorer lines are indica varieties and are recalcitrant in transformation, many herbicide resistant near-isogenic restorer lines were developed by sexual hybridization of indica and japonica varieties and backcross with indica restorer lines later, such as Ce 64, Minghui 63, Teqing, Milyang 46, R402 and 9311, in which 9311 is a restorer line of two-line system. The elite photoperiod-sensitive/thermo-sensitive genic male sterile lines, such as Pei'ai 64S, P88S, 4008S and 7001S, were transformed with herbicide resistance genes. A few herbicide resistant male sterile lines were developed through sexual hybridization and subsequently systemic selection, such as Bar1259S, Bar2172S, 05Z221A and 05Z227A. With the employment of herbicide resistant male sterile lines or herbicide resistant restorer lines, a few herbicide resistant hybrid rice combinations were developed, such as Xiang 125S/Bar 68-1 and Pei'ai 64S/Bar 9311. Based on herbicide resistance, the research was marching on to investigate the parental lines of hybrid rice with insect resistance, drought tolerance, etc.     

抗除草剂基因导入培矮64S实现杂交水稻制种机械化的初步研究

 利用农杆菌介导的高效遗传转化技术，成功地将Bar基因转入两系杂交稻的不育系培矮64S。转基因植株具有明显的Basta抗性，分子检测进一步证明Bar基因已整合到水稻染色体上。模拟制种的成功，表明机械化制备杂交水稻种子是可行的。讨论了实现机械化制备杂交水稻种子所需解决的各种问题。     

一个除草剂安全剂AD-67诱导的水稻基因片段的克隆和分析

为寻找化学诱导表达基因,利用mRNA差异显示方法,从除草剂安全剂AD 67（苯叉酰胺）诱导处理的水稻品种9311的幼苗中筛选分离得到1个诱导表达增强的cDNA片段。该片段在处理后6 h表达明显增强,直到第5天仍然有表达。从poly gel上回收片段并测序,核酸同源序列分析显示它与水稻多个EST和cDNA序列一致性达到100%,氨基酸序列分析表明它与水稻、四膜虫、隐球酵母等真核生物的Yippee基因相似性达60%~84%。序列经延伸得到831 bp的cDNA,由此设计引物并进行RT PCR分析,结果说明该基因在除草剂安全剂AD 67诱导后表达增强。     

Nutritional evaluation of genetically modified rice expressing human lactoferrin gene

The nutritional quality of a new strain of genetically modified rice (Oryza sativa L.) expressing human lactoferrin gene (hLF rice) was evaluated on the basis of components, nutrient digestibility in pigs, protein availability in rats and protein digestibility corrected amino acid scores (PDCAAS), and compared to its parental rice variety (PR rice). Although exogenous human lactoferrin gene was introduced, it did not interfere with the digestibility of protein, carbohydrates, fat and crude fiber. The revised protein efficiency ratio of hLF rice was increased to 2.50, which was significantly higher than that of PR rice. The PDCAAS of PR rice was 52.66 and its first limiting amino acid was lysine, while the PDCAAS of hLF rice was improved to 54.06 and its first limiting amino acid was tryptophan. Thus, it can be concluded that the nutritional quality of hLF rice is superior to PR rice according to the results of availability experiments and PDCAAS, and the hLF rice would be a superior strain of rice based on protein composition of the grain.     

基因枪法介导转人工合成Rs-AFP2基因小麦的获得和检测

萝卜（Raphanus sativus）抗菌肽Rs—AFP2在体外强烈抑制小麦赤霉病菌（Fusarium graminenrum）、黄色镰孢菌（Fusarium culmorum）、烟草赤星病（Alternaria longipes）的菌丝生长。为了研究Rs-AFP2在小麦抗病育种上的应用潜力,人工合成了Rs-AFP2基因。通过基因重组技术（包括限制性内切酶酶切和连接）,将人工合成的R以FP2基因替代单子叶高效组成型表达栽体pAHC25中的GUS基因,构建了R￡AFP2基因的单子叶高效表达栽体pUAFP2。该栽体除携带受Ubiquitin启动子控制的Rs-AFP2基因表达盒外,还具有1个受Ubiqutin启动子控制的Bar基因表达盒,后者可为后续利用除草剂Bialaphos筛选转化再生植株提供抗性;采用基因枪法轰击小麦品种扬麦12、济麦19、晋麦47和豫麦34幼胚共4042个,经过2～3次Bialaphos筛选,最终获得扬麦12再生植株316株;利用高效表达栽体pUAFP2的Bar和Rs-AFP2基因的特异引物对上述成活的转化植株进行PCR检测,获得Bar和Rs-AFP2基因均为阳性的植株58株,转化率为1．43％。     

Prokaryotic Expression of Rice Ospgip1 Gene and Bioinformatic Analysis of Encoded Product

Using the reference sequences of pgip genes in GenBank,a fragment of 930 bp covering the open reading frame(ORF) of rice Ospgip1(Oryza sativa polygalacturonase-inhibiting protein 1) was amplified.The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani,the causal agent of rice sheath blight,and reduced its polygalacturonase activity.Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point(pI) of 7.26.The...     

The effect of wild type P53 gene transfer on growth properties and tumorigenicity of PANC-1 tumor cell line

BACKGROUND: The p53 protein function is essential for the maintenance of the nontumorigenic cell phenotype. Pancreatic tumor cells show a very high frequency of p53 mutation. To determine if restoration of wild type p53 function can be used to eliminate the tumorigenic phenotype in these cells, pancreatic tumor cell lines, PANC-1 and HTB80, differing in p53 status were stably transfected with exogenous wild type p53 gene. METHODS: The transfection was performed using Polybrene/DMSO-Assisted Gene Transfer method. The wild type p53 gene integration into genomic DNA was detected by Southern blot and PCR. Furthermore, the expression of wild type p53 protein was detected in selected clones by immunohistochemistry and Western blot. RESULTS: While HTB80 cell line failed to produce a stable p53 expressing clone, the PANC-1 cells produced stable lines. Following characterization of clones, the growth rate and tumorigenicity of PANC-1 wild type p53 clones were compared to the control cells. Our data showed that the expression of wild type p53 decreased the growth rate of PANC-1 cells. It was also observed that the expression of wild type p53 in PANC-1 cells suppressed its potential for tumor formation in nude mice, completely, while the parental line leads to the formation of a relatively large tumor. CONCLUSION: Our results suggest that gene therapy based on restoration of wild type p53 protein function in pancreatic tumor cells with high amount of mutant p53 is a feasible option in pancreatic cancer treatment.