外源基因在转基因抗虫油菜中的遗传行为研究

为了选育出稳定的转基因抗虫油菜新品种,采用卡那霉素抗性分析和PCR检测技术对转基因抗虫油菜中Bt杀虫蛋白基因的遗传行为进行了研究.连续三代的研究结果表明Bt杀虫蛋白基因以单拷贝、杂合地整合到转基因植株(T01,T02,T03,T05,T06,T07,T08)的基因组中,并稳定地遗传给后代.纯合转基因株系杂交分析表明,在不同T0转基因植株中Bt杀虫蛋白基因整合位点是不同的,T01和T05或T03和T08的Bt杀虫蛋白基因整合位点是同源染色体的不同座位,而其他转基因植株的Bt杀虫蛋白基因整合位点是在非同源染色体上.     

外源基因在转基因抗虫油菜中的遗传行为

为了选育出稳定的转基因抗虫油菜新品种，采用卡那霉素抗性分析和PCR检测技术对转基因抗虫油菜中Bt杀虫蛋白基因的遗传行进行了研究，连续三代的研究结果表明，Bt杀虫蛋白基因以单拷贝、杂合地整合到转基因植株（T01，T02，T03，T05，T06，T07，T08）的基因组中，并稳定地遗传给后代，纯合转基因株系杂交分析表明，在不同T0转基因植株中Bt杀虫蛋白基因整合位点是不同的，T01和T05或T03和T08的Bt杀虫蛋白若整位点是同源染色体的不同座位，而其他转基因植株的Bt杀虫蛋白若整位点是在非同源染色体上。     

抗虫基因cry1Ab／Ac在水稻杂交转育后代中的表达效应

以转入了Bt基因cry1Ab/Ac的水稻品系TT51为供体,与10个恢复系杂交。通过分子检测,并结合田间的抗虫性观察,筛选出农艺性状良好的转基因纯合株系。抗虫基因在所有的杂交组合后代中按照孟德尔规律遗传,但是不同遗传背景下的表达量有很大差异。 Cry1Ab/Ac蛋白含量维持原有水平的株系对螟虫表现出良好的抗性,蛋白含量降低的株系则受到轻微的危害。纯合株系的部分农艺性状发生变异,其程度与Cry1Ab/Ac蛋白含量有关。结果为利用杂交转育培育优良抗虫水稻提供依据。     

农杆菌浸蘸柱头创制高品质抗虫棉研究

选育高品质杂交棉主要利用双亲纤维品质的平均优势,要求双亲均为高品质棉。而选育高品质抗虫杂交棉要求至少有一个高品质亲本包含抗虫基因。目前,由于缺乏高品质抗虫资源限制了高品质抗虫杂交棉选育。为了获得更多高品质抗虫棉种质,以10个高品质陆地棉品种(系)作为受体,用农杆菌菌液浸蘸棉花柱头的转化方法将Bt+Sck双价抗虫基因导入高品质陆地棉基因组,获得了63个转基因株系,通过卡那霉素抗性检测、PCR和免疫试纸检测,证明目的基因已整合到受体基因组中,该检测方法结合系统选育获得了多个转基因纯合系。对最早获得的3个纯合系进行了可靠的Southern验证、抗虫性检测和纤维品质测定,其转基因株系具有较高抗虫性和优良纤维品质。这些材料的获得为下一步进行高品质抗虫杂交棉的培育奠定了坚实基础。     

Bt基因导入形态抗蚜棉研究

野生棉种多茸毛是一种形态抗蚜性状,其抗虫机理在于阻碍害虫特别是刺吸式害虫的取食,对幼虫的移动产生机械障碍作用,对棉蚜、棉叶螨、棉叶蝉、红铃虫等棉花害虫具有抗性。通过远缘杂交方式将野生棉种的多茸毛性状转育到栽培种陆地棉上,采用花粉管通道法将Bt基因导入形态抗蚜棉,获得转Bt基因形态抗虫棉纯合系;对纯合系2种害虫的抗性检测表明,通过转基因技术可以将高抗棉铃虫特性导入到形态抗蚜棉,但转化株系仍然保留了受体材料的抗蚜性状。表明通过远缘杂交与转基因技术可以实现多茸毛抗蚜性状与转基因抗棉铃虫性状的融合,并可有效解决棉花生产上的主要虫害。     

转Bt基因油菜的抗虫性分析

利用农杆菌介导的遗传转化分别将Cry1C和Cry2A的2个单价Bt基因转入油菜(Brassica napus L.),以两个纯合的转基因抗虫油菜为亲本,通过有性杂交的方法将不同Bt基因聚合,培育双价Bt抗虫油菜,并对其抗虫性和种子品质性状进行评价。结果表明,Cry1C、Cry2A在聚合后均能稳定表达,和单价Cry1C转基因植株相比,双价株系中蛋白质含量明显降低,以Cry2A为母本的聚合株系蛋白质含量降低更多,Cry1C在遗传上存在母本效应。室内接种小菜蛾二龄幼虫结果显示,转化单价和双价聚合Bt基因的抗虫性增强,其中转化单价Cry1C的抗虫性优于双价聚合Bt基因和单价Cry2A基因的转基因植株。玻璃温室栽培转基因植株,单价Bt和双价聚合Bt基因的转基因植株能生存而非转基因植株受到严重虫害而死亡。对抗性优良的单价和双价聚合的转基因植株种子品质测定发现,与未受到虫害的受体材料双低优良恢复系7-5的含油量和硫苷含量差异不显著,从而达到抗性改良的目的。     

粳稻抗虫转基因保持系的研究

利用基因枪转化法,以优质粳稻保持系早花二B为受体,获得了含有GNA和Hyg基因的转基因植株。对T2产生的6个纯合株系进行多代潮霉素筛选、PCR分子检测及稻飞虱接种鉴定,表明标记基因在受体中稳定遗传,并高效表达,且一经纯合不再分离;而目的基因表达效果不一样,只有T-10(克隆号A5-32)表达效果最好,其抗虫性达到中抗(MR)以上水平,比受体抗性(S)明显提高。将转基因保持系与不育系早花二A回交,获得的转基因不育系高抗潮霉素,且抗性也完全纯合,表明转基因保持系的抗性基因已转化到不育系中,并得到高效表达。     

转rolC基因八棱海棠组培苗生物学特性的研究

【目的】通过研究转rolC基因八棱海棠不同株系的转基因拷贝数，在转录水平上的表达丰度，以及转基因组培苗的生物学特性，为进一步深入阐明rolC基因在八棱海棠中的表达机制和培育优良的苹果砧木奠定理论基础。【方法】以通过gus染色和PCR检测的3个转rolC基因八棱海棠株系组培苗为试材。Southern杂交鉴定rolC基因整合拷贝数。Northern杂交鉴定rolC基因在转录水平上的表达丰度。调查转基因植株组培苗在含有不同种类和浓度的植物生长调节剂的培养基上茎段增殖、叶片再生、生根能力等生物学特性；将生根后的组培苗进行炼苗，移入温室4个月后，研究株高、节间数、节间长度、叶面积等生物学特性。【结果】Southern杂交结果表明，rolC基因分别整合进入3个八棱海棠株系基因组，其中株系20a和33a分别获得了1个rolC基因拷贝，株系20b获得了2个rolC基因拷贝。Northern杂交结果表明，3个转基因株系中的rolC基因均在转录水平上得到了表达，且该基因在单拷贝株系的表达丰度高于双拷贝株系。转基因组培苗生物学特性研究结果表明：（1）3个转rolC基因八棱海棠株系茎段增殖系数、叶片再生率、生根所需外源激素浓度均显著低于对照植株，单拷贝株系的以上指标亦显著低于双拷贝株系。（2）3个转基因八棱海棠株系组培苗平均生根数显著高于转基因株系，双拷贝株系显著低于单拷贝株系；根长情况正好相反；对照植株根粗和转基因株系根粗之间均无显著差异。（3）3个转基因八棱海棠株系的株高、节间长度、节间数、叶面积均显著小于对照植株。其中单拷贝株系显著小于双拷贝株系。【结论】rolC基因整合进入3个八棱海棠转基因株系基因组，并分别在转录水平上得到表达。外源rolC基因的表达导致转基因植株体内内源激素含量和植株形态学的改变，且rolC基因的整合拷贝数对它的表达有一定的影响。     

农杆菌介导BtCry1Ac、NTHK1双价基因转化烟草植株的研究

为探讨多基因植物转化载体p096899中外源基因BtCry1Ac和NTHK1在转基因植株中的表达情况,通过根癌农杆菌介导的叶盘转化法转化野生烟草,经卡那霉素筛选,初步获得了10株转基因烟草植株.经PCR鉴定,证明目的基因已被整合到烟草基因组中.利用荧光定量PCR和ELISA分析目的基因在转录水平和翻译水平的表达量,并对转基因烟草株系进行了抗虫试验和耐盐性试验.结果表明,目的基因在转录水平和翻译水平分别得到表达,但表达量存在差异,其中2号转基因株系毒蛋白表达量最高,其含量达可溶性总蛋白的0.028 5％;饲虫试验中,部分转基因烟草株系对斜纹夜蛾的毒杀作用明显高于未转化烟草,其中2号转基因株系的抗虫性最高,饲喂第6天斜纹夜蛾幼虫死亡率达到75.56％,与Bt毒蛋白表达量相一致;在盐胁迫下,转基因烟草的叶片生长状况均好于对照(非转基因)烟草的生长.证明BtCry1Ac基因和NTHK1基因的转入在一定程度上提高了烟草的抗虫性和耐盐能力.     

多抗转基因水稻植株的获得及其抗性鉴定

利用转Xa21基因(抗白叶枯病)纯合稳定的圣稻301与转Bt基因(抗螟虫)、转car基因(抗除草剂)的纯合稳定株系GK-1杂交,依据田间抗病虫性表现,利用标记基因选择,分子标记辅助选择,获得了多抗转基因水稻植株,其中某些多抗转基因水稻株系表现出良好的农艺性状和丰产潜力。     

利用分子标记辅助选择技术培育抗虫水稻品种的研究

Bt基因是从苏云金芽孢杆菌中克隆出的针对鳞翅目害虫的抗虫基因,经过基因工程方法人工改造已经获得Cry1Ab/Ac、Cry1C等抗虫基因。以云南主栽水稻品种楚粳28为受体,以携带Bt抗虫基因的"华恢1号"、"RJ-5"和"T1C-19"分别作为供体,利用分子标记辅助选择(MAS)技术进行回交育种,选育成携带Bt抗虫基因水稻新品系。结合农艺性状表现,获得了云抗虫稻1号、云抗虫稻2号、云抗虫稻3号Bt蛋白高表达的水稻抗虫品系。这些抗虫品系为云南省抗虫水稻的遗传改良和生产应用提供了基因资源和应用基础。     

转Cry1Ab水稻纯合体快速准确的PCR鉴定方法

转Cry1Ab基因水稻Bt22为一种新型的转基因抗虫水稻,本研究介绍了一种筛选纯合转基因植株Bt22的PCR方法。根据外源序列在Bt22中插入位点的两侧旁邻序列设计引物,建立了转基因抗虫水稻Bt22品系特异的定性PCR检测方法,该方法灵敏度可达0.01 ng,在此基础上组合各引物建立了筛选转基因水稻Bt22纯合体的多重PCR方法,结果表明该方法简单高效,便于操作。     

Selection of Homozygous Cotton Lines Transformed with Two Insect-Resistant Genes

A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API-B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera, nine transgenic homozygous cotton lines with insect-resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.     

转两类抗虫基因棉花优良纯合品系的选育

 用含合成的Cry1Ac杀虫蛋白嵌合基因 (Bt2 9K)及慈菇蛋白酶抑制剂B(API B)基因表达框的双抗虫基因植物表达载体 ,通过根癌农杆菌介导转化棉花生产品种冀合 32 1,获得了一批抗卡那霉素的转化再生植株。在对转化再生植株进行PCR、卡那霉素抗性和抗虫性测定的基础上 ,经 6代筛选培育出棉铃虫抗性 90 %以上且农艺性状优良的 9个转双抗虫基因棉纯合品系。各纯合株系子代植株的抗虫性及部分序列检测结果进一步表明上述Cry1Ac嵌合蛋白基因是能稳定遗传的。这些品系具有很好的农艺性状 ,其结铃性和纤维比强度明显高于转化受体冀合 32 1,这些纯合系可直接用于生产或作为双抗虫棉种质利用。     

Cry1Ab rice does not impact biological characters and functional response of Cyrtorhinus lividipennis preying on Nilaparvata lugens eggs

One concern about the use of transgenic plants is their potential risk to natural enemies. In this study, using the eggs of the rice brown planthopper, Nilaparvata lugens, as a food source, we investigated the effects of Cry1 Ab rice on the biological characteristics and functional response of an important predator Cyrtorhinus lividipennis. The results showed that the survival ability(adult emergence rate and egg hatching rate), development(egg duration, nymphal developmental duration), adult fresh weight, adult longevity and fecundity of C. lividipennis on Bt rice plants were not significantly different compared to those on non-Bt rice plants. Furthermore, two important parameters of functional response(instantaneous search rate and handling time) were not significantly affected by Bt rice. In conclusion, the tested Cry1 Ab rice does not adversely impact the biological character and functional response of C. lividipennis.     

Thrips-mediated impacts from transgenic rice expressing Cry1Ab on ecological fitness of non-target predator Orius tantilus(Hemiptera:Anthocoridae)

Various rice lines have been genetically modified with genes from Bacillus thuringiensis(Bt) to continuously produce Bt insecticidal proteins against lepidopteran larvae. The Bt insecticidal protein constantly expresses in the plants to create an opportunity for non-target herbivores to acquire and convey the protein to their predators or parasitoids across trophic levels. This paper evaluates the effects of Bt rice(namely, Kemindao 1(KMD1) and Kemindao 2(KMD2)) expressing Cry1 Ab as compared to its non-Bt control line, Xiushui 11 on non-target predator Orius tantilus(a generalist predatory anthocorid of thrips) under laboratory and field conditions. To measure several biological parameters such as total nymphal duration and fecundity of this bug, it was reared on thrips and pollens of KMD1 and KMD2 as compared to their control under laboratory conditions. By comparison with the control, Bt rice did not significantly affect main life-history characteristics(total nymphal duration, female adult longevity, oviposition period and fecundity) of this anthocorid preying on Bt rice-fed thrips along with Bt rice pollens, except that the fecundity of this predator for KMD1 was distinctly lower as compared with KMD2 or the control. Enzyme-linked immunosorbent assay(ELISA) results showed that no Cry1 Ab protein was detected in this predator fed on thrips or rice pollen from Bt rice but was in Bt rice pollens. With the beat plate, plastic bag and color trap sampling methods, two-year field monitoring of O. tantilus abundance demonstrated that Bt rice had no significant detrimental effects on the population dynamics and seasonal average densities of this predatory anthocorid as compared with the control. Thus, it is suggested that growing our tested Bt rice(KMD1 and KMD2) producing Cry1 Ab will pose a negligible risk to the anthocorid, O. tantilus.     

用Envirologix Cry1Ab/Cry1Ac试剂盒快速测定转基因水稻Bt杀虫蛋白含量的研究

 用 Envirologix Cry1Ab/Cry1Ac平板试剂盒检测了两个转 cry1Ab的水稻品系克螟稻 1号 ( KMD1)、克螟稻 2号 ( KMD2 ) ,及未转化的对照品种秀水 11米粒中的 Bt杀虫蛋白含量。结果表明 ,该试剂盒标样制作的标准曲线相关系数为 0 .985～ 0 .998,在 0 .0 5或 0 .0 1水平上显著。同批次试剂盒的不同试验及不同批次试剂盒的测定值均差异不显著。最低检测剂量达 0 .5 ng/g,每次测试时间比 Western dot blotting方法缩短约 2 h。试验表明 ,该试剂盒是定量检测转基因水稻 Bt毒蛋白表达的一种理想产品     

Studies on Rapid Quantitative Analysis of Bt Toxin by Using Envirologix Kits in Transgenic Rice

Investigations were done on the usefulness of Envirologix Cry1Ab/Cry1Ac Plate kits for quantitative analysis of Bt toxin content in transgenic rice grains. Two transgenic rice lines: Kemingdao 1 (KMD1) and Kemingdao 2 (KMD2), transformed with a cry1Ab gene, and their parental variety, cv.Xiushui 11, were used as positive and negative samples. Results showed that the correlation coefficients as high as 0. 985 - 0. 998, significant at probability level of 0.05 or 0.01, were obtained for linear regression equations by using the appended calibrators of the kits. No significant differences were detected for values of same rice sample obtained from different trials or by using different lots of the product (kit). The detectable Bt toxin content by this method could be as low as 0.5 ng/g. The Envirologix Kit could be useful for rapid quantitative detection of Bt toxin in rice grains because of its preciseness, simplicity and time saving.     

转Bt基因水稻稻米的诱变活性研究

利用微核、精子畸变和Ames试验检测了转Bt基因水稻稻米对小鼠骨髓嗜多染红细胞(polychromatic erythrocytes,PCE)微核率(micronucleus frequency,MN%)和小鼠精子畸形发生率(sperm malformation rate)及组氨酸营养缺陷型鼠伤寒沙门氏菌株回变菌落数的影响。结果表明,在本实验条件下,Bt水稻稻米对小鼠体细胞和性细胞均无诱变活性,且无剂量效应;该受试物对TA97a、TA98、TA100和TA102菌株无论在有无代谢活化系统存在的条件下,各剂量组回变菌落数均处于正常水平。