Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene

Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluated using PCR amplification, test paper evaluation, insect resistance evaluation in both the laboratory and paddy fields, nursery evaluation of rice blast resistance and pedigree selection of agronomic traits. Larval mortalities on Bt5198 and Bt Minghui 63 were 100% when rice culms were inoculated with the eggs of the striped stem borer （SSB） in the laboratory. Bt5198 was highly resistant against SSB and the yellow stem borer （YSB） under field conditions. The F1 hybrids derived from Bt5198 and four cytoplasmic male sterile （CMS） lines were also highly resistant to SSB and YSB and had a significant heterosis. Two-year evaluation of rice blast resistance confirmed that the resistance levels of Bt5198 to leaf blast and neck blast were similar to those of Chenghui 177 and significantly better than those of Bt Minghui 63. Seed germination ability and pollen yield of Bt5198 were similar with Chenghui 177, suggesting that the introduction of the Bt gene into the new restorer line had no significant effects on seed vitality or the yield of seed production. To identify the presence of the Bt gene, it was effective to combine test paper examination with the evaluation of insect-resistance, both in the laboratory and under field conditions.

Genetic Transformation of Brassica campestris L.ssp.pekinensis via Agrobacterium-with Bt Gene

By means of Agrobacterium-mediated transformation, 43 kanamycin-resistant buds of Chinese cabbage were got. PCR, PCR-Southern blot and dot blot analysis were used to identify and characterize the putative transgenic plants. 26 plants had the predicted bands of the fragment of npt Ⅱ gene. Insect bioassays of 4 transformants showed that toxic protein had been translated and the translation levels were different among these transformants.

Expression Characteristics and Borer Resistance of Bt Gene of Transgenic cry1C~* Super Japonica Rice

[Objective] The paper was to study the expression characteristics and borer resistance of Bt gene of Songjing 9 （1C^＊）. [Method] With super japonica rice Songjing 9 as the receptor, transgenic crylC^＊ super japonica rice Songjing 9 （1C^＊） regulated by ubi promoter was created by agrobacterium-mediated genetic transformation method. Ten strains of transgenic super japonica rice formed from different transformation events were planted in the field; the Tt-mRNA of leaf, stem-sheath and young spikelet at booting stage were detected by real-time fluorescent quantitative PCR; the Bt protein content of leaf, stem-sheath and young spikelet and harvested brown rice were detected by enzyme-linked immunosorbent assay （ELISA）; the white head rate damaged by Chilo suppressalis was manually investigated. [Result] The Bt-mRNA and Bt protein content of various organs and Bt protein content of mature brown rice at booting stage of Songjing 9 （1C^＊） significantly differed among strains; there was no correlation relationship in Bt-mRNA and Bt protein content between leaf and stem-sheath, between leaf and young spikelet, and between stem-sheath and young spikelet; there was no relation between Bt-mRNA or Bt protein content of leaf, stem-sheath and young spikelet and Bt protein content of brown rice; there was positive correlation between Bt-mRNA and Bt protein content in the same organ such as leaf, stem-sheath and young spikelet at booting stage; such strains as Songjing 9 （1C^＊）, whether the Bt gene expression was high or low, showed good resistance against C.suppressalis. This indicated that there were differences in Bt gene expression in different strains of transgenic cry1C^＊ super japonica rice or different organs of the same strain; there was no relation in Bt gene expression among different organs; the Bt-mRNA and protein content of the same organ usually had consistent expression; the Bt gene expression of various organs had nothing to do with the Bt protein content of brown rice. [Conclusion] The Bt gene and its expression cassette can meet the requirement of cultivating japonica rice varieties against stem borers.

Agrobacterium-meditated Genetic Transformation of an Upland Cotton(Gossypium hirsutum cv Coker 310) Using a Novel Bt Gene Cry2Ac

The development of transgenic cotton varieties resistant to bollworms has been a major success of applying plant genetic engineering technology to agriculture,evidenced by phenomenal increase in

Introgression of Bt Genes in Novel Germplasm and Contribution to Indian Cotton Economy

Emergence of transgenic Bt-cotton technology has opened up a new chapter in Indian cotton production in 21st century.The cry1Ac gene of Monsanto derived from American Upland Coker-312 background was not directly suitable for varied cotton growing situations in India.Delivery of

深入了解目的基因(Bt基因)的检测与鉴定

目的基因的检测与鉴定是基因工程基本操作程序的最后一步,用以确定目的基因是否真正在受体细胞中稳定表达其遗传特性。本文结合实际教学案例,介绍目的基因的检测和鉴定方法,为理解基因工程提供一定的参考。

The Research of Bt and OC Gene Cotransformation in Tobacco Chloroplast

The Bt Cry IA (C) chloroplast expression cassette and OC chloroplast expression cassette were constructed. The Bt expression cassette contained the 3.5 kb wild type Bt Cry I4(C) gene under the control of the strong light-induced psbA promoter and terminator from rice (Oryza saliva . L) chloroplast, the gene: trnH-psbA-trnk from tobacco (Nicotiana tabacum. L) as the homologous fragment. The OC chloroplast expression cassette contained the OC gene under the control of 16S promoter and terminator from tobacco, the tobacco gene: psbA-ORF512 as homologous fragment. The two cassettes both had the aadA gene expression cassette as the selectable marker. Leaves of tobacco were cotransformed with the particle bombardment method. After selection by spectinomycin, the transformants were obtained. The integration of Bt and OC gene were confirmed by Southern-blotting analysis, and Western-blotting analysis. Proteinase inhibitor assays showed that the Bt and OC gene had expressed. Bioassays showed that the transgenic tobacco had a significant resistance to the larvae of cotton bollworm (helicoverpa zea).

Introducing Bt Gene Into Maize With Ovary Injection

It is reported here that Bt toxin gene has been successfully transferred into maize inbred line by ovary injection for the first time both at home and abroad. One transgenic plant (To) has been confirmed by Southern blotting and PCR test, and 71 progenies (T1) from T0 have been obtained through self-pollination. Of these 71 progenies, seven plants demonstrated positive results in the PCR test; four were used to feed Asian corn borer, and certain effect of insect-resistance was observed. The experiments on the ovary injection in Hainan Province have also been repeated, thus providing new chance to the application of genetic engineering to the maize improvement.

Introduction of Bacillus thuringiensis(Bt)gene does not reduce potassium use efficiency of Bt transgenic cotton(Gossypium hirsutum L.)

Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.

Effects of Exogenous Jasmonic Acid on Concentrations of Direct-Defense Chemicals and Expression of Related Genes in Bt (Bacillus thuringiensis) Corn (Zea mays)

Bt corn is one of the top three large-scale commercialized transgenic crops around the world. It is increasingly clear that the complementary durable approaches for pest control, which combine the endogenous defense of the crop with the introduced foreign genes, are promising alternative strategies for pest resistance management and the next generation of insect-resistant transgenic crops. In the present study, we tested the inducible effects of exogenous jasmonic acid （JA） on direct-defense chemical content, Bt protein concentration, and related gene expression in the leaves of Bt corn cultivar 34B24 and non-Bt cultivar 34B23 by chemical analysis, ELISA, and RT-PCR. The results show that the expression of LOX, PR-2a, MPI, and PR-I genes in the treated leaf （the first leaf） was promoted by exogenous JA both in 34B24 and 34B23. As compared with the control, the concentration of DIMBOA in the treated leaf was significantly increased by 63 and 18% for 34B24 and 34B23, respectively. The total phenolic acid was also increased by 24 and 12% for both 34B24 and 34B23. The Bt protein content of 34B24 in the treated leaf was increased by 13% but decreased significantly by 27% in the second leaf. The induced response of 34B24 was in a systemic way and was much stronger than that of 34B23. Those findings indicated that there is a synergistic interaction between Bt gene and internally induced chemical defense system triggered by externally applied JA in Bt corn.

Cloning and Expression of Conserved Sequences of cry Gene in E.coli Strain Rosetta(DE3)

[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.

陆地棉抗虫与抗草甘膦基因的分子聚合及经济性状相关分析

培育抗棉铃虫兼抗草甘膦的棉花,可以同步提高品种的抗虫与抗除草剂能力,降低植棉的除虫、除草用工成本,提升植棉综合经济效益。本研究利用自育的高产优质陆地棉品系、国产转Bt基因抗虫棉品系以及转GR79+GAT基因的抗草甘膦陆地棉品系为杂交亲本,配置复交组合。在繁殖的复交分离群体后代,通过在苗床喷洒0.2%草甘膦,去除不抗草甘膦棉苗;并在大田不防治棉铃虫的条件下,筛选抗虫棉株。而后在实验室利用特异引物对抗虫基因与抗草甘膦基因进行分子跟踪检测,将同时具有Bt基因与GR79+GAT基因的单株繁殖成株系,经大田筛选获得9个抗虫兼抗草甘膦的优良品系。然后对育成品系进行产量比较试验,结果显示,其中品系BG-6不但实现了抗虫与抗草甘膦基因的聚合,而且该品系的皮棉产量水平较高、纤维品质表现良好:纤维长度达30.9 cm、比强度30.1 cN tex^(-1)、马克隆值4.9。对产量性状与纤维品质性状相关分析显示,只有衣分与棉纤维整齐度呈极显著负相关(r=-0.838**),其他产量性状与纤维品质性状均不存在显著相关性。文章最后得出结论:在复合杂交组合的分离后代,通过在苗床喷洒草甘膦筛选抗性棉苗,再结合大田不治虫筛选抗虫单株;而后在室内对2种抗性基因进行PCR分子跟踪检测,再在大田对产量性状与品质性状进行系统选育,可实现抗虫与抗草甘膦基因以及高产与优质性状的多元聚合。

转Bt基因和蛋白酶抑制剂基因杨树抗虫性的研究

虫害是杨树造林生产中所面临的一大难题。通过农杆菌介导 ,将经过人工改造的苏云金杆菌杀虫结晶蛋白 (Bt)基因转化杨树 ,得到杨树再生植株 ,然后 ,再将蛋白酶抑制剂基因 (PI)导入已含Bt基因的转基因杨树 ,经PCR检测和Southern杂交分析证明 ,最终获得既含有Bt基因又含有蛋白酶抑制剂基因的转基因杨树植株。利用这种杨树叶片饲喂舞毒蛾幼虫的杀虫试验结果表明 ,转基因杨树具有明显的杀虫活性 ,同时表明含有Bt基因和蛋白酶抑制剂基因双基因的植株其抗虫能力明显高于仅含单一Bt基因的植株。为探索培育高抗虫性杨树开辟了一条新途径。

转基因棉Bt毒性表达的时空动态及对棉铃虫生存、繁殖的影响

转Ｂｔ基因棉３１０品系在不同发育阶段，对棉铃虫的毒性表达测定表明，（１）棉株各空间组织在不同发育阶段均能强力阻止棉铃虫取食，表达出较高的抗虫效果。（２）棉株不同空间组织在同一时期，毒性表达存在差异，以营养组织，中、小蕾高于大蕾和铃。（３）同一组织毒性表达随着棉株生长、发育、成熟、衰老逐渐呈动态下降。（４）前后各阶段，同一组织饲喂棉铃虫，死亡所需要天数呈动态增加，但最终生存率降低到一个较低水平。存活老熟幼虫重量、蛹重及蛹羽化率比对照棉铃虫下降３０％～５０％，羽化的蛾子进行自交或异交均能产下受精率、孵化率很高的卵。取食Ｂｔ转基因棉的雌蛾产卵量比对照减少５０％～７０％。

苏云金芽孢杆菌(Bt)晶体毒蛋白基因在烟草叶绿体中的表达

将全长３．５ｋｂ的Ｂｔ基因３'端缺失，得到长为２．１ｋｂ、１．８ｋｂ的基因。分别将这３个长度 （１．８ｋｂ２．１ｋｂ、３．５ｋｂ）的基因置于水稻叶绿体ｐｓｂＡ基因的启动于和终止子调控之下，并与选择 标记基因ａａｄＡ（编码氨基糖苷－３'－腺苷酸转移酶，具壮观霉素抗性）表达盒相连５以烟草叶绿 体基因ｔｒｎＨ－ｐｓｂＡ－ｔｒｎＫ为同源片段，构建成叶绿体转化载体ｐＢＴ３、ｐＢＴ８和ｐＢＴ２２。用基因枪 把Ｂｔ基因导入烟草叶绿体中，以壮观霉素筛选，获得转化再生植株。经Ｓｏｕｔｈｅｒｎ、Ｗｅｓｔｅｒｎ检测 分析证明Ｂｔ基因已整合进入烟草叶绿体基因组中并得到表达。且子代呈现壮观霉素抗性，即 外源基因得到稳定的遗传。利用转基因植株叶片对棉铃虫进行杀虫实验，有些转化植株表现 出较强的抗虫性。总体上来说，转Ｂｔ全长基因的烟草植株，其杀虫效果最好，其余两种差异不 大。首次报道将Ｂｔ基因成功转入高等植物叶绿体并获得表达。

Bt转基因抗虫植物研究进展

目前Bt成为世界上植物抗虫基因工程中研究和应用最多的基因。已经有多种转单一Bt和复合Bt的转基因玉米、棉花、马铃薯等作物得到大面积种植。还有许多新的具有良好抗虫性的Bt转基因植物,如水稻、大豆、油菜、苜蓿、花椰菜、蓖麻等已经试验成功,并逐渐推广种植。Bt转基因抗虫植物的培育为提高产量,减少杀虫剂的使用和保护环境做出了举足轻重的贡献。就Bt的分类、杀虫机理、Bt转基因抗虫植物的发展状况以及种植Bt抗虫植物对环境的影响进行了概述。

应用近等基因系研究Bt基因对水稻性状表现的影响

以密阳46(MY46)为轮回亲本,采取连续回交,结合GUS标记基因辅助选择技术,育成了Bt基因近等基因系抗螟虫密阳46(RIMY46).经考察,RIMY46及其所配杂种II32A/RIMY46的GUS反应和PCR检测均为阳性,且高抗螟虫.在早期营养生长阶段,RIMY46苗高比MY46低,分蘖数比MY46少,表明纯合态Bt基因对纯系植株的前期生长进程有一定延缓作用,这种影响随着植株长大而减小,最终除导致抽穗延迟和整精米率降低外,对其余8个农艺性状和9个稻米品质性状的影响则均不显著.比较Bt近等基因系所配杂种II32A/RIMY46和II32A/MY46有关性状的表现差异,结果则发现杂合态Bt基因对杂种F1性状表达均无明显的负作用.

子房注射法将Bt基因导入超甜玉米

用子房注射法将Bt基因导入超甜玉米品种粤甜3号,获得了1株整合有Bt基因的转基因植株,定名为ZT11,以发芽成苗的植株计算,基因转化效率达9.09%。经PCR和Southernblot分析,证明抗虫基因已经整合在超甜玉米基因组中,并且为单拷贝。T1代植株PCR检测结果表明,Bt基因能够在转基因后代中稳定遗传,基因分离符合1∶1的孟德尔遗传分离规律。

通过农杆菌介导法将Bt(cryIA)基因导入大豆

采用农杆菌介导的大豆子叶节转化系统成功地将Bt基因 (cryIA)导入大豆。从发芽 5 - 7天的大豆无菌苗切取子叶节外植体 ,经农杆菌感染和共培养后 ,在选择培养基上 4周左右出现抗性不定芽。将不定芽转移到芽伸长培养基上 ,4 - 6周后再生苗长至 2 .5 - 3cm高。再将再生苗切下转入生根培养基 ,2周左右生根。生根后的再生植株经逐步锻炼移入盆中 ,所有植株均能正常开花结荚。在移栽成活的 8株T0 植株中有 7株PCR检测呈阳性反应 ;在 7个T1株系中有 4个株系存在PCR阳性植株。取 4个稳定遗传的T1代株系内的阳性植株的叶片提取DNA ,用地高辛标记的Bt基因探针进行Southern杂交分析 ,结果 4个株系均呈现阳性 ,证明Bt基因已整合到受体大豆的基因组内并能传递给后代。

Bt转基因抗虫粳稻品种选育中若干问题研究

以Bt转基因水稻“克螟稻”为供体,与农艺性状优良的粳稻品种杂交,对杂交后代抗虫品种选育中GUS组织染色辅助选择、农艺性状选择对Bt基因纯合影响、混合选择与外源基因传递、及自然抗虫鉴定选择效果等方面作了研究,结果发现采用系谱法并结合GUS组织染色辅助选择的Bt转基因粳稻育种效果为最佳.经8代系谱选择,育成了3个集高产和抗虫于一体的优良粳稻新品系.