Resistance identification of bivalent fungi-resistant genes transformed soybean to Phytophthora sojae

Soybean is one of the most important sources of edible oil and proteins in the world.However,it suffers from many kinds of fungal diseases which is a major limiting factor in soybean production.The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation.In this study,the resistance to Phytophthora sojae of five bivalent transgenic soybean lines was identified using the hypocotyls inoculation technique.The lines were the T,of the transgenic soybean which were transformed with kidney bean chitinase gene and barley ribosome inactivating protein gene,and were positive by Southern Blot analysis.The resistance difference was studied through comparing the death percentage of transgenic soybean with the control.The results showed that four lines were more resistant to P.sojae,whereas other one had no significant difference in comparison with the control.These transgenic soybean lines with enhanced resistance to P.sojae will be useful in soybean resistance breeding.     

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha~(–1) of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.     

Constitutive expression of feedback-insensitive cystathionine γ-synthase increases methionine levels in soybean leaves and seeds

Soybean(Glycine max(L.) Merr.) is a major crop that provides plant-origin protein and oil for humans and livestock.Although the soybean vegetative tissues and seeds provide a major source of high-quality protein,they suffer from low concentration of an essential sulfur-containing amino acid,methionine,which significantly limits their nutritional quality.The level of methionine is mainly controlled by the first unique enzyme of methionine synthesis,cystathione γ-synthase(CGS).Aiming to elevate methionine level in vegetative tissues and seeds,we constitutively over-expressed a feedback-insensitive Arabidopsis CGS(At D-CGS) in soybean cultivars,Zigongdongdou(ZD) and Jilinxiaoli 1(JX).The levels of soluble methionine increased remarkably in leaves of transgenic soybeans compared to wild-type plants(6.6-and 7.3-fold in two transgenic ZD lines,and 3.7-fold in one transgenic JX line).Furthermore,the total methionine contents were significantly increased in seeds of the transgenic ZD lines(1.5-to 4.8-fold increase) and the transgenic JX lines(1.3-to 2.3-fold increase) than in the wild type.The protein contents of the transgenic soybean seeds were significantly elevated compared to the wild type,suggesting that the scarcity of methionine in soybeans may limit protein accumulation in soybean seeds.The increased protein content did not alter the profile of major storage proteins in the seeds.Generally,this study provides a promising strategy to increase the levels of methionine and protein in soybean through the breeding programs.     

Establishment of PCR-ELISA for Detecting Glyphosate Resistant Transgenic Soybean

A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows: the concentration of digoxin tag probe was 0.5 μmol · L-1, the time of hybridization reaction was 15 min and the chromogenic reaction should last for 30 min. The sensitivity and the repeatability of our PCR-ELISA method were evaluated, and the results showed that it could be detected when the concentration of DNA template from transgenic soybean samples was 0.01% or higher, and the coefficient of variation of this method was less than 5% in our research condition. These results suggested that PCR-ELISA method establishment in this study had good repeatability and high precision for detecting the transgenic soybean samples.     

Impact of Crop Rotation on Pathotype and Genetic Structure of Phythophthora sojae in Fields

To estimate the impact of crop rotation on the pathotype and genetic structure ofPhythophthora sojae in fields, 372 isolates ofP. sojae were obtained from long-term localisation experimental fields in Heilongjiang Province of China. The hypocotyl inoculation method was used to characterize the virulence ofP. sojaeon 13 differential cultivars, and the amplified fragment length polymorphism (AFLP) technique was used to analyze difference in the genetic structure ofP. sojae. The results indicated that an abundant diversity of genetic structures and pathotypes ofP. sojae, a more uniform distribution of pathotypes and less dominance of pathotypes occurred in corn-soybean and wheat-soybean rotation fields than in a continuous soybean mono-cropping field. These findings suggested thatP. sojae did not easily become the dominant race in rotation fields, which maintain disease resistance in soybean varieties. Therefore, the results of this study suggested that Phytophthora stem and root rot of soybeans could be effectively controlled by rotating soybeans with non-host crops of corn and wheat.     

Gene flow from transgenic roundup-ready soybean to wild soybean

A study was conducted in the field of the Institute of Vegetable Crops, Jiangsu province from July 2000 to August 2003. The transgenic roundup-ready soybean was sown in the middle of the field in a circular manner for 5 circles, with the distance of 3 m, from one circle to another. Then the wild soybean was planted in plots as the rays of the circles in 8 directions （N, E, W, S, NE, NW, SE and SW）, spaced every 5 m until 50 m. Each plot comprised 25 plants. In the second year, the wild soybean seeds from the first year were planted in the field together with the original wild soybean as check. Before flowering time, high concentrations of roundups （about 4-5 times of the normal dose） were sprayed on the plants and the surviving plants were identified. The leaves were taken to the lab for DNA extraction to determine the unique DNA for roundup-ready soybean （CTAB method）. About 2% of the plants survived, but some leaves were yellow. One plant of wild soybean was found to have the roundup-ready gene from the original roundup-ready soybean. The other surviving wild soybeans should also had some fragments of the roundup tolerance gene. However, the DNA bands were not very clear in the PCR map.     

Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated crylAh Gene via Agrobacterium-Mediated Transformation

A novel insecticidal gene crylAh was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active CrylAh toxin has a toxicity level similar to that of the full-length CrylAh toxin. In this study, plant expression vector pMhGM harboring truncated crylAh gene was transformed into maize （Zea mays L.） immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions （madMARs） were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer （Ostriniafurnacalis）. These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the crylAh gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene crylAh was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T l-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.     

Study of the Protective Effects in PEPC Transgenic Rice

The diurnal course of chlorophyll fluorescence parameter and active oxygen metabolism of flag leaves in PEPC transgenic and untransformed rice Kitaake were studied. The results showed that the photosynthetic rate under high light intensity has been increased by 50% and photoinhibition of photosynthesis in PEPC transgenic was alleviated after the introduction of PEPC gene from maize into rice. It was demonstrated that the increment of photosynthesis in PEPC transgenic was related to the introduction of PEPC gene using specific inhibitor of PEPC. Photoinhibition of photosynthesis in different genotypes exists at noon under natural condition. PEPC transgenic rice exhibited a less decrease in Fv/Fm, a less photoinhibition and a higher efficiency of light energy conversion to chemical energy and lower thermal energy dissipation.These results provided the physiological basis on the mechanism of tolerance to photoinhibition and rice breeding with high photosynthetic efficiency.     

Screening Method for Transgenic Maize with Kanamycin Resistance

[Objective]The paper was to establish simple and effective method to screen marker gene in maize with kanamycin resistance.[Method]Using inbred line "Chang 7-2" and hybrid "Zhengdan 958" of maize as test materials,their seeds were soaked with different concentrations and volumes of kanamycin for3 and 4 d,respectively,the rate of albino seedlings and average seedling height after sowing for10 d were investigated.[Result]The rate of albino seedlings not only was related to kanamycin concentration,but also had relationship with solution volume during soaking process.The difference between inbred line and hybrid was no significant.When 100 ml of kanamycin solution with concentration of 200 mg/L was used to soak seeds for 3 d,the rate of albino seedlings basically could reach 100%.When 100 ml of kanamycin solution with concentration of 100 mg/L was used to soak 20 seeds for 3 d to carry out resistance screening,the accuracy was up to 84.8% after verifying the screening test of T1 transgenic maize plants.[Conclusion]The method was feasible,which could be used as a simple method for screening transgenic gene maize with kanamycin resistance.     

Development and identification of glyphosate-tolerant transgenic soybean via direct selection with glyphosate

Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.     

锰胁迫对大豆幼苗POD活性及其同工酶的影响

[目的]在分子水平上探讨不同品种大豆在锰毒害下的一些遗传特性和基因差异的本质原因。研究不同锰浓度（0～120mg/L）对大豆幼苗水栽培,苗龄10d的根系、茎部以及叶片的过氧化物酶（POD）活性的影响以及其同工酶谱的变化。[结果]随锰浓度的提高,大豆幼苗的根系、茎部以及叶片的过氧化物酶（POD）活性均表现为先上升后下降的变化趋势。不同器官POD同工酶酶谱对锰胁迫的反应存在一定差异,其中根系同工酶的酶带数和活性变化较大,茎部与叶片的同工酶的酶带数和活性较稳定。[结论]大豆幼苗不同器官的同工酶酶谱的变化与其对锰胁迫的抗性有关。     

Taqman定量PCR技术检测转基因大豆中外源基因拷贝数

[目的]采用Taqman定量PCR技术检测转基因杂交大豆中外源nos终止子基因的拷贝数。[方法]以大豆凝集素基因为内参照基因,以非转基因大豆基因组DNA为内参照基因标准品,通过梯度稀释法分别求取了内参照基因和质粒DNA的Ct值与拷贝数对数值的相关性标准曲线方程,并通过将得到的Ct值代入标准曲线方程求取了样品的拷贝数。[结果]内参照基因标准曲线方程为y=-3.422x＋35.201,R2=0.998;外源基因标准曲线方程为y=-3.348x＋34.890,R2=0.999。nos终止子基因及其下游边界序列在转基因杂交大豆中为单拷贝。[结论]该研究为确定转基因大豆外源基因拷贝数提供了理论依据。     

抗虫基因Cry1C转化大豆的研究

[目的]将对鳞翅目害虫具有抗性的抗虫基因Cry1C转入大豆中,获得对田间鳞翅目害虫具有抗性的大豆材料.[方法]利用农杆菌介导法将抗虫基因CrylC转入大豆品种Williams82中,并通过Bar试纸条及PCR方法对所获得的转化苗进行鉴定.[结果]获得28株阳性大豆转化植株.[结论]初步认定CryIC基因已整合到大豆基因组中.     

转cry1Ab13基因玉米新种质的创制

【目的】构建包含抗鳞翅目害虫基因cry1Ab13的重组植物表达载体,并利用其创制对玉米螟Ostrinia furnacalis具有优良抗性的转基因玉米Zea may L.新种质。【方法】利用同源重组法将cry1Ab13基因连接到表达载体pCAMBIA3300-Bar上,获得以抗除草剂Bar基因为筛选标记的植物表达载体pCAMBIA3300-cry1Ab13-Bar。通过农杆菌介导法转化玉米自交系H99幼胚,对再生植株进行逐代除草剂筛选、PCR检测及T2代植株的Southern blotting检测、实时荧光定量PCR检测,并对转基因植株进行田间及室内抗虫性鉴定。【结果】构建了cry1Ab13基因的植物表达载体,转化玉米获得3株高抗玉米螟和1株抗玉米螟的T2代转基因植株。Southern blotting证明cry1Ab13基因已经整合到玉米基因组中,实时荧光定量PCR结果表明cry1Ab13基因已经在玉米植株内表达。抗虫性鉴定结果表明,与对照相比转基因植株对玉米螟的抗性显著提高。【结论】将cry1Ab13基因导入玉米并成功表达,显著提高了转基因玉米对玉米螟的抗性,为抗虫玉米新种质的创制奠定了基础。     

转基因抗虫棉Bt毒蛋白含量时空变化及土壤降解研究

[目的]研究转基因抗虫棉Bt毒蛋白含量的时空变化及其土壤降解。[方法]采用ELISA法(酶链免疫法)研究和分析了转Bt-cry1Ac基因抗虫棉的根、茎和叶片组织在不同发育时期毒蛋白的含量变化及转Bt基因抗虫棉(GK45)和非转基因棉花(新陆早36号)在根际土壤、表层土壤和后茬种植区土壤中Btcry1Ac毒蛋白的年平均含量变化。[结果]BtCry1Ac毒蛋白含量在抗虫棉生长过程中均呈动态下降趋势,而根中下降的速率最快,茎和叶片次之;棉花种植区土壤表层中均检测到Btcry1Ac毒蛋白,且后茬种植区中表层毒蛋白的含量增加,而根际土中含量极低。[结论]Btcry1Ac毒蛋白的含量检测为种植转基因作物的风险评价及转基因作物的土壤生态系统安全性评价提供了科学依据。     

黑豆主要营养成分分析

[目的]分析研究黑豆的各种化学成分。[方法]用GC9800气相色谱仪、高效液相色谱仪、电热鼓风干燥箱等仪器对黑豆粉的灰分、粗脂肪、蛋白质的含量,脂肪酸的组成,维生素E的含量进行测定。[结果]黑豆中灰分含量为4.47%,高于其他豆类;黑豆中蛋白质含量为45.70%,高于黄豆;粗脂肪的含量为15.60%,油脂中所检出的不饱和脂肪酸成分为83.00%,其中软脂酸、油酸、亚油酸的含量较高,人体不能自身合成的必需脂肪酸亚油酸和亚麻酸的相对含量分别为49.19%和8.41%。黑豆中富含维生素E,100 g黑豆粉中含21.9mg维生素E,具有较强的抗氧化能力。[结论]黑豆营养成分丰富,开发黑豆食品对改善人类膳食结构具有重要意义。     

吉林省中南部区不同类型大豆的筛选研究

[目的]筛选吉林省中南部地区不同类型大豆,为明确吉林省中南部地区适宜种植何种类型大豆提供理论参考。[方法]选用3种类型的9个大豆品种在吉林省公主岭市开展试验研究,分析不同类型大豆品种的产量和品质。[结果]确定在吉林省中南部区种植大豆,高脂肪类型品种的产量优于高蛋白品种,与普通大豆品种差异不显著。[结论]吉林省中南部大豆产区适宜推广种植高脂肪类型大豆品种。     

锰胁迫对大豆幼苗POD活性及其同工酶的影响

[目的]在分子水平上探讨不同品种大豆在锰毒害下的一些遗传特性和基因差异的本质原因。[方法]研究不同锰浓度（0～120.0mg/L）对大豆幼苗水栽培,苗龄10 d的根系、茎部以及叶片的过氧化物酶（POD）活性的影响以及其同工酶谱的变化。[结果]随锰浓度的提高,大豆幼苗的根系、茎部以及叶片的POD活性均表现为先上升后下降的变化趋势。不同器官POD同工酶酶谱对锰胁迫的反应存在一定差异,其中根系同工酶的酶带数和活性变化较大,茎部与叶片的同工酶的酶带数和活性较稳定。[结论]大豆幼苗不同器官的同工酶酶谱的变化与其对锰胁迫的抗性有关。     

Bt抗虫水稻转育后代两种筛选方法的比较

[目的]通过比较分子标记辅助选择和除草剂抗性筛选两种检测方法的优缺点,为快速、准确、经济有效地选择抗螟虫水稻转育后代提供依据.[方法]用分子标记检测和除草剂抗性筛选两种方法,对不同组合的抗螟虫转育后代进行筛选,同时采用ELISA检测方法对筛选的抗性株系测定杀虫蛋白含量,比较两种筛选方法的准确性.[结果]在以T1C-19为供体亲本的4个组合169个单株中,两种筛选方法的符合率达到98.2%;而以T2A-1为供体亲本的3个组合141个单株中,两种筛选方法的符合率达到98.6%.在随机选择的经两种筛选方法鉴定均为阳性的144个单株中,均含有对应Bt抗虫基因的杀虫蛋白表达量,与亲本T1C-19、T2A-21相比,Bt抗虫水稻转育后代中个别单株的Bt蛋白含量出现大幅度下降或上升的现象.[结论]分子标记检测和除草剂抗性筛选对抗螟虫水稻转育后代的选择均有很高的准确性:分子标记检测准确可靠,且不受环境影响,但费用相对高;除草剂抗性筛选法耗费低、简便易行,但易受雨天影响.在筛选的过程中可根据现有的条件做出适宜选择.     

抗虫转基因欧洲黑杨鲜叶对小鼠的急性毒性

[目的]为了研究抗虫转基因欧洲黑杨对小鼠的急性毒性,为其生物安全性评估提供依据。[方法]以转基因欧洲黑杨鲜叶和非转基因欧洲黑杨鲜叶作为受试物,采用限量法测定小鼠的急性经口毒性反应。[结果]灌胃后7 d时间内所有处理组无小鼠死亡,一般状况观察和大体解剖也未见明显异常和毒性反应,小鼠LD5010.0 g/kg体重,表明其属于"实际无毒"级别。组织病理学切片检查发现转基因灌胃组中有一雌性小鼠肺部出现间质性炎症及一雄性小鼠肝部出现局部胞浆淡染、细胞轮廓不清及空泡样变的现象,具体原因需进一步深入研究。[结论]转基因欧洲黑杨和非转基因欧洲黑杨鲜叶对小鼠均无明显急性毒性作用。