Field evaluation of antisense RNA mediated inhibition of GBSS gene expression in potato

Summary Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Analysis of antisense RNA mediated inhibition of GBSS gene expression in large numbers of tubers from in vitro grown, greenhouse grown and field grown transgenic potato plants revealed stable and total inhibition of GBSS gene expression in one clone. In three other transgenic genotypes partial and unstable inhibition was found. In these genotypes both GBSS activity and amylose content were remarkably reduced compared with the non-transformed control genotype. No relationship was found between the level of inhibition of GBSS gene expression and yield and dry matter content.     

腹泻抗原融合基因CTB-CS3对胡萝卜的遗传转化

CS3是CFA(产肠毒素型大肠杆菌引起腹泻时产生两类毒性因子之一)阳性株的保护性抗原。临床研究结果表明,以CS3作为疫苗可有效地防止由相关细菌导致的腹泻(董自正等,     

农杆菌介导的地被菊遗传转化体系的优化

本研究以地被菊‘北林黄’无菌苗叶片外植体为受体材料,以卡那霉素抗性基因为选择标记基因,对农杆菌介导的地被菊‘北林黄’的遗传转化体系进行优化。研究结果表明:选取植株中部叶片为转化受体,外植体在经过1d的预培养,用活化的OD600为0.6的农杆菌侵染10min,共培养2d,再经过3d延迟培养后转移到筛选培养基上培养,有利于提高遗传转化效率,外植体的愈伤组织获得数和抗性芽数均最高。在筛选后期,不定芽生根阶段使用的Kanamycin(Kan)选择压力为25mg/L。获得的部分Kan抗性植株经PCR检测,扩增出特异条带,初步证明目的基因已整合到‘北林黄’基因组DNA中。本研究将为进一步利用分子手段对地被菊进行遗传改良,并获得综合抗逆性提高的优异种质奠定基础。     

The Efficient Transformation of Wheat in Planta by Agrobacterium tumefaciens

Transformation of wheat was performed by pipetting spikelets with Agrobacterium tumefaciens which contained expression vectors using Npt Ⅱ as reporter gene. Transformants were identified through kanamycin resistance, PCR and Southern blot. The results showed that transformation efficiency was within 2.0 to 3. 2 % in all tested varieties of wheat. Then the simple and efficient protocol of wheat transformation by Agrobacterium tumefaciens in planta was primarily established.     

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies （PLBs） has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics （including ampicillin, carbenicillin, cefotaxime, and cefoperazone）, meropenem showed the highest activity in suppressing all tested A. tumefaciens strains （minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]）. Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.     

农杆菌介导的海岛棉茎尖再生体系及其遗传转化影响因子的研究

采用根癌农杆菌LBA4404和EHA105介导,对海岛棉的茎尖再生体系及其转化影响因子进行了研究。茎尖培养与棉花体细胞胚胎发生相比,不同基因型的茎尖再生频率差异不明显;在9种不同的激素组合中,以6-BA0.5 mg/L NAA0.1 mg/L与1/2MSB5 IBA 0.1 mg/L对茎尖的再生效果较好;获得较高转化频率的海岛棉培养条件为:海岛苗茎尖在OD600值为0.6~0.8农杆菌菌液中感染20~30 min,共培养3 d后进行转接,在含有不同激素和抗生素的各类培养基中进行抑菌和筛选继代培养,选择压以50~150 mg/L梯度进行;农杆菌以EHA105菌株的转化效果较好;抗性苗用SDS-CTAB法提取其总DNA,进行PCR扩增检测,有7株PCR检测呈阳性反应。由此,初步证明外源抗虫基因已经进入到再生植株的细胞核中。     

农杆菌介导的大花蕙兰转ICE1基因

大花蕙兰属热带植物,在北方组培成本很高,从拟南芥的RNA中克隆到耐寒基因(COR)的转录激活因子ICE1,连上CaMV35S启动子构建成植物表达载体pCAM BIA1300-35S.ICEl-poly,通过农杆菌介导的方法转化大花蕙兰.经过抗性筛选、诱导生芽、诱导生苗、诱导生根等过程.获得了70多棵潮霉素抗性植株,PCR鉴定12.9%为阳性.且部分PCR阳性植株通过了Southern检测,确定为转基因植株.     

Regular Production of Transgenic Wheat Mediated by Agrobacterium tumefaciens

Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0-1.5mm in size, C58c1 of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production,shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptⅡ ELISA (nptⅡ enzyme assay), PCR, Southern blot and leaf bleach,29 positive plants were identified from two genotypes of Bobwhite and Yanglmai 10, with an average transformation efficiency of 0.82 %. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.     

农杆菌介导法将FPFJ基因导入油菜的研究初报

将拟南芥早花基因FPF1（flowering promoting factor 1)插入双元高效表达载体pBI 121中,构建了植物表达载体pBIUbi-FPF1。以“双低”甘蓝型晚熟油菜品种2000 F4102,2000 F4153的下胚轴为转化受体,通过农杆菌介导的遗传转化获得抗卡那霉素的再生转基因植株,并探讨了影响油菜再生频率以及遗传转化效率的几个因素。对部分经卡那霉素筛选得到的再生植株进行了PCR检测,其中70％以上的卡那霉素抗性植株表现阳性,初步证明FPF1基因已整合到油菜细胞核基因组中。     

提高马铃薯遗传转化体系再生频率的研究

为提高马铃薯遗传转化体系的再生频率,改进了受体薯片的消毒方法,解决了外植体易污染问题,使污染率几乎降低为零;共浸染时加入适量的乙酰丁香酮可以提高再生频率;适当降低芽分化培养基的激素水平,在外植体分化正常的情况下,节省了在激素上的资金投入;不同品种马铃薯片转化后的植株再生情况不同,津引薯8号微型薯出苗量大,而S1微型薯首批苗更好。综合以上改进措施,使再生频率由29%提高到60%。