龙牙百合的植株再生与遗传转化

以龙牙百合鳞片叶切块为外植体，通过多种培养基的比较试验，得出MS 2，4-D 2mg／L 6-BA 0．2mg／L是诱导愈伤的最佳培养基，MS 6-BA 1mg／L NAA 0．05mg／L是不定芽诱导及伸长的适宜培养基。MS N_AA2mg／L是生根的最佳培养基。本试验已建立了适用于龙牙百合遗传转化的快速高频再生系统。用携带有几丁质酶基因和β—1、3葡聚糖酶基因的工程菌，通过农杆菌介导法和基因枪转化法转化龙牙百合，经PCR和点杂交检测证明外源基因已经整合到植物染色体中。同时对农杆菌介导法和基因枪法进行比较，发现农杆菌介导法的转化率为16．7％，基因枪法的转化率为50％，因此可能基因枪转化法更适于龙牙百合的遗传转化。     

农杆菌介导的马铃薯茎段遗传转化体系优化研究

以甘肃主栽品种的茎段为外植体,研究了不同转化条件(菌液浓度、感染时间、预培养及共培养时间)对转化效率的影响,以及选择压和培养基对抗性出芽的影响,初步建立了马铃薯品种“陇薯3号”和“台湾红皮”的遗传转化体系并获得了转化植株。卡那霉素生根筛选和PCR鉴定结果表明,再生的转化植株假阳性率比较高,要获得预期的转基因植株群体需加大遗传转化工作中转基因植株的数量。     

The Prospects of Tissue Culture and Genetic Engineering for Strawberry Improvement

Abstract

Strawberry shoot meristem cultures have been used for producing virus-free strawberry plants. Plantlet regneration from leaf mesophyll protoplasts, and Agrobacterium tumefaciens mediated genetic transformation, mark the beginning of strawberry improvement with biotechnologies. The use of biotechnological tools has a greater potential in producing strawberry plants with traits such as early maturation, frost and disease resistance, mite and nematode resistance, high yield, better quality, suitability for mechanical harvesting, shipping ability, ellgaic acid content, aroma compounds and cost-effective micropropagation. Gene identification and mapping with RFLPs or RAPDs would be desirable. Transgenic strawberries should be tested for their quality before releasing to the consumers.     

杜氏盐藻MAPK基因植物表达载体的构建及对烟草的转化

以载体pBI121为基础,经SmaI和BamHI单酶切后,构建了植物表达载体pBI121-MAPK,并通过农杆菌介导采用叶盘转化法将其转入烟草中,经卡那霉素抗性筛选,获得转基因抗性植株。经PCR检测表明pBI121-MAPK已整和到烟草基因组中。     

根癌农杆菌介导Cry1Ac基因转化‘雪青’梨获得转基因植株

 以‘雪青’梨叶片愈伤组织为外植体, 经根癌农杆菌介导将CaMV35S启动子调控下的Cry1Ac基因导入‘雪青’梨。698块愈伤组织和231个Kan^r芽与携带表达载体质粒pBX203的根癌农杆菌菌株LBA4404共培养3 d后, 转入含50 mg·L^-1 Kan的筛选培养基培养30 d, 15 d转接1次。结果表明,在MS + 5 mg·L ^-1 6-BA + 0.1 mg·L^-1 IAA筛选培养基中, Kan^r芽率为34.24% , 在1/2MS + 2 mg·L^-1 IBA+ 0.5 mg·L^-1 6-BA + 500 mg·L^-1AC筛选培养基中, 15.58% Kan^r芽生根。共获得再生植株32株, 经PCR和Southern-blot分析证明, 其中4株的基因组已成功导入和整合Cry1Ac基因。     

Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD

Improved protocol for Agrobacterium mediated transformation of tomato (Lycopersicon esculentum) Micro-Tom was developed to use in corporation of the carotenoid biosynthetic genes CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase). From these experiment, a transformation methodology using explants from cotyledons cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/L kanamycin and 500 mg/L carabenillin for 6–8 weeks and resulted in a greater than 20% transformation efficiency in the concentration of Agrobacterium OD600 = 0.2 tested. In this transformation method, no feeder layers were used and the subculture media was minimal. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efficiency, 20.87%, was obtained by using OD600 = 0.2, which was significantly higher than that of OD600 = 1.0. The presence of the inserted target genes was checked using a rapid and efficient PCR test. The protocol was successfully employed in the production of transgenic Micro-Tom tomato containing the carotenoid biosynthesis CsZCD under constructive promoter. This procedure represents a simple, efficient and general means of transforming tomato.     

稻瘟病菌T-DNA插入的突变表型分析

PCR技术检测28个形态发育和致病性相关T-DNA插入突变体，结果所有突变体均含磷酸转移酶基因序列。对这些突变体展开进一步生物学性状观察，发现15个颜色异常，8个菌落生长缓慢，2个分生孢子形态异常，2个附着胞形态异常，3个不能形成附着胞。致病性测定结果，9个突变体完全不能导致抗瘟（C101）和感瘟（日本晴）水稻苗致病，病害级别均为0级。用标准菌株1528和P131测定突变体有性世代的形成能力，结果发现， Y34-0211、Y34-1469和Y34-0635 3个突变体完全丧失产生有性世代的能力。     

草莓果实外源基因瞬时表达系统的建立

将东北红豆杉紫杉烷13α-羟基化酶(Taxane13α-hydroxylase,13OH)基因全长cDNA正向插入到pCAMBI-A1304,构建其植物表达载体pCT13OH;将水稻小RNA169d(microRNA169d,miRNA169d)基因前体(precursor)全长DNA正向插入到pCAMBIA1305.1,构建其植物表达载体pCO169d。用电击法把它们导入根癌农杆菌GV3101,获得有关工程菌株。用1mL无菌注射器分别将这2种工程农杆菌悬浮液注射到草莓(Fragaria×ananassa)授粉后1周、2周、3周的果实中,发现授粉后2周果实适宜注射,可用于瞬时表达。对授粉后2周的果实进行不同注射部位、根癌农杆菌不同活化时期和根癌农杆菌悬浮液不同注射量试验,以蒂部注射、根癌农杆菌活化12h和0.5mL悬浮液注射量的效果最好,与结构基因13OH和调节基因miRNA169d相融合的GUS基因都得到表达,瞬时表达成功。     

发根农杆菌(Agrobacterium rhizogenes) Ri T-DNA遗传转化丝石竹(Gypsophila paniculata)的研究

近年来,已发展出遗传转化高等植物的一些新技术,其中有些技术如脂质体融合,微注射技术和电击导入都是基于动物细胞培养方面的工作,而另一些技术是来自于植物界独特的天然转化系统,其中包括已知能遗传转化高等植物的农杆菌(Agrobacterium)的二个种,即致瘤农杆菌(A.tumefaciens)和发根农杆菌(A.rhizogenes),这二个种都能够将其致病质粒Ti或Ri所携带的DNA序列(T-DNA)插入到双子叶植物细胞核基因组中。pTi诱发寄主产生根基肿瘤,pRi诱发寄主产生毛状根。二者的差别可能是毛状根可以从毛状根培养物获得具有完整的T-DNA序列的有生育能力的再生植株,而从致病农杆菌(A.tumefaciens)菌株所诱发的肿瘤很难获得再生植株。因此,利用发根农杆菌(A.rhizogenes)pRi作为遗传转化高等植物的基因克隆载体的研究和应用日益受到重视。     

提高根癌农杆菌介导的香石竹遗传转化效率的研究

以无菌苗叶片为外植体,在根癌农杆菌介导下建立并优化了香石竹5个品种的遗传转化体系。预培养2d可明显提高转化率;香石竹品种间在转化上存在差异;培养基中添加20μmol的AgNO3抑制不定芽的分化。转化植株在含25mg·L-1卡那霉素的生根培养基上培养,生根率为72 1%,GUS检测结果55%的转化植株呈蓝色,PCR扩增表明阳性率为32 2%,Southern杂交证实外源基因已整合到植物基因组中。