发根农杆菌介导的白花草木樨毛状根转化体系的建立

白花草木樨（Melilotus albus Medic.ex Desr.）是草木樨最常见的栽培用种之一，具有较强的抗逆性和固氮能力。为了开发一套高效的草木樨转化体系，本研究以白花草木樨为材料，以结瘤基因MaROP6为目的基因，建立了发根农杆菌（Agrobacterium rhizogenes）介导的高效白花草木樨毛状根转化体系。结果表明：利用该方法可以在两周左右获得毛状根，其发生率可达78%，MaROP6基因的毛状根转化率可达70.8%。qRT-PCR鉴定结果表明，MaROP6转基因毛状根的平均相对表达量为对照的36.87倍。毛状根转化体系的建立为白花草木樨基因功能的验证奠定了基础。     

油菜黑胫病菌T-DNA插入诱变因素优化及突变体筛选

油菜黑胫病是由Leptosphaeria biglobosa引起的一种真菌病害。这种病害在我国油菜产区广泛发生，并造成一定的经济损失。为通过获取突变体来研究L. biglobosa的生态适应性机制及致病机制，本文优化了影响农杆菌介导转化（ATMT）油菜黑胫病菌L. biglobosa菌株Lb731的因素，评估转化子质量，并筛选相关突变体。结果明确了农杆菌介导转化菌株Lb731的最佳因素：潮霉素B浓度为50 μg/mL，转化受体（分生孢子）培养时间为15 d (20℃)，浓度为2 × 10^7~8孢子/mL，农杆菌-受体共培养温度为25℃，共培养时间为72 h。在最适条件下的转化效率达到80个转化子/百万分生孢子。T-DNA插入基因组的频率为100%，单拷贝插入频率为72.7%，转化子抗潮霉素性状能稳定遗传。从2136个转化子中获得了11个菌丝生长减缓突变体，7个色素产生缺陷突变体和14个分生孢子产生缺陷突变体，并从这些突变体中鉴定出7个致病力丧失突变体。采用hiTAIL-PCR技术，从3个突变体中获得了T-DNA插入位点侧翼序列。上述结果为深入研究L. biglobosa的生态适应性机制及致病机制提供了材料和线索。     

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。     

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。     

不同因素对农杆菌介导的杉木转化效率的影响

以杉木试管苗靠近茎尖的茎段为受体材料,研究6个影响杉木遗传转化效果的因素,初步确定杉木茎段较为合适的转化体系为:预培养1～3 d,OD600 nm为0.1～0.4的菌液浸染10～15 min,共培养3～5 d,共培养基附加乙酰丁香酮(AS)80 μmol·L-1,共培养后延迟3 d筛选.在初次筛选培养基上共得到186个卡那霉素(Km)抗性芽,在二次筛选培养基上获得39个Km抗性芽.PCR检测有2个Km抗性芽呈稳定阳性,初步证明外源天麻抗真菌蛋白(GAFP)基因已整合到这2个Km抗性芽的基因组DNA中.     

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。     

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。     

农杆菌介导遗传转化在水稻基因工程育种中的应用

随着生物技术的发展，水稻基因工程育种在水稻育种中发挥着越来越重要的作用。在诸多的转基因方法中，农杆菌作为一种天然的植物基因转化系统，以其独特优点而倍受重视。本文概述了农杆菌介导转化水稻的原理，农杆菌介导转化水稻的优点并且介绍了农杆菌菌株、受体材料和培养基成分的不同对农杆菌介导转化水稻的影响。另外对农杆菌介导遗传转化在水稻抗虫基因工程育种、抗病基因工程育种、抗逆基因工程育种、优质基因工程育种、耐除草剂基因工程育种及提高水稻光合效率和延缓衰老方面的研究进展进行了综述。最后对农杆菌介导遗传转化在水稻基因工程育种中存在的问题和发展前景做了简要说明。     

Introduction of new traits into cotton through genetic engineering: insect resistance as example

The main goal of gene transfer into cotton is the development of insect-resistant varieties. The stakes are important since cotton protection against insects uses almost 24% of the world's chemical insecticides market, which is not without consequences on the environment. The first approach was to introduce and express in the cotton genome, genes from the bacterium Bacillus thuringiensis (B.t.) which produces entomopathogenic toxins. The development of an efficient Agrobacterium tumefaciens mediated transformation system was the first step. The expression of B.t. genes was studied and synthetic genes more adapted to a plant genome have been constructed. Studies on their expression in cotton is underway. The second focus was to develop strategies that would minimize the risks of inducing insect resistance. The main approach is to associate several genes coding for entomopathogenic proteins with different modes of action. Genes encoding protease inhibitors were chosen. One possibility is to associate a B.t. gene and a gene encoding a protease inhibitor. Several protease inhibitors were tested in artificial diets on major pests of cotton. The corresponding genes have been introduced into the cotton genome. These various orientations of the research program will be presented. This revised version was published online in July 2006 with corrections to the Cover Date.     

A melon genotype with superior competence for regeneration and transformation

Transformation efficiency of melon is low and is still regarded as a challenge. In this paper, the regeneration and transformation response of ‘BU‐21/3′, a newly characterized melon breeding line, is described. The line seems to be superior in this regard to previously evaluated genotypes. Agrobacterium‐mediated delivery of the GUS or GFP reporter genes into cotyledon explants was used to evaluate efficiency of transient and stable transformation. Good transient expression was observed, and stable transformation frequencies of 0.4‐1.5 transgenic shoots per explant were obtained. Transgenic plantlets were transferred to a contained greenhouse as early as 8‐10 weeks after transformation. Transgenic plants are fertile and exhibit a true‐to‐type phenotype. The ‘BU‐21/3’ line may become a useful tool for the facilitation of transgenic breeding in melon.