美洲商陆毛状根诱导及其离体培养的影响因素

为了探讨利用美洲商陆毛状根生产其药用成分的可能性,研究了美洲商陆毛状根诱导及其离体培养的影响因素。结果表明,美洲商陆叶片外植体被发根农杆菌ATCC 15834感染约18 d后,从其叶片外植体形态学下端叶脉切口处产生毛状根,其中以预培养1 d,农杆菌感染20 min,共培养4 d时的毛状根诱导率最高,达到70%。PCR扩增和硅胶薄层层析结果显示,发根农杆菌Ri质粒的rol C基因以及冠瘿碱合成酶基因已在美洲商陆毛状根基因组中整合和表达。所获得的美洲商陆毛状根系都能在无外源激素的MS固体培养基上快速自主生长;其中以毛状根根系2的生长速度最快、分生侧根能力最强和根表面的根毛密度最高;毛状根根表面呈紫红色或呈白色。在供试的MS、1/2MS、B5和6,7-V液体培养基中,以无外源激素的MS培养基最适合美洲商陆毛状根根系生长。与无外源激素的MS培养基相比,6,7-V培养基更有利于毛状根中商陆皂苷甲的合成与积累。本文所建立的美洲商陆毛状根诱导及其离体培养的适宜条件为今后利用其毛状根株系的规模培养来生产其药用有效成分商陆皂苷甲奠定了实验和技术基础。     

Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1‐family effectors relies on EDS1‐dependent immunity

Many Gram‐negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type III secretion system during infection. In Nicotiana benthamiana, recognition of XopQ/HopQ1 proteins induces an effector‐triggered immunity (ETI) reaction which is not associated with strong cell death but renders plants immune against Pseudomonas syringae and Xanthomonas campestris pv. vesicatoria strains. Additionally, XopQ suppresses cell death in N. benthamiana when transiently co‐expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the TIR‐NB‐LRR class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed Agrobacterium‐mediated protein expression experiments in wild‐type and EDS1‐deficient (eds1) N. benthamiana leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of Agrobacterium and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in eds1 plants. We conclude that XopQ‐mediated cell death suppression in N. benthamiana is due to the attenuation of Agrobacterium‐mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co‐expression assays for elucidation of in planta effector activities, at least under conditions of ETI induction.     

影响农杆菌介导玉米愈伤组织遗传转化因素的研究

用农杆菌介导玉米愈伤组织的转化,其筛选的结果得到的抗性愈伤组织受玉米愈伤组织的继代时 间、浸染的农杆菌菌液浓度、共培养的温度以及其共培养时间等因素的影响。玉米愈伤继代后7～9d,农杆菌 浓度为OD600值0.3左右、共培养温度约22℃、培养时间3d时,抗性愈伤的获得率最高。     

有害疣孢霉Hypomyces perniciosus遗传转化体系的建立及其突变体库的构建

有害疣孢霉Hypomyces perniciosus是引起双孢蘑菇Agaricus bisporus湿泡病的病原真菌,目前其致病分子机理尚不清楚,而高效稳定的遗传转化体系和突变体库构建是挖掘和研究病原菌致病基因的基础和有效手段。因此,本实验以高致病力的有害疣孢霉菌株WH001为研究对象,采用冻融法将双元载体pBHt1转入农杆菌AGL-1中,建立并优化根癌农杆菌介导的遗传转化体系,并利用其构建T-DNA插入突变体库。结果表明有害疣孢霉菌株WH001的潮霉素（Hygromycin,Hyg）耐受浓度为250ng/L,当农杆菌侵染液浓度OD₆₀₀=1,侵染时间为30min,乙酰丁香酮（Acetosyringone,AS）浓度为1.5mg/mL,共培养时间为3d时,转化体系效率最高。然后利用该优化体系构建有害疣孢霉的突变体库,通过PCR检测和形态学鉴定获得若干表型发生改变、稳定遗传的T-DNA插入突变体,与原菌种WH001相比,突变体在菌丝形态、生长速率、色素分泌和致病力等方面发生改变。本研究为进一步挖掘有害疣孢霉未知基因功能、解析生物学性状、探讨致病分子机制奠定基础。     

Media development for large scale Agrobacterium tumefaciens culture

A chemically defined media was developed for growing Agrobacterium tumefaciens at large scale for commercial production of recombinant proteins by transient expression in plants. Design of experiments was used to identify major and secondary effects of ten media components: sucrose, ammonium sulfate ((NH₄)₂SO₄), magnesium sulfate heptahydrate (MgSO₄*7H₂O), calcium chloride dihydrate (CaCl₂*2H₂O), iron (II) sulfate heptahydrate (FeSO₄*7H₂O), manganese (II) sulfate monohydrate (MnSO₄*H₂O), zinc sulfate heptahydrate (ZnSO₄*7H₂O), sodium chloride (NaCl), potassium chloride (KCl) and a sodium/potassium phosphate buffer (Na₂HPO₄/KH₂PO₄). Calcium and zinc were found to have no detectable impact on biomass concentration or transient expression level, and concentrations of the other components that maximized final biomass concentration were determined. The maximum specific growth rate of Agrobacterium strain C58C1 pTFS40 in this media was 0.33 ± 0.01 h^?1 and the final biomass concentration after 26 h of batch growth in shake flasks was 2.6 g dry cell weight/L. Transient expression levels of the reporter protein GUS following infiltration of a recombinant Agrobacterium strain C58C1 into N. benthamiana were comparable when the strain was grown in the defined media, Lysogeny Broth (LB) media, or yeast extract‐peptone (YEP) media. In LB and YEP media, free amino acid concentration was measured at three points over the course of batch growth of Agrobacterium strain C58C1 pTFS40; results indicated that l ‐serine and l ‐asparagine were depleted from the media first, followed by l ‐alanine and l ‐glutamic acid. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1218–1225, 2017     

高通量测序检测金针菇RNAi转化子插入位点及拷贝数

本研究对金针菇Flammulina velutipes的一个RNAi转化子菌株1382R3进行了高通量测序,以本实验室先前获得的野生型W23基因组数据为参考,分析了该转化子的基因插入位点以及拷贝数。转化子菌株1382R3是通过农杆菌介导将fv-hmg1-RNAi载体转化至金针菇菌株并通过PCR检测筛选标记而得到。通过BLAST将转化子测序的reads对外源载体和基因组定位,找到具有基因组序列（GS）和外源载体序列（ES）两种序列的临界reads,并据此使用PERL语言程序成功在转化子1382R3菌株中找到两个插入位点。对两个插入位置的序列分析表明：在插入位点1,T-DNA片段部分插入;在插入位点2,T-DNA全部插入到基因组。两个插入位点都对基因组内源基因的表达造成了一定的干扰。此方法拓宽了高通量测序技术的应用范围,将其运用到遗传转化插入位置和拷贝数的研究中,有利于食用菌的功能基因组及基因工程研究。     

FMDV vp1基因在豆科牧草百脉根中的转化与表达

通过根癌农杆菌介导法,将FMDV阿克苏(Akesu/O/58)株结构基因vp1转化豆科牧草百脉根子叶和子叶柄,其愈伤、芽和生根等过程经50 mg/L Kan筛选后,获得Kan抗性百脉根植株.对抗性植株进行vp1基因的PCR、RT-PCR检测和VP1蛋白的Western-blotting杂交.结果表明vp1基因转入百脉根中,检测有转录活性;目的蛋白获得了正确表达;扩繁和移栽后获得了批量转基因百脉根,为下一阶段的动物试验提供了实验材料.     

大豆体细胞胚胎发生与农杆菌介导的遗传转化

以55个大豆基因型未成熟子叶为外植体,用高浓度2,4-D诱导大豆体细胞胚胎发生与植株再生,并对生产上种植面积大、体细胞胚胎发生率高的大豆基因型用农杆菌介导法进行遗传转化。结果表明,东北地区主栽的大豆基因型中有14个基因型体细胞胚胎发生率超过40%。用含有pGBI121S4ABC质粒的LBA4404农杆菌侵染5个东北地区主栽大豆基因型的2147个未成熟子叶,经卡那霉素抗性筛选得到12株PCR阳性植株。 Abstract：Somatic embryogenesis was induced and the regenerated plants were obtained by higher concentrations of auxins with immature cotyledon of 55 genotypes in soybean. Bivalent insect resistant genes were transformed into immature cotyledon of soybean which have high frequency of somatic embryogenesis via Agrobacterium-mediated. The results showed that 14 genotypes possessed high frequency of somatic embryogenesis (more than 40%) among soybean genotypes from Northeast area. 2147 immature cotyledons of 5 different soybean genotypes cultured in Northeast area were inoculated with LBA4404 (including pGBI121S4ABC plasmid). 12 regenerated plants selected by Kanamicy gave positive PCR reaction.     

农杆菌介导的大豆高频遗传转化

大豆(Glycine max(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题.为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2 mg/L 6-BA和5 mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01 mg/L TDZ和2mg/L glufosinate的液体培养基中筛选,并每周更换一次培养液.得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1～2个基因拷贝数.该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论.