团头鲂β-actin启动子在家蚕中活性分析

利用双式荧光报告基因检测团头鲂β-actin启动子在家蚕BmN细胞和在家蚕体内的活性。通过脂质体介导法将转基因载体pigA3GFPAβdsRed和辅助质粒(1∶3)混合均匀后转染BmN细胞,48h后,可以检测到同时发绿色荧光和红色荧光的家蚕细胞,表明来自团头鲂的β-actin启动子在家蚕BmN细胞具有活性,能驱动DsRed基因在家蚕BmN细胞中表达。以gfp、dsred的特异引物,可从pigA3GFPAβdsRed转化家蚕BmN细胞基因组内扩增出gfp、dsred相应大小的片段,提示外源基因已经插入到细胞基因组中。随着转化细胞的传代,绿色荧光稳定存在,而红色荧光会逐渐减弱直至检测不到,暗示β-actin启动子在家蚕细胞中容易受到家蚕BmN细胞因子的影响而活性减弱。将转基因载体pigA3GFPAβdsRed和辅助质粒(1∶3)利用精子介导法导入家蚕受精卵,7天后,在部分受精卵中可观察到绿色荧光和红色荧光,进一步证明了β-actin启动子在家蚕体内具有活性。结果显示,含双式荧光报告基因的转基因载体为鉴定β-actin启动子等外源启动子在家蚕中的活性提供了快捷准确的鉴定方法。     

转座子随机突变芽孢杆菌的研究进展

近年来,随着功能基因组学的发展,转座子随机突变技术因操作简便易行而被广泛应用.某些具有随机转座特性的转座子及其衍生物,已成为发现新基因、克隆功能基因、发掘已知基因的新功能以及研究蛋白功能的有效工具,其中转座子Tn917、Tn10及mariner类家族转座元件已被改造并成功应用于芽孢杆菌突变体库的构建及基因功能的研究中.本文介绍上述3种转座子的结构、转座特点及在芽孢杆菌中的应用,并分析了mariner类家族转座元件的优势及应用前景.     

Ac/Ds转座子系统及其在玉米突变体库构建中的应用

Ac/Ds转座子系统是研究玉米功能基因组学的重要工具。Ac/Ds转座子系统以其在基因组中的低拷贝、倾向插入基因外显子、可以在同一基因不同位点造成突变等独特优势,在构建玉米突变体库和进行基因功能鉴定方面具有重要应用前景。综述了Ac/Ds转座子系统的插入特点、转座机制、在玉米突变体库构建中的应用以及我国玉米突变体库的现状,并对我国玉米Ac/Ds突变体库的研究提出展望。     

植物青枯病原细菌胞外蛋白相关基因的克隆

 用含有转座子Tn5的铜绿假单胞杆菌PAO1826(pMO75∷Tn5,Km^r)诱变我国马铃薯青枯菌小种3号菌株PO41,获得6000多个胞外多糖正常产生的接合子。经SDS-PAGE电泳,筛选出2个胞外蛋白减少9种的突变株PM2644和PM239,其可以引起烟草叶片典型的过敏性反应,致病力比野生型菌株显著降低,皆为原养型菌株。Southern杂交的结果表明,突变株染色体上只有1个转座子插入位点。标记交换证明,9种胞外蛋白缺失与转座子插入相偶联的频率为100%。以聚半乳糖醛酸酶和内葡聚糖酶2种胞外酶为指示蛋白进行定量测定的结果表明,在突变株上清液中和菌体细胞内都没有这2种胞外酶的存在。以突变株PM2644为受体菌,通过基因功能互补的方法,从野生型青枯菌基因文库筛选到2个阳性克隆pPSP1和pPSP2,它们既能恢复2个突变株产生胞外蛋白的能力,也能将2个突变株的致病力恢复到接近野生型菌株的水平。     

玉米Mutator转座子突变体库研究进展

Mutator转座子以其高的正向突变率、倾向插入基因富含区和低拷贝序列区等独特的遗传特性,使其在玉米基因功能的研究以及突变体库的构建中发挥了重要的作用。本文从群体大小、群体优缺点以及利用途径等方面对Mutator介导的玉米突变体库作了详细的介绍,以期能为我国玉米基础研究工作者提供启示和借鉴。     

Establishing a zebrafish transgenic line expressing tilapia lysozyme with enhanced antibacterial activity

In recent years, infectious disease caused by Streptococcus agalactiae has increased in aquaculture, threatening the healthy development of tilapia and resulting in substantial economic losses. Cultivating disease‐resistant tilapia by transgenic technology is one strategy that has been developed to address this issue. Here, we used a transposon system to investigate gene expression and tissue lytic activity in transgenic zebrafish expressing the tilapia lysozyme. The transpose vector contained the Tgf2 transposon skeleton, tilapia heat shock protein 70 promoter (Hsp70) and two target genes (tilapia C‐type lysozyme 3 [lysozyme‐C3] and green fluorescent protein [GFP]). The transgenic zebrafish F₀ generation was obtained by microinjecting the donor vector and transposase mRNA into fertilized zebrafish eggs. There was 45% green fluorescence in the zebrafish F₀ generation, which was spread over the entire body based on microscope observation. The F₀ generation was reared to sexual maturity, at which point individuals were mated with wild‐type zebrafish to produce the F₁ generation. Tilapia lysozyme‐C3 expression was detected in the liver of F₁ generation by RT‐PCR and western blotting, but was not detected in the skin, intestine, or muscle. Significantly higher liver bacteriolysis activity was detected in transgenic zebrafish compared with wild‐type zebrafish. Therefore, transgenic zebrafish may have an increased potential for bacterial resistance.     

4种鲇基因组中Tc1/Mariner转座子的鉴定与演化

为了探讨Tc1/Mariner转座子在4种鲇(沟鲇、黑斑原、鲇和南方鲇)中的特征及在演化过程中的作用,实验使用de novo预测和同源预测两种方法鉴定了4种鲇基因组中的Tc1/Mariner转座子并进行演化分析。通过对所获得序列的统计及分类,结果显示,4种鲇基因组中Tc1/Mariner转座子的含量分别为9.46%、2.70%、7.83%和8.54%,并且分别被分为38、20、43和55个家族。基于转座酶催化区域的结构和系统发育分析的结果,4种鲇的Tc1/Mariner转座子总共可以分为5个不同的亚类群:DD34E(Tc1)、DD35E、DD36E、DD37E和DD35D(pogo)。系统发育分析表明,多数支系中鲇和南方鲇的转座子聚为姐妹群。插入时间分析表明,Tc1/Mariner转座子的插入时间主要分布在0~5百万年前。大多数Tc1/Mariner转座子在0~1百万年前或2~3百万年前有一个突然的扩增。另外,4种鲇的Tc1/Mariner转座子大部分插入到基因的内含子中。本实验通过对4种鲇中Tc1/Mariner转座子的鉴定、比较与演化分析,为Tc1/Mariner转座子的深入研究奠定了基础。     

甜瓜细菌性果斑病菌致病性突变体筛选与hrcR基因的克隆

 细菌性果斑病(Bacterial Fruit Blotch,BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌株MH21,经鉴定为燕麦食酸菌西瓜亚种(Acidovorax avenae subsp. citrulli)。利用转座子Mini-Tn5构建MH21菌株的突变体库,Southern印迹杂交结果显示Mini-Tn5在菌株MH21染色体上可随机、单拷贝插入。通过浸种处理甜瓜种子进行突变体的致病性检查,筛选得到1株致病性完全丧失的突变体M543。对M543中转座子插入基因的克隆和测序表明其突变基因为燕麦食酸菌Ⅲ型分泌系统(TTSS)中的保守基因hrcR。推测菌株MH21中hrcR基因编码的蛋白定位于细菌内膜,是分泌通道主要成分之一。hrcR基因互补菌株的致病性检查结果显示其致病力恢复到野生型的84%。研究同时发现hrcR基因突变后MH21菌株丧失了在烟草上诱导过敏性坏死反应的能力。本研究从遗传学角度说明TTSS是西甜瓜果斑病菌致病性的重要因子。     

北京地区草莓灰霉病菌的转座子及其分布频率

为了解北京地区草莓灰霉病菌的转座子类型及其分布,本研究用转座子Boty和Flipper的特异性引物对北京地区2012-2013年从12个草莓园采集和分离的60株草莓灰霉病菌进行PCR扩增。结果表明,北京地区草莓灰霉病菌群体中共存在3种转座子类型:transposa型、Boty型和Flipper型。其中,以transposa型菌株最多,占供试菌株的63.3%,Boty型菌株占供试菌株的28.3%,Flipper型菌株最少,仅占8.4%,未检测到vacuma型菌株。选取属于不同转座子类型的18株菌株测定其对草莓叶片的致病力,结果显示Boty型菌株所致病斑的平均直径显著大于Flipper型。草莓灰霉病菌转座子类型与致病力的关系有待进一步研究。转座子类型的检测为进一步研究灰葡萄孢的遗传多样性及遗传变异提供了基础。     

The role of siderophores in potato tuber yield increase by Pseudomonas putida in a short rotation of potato

The effect of treatment of potato seed tubers withPseudomonas putida isolate WCS358 on tuber yield was studied in different crop rotations at the Experimental Farm De Schreef, near Lelystad. With untreated, tuber yield in a 1:3 (short) rotation compared to yield in a 1:6 (long) rotation of potato was decreased by 11% at 86 days (seed tuber harvest) and by 14% at 130 days (ware potato harvest) after seeding. Seed tuber treatment with the wild-type isolate WCS358 increased tuber yield with 13% in a short rotation of potato 86 days after seeding, whereas a siderophore-negative Tn5 transposon mutant of this isolate had no effect on tuber yield. Seed tuber treatment with the wild-type isolate or the siderophore-negative mutant in a long rotation of potato had no effect on tuber yield. At 130 days after seeding no effect of any of the seed tuber treatments was found in both short and long rotations of potato.Root colonization by siderophore-producing Tn5 transposon mutants of WCS358 was decreased at the end of the growing season. No difference in root colonization between siderophore-producing and siderophore-negative Tn5 transposon mutants was found at 130 days after seeding.Siderophore production seems to be a prerequisite in potato tuber yield increase by WCS358 under field conditions. This is the first time that the involvement of siderophores in growth stimulation has been demonstrated in the field.Samenvatting De invloed van een behandeling van aardappelpootgoed metPseudomonas putida isolaat WCS358 op de knolopbrengst werd onderzocht in verschillende gewasrotaties on een proefveld van proefboerderij De Schreef, Flevopolder. In de controlebehandelingen werd in een nauwe aardappelrotatie (1:3) een reductie van 11% in opbrengst van pootaardappelen (86 dagen na het poten) geconstateerd ten opzichte van een ruime aardappelrotatie (1:6); 130 dagen na het poten werd een vermindering met 14% gevonden in de opbrengst van consumptieaardappelen.Pootgoedbehandeling met het siderofoorproducerende isolaat WCS358 verhoogde de opbrengst van pootaardappelen in de 1:3-rotatie met 13%. Een Tn5-transposonmutant van dit isolaat die het vermogen sideroforen te produceren had verloren, had geen effect op de opbrengst. In de 1:6-rotatie had behandeling van pootgoed met WCS358 geen effect op de opbrengst van pootaardappelen.Zowel in de nauwe (1:3) als in de ruimte (1:6) rotatie werd (130 dagen na het poten), geen effect van behandeling van pootgoed met WCS358 op de opbrengst van consumptieaardappelen gevonden.Wortelkolonisatie door siderofoorproducerende Tn5-transposonmutanten van WCS358 nam aan het eind van het seizoen af. Er werd, 130 dagen na het poten, geen verschil in wortelkolonisatie geconstateerd tussen siderofoorproducerende en siderofoornegatieve Tn5-transposonmutanten.Siderofoorproduktie blijkt een voorwaarde te zijn voor verhoging van de knolopbrengst door WCS358 onder veldomstandigheden. De verhoging van de knolopbrengst treedt alleen op in de nauwe aardappelrotatie. Dit is de eerste keer dat de betrokkenheid van sideroforen bij groeistimulatie onder veldomstandigheden is aangetoond.