Ac/Ds转座子及其作为插入序列标签的应用

简要介绍了Ac/Ds转座子作为插入序列标签的构建形式及其在植物基因组中的插入特点、影响Ds转座子跳跃的因素,并且对其在植物功能基因组的发展前景作了分析。     

piggyBac转座子介导的家蚕细胞转基因研究初探

为探讨稳定转化家蚕细胞表达外源基因的新方法,以家蚕核型多角体病毒(BmNPV)极早期蛋白基因(ie-1)启动子元件驱动新霉素抗性基因(neor),并克隆至具有绿色荧光蛋白基因(gfp)标记的昆虫转座子载体piggyBac中,构建转基因载体pigA3-IE-Neo。以该载体转染家蚕BmN细胞,用终浓度800μg/mL的遗传霉素(geneticin,G418)筛选3个月,获得了稳定转化的细胞,呈现绿色荧光的细胞数达80%以上。通过PCR鉴定证实了细胞基因组DNA中neor基因和gfp基因的存在。     

piggyBac转座酶的瞬时表达对莱茵衣藻体内piggyBac转座子的作用

piggyBac转座系统作为一种遗传修饰的工具,在很多生物体如哺乳动物、昆虫、酵母中已经得到了广泛应用.然而,目前piggyBac转座系统在绿藻一莱茵衣藻(Chlamydomonas reinhardti)中是否有活性很少有入研究.本研究构建含有一个拷贝或多个拷贝并且能够稳定表达piggyBac转座元件的莱茵衣藻藻株.然后在含有转座元件的藻株中瞬时表达普通的piggyBac转座酶和密码子优化后的piggyBac转座酶,通过限制性片段长度多态性(restriction fragment length polymorphism,RFLP)分析piggyBac转座元件的转座现象.转座酶转入莱茵衣藻细胞内一周和两周之后与转入之前的RFLP图谱对比,有很明显的差异.转入转座酶一周后的TR1藻株原有的1.5kb大小的片段消失,同时出现了3条新的片段:2.8、4.2和6.5 kb.两周之后,这3条片段又发生了变化,表明转座现象存在并且持续发生.转入密码子优化后转座酶一周后的TR3藻株中,1.5、3和4kb大小的片段没有发生改变,但是6.5 kb大小的片段消失了,与此同时新出现了一个2.8 kb大小的片段.两周之后,TR3的限制性片段多态性图谱并没有发生变化.为了检测转座酶是否成功转入细胞中,我们在新的接头序列两侧进行侧翼PCR,同时对PCR产物进行序列比对分析,结果显示,piggyBac转座子偶尔会在非常规性位点5’-TTT-3’和5’-ACGCAG-3’处发生剪切,而常规性剪切位点发生在5’-TTAA-3’处.研究结果表明,piggyBac转座酶对莱茵衣藻体内piggyBac转座子具有转座作用,piggyBac转座系统可以用于莱茵衣藻的遗传学修饰.     

Genetic diversity of Botrytis in New Zealand vineyards and the significance of its seasonal and regional variation

Species‐ and population‐specific differences in fungicide resistance and aggressiveness within Botrytis makes basic data on genetic diversity important for understanding disease caused by this fungus. Genetic diversity of Botrytis was surveyed between 2008 and 2012 from grapes from five New Zealand wine‐growing regions. A total of 1226 isolates were gathered from symptomless flower buds at the start of the growing season and 1331 isolates from diseased fruit at harvest. Two species were found, B. cinerea and B. pseudocinerea. Botrytis pseudocinerea was common in both Auckland vineyards sampled, and infrequent elsewhere. However, even in Auckland, it was rarely isolated from diseased fruit. The presence of the Boty and Flipper transposons was assessed. Isolates with all four transposon states (Boty only, Flipper only, both Boty and Flipper, no transposons) were found for both species. Both vineyards in the Auckland region had high numbers of Flipper‐only isolates at flowering; both vineyards from the Waipara region had high numbers of Boty‐only isolates at flowering. Most isolates from diseased fruit at harvest contained both transposons. These observations suggest that B. pseudocinerea, and isolates with one or both of the transposons missing, may be less aggressive than B. cinerea, or than isolates with both transposons present. Two clades were resolved within B. pseudocinerea, only one of which has been reported from European vineyards. Phylogenetic diversity within B. cinerea in New Zealand was similar to that known from Europe, including isolates that appear to match Botrytis ‘Group S’. The taxonomic implications of this genetic diversity are discussed.     

热不对称PCR(TAIL-PCR)和Tn5转座子质粒拯救法快速分离水稻条斑病菌致病相关基因

构建植物病原菌突变体库是进行新基因发掘和功能基因组学研究的有效途径之一.为快速准确地鉴定Tn5的插入位点和相应的功能基因,研究采用热不对称PCR(TAIL-PCR)和Tn5转座子质粒拯救两种技术,鉴别了在筛选水稻条斑病菌(Xanthomonas oryzae pv.oryzicola)Tn5插入突变体库过程中获得的8个在水稻(Oryza sativa)上毒性减弱的突变体.结果显示,TAIL-PCR和Tn5质粒拯救结合使用,可有效地鉴别Tn5的插入位点和相应的功能基因.TAIL-PCR鉴别的5株突变体是Tn5分别插入在分泌系统结构蛋白F基因(gspF)、cAMP调控蛋白、膜镶嵌蛋白酶酶原、S-蛋硫氨酸脱羧酶亚基和gspJ基因上;Tn5质粒拯救法鉴别的3株突变体是Tn5分别插入在致病性蛋白、功能未知的保守蛋白和鞭毛特异性ATP合酶基因上.序列同源性分析发现,8株突变体Tn5插入的基因与基因组测序的BLS256菌株中的对应基因同一性达100%.这表明,TAIL-PCR和Tn5质粒拯救技术可有效地应用于植物病原菌Tn5突变体库的鉴别和目标基因的分离.     

稳定转化家蚕BmN细胞表达人粒细胞-巨噬细胞集落刺激因子

为了建立稳定转化家蚕细胞持续表达外源基因的技术体系,构建了基于piggyBac转座子的带有人粒细胞-巨噬细胞集落刺激因子基因(hGM-CSF)和新霉素抗性基因(neo)表达元件的转基因载体pigA3GFP-IE-neo-FH-hGM-CSF-polyA-fib-L-intron1,以及带有hGM-CSF和吉欧霉素(zeocin)抗性基因的昆虫细胞转化载体pIZT-IE-hGM-CSF,分别转染家蚕卵巢BmN细胞,并以含相应抗生素G418或zeocin的培养液筛选,得到稳定的转化细胞系FH-hGM-CSF和IE-hGM-CSF。ELISA检测结果显示,hGM-CSF在FH-hGM-CSF和IE-hGM-CSF转化细胞系的表达水平分别为1.534 55 fg/个细胞和2.227 38 fg/个细胞。     

PiggyBac转座子介导的诱导细胞永生化转基因载体的构建及其整合效率的检测

【目的】构建在小鼠水平上实现多个目的基因的表达以及标记基因安全删除的转基因载体。【方法】以载体为模板,分别扩增SV40P neo、IRES、tk-PloyA,通过overlap PCR连接,并在两端添加方向相同的LoxP序列,构建转基因基本载体PB-NIT;然后通过overlap PCR扩增获得Tet-CMV-SV40 T-T2A-p 53-PolyA基因表达盒,将其插入PB-NIT中,构建载体PB-NIT-STP;最后将转录因子激活域 rtTA 通过同尾酶连接插入PB-NIT-STP,构建载体PB-rtTA-NIT-STP。【结果】经酶切和测序等鉴定,以上载体均正确;经转座活性鉴定,共转染转座子载体PB-rtTA-NIT-STP和转座酶载体比只转染转座子载体获得的阳性克隆数提高了20倍。【结论】得到了基于PiggyBac转座子的可用于正负向筛选和诱导目的基因表达的转基因载体。     

Genome‐wide identification of pathogenicity genes in Xanthomonas oryzae pv. oryzae by transposon mutagenesis

A transposon mutant library was constructed from the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) KACC10331 by Tn5 transposon mutagenesis. The susceptible rice cultivar Milyang 23 was inoculated with a total of 24 540 mutants resistant to kanamycin and 67 avirulent or reduced‐pathogenicity mutant strains were selected for study. Southern hybridization verified that 84 mutant strains had single‐copy insertions and their single‐transposon insertion sites were identified by sequencing analysis combined with thermal asymmetric interlaced (TAIL)‐PCR. The single‐transposon‐tagged sequences of 21 mutant strains belonged to pathogenicity‐related genes previously reported in Xanthomonas species, while the other 46 single‐transposon‐tagged sequences included diverse functional genes encoding, five cell‐wall‐degrading enzymes, three fimbrial and flagella assembly regulators, five regulatory proteins, 15 metabolic regulators and 18 hypothetical proteins, which were identified as novel pathogenicity genes of Xoo.     

转座子mini-Tn10介导的运动突变对溶藻弧菌毒力的影响

采用转座子mini-Tn10/Km构建了致病性溶藻弧菌的突变库,采用半固体双层平板初筛到73株运动缺陷突变株,初筛的突变株纯化及重复筛选后获得运动性缺陷表型稳定的突变株ND-01MM,Southern鉴定确定其为单位点插入。野生型菌株ND-01和运动缺陷突变株ND-01MM对致病性溶藻弧菌的天然宿主大黄鱼粘液的趋化、粘附以及在大黄鱼吞噬细胞内存活能力等生理功能的比较研究发现,溶藻弧菌运动缺陷后趋化及粘附能力均极显著下降(P<0.01),但在吞噬细胞内的存活能力则与野生型菌株差异不显著(P>0.05)。采用腹腔注射和浸泡两种方式感染大黄鱼,结果发现运动缺陷对浸泡感染的影响较为明显,但对腹腔注射的感染方式影响不大。     

Temperature‐responsive genetic loci in pectinolytic plant pathogenic Dickeya solani

Fifty‐four Dickeya solani thermoregulated genes were identified using Tn5 transposon mutagenesis with an inducible gusA reporter system; 45 genes were up‐regulated at 37 °C, whereas nine were up‐regulated at 18 °C. The relative level of gene up‐regulation ranged from 2–1200 and 5–650 U/mg total proteins at 18 and 37 °C, respectively. Among the temperature‐regulated loci, genes coding for proteins involved in fundamental bacterial metabolism, membrane‐related proteins and pathogenicity‐corresponding factors and several hypothetical unknown proteins were found. The mutants were tested for their pathogenicity in planta and for features known to be important for D. solani virulence viz. production of pectinolytic enzymes, cellulases, proteases, siderophores and auxins as well as for motility and the ability to form a biofilm. Eight Tn5 mutants, four up‐regulated at high and four up‐regulated at low temperature, expressed visible phenotypes including the decreased ability to cause symptoms on potato tubers and chicory leaves, impairment in phospholipase production and/or deficiency in biofilm formation. The implications of environmental temperature on the ability of D. solani to cause disease symptoms in potato are discussed.