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.
Transposition of PiggyBac (PB) Element is Attributed to Transient Expression of PB Transposase in Chlamydomonas reinhardtii
The cut-and-paste piggyBac (PB) transposon system has been shown to be a useful tool for genetic modification in a number of organisms including mammals,insects,and yeast.However,little is known whether it is active in the green microalga Chlamydomonas reinhardtii.In this study,we constructed stable PB element-containing C.reinhardtii strains bearing either a single copy or multiple copies of PB element.Subsequently,we transiently expressed TPase or codon-optimized crTPase in the PB element-containing strains to assay for transposition of PB element using restriction fragment length polymorphism (RFLP) methodology.Our analyses show that initial RFLP patterns of PB element are altered in cells one week and two weeks after plasimds containing TPase or crTPase expression cassette.The size of PB element-containing fragment remained unchanged in TR1 cells after transformation of the pJR38 plasmid containing no TPase or crTPase sequences.This result indicated that PB element was stable without TRpase or crTPase activity in cells.On the contrary,RFLP patterns of the PB element were altered 1 week and 2 weeks after transient expression of TPase or crTPase in TR1 or TR3 cells.For example,a fragment of 1.5 kb containing PB element was present in TR1 prior to transformation of TPase-containing plasmid.One week after transformation,one initial fragment of 1.5 kb and 3 new fragments of 2.8,4.2 and 6.5 kb disappeared and appeared,respectively,suggesting transposition of the PB element occurred in TR1 cells.Two weeks after transformation,the size of all 3 fragments changed again,implying that transposition persists.In TR3,1 week after the transformation,3 fragments of 1.5,3 and 4 kb and 1 fragment of 6.5 kb remained unchanged and disappeared,respectively.Meanwhile,a new fragment of 2.8 kb appeared.The RFLP pattern was hardly altered again 2 week after the transformation.These results indicated that the sizes of PB element-containing fragments changed,implying the PB element transposed after introducing the TPase and crTPase activities in C.reinhardtii TR1 and TR3 strains.Furthermore,to test whether PB element had been transposed in these cells,we examined the presence of a new joint generated by excision of the PB element in the genome.To this end,primers flanking to the new joint were applied for PCR analysis using the genomic DNA derived from the colonies.Clearly,excision event of the PB element was detected.This result supports the results that PB transposon system is active in C.reinhardtii.To find out the reason that the cells could survive the transposition of PB element,PCR fragments containing the PB element excision joint were subjected to nucleotide sequence analysis.Alignment of the sequences indicated that cells from the colonies suffered from illegitimate excision of PB element.The study shows that illegitimate excision of PB element occasionally occurs at the short repeat sequences 5'-TTT-3'and 5'-ACGCAG-31,but the standard excision site 5'-TTAA-3'.This study shows that the TPase and crTPase are active in transposition of PB element in C.reinhardtii.Hence,we propose that PB transposon systems can be adopted for genome modification in C.reinhardtii.
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维普
