运用cDNA-AFLP技术初步鉴定两种方法合成的cDNA的指纹图谱

Identification of cDNA Synthesized by Different Methods Using cDNA-AFLP Fingerprinting Technology

在植物基因克隆以及构建cDNA文库时,都需要合成高质量的双链cDNA,并要达到一定的数量。然而研究者经常受到试验样品量的限制。特别是比较稀少的植物材料,例如植物的根尖、茎尖或花的雌、雄蕊等,难以获得足够的RNA,以致影响cDNA的合成量,无法开展下游的实验。以PCR为基础合成第二链cD-NA的Smart技术(LD-PCR),能够以50ng的总RNA为反转录模板合成高质量的双链cDNA。但研究者对第二链采用PCR方法是否有些基因信息丢失或丰度上发生很大的变化存有疑虑。针对以上存在的问题,通过置换合成和长距离PCR(LD-PCR)两种方法合成3个月的梭梭幼苗茎尖双链cDNA,EcoRI和MseI限制性内切酶双酶切后,用16对选择性扩增引物对两种cDNA进行cDNA-AFLP的指纹图谱分析。结果表明,置换合成和LD-PCR两种方法合成的cDNA指纹图谱中,分别有条带约495条和470条。其中,相同的条带共计433条,不同的条带分别有62条和37条,分别为各自合成方法总指纹数的12.5%和7.87%,相同的指纹信息达87%以上。这说明两种方法合成的cDNA存在着较大的差异,合成方法对cDNA合成质量的影响较大,为研究人员选用何种cDNA合成方法提供了借鉴。

High-quality and adequate eDNAs are the premise when plant gene cloning and eDNA library constructing are carried out. But the limited experimental samples, such as root and shoot tips, stamen and pistil of flowers, influence eDNA synthesis and further restrict the downstream studies. Based on the Long-Distance PCR （LD-PCR） technology, enough double strands eDNAs can be synthesized by the least 50ng total RNA. However, researchers are not sure if some gene information have lost or changed by LD-PCR method. To resolve the problems mentioned above, double strands eDNAs of three-month Haloxylon Ammodendron young seedlings were synthesized by two methods--replacement synthesis and LD-PCR. After digested by the restriction enzyme of EcoR Ⅰ and gse Ⅰ, eDNA-AFLP fingerprinting of two eDNAs was analyzed. The results demonstrated that there are 495 and 470 bands respetively in above two eDNAs, the equal has 433 bands, the different have 62 and 37 bands respectively, the same fingerprinting accounts for 87%. Which showed there exists the differences of two eDNAs synthesized by the above methods.