跨界原生质体融合菌Xhhh基因组分子鉴定

Molecular identification of Xhhh strain's genome from inter-kingdom protoplast fusion

Xhhh是由真核、原核两界生物3个亲株PC、SC、XZ的原生质体跨界融合而成的基因工程茵。本研究以Xhhh及其三亲株(PC、SC、XZ)的基因组DNA为扩增模板,筛选出的38条引物,应用随机扩增多态性DNA(RAPD)技术扩增出739条清晰条带,平均每条引物扩增出了7条清晰可重复的条带。通过聚类分析,结果显示Xhhh与PC、SC与XZ的遗传相似指数SI分别为36.21%、37.73%和37.48%,提示Xhhh与三亲株间存在明显的亲缘关系。研究同时根据已公布的mnp、lip以及FLO1核酸序列设计了相应的功能基因引物,并从Xhhh及其亲株中扩增出了mnp、lip以及FLO1功能基因片段,表明RAPD结合功能基因PCR扩增技术可以快速、经济、准确的用于跨界原生质体融合菌株的分子鉴定,以及其它融合子的鉴定。

Xhhh was a new gene-engineering strain constructed through inter-kingdom protoplast fusion from PC, SC and XZ. Randomly amplified polymorphic DNA （RAPD）was used to analyze nuclear genomes, identify hybrids and understand the relationship between parents and hybrid. A total of 739 clear and consistent bands were observed in RAPD analysis with 38 primers. The genetic similarity index between Xhhh and PC, SC, XZ was 36.21%, 37.73% and 37.48 % respectively. RAPD fingerprinting results showed that Xhhh had very near genetic relationship with its three parents. According to the sequences of Phanerochaete chrysosporium （ mnp, lip） genes and Saccharornyces cerevisiae （FL01） gene in the Genl3ank, the PCR products of the functional genes showed that Xhhh inherited the DNA fragments of mnp, lip and FLO1 genes from its parental strains. The methodology used in this paper was full-scale, precise, sen- sitive and cost effective for fusant identification. It also seemed to have a wide applicability, as there was no requirement for fusion parents.