Construction Of A Giant Panda (Ailuropoda Melanoleuca) Gene Resource Library And Creation Of A Method Of Extracting Highly Quility DNA From Feces

1. Genomic library is the recombinant DNA clones containing entire random fragments of a certain organism. It is essential to genome structure and function research. Thereinto, bacterial artificial chromosome(BAC) library is the most widely used one at present due to its higher transformation efficiency, lower chimerism level, easier insert fragments isolation and larger capability.The library is constructed by using fiberoctye and liver cells from Giant Panda(Ailuropoda melanoleuca) and pBeloBACll vector. It consists of 93,700 clones, which have been arrayed in 977 96-well microriter plates. The average insert size in the BAC vector is calculated to be 160kb based on the examination of 100 randomly selected clones, indicating that the library contained nearly 5-fold coverage of Giant Panda genome. Restrict analysis of 10 BAC clones show that these inserts are stable even reproduced for at least 100 cell generations.2. To clone the specific expression genes two full-length cDNA libraries of liver and brain of giant panda(Ailuropoda melanoleuca) were constructed by using the SMART cDNA library construction kit (Clontech). Total RNA was prepared fromliver and brain using RNeasy Mini Kit, and Oligotex (QIAGEN) was used to separate the mRNA of liver and brain from total RNA. The titer of un-amplified cDNA libraries was about 5.0><106 for liver library and 3.5><106 for brain library. The cDNA insert sizes were from 400bp to 4kb. The results obtained indicated that the quality of the cDNA libraries of liver and brain of giant panda is good enough for further cloning of the liver and brain specific expression genes. 3. The technique system of extracting highly qulity DNA from feces of giant panda was set up through the study: (1) Enough quantity of feces were needed to extract highly qulity DNA;(2) The effect of the PCR stayer can be effectively reduced by adding bovine serum albumin (BSA);(3) The alimentary tract epithelia can be riched by the method of grade centrifuge. It also showed that a large DNA fragment can be extracted from the Formalin-.xed feces samples. The established technique system can greatly accelerate the conservation genetics research on giant panda, and also it can be used for technique reference for conservation genetic research on other protcted widlife.