Analysis Of The Intestinal Microflora Of The Reintroducting Giant Panda By PCR-DGGE

The giant panda is an endangered species. Due to habitat fragmentation, their populations has become isolated, and even there is the extinction of small populations.In order to supplement or rejuvenation of small populations, the reintroduction become an important means to save their populations. In the reintroduction process, to determine the time of reintroduction, in addition to observe the giant pandas’behavior characteristics and other biological data, understanding the condition of the intestinal flora is one of the important indicators for evaluating their wild state. The methods of studying the intestinal flora is mainly divided into the traditional identification method and the molecular biological identification method. The molecular identification method is a useful complement and expansion to the traditional method. It provides a more accurate tool for the study of intestinal flora, and PCR-DGGE technique is a more practical, convenient and efficient way with high-resolution spectra.The trial was the first continuous tracking to capture faeces samples in the reintroduction process. The samples were collected in the period of15months from September2011to November2012. The faeces samples of the wild giant pandas, the captive giant pandas and the giant pandas reintroduced into the wild were8,21and39.The faeces samples were divided into five groups in accordance with the four seasons. The samples were extracted complete DNA and enlarged the V3region gene fragment in16S rRNA by Nested PCR and Touchdown PCR, and then to get diversity fingerprint by DGGE, analyzed the fingerprint to get lane images, similarity matrix and phylogenetic tree by quantity one and other software, and then we utilized the data to make the bioinformatic analysis of the samples. In the experiments we also made ERIC-PCR as a reference to know the advantages and disadvantages of the two methods in the area of giant pandas intestinal flora diversity analysis. Compared with ERIC-PCR, PCR-DGGE had more advantages in reflecting the diversity of animal intestinal flora. We could get higher quality maps, more comprehensive diversity and more accurate data by PCR-DGGE. The advantage in analysis of the giant panda intestinal flora diversity was obvious higher than ERIC-PCR.It could be drawn through the analysis and the results of the test, in the five quarters from Fall2011to Fall2012, the structural similarity average of the intestinal flora of the giant pandas reintroduced into the wild and the pandas in the wild was47.0%,50.1%,55.7%,44.8%and49.8%, respectively, and the structural similarity average of the intestinal flora of the giant pandas reintroduced into the wild and the captive giant pandas, not including the captive giant pandas in Changsha Zoo, was53.6%.46.7%,53.5%,40.8%and48.6%, respectively. It could be drawn through the test results, in the fall of2011and fall of2012, the structural similarity of the intestinal flora of the giant pandas reintroduced into the wild and the pandas in the wild increased from47.0%to49.8%, however, comparing the intestinal flora of the giant pandas reintroduced into the wild and the captive giant pandas, the structural similarity reduced from53.6%to48.6%. The experiments showed that in the reintroduction process, the intestinal flora of the giant pandas reintroduced into the wild and the pandas in the wild was becoming more and more consistent and the reintroduction achieved certain results. In the five quarters, the structural similarity of the intestinal flora of the giant pandas reintroduced into the wild and the pandas in the wild, in the spring, was the highest one. The similarity was55.7%. The external environment in the spring had relatively small impact on the giant panda intestinal flora, and it was a wise choice to release the giant panda into the wild at this time.The results showed that, the structural similarity of the intestinal flora of Caocao, a giant panda reintroduced into the wild, and the pandas in the wild increased from45.5%to59.7%.,while, the structural similarity of the intestinal flora of Jinzhu, like Caocao, and the pandas in the wild reduced from53.9%to52.9%.It showed that, under the same conditions, the structural similarity of the intestinal flora of the giant panda individuals also had very significant differences. The structural similarity average of the intestinal flora of Caocao, Qianqian, Jinzhu, Taotao and Zhangka, the giant pandas reintroduced into the wild,and the pandas in the wild was respectively57.2%,50.5%,53.8%,41.6%and57.4%, respectively in the winter, yet35.3%,54.6%,59.5%,52.4%and43.0%, respectively in the summer. The similarity between the two types of panda was no obvious trend. It was reasoned that individual factors had led to the giant pandas reintroduced into the wild not fully adapting to poor weather like the pandas in the wild.In short, we believed that from the perspective of intestinal flora similar batch of giant pandas after field training, it is appropriate to choose spring reintroduction.