Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat 388 001, India.
Curr Microbiol. 2011 Sep;63(3):281-8. doi: 10.1007/s00284-011-9975-4. Epub 2011 Jul 10.
The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.
采用扩增核糖体 DNA 限制分析、18S rDNA 序列同源性和系统发育以及实时 PCR 分析方法研究了苏拉蒂水牛瘤胃原生动物的遗传多样性。三头动物饲喂由绿色饲料 Napier bajra 21（Pennisetum purpureum）、成熟牧场草（Dicanthium annulatum）和浓缩混合物（20%粗蛋白、65%总可消化养分）组成的日粮。使用原生动物特异性引物（P-SSU-342f）和真核生物特异性引物（Medlin B）从瘤胃液中扩增编码原生动物小亚基（SSU）核糖体 RNA 的 1,360 bp 片段的 DNA。共检查了 91 个克隆，并根据 PCR-RFLP 模式鉴定了 14 种不同的 18S RNA 序列。这些 14 种系统发育型基于 18S rDNA 数据库序列分为四个属，并鉴定为 Dasytricha（57 个克隆）、Isotricha（14 个克隆）、Ostracodinium（11 个克隆）和 Polyplastron（9 个克隆）。系统发育分析也用于推断苏拉蒂水牛瘤胃中原生动物群落的组成。在 14 个序列中，8 个序列（69 个克隆）与 Dasytricha ruminantium 样克隆聚类，4 个序列（13 个克隆）在系统发育上也与 Isotricha prostoma 样克隆聚类。此外，2 个系统发育型（9 个克隆）与 Polyplastron multivesiculatum 样克隆有关。此外，Dasytricha ruminantium（纤毛虫原生动物的 0.05%）的 18S rDNA 基因拷贝数高于 Entodinium sp.（2.0×10(5)比 1.3×10(4)）。
