Diversity Analyze Of Active Ureolytic Bacterial Community In The Rumen Of Dairy Cows

Ureolytic bacteria produce urease which hydrolyzes urea to ammonia,and the ammonia is an important N source for microbial growth and reproduction in rumen.Therefore,Ureolytic bacteria play a key role in the urea nitrogen cycle.The diversity of ureolytic bacteria have been explored based on in vitro culture and at gDNA level.However,there is no information at the RNA level which reflects the active status of bacteria.The purpose of this study was to reveal the diversity of active ureolytic bacteria in rumen.In order to extract high-quality RNA from rumen microorganisms,this study optimized the sample pretreatment method.Four factors during sampling were selected,and combined to five treatment groups.After the pretreatment,RNA was extracted with Trizol and then RNA concentration and integrity(RIN)was detected.The results showed that the concentration and integrity of RNA extracted from rumen contents and rumen bacteria was no significant difference.The concentration and integrity of RNA extracted from samples preserved in liquid nitrogen were significantly higher than those preserved in RNA protection solution.There was no significant difference in the effect of thawing treatment on RNA concentration,but the integrity of RNA extracted by rumen fluid with unfreeze was significantly higher than thawing treatment.The concentration of RNA extracted by liquid nitrogen frozen homogenized was significantly higher than that by room temperature,but there was no significant difference in RNA integrity.In order to analyze the diversity of active ureolytic bacteria in rumen,rumen fluid was collected from four Chinese Holstein dairy cows with rumen fistulas at 0,2 and 6 h after morning feeding.Total microbial gDNA and RNA was isolated,and the RNA was reverse-transcribed into cDNA.The ureC gene amplicon of gDNA and cDNA were produced and sequenced by Miseq.The results showed that it had a distinct ureolytic bacterial community profile for gDNA and cDNA.Approximately 43% ureC sequences from cDNA could not be confidently classified at phylum level.The sampling time had no significant effect on the alpha and beta diversity of ureC gene.Shannon diversity of ureC gene for cDNA was greater than that for gDNA(P < 0.05).Beta diversity of ureC gene showed significant difference between gDNA and cDNA(P < 0.01),which indicates a shift of community for active ureolytic bacteria.The active ureolytic bacteria were mainly from Helicobacter,Herbaspirillum,Clostridium,Paenibacillus,Synechococcus and Sphingobacterium,etc.The changes of OTUs revealed that the top abundant ureC genes were mostly consistent between gDNA and cDNA,and most of difference happened in low abundant diverse ureC genes.In this study,a pre-treatment method for extracting high-quality rumen microbial RNA was established,and the diversity of active ureolytic bacteria in the rumen was revealed,providing a theoretical basis for regulating the utilization efficiency of urea nitrogen in the rumen.