Study On The Interaction Between Brucella Melitensis SRNA AbcR1 And Targets

Brucella is a species of Gram-negative facultative intracellular bacteria which can cause brucellosis of livestocks and human. Brucellosis is a zoonotic disease that is able to spread via skin, mucosa, inhalation and ingestion. When infected, goats, cattle and swine will suffer infectious abortion and sterility caused by placentitis while human will fall victim to epididymitis, orchitis, Malta Fever and hyperhidrosis. Once invading relevant cells, brucella can establish chronic infection. Intriguingly, it shows a characteristics to avoid the host immune system through unknown mechanism. Recent research indicates that, s RNA(small non-coding RNA), which exists among bacterias, has potential functions about regulation of metabolism and virulence. Previous research of RNA-seq in this lab reveals that s RNA of brucella melitensis Abc R1 and Abc R2 might have potential of regulatory funtions.The s RNA of brucella binds its corresponding m RNA and then perform its function. According to homologous alignment and bioinformatics prediction we found a series of potential target genes of this s RNA such as BMEII0923, BMEII0372, BEMI1250, which relates to Mer R family transcriptional regulator, short chain dehydrogenase, and periplasmic protein. First, rapid-amplification of c DNA end(RACE) was utilized to find the TSS of these targets. Then these targets were cloned to p XG-10 SF vector which can express superfolder GFP protein infused with targets at the carboxylated terminal, meanwhile we cloned the s RNA to Amplicillin resistance vector p ZK001. The vector, which is of chloromycetin resistance, contains GFP, is using to merge target. After cotransformation of these two plasmids, bacterial colonies with both chloromycetin and Amplicillin resistances were checked for fluorescence under stereomicroscope; Then ultrasonic wave was used to lyse bacteria, and Western blot was performed to detect the expression of GFP. Finally, the transcription of GFP was quantified by q RT-PCR. The experiments was summarized as following:1. Target RNA2, which is focus on bacteria s RNA targets finding, can be used for Brucella s RNA targets analysis, for instance, Abc R1, but the prediction need further validation by experiments.2. Brucella melitensis’ s s RNA Abc R1 is able to down regulate BMEII0923’s expression, and up-regulate the expression of BMEII0372 in vitro. For BMEI1250, results shows that this gene is not a target of s RNA Abc R1.3. The GFP reporting system can be used to validate interaction between s RNA and its targets instantly and directly in brucella melitensis.Compared to conventional methods such as western blot and q RT-PCR, this GFP reporting system is more quickly and convenient and can be used directly to validate s RNA and predicted targets visually. This approach may benefit to save time and energy to find new insights to regulation of s RNA on targets of brucella about virulence and metabolism.