Detection Of Vibrio Harveyi Gene Expression In The Viable But Non-culturable State By Real-time PCR

Vibrio harveyi is a Gram-negative, luminous bacterium found in marine environments. It can infect many marine invertebrate and vertebrate species, and it is a primary pathogen that causes vibrisis in marine animal. The viable but nonculturable state (VBNC) is a kind of special survival state when bacteria are under harsh surroundings conditions. When entered into this state, the bacteria cells become coccoid and decrease in size. The VBNC cells no longer able to grow and form colonies on conventional culture media but still maintain metabolic activity and pathogenicity. However, when optimal conditions are restored, the VBNC cells may return to culturable state. It has been described that V. harveyi can enter the VBNC state at low temperature under poor nutrition conditions. The aim of this study was to analyse the six virulence-related and stress-related gene(vhh, hsp, cysS, oppA, MnSOD, rpoS) expression of V. harveyi in the whole process from cold inducement to resuscitation using real-time PCR technique. And the influence of heat shock and cold shock on these six gene expression was also investigated.V. harveyi SF-1 was incubated at 4℃for Oh, 1h,2h,4h,8h,12h and the gene expression of V. harveyi was detected by Real-time PCR technique. The results showed that the expression of vhh and cysS genes decreased during the cold shock treatment. Expression of oppA gene did not show significant regularity. Expression of MnSOD, rpos and hsp genes increased obviously during the whole process of cold shock.V. harveyi SF-1 was incubated in 40℃water bath Omin, 10min,20min,3 Omin, 50min and the gene expression of V. harveyi was detected by Real-time PCR technique. The results showed that the hsp gene expression increased obviously during the heat shock treatment, especially at the the earlier stage of treatment. Expression of MnSOD gene increased to 9.641 times after 10min treatment, and then decreased slowly. Expression of MnSOD gene restored to normal level after 50min treatment. Expression of oppA gene decreased obviously. Expression of vhh, cysS and rpoS genes increased a little at the earlier stage of heat shock, and then decreased obviously.V. harveyi SF-1 were incubated in artifical sea water microcosm at 4℃. The result showed that large population of the V. harveyi became nonculturable after 84d cultured in artifical sea water at 4℃. Real-time PCR technique was used to detect six virulence-related and stress-related gene expression ofⅤ. harveyi in the whole process from cold inducement to resuscitation. The result showed that expression of rpos, cysS and vhh genes increased during the earlier stage of low temperature induction, and then decreased slowly with the inducement time prolonged. The expression of rpos, cysS and vhh genes respectively decreased to 0.037,0.055 and 0.232 times than normal level. After resuscitated from VBNC, expression of rpos gene up to 0.674 times, cysS and vhh gene expressed normally. The expression of Hsp, oppAand MnSOD gene decreased during the low temperature induction and VBNC state. When the cells were resucitated from the VBNC state, expression of these genes increased to 1.765,0.232 and 2.593 times respectively. Virulence-related gene expression decreased whenⅤ. harveyi entered into VBNC state. However, the gene expression restored to the normal level after they were resuscitated from VBNC, which may be one of the important reasons that the resuscitative V. harveyi retain their pathogenicity. Key words:Vibrio harveyi; VBNC; Resuscitation; Real-time PCR; Cold shock; Heat shock; Gene expression...