水产科学
水產科學
수산과학
FISHERIES SCIENCE
2014年
12期
826-830
,共5页
王佩%吕艳杰%吕黎%杨洪%宁黔冀
王珮%呂豔傑%呂黎%楊洪%寧黔冀
왕패%려염걸%려려%양홍%저검기
咪唑类物质K K-42%日本沼虾%日本沼虾几丁质酶基因-3%嗜水气单胞菌%基因表达
咪唑類物質K K-42%日本沼蝦%日本沼蝦幾丁質酶基因-3%嗜水氣單胞菌%基因錶達
미서류물질K K-42%일본소하%일본소하궤정질매기인-3%기수기단포균%기인표체
imidazole derivative KK-42%Macrobrachium nipponense%M . nipponense chitinase-3%Aero-monas hydrophila%expression
为探讨咪唑类物质K K‐42提高感染嗜水气单孢菌的日本沼虾幼虾成活率的机制，首次克隆了日本沼虾几丁质酶基因‐3部分序列，研究了其时空表达及酶活力的变化。将体长3．5～5．0cm的日本沼虾幼虾随机分为两组，分别用1．95×10‐4 mol／L的咪唑类物质KK‐42溶液（试验组1）或不含咪唑类物质KK‐42的溶液（对照组1）浸泡处理1 min ，12 h后，分别注射嗜水气单胞菌菌悬液（菌攻毒试验组和菌攻毒对照组）或生理盐溶液（试验组2和对照组2），不同时间测定幼虾的成活率，分析几丁质酶基因‐3 mRNA水平及其酶活力的变化。结果显示，12～48 h时，菌攻毒试验组的成活率比菌攻毒对照组增加133％以上。克隆的日本沼虾几丁质酶基因‐3部分mRNA序列与其他甲壳类具有90％以上同源性。几丁质酶基因‐3表达水平在肝胰腺中最高，以蜕皮前期最为显著。与对照组1相比，在12 h ，试验组1几丁质酶基因‐3 mRNA水平和酶活力分别增高了150％和75％；菌攻毒试验组mRNA水平在12～48 h明显高于菌攻毒对照组，尤其在24 h ，增幅高达304％，酶活力在24 h比菌攻毒对照组增高48％。咪唑类物质K K‐42预处理能显著上调感染嗜水气单胞菌的日本沼虾幼虾肝胰腺几丁质酶基因‐3的转录、诱导酶活力增强，这可能是咪唑类物质K K‐42提高幼虾成活率的机制之一。
為探討咪唑類物質K K‐42提高感染嗜水氣單孢菌的日本沼蝦幼蝦成活率的機製，首次剋隆瞭日本沼蝦幾丁質酶基因‐3部分序列，研究瞭其時空錶達及酶活力的變化。將體長3．5～5．0cm的日本沼蝦幼蝦隨機分為兩組，分彆用1．95×10‐4 mol／L的咪唑類物質KK‐42溶液（試驗組1）或不含咪唑類物質KK‐42的溶液（對照組1）浸泡處理1 min ，12 h後，分彆註射嗜水氣單胞菌菌懸液（菌攻毒試驗組和菌攻毒對照組）或生理鹽溶液（試驗組2和對照組2），不同時間測定幼蝦的成活率，分析幾丁質酶基因‐3 mRNA水平及其酶活力的變化。結果顯示，12～48 h時，菌攻毒試驗組的成活率比菌攻毒對照組增加133％以上。剋隆的日本沼蝦幾丁質酶基因‐3部分mRNA序列與其他甲殼類具有90％以上同源性。幾丁質酶基因‐3錶達水平在肝胰腺中最高，以蛻皮前期最為顯著。與對照組1相比，在12 h ，試驗組1幾丁質酶基因‐3 mRNA水平和酶活力分彆增高瞭150％和75％；菌攻毒試驗組mRNA水平在12～48 h明顯高于菌攻毒對照組，尤其在24 h ，增幅高達304％，酶活力在24 h比菌攻毒對照組增高48％。咪唑類物質K K‐42預處理能顯著上調感染嗜水氣單胞菌的日本沼蝦幼蝦肝胰腺幾丁質酶基因‐3的轉錄、誘導酶活力增彊，這可能是咪唑類物質K K‐42提高幼蝦成活率的機製之一。
위탐토미서류물질K K‐42제고감염기수기단포균적일본소하유하성활솔적궤제，수차극륭료일본소하궤정질매기인‐3부분서렬，연구료기시공표체급매활력적변화。장체장3．5～5．0cm적일본소하유하수궤분위량조，분별용1．95×10‐4 mol／L적미서류물질KK‐42용액（시험조1）혹불함미서류물질KK‐42적용액（대조조1）침포처리1 min ，12 h후，분별주사기수기단포균균현액（균공독시험조화균공독대조조）혹생리염용액（시험조2화대조조2），불동시간측정유하적성활솔，분석궤정질매기인‐3 mRNA수평급기매활력적변화。결과현시，12～48 h시，균공독시험조적성활솔비균공독대조조증가133％이상。극륭적일본소하궤정질매기인‐3부분mRNA서렬여기타갑각류구유90％이상동원성。궤정질매기인‐3표체수평재간이선중최고，이세피전기최위현저。여대조조1상비，재12 h ，시험조1궤정질매기인‐3 mRNA수평화매활력분별증고료150％화75％；균공독시험조mRNA수평재12～48 h명현고우균공독대조조，우기재24 h ，증폭고체304％，매활력재24 h비균공독대조조증고48％。미서류물질K K‐42예처리능현저상조감염기수기단포균적일본소하유하간이선궤정질매기인‐3적전록、유도매활력증강，저가능시미서류물질K K‐42제고유하성활솔적궤제지일。
This study was designed to determine the possible molecular mechanism of imidazole derivative KK‐42 acting for increasing survival rate of oriental river prawn Macrobrachium nipponense infected with Aeromonas hydrophila .The part sequence of oriental river prawn chitinase‐3 was firstly cloned and the spatio‐temporal expression of the gene as well as its activity were measured .The prawn with body length 3 .5—5 .0 cm was soaked for 1 min in KK‐42 solution at a concentration of 1 .95 × 10‐4 mol/L (KK‐42 treat‐ment group 1) or 0 (control group 1) ,separatively .The prawn in KK‐42 treatment group 1 was injected individually with A .hydrophila suspension (KK‐42 treatment group‐A .hydrophila) or saline (KK‐42 treatment group 2) into the ventral sinus at 12 h after KK‐42 treatment and the prawn in control group 1 was divided into control group‐A .hydrophila and control group 2 ,according to the same way .Then the survival rate was surveyed at different time ,and the prawn chitinase‐3 mRNA level and activity derived from different tissues were measured .Results showed that the survival rate was dramatically increased by 133% in KK‐42 treatment group‐A .hydrophila than that in the control group‐A .hydrophila .Sequence comparison indicated that the prawn chitinase‐3 deduced amino acid sequence of M .nipponense had an o‐verall similarity of 90% to that of other crustaceans .Real‐time PCR analysis revealed that the praw n chiti‐nase‐3 was mainly expressed in hepatopancreas ,and higher at premolt .Compared with the control group 1 ,the changes in the prawm chitinase‐3 mRNA level and activity from KK‐42 treatment group was found to be an up‐regulation about 150% and 75% ,respectively .In KK‐42 treatment‐A .hydrophila group ,the prawn chitinase‐3 mRNA transcripts were significantly higher at 12—48 h ,the maximum at 24 h with an increase by 304% ,than that in control group‐A .hydrophila and its activity increased by 48% at 24 h .In conclusion ,imidazole derivative KK‐42 pretreatment can significantly up‐regulate the prawn chitinase‐3 ex‐pression as well as its activity in hepatopancreas of prawn infected with A .hydrophila ,which is likely one of the molecular mechanisms of imidazole derivative KK‐42 acting for increasing survival rate of the prawn infected with A .hydrophila .
