海洋科学
海洋科學
해양과학
MARINE SCIENCES
2014年
5期
72-80
,共9页
张敏智%李广丽%朱春华%邓思平
張敏智%李廣麗%硃春華%鄧思平
장민지%리엄려%주춘화%산사평
金钱鱼(Scatophagus argue Linnaeus)%温度%鱼油%芳香化酶%mRNA表达
金錢魚(Scatophagus argue Linnaeus)%溫度%魚油%芳香化酶%mRNA錶達
금전어(Scatophagus argue Linnaeus)%온도%어유%방향화매%mRNA표체
Scatophagus argus%temperature%fish oil%Cyp19al%mRNA expression
为了探讨 Cyp19a1b 基因表达与温度及鱼油添加量的相互关系,奠定金钱鱼人工繁育理论基础,作者采用 RT-PCR 和 RACE 法,克隆了金钱鱼(Scatophagus argue Linnaeus)脑型芳香化酶基因(Cyp19a1b)；采用实时荧光定量PCR法,检测了不同温度(23、26和29℃)和不同水平鱼油(0、2%和6%)处理后二龄雌性金钱鱼脑垂体中Cyp19a1b mRNA表达。结果表明：Cyp19a1b cDNA全长2409bp,5′端非编码区164 bp,3′端(不包括poly(A))712 bp,开放阅读框(ORF)1506 bp,编码501个氨基酸,推测其蛋白质分子量为56.697 kDa。序列分析及分子系统进化树结果表明：金钱鱼Cyp19a1b氨基酸序列与黄锡鲷和舌齿鲈 Cyp19a1b 同源性较高,分别为86.2%、86.5%,与鲤鱼(Cyprinus carpio)、稀有鲫(Gobiocypris rarus)、斑马鱼(Danio rerio)、赤点石斑鱼(Epinephelus akaara)、大黄鱼(Larimichthys crocea)、南方大口鲇(Silurus meridionalis)和鲻鱼(Mugil cephalus)Cyp19a1b同源性为64.1%~84.4%,但与上述7种鱼性腺芳香化酶(Cyp19a1a)同源性低于63.5%,同源性分析结果与根据传统形态学和生化特征分类的结果相一致。荧光定量 PCR 结果显示,随处理时间的延长,26℃和29℃组金钱鱼脑垂体中Cyp19a1b mRNA的表达量都逐渐降低,但29℃组显著低于26℃组(P<0.05),而23℃组在6周时则显著高于26℃和29℃组(P<0.05),约为26℃组2倍的表达量；鱼油处理3个投喂组脑垂体中 Cyp19a1b mRNA 的表达量均逐渐降低,但鱼油对金钱鱼脑垂体中 Cyp19a1b mRNA 的表达量无显著影响(P>0.05)。以上结果表明：随着卵巢发育或鱼油含量的提高,可降低雌性金钱鱼脑垂体中Cyp19a1b mRNA的表达,这种降低可能是E2反馈调节所致；水温高于26℃将抑制Cyp19a1b mRNA的表达。
為瞭探討 Cyp19a1b 基因錶達與溫度及魚油添加量的相互關繫,奠定金錢魚人工繁育理論基礎,作者採用 RT-PCR 和 RACE 法,剋隆瞭金錢魚(Scatophagus argue Linnaeus)腦型芳香化酶基因(Cyp19a1b)；採用實時熒光定量PCR法,檢測瞭不同溫度(23、26和29℃)和不同水平魚油(0、2%和6%)處理後二齡雌性金錢魚腦垂體中Cyp19a1b mRNA錶達。結果錶明：Cyp19a1b cDNA全長2409bp,5′耑非編碼區164 bp,3′耑(不包括poly(A))712 bp,開放閱讀框(ORF)1506 bp,編碼501箇氨基痠,推測其蛋白質分子量為56.697 kDa。序列分析及分子繫統進化樹結果錶明：金錢魚Cyp19a1b氨基痠序列與黃錫鯛和舌齒鱸 Cyp19a1b 同源性較高,分彆為86.2%、86.5%,與鯉魚(Cyprinus carpio)、稀有鯽(Gobiocypris rarus)、斑馬魚(Danio rerio)、赤點石斑魚(Epinephelus akaara)、大黃魚(Larimichthys crocea)、南方大口鲇(Silurus meridionalis)和鯔魚(Mugil cephalus)Cyp19a1b同源性為64.1%~84.4%,但與上述7種魚性腺芳香化酶(Cyp19a1a)同源性低于63.5%,同源性分析結果與根據傳統形態學和生化特徵分類的結果相一緻。熒光定量 PCR 結果顯示,隨處理時間的延長,26℃和29℃組金錢魚腦垂體中Cyp19a1b mRNA的錶達量都逐漸降低,但29℃組顯著低于26℃組(P<0.05),而23℃組在6週時則顯著高于26℃和29℃組(P<0.05),約為26℃組2倍的錶達量；魚油處理3箇投餵組腦垂體中 Cyp19a1b mRNA 的錶達量均逐漸降低,但魚油對金錢魚腦垂體中 Cyp19a1b mRNA 的錶達量無顯著影響(P>0.05)。以上結果錶明：隨著卵巢髮育或魚油含量的提高,可降低雌性金錢魚腦垂體中Cyp19a1b mRNA的錶達,這種降低可能是E2反饋調節所緻；水溫高于26℃將抑製Cyp19a1b mRNA的錶達。
위료탐토 Cyp19a1b 기인표체여온도급어유첨가량적상호관계,전정금전어인공번육이론기출,작자채용 RT-PCR 화 RACE 법,극륭료금전어(Scatophagus argue Linnaeus)뇌형방향화매기인(Cyp19a1b)；채용실시형광정량PCR법,검측료불동온도(23、26화29℃)화불동수평어유(0、2%화6%)처리후이령자성금전어뇌수체중Cyp19a1b mRNA표체。결과표명：Cyp19a1b cDNA전장2409bp,5′단비편마구164 bp,3′단(불포괄poly(A))712 bp,개방열독광(ORF)1506 bp,편마501개안기산,추측기단백질분자량위56.697 kDa。서렬분석급분자계통진화수결과표명：금전어Cyp19a1b안기산서렬여황석조화설치로 Cyp19a1b 동원성교고,분별위86.2%、86.5%,여리어(Cyprinus carpio)、희유즉(Gobiocypris rarus)、반마어(Danio rerio)、적점석반어(Epinephelus akaara)、대황어(Larimichthys crocea)、남방대구점(Silurus meridionalis)화치어(Mugil cephalus)Cyp19a1b동원성위64.1%~84.4%,단여상술7충어성선방향화매(Cyp19a1a)동원성저우63.5%,동원성분석결과여근거전통형태학화생화특정분류적결과상일치。형광정량 PCR 결과현시,수처리시간적연장,26℃화29℃조금전어뇌수체중Cyp19a1b mRNA적표체량도축점강저,단29℃조현저저우26℃조(P<0.05),이23℃조재6주시칙현저고우26℃화29℃조(P<0.05),약위26℃조2배적표체량；어유처리3개투위조뇌수체중 Cyp19a1b mRNA 적표체량균축점강저,단어유대금전어뇌수체중 Cyp19a1b mRNA 적표체량무현저영향(P>0.05)。이상결과표명：수착란소발육혹어유함량적제고,가강저자성금전어뇌수체중Cyp19a1b mRNA적표체,저충강저가능시E2반궤조절소치；수온고우26℃장억제Cyp19a1b mRNA적표체。
In this study, in order to investigate the relationship between Cyp19a1b gene expression and the temperature or fish oil amount, laying the theoretical foundation for the artificial breeding of Scatophagus argus, a cDNA encoding Cyp19a1b was derived from S.argus, using RT-PCR and RACE methods. Meanwhile, we investigated the effect of different temperature (23℃, 26℃, and 29℃) and concentration of dietary fish oil (0, 2%, and 6%) on pituitary Cyp19a1b mRNA expression in 2-year-old female S.argus. Results showed that the cDNA was 2409 bp with 164 bp 5′UTR, 712bp 3′UTR (excluding poly (A)) and 1506 bp ORF, which encoded 501 amino acids and had a predicted mol wt of 56.697 kDa. Sequence and phylogenetic analysis indicated that the S.argus Cyp19a1b shared 86.2% and 86.5% sequence identity with Rhabdosargus sarba and Dicentrarchus labrax respectively, and between 64.1%~84.4% with that of Cyprinus carpio, Gobiocypris rarus, Danio rerio, Epinephelus akaara, Larimichthys crocea, Silurus meridionalis, and Mugil cephalus. However, it shared lower sequence identity (below 63.5%) with Cyp19a1a of the fish above. These results were consistent with those analyzed by traditional morphology and biochemistry in their evolution status. The fluorescent real time quantity PCR analysis revealed that pituitary Cyp19a1b mRNA expressions were found gradually decreasing with treatment time both in 26℃ and 29℃ group, but that in 29℃ group was significantly lower than that in 26℃ group (P <0.05). However, higher pituitary Cyp19a1b mRNA expression was found in 23℃ group at 6 weeks compared to that in 26℃ and 29℃group, and was about 2 times higher than that in 26℃ group. In addition, pituitary Cyp19a1b mRNA expressions were found gradually decreasing with feeding time in 3 groups of different concentration of dietary fish oil, but no significant difference was found among the 3 groups (P >0.05). Results showed that pituitary Cyp19a1b mRNA expression would gradually decease with ovarian development or increasing of fish oil concentration in female S.argus, which might be caused by estradiol feedback regulation, and temperature more than 26℃ would restrain pituitary Cyp19a1b mRNA expression.
