冷应激对雏鹅HPA和HPT轴活性影响及其相关基因表达调控的研究
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摘要
应激是机体对各种刺激所产生的一种非特异性应答反应。劣性应激可引起畜禽生产性能下降,诱发多种疾病,甚至造成死亡。寒冷是雏禽最普遍的应激原之一,由此引起的直接和间接经济损失是制约北方畜牧业发展的重要因素,研究动物冷应激发生机制,探索科学的应激监测方法,对提高应激预防水平,保障畜牧业发展有重要意义。冷应激对动物HPA和HPT轴为主的神经内分泌系统产生重要影响。然而未见冷应激对雏鹅HPA、HPT轴的影响及其相关基因表达调控的研究报道。本研究以7日龄皖西白鹅为试验对象,研究于12℃±1℃条件下进行急性冷应激处理时对HPA、HPT轴的影响。利用放射免疫法、FQ-RT-PCR等技术动态考察冷应激反应中血液生理生化指标、应激激素相关基因TRH、TSH、CRH、ACTH和HSP70基因mRNA的变化规律。同时采用RT-PCR技术克隆测序HSP70基因cDNA序列,并进行生物信息学分析。为深入研究动物冷应激机理,动物冷应激的早期监测和防治提供重要实验依据。研究主要取得如下成果：
1.雏鹅受到冷应激时血相指标变化的测定结果
以7日龄皖西白鹅为试验对象,研究于12℃±1℃条件下进行急性冷应激对皖西白鹅雏鹅某些血相指标变化的影响。研究结果表明,全血中红细胞(RBC)、红细胞压积(HCT)、血红蛋白(HGB)、红细胞分布宽度(RDW)总体呈减少趋势,且RBC在冷暴露3h、6h和冷暴露结束后0.5h和3h极显著低于冷应激前的水平；HCT和HGB在冷暴露3h、6h和冷暴露结束后3h时显著低于冷应激前的水平；RDW在冷暴露6h后至结束冷暴露后12h期间均极显著低于应激前水平(P<0.01)。白细胞(WBC)在冷暴露12h和24h时显著升高。淋巴细胞绝对值(LYM)在冷暴露6h时显著低于应激前的水平(P<0.05),淋巴细胞百分比(LYM%)在冷暴露后3h、6h和12h时显著升高(P<0.01),但冷应激24h时淋巴细胞百分比已经恢复正常,雏鹅已经开始适应。首次获得了皖西白鹅血液生理指标参考值,为兽医临床诊断与治疗提供了重要依据之一
2.雏鹅冷应激时HPA、HPT轴应激激素测定的结果
研究于12℃±1℃条件下进行急性冷应激处理时对雏鹅下丘脑-垂体-肾上腺皮质(HPA)轴和下丘脑-垂体-甲状腺(HPT)轴的影响。结果表明,FT3在冷暴露6h和12h时显著升高。FT4在冷暴露3h显著升高(P<0.01),随后逐渐下降,至结束冷暴露后12h显著降低(P<0.05)。T3在冷暴露过程中逐渐升高,在冷暴露6h和结束冷暴露后0.5h和3h时明显高于应激前水平。T4在冷暴露6h和结束冷暴露后6h时达到峰值。Cor在冷暴露3h时显著升高(P<0.05),并在冷暴露期间保持高水平。血清中ACTH随冷暴露时间延长而逐渐升高,在冷应激6h时显著高于应激前的水平(P<0.01),至冷暴露结束后3h时,ACTH再次明显升高(P<0.05)。ACTH、Cor和FT3反应敏感,初步认为它们可以作为评估冷应激程度的辅助指标。
3.雏鹅冷应激反应中HPA、HPT轴应激激素相关基因表达调控规律
采用RT-PCR技术对HPA、HPT轴应激激素相关基因TRH、TSH、CRH和ACTH克隆、测序,同源性比较,鹅TSH基因测序序列与GenBank上鹅TSH序列同源性为100%。鹅TRH、CRH、ACTH基因序列与GenBank上鸡TRH、CRH、ACTH基因序列同源性分别为94%、95%和95%。利用实时荧光定量逆转录PCR技术分析这些基因的表达调控规律。下丘脑TRH基因mRNA转录量在冷应激0.5h、3h、6h、9h、18h与应激结束后1h、6h和12h显著升高,其他时间内均下降到室温时的转录水平。垂体TSH基因nRNA转录量在冷应激1.5h到12h期间显著升高,应激12h时转录水平达到最高点,而其他应激时间均降低到应激前的水平,应激结束后1h到6h期间垂体TSH基因mRNA转录水平也被显著升高,冷处理结束后9h-24h已恢复到应激前的正常水平。下丘脑CRH基因nRNA转录量在冷应激3h、9h、18h时与应激结束后0.5h、1h和9h显著升高,其他时间点降到应激前的正常水平。肾上腺ACTH基因mRNA转录水平在冷应激1.5h-9h、18h时显著升高,其他时间点均迅速下降到应激前水平；冷处理结束后0.5h和3h,由于又突然受到热的室温应激,ACTH基因mRNA转录量又显著升高,其他时间点降到应激前的正常水平。
下丘脑TRH基因mRNA表达呈现波动性变化趋势,但表达量均不超过1.5,说明下丘脑TRH基因是负调控的。下丘脑CRH、垂体TSH和肾上腺ACTH基因1nRNA的表达量分别在2.27-7.76、1.75-6.17、5.5-14.83范围内,总体呈上升趋势,说明TSH、CRH和ACTH基因均是冷应激反应时的正调控基因。
4.鹅HSP70基因的克隆和生物信息学分析
首次克隆了皖西白鹅HSP70基因cDNA序列2181bp,包括CDS全序列1905bp和3’UTR序列276bp,编码634个氨基酸。与GenBank上乌棕鹅HSP70基因cDNA序列进行比对分析,皖西白鹅HSP70基因共存在15个核苷酸变异(均在CDS区),只有1个蛋白质位点的差异,编码区同源性序列比对,与乌棕鹅同源性达99%,与家鸭、鸡、日本鹌鹑HSP70基因编码区同源性序列同源性分别达97%、92%和92%,与哺乳动物同源性均高于82%,进一步证实了HSP70在不同生物间的高度保守性。
利用生物信息学预测鹅HSP70蛋白质的结构与功能,结果表明,HSP70蛋白质以亲水性区域为主；该蛋白具有丰富的B细胞抗原位点；抗原表位区域、柔韧性区域和表面可能性区域出现了较多的重叠区,这些区域相对易于形变,便于抗原、抗体的自由结合,可能是抗原位点的富集区；该蛋白二级结构以α-螺旋和无规则卷曲为主,无明显的跨膜螺旋,含有34个磷酸化位点,无糖基化位点,推测其可能在细胞信号转导与调控中发挥重要作用。
5.雏鹅冷应激反应中HPT、HPTA轴HSP70mRNA的动态表达规律
实时荧光定量逆转录PCR法检测结果表明,下丘脑HSP70mRNA转录量在冷应激(12℃±1℃)1.5h、6h和9h显著升高,其他时间点均恢复到正常水平；在冷处理结束后6h内HSP70mRNA转录水平均显著升高,应激结束后6h转录水平最高,随后又迅速下降到室温下的转录水平。垂体HSP70mRNA转录量在冷应激0.5h、1.5h、6h和12h时显著降低,其他应激时间均升高到应激前的水平,应激结束后0.5h、3h和6h转录水平也被显著降低,随后逐渐升高到应激前的正常水平。甲状腺HSP70mRNA转录量在冷应激0.5h时显著升高,随后迅速下降到应激前的水平,应激3h、6h和12h时显著降低。应激结束后0.5h和3h转录量显著升高,其他时间点逐渐恢复到正常水平。肾上腺HSP70基因mRNA转录水平在冷应激1.5h、6h、9h时和冷处理结束后3h到6h期间表达水平被显著降低,其他应激时间恢复到应激前的水平。冷应激期间及结束冷处理后一段时间,肾上腺HSP70基因mRNA表达水平都很低,在0.02-0.57范围内,HSP70基因mRNA表达量仍然不超过1,推测HSP70基因也是肾上腺组织细胞表达的下调基因,是负调控的。雏鹅冷应激反应中,HSP70基因在下丘脑、垂体、甲状腺和肾上腺中的表达规律是不同的,在不同的组织中有不同的调控规律。下丘脑HSP70mRNA转录水平最高,表达量总体呈现明显上升趋势,是冷应激反应时的正调控基因：而它在垂体、甲状腺和肾上腺中的表达总体是被抑制的,是垂体、甲状腺和肾上腺冷应激反应时的负调控基因。初步认为可以将HSP70作为动物冷应激的分子生物标志。
Stress is a nonspecific response to various kinds of stimulates in organism which can cause performance descends, induce various diseases, even result in death in livestock and poultries. The cold is one of the most important factor to restrict livestock farming development owing to the direct and indirect economic loss. Studying'the animal cold stress mechanism and investigating scientific stress monitor method have important meaning to elevate preventative level of stress and ensure livestock farming development. Cold stress has important effect on the HPA and HPT axis of nerve internal system, however, there was no correlated study in young goose. In this study,7d Wanxi White geese were used to study the effects on hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-thyroid (HPT) axis under cold stress (12℃±1℃), using the radioimmunity, fluorescence quantitative RT-PCR etc.
Dynamic change rule of blood physiological and biochemical indicators, stress hormone, TRH, TSH, CRH, ACTH, HSP70mRNA by different times during cold exposure0,0.5,3,6,9,12,18,24hour and after terminate cold exposed0,0.5,3,6,9,12,24hour using radioimmunity, fluorescence quantitative PCR. Moreover, HSP70gene was cloned from Wanxi white goose by RT-PCR, the protein structure and function were further predicted by bioinformatics. The studies can provide experimental evidence for investigating reaction mechanism and earlier monitoring of animal cold stress. The main results were showed as following:
1. Analysis of blood routines
7d Wanxi White geese were used to study the influence on some parameters of blood under cold stress (12℃±1℃). The results were as follows. The Red blood cell (RBC), Haematocrit (HCT), Hemoglobin (HGB), Red blood cell distribution width (RDW) were decreased collectively. RBC was significant lower at3h,6h and at0.5h,3h after terminating cold stress than pre-cold stress. HCT and HGB were obviously decreased at3h,6h during cold stress and at3h after terminating cold stress. RDW was lower at6h during cold stress and at12h after end cold stress than pre-cold stress significantly (P<0.01). WBC was increased at12h,24h during cold stress. The absolute value of Lymphoid cell (LYM) was lower at6h during cold stress than pre-cold stress significantly (P<0.05). LYM%was increased at3h,6h and12h after cold stress (P<0.01). however, at normal level at24h after cold stress. Collecting detected the hematological results from all test groups during cold stress, obtaining Wanxi white goose blood physiological and biochemical reference values, which can provide important proof for veterinary clinical diagnosis and therapy.
2. Analysis of stress hormone in HPA and HPT anxis
The effect on HPA axis and HPT axis was analyzed under cold stress (12℃±1℃) in Wanxi White geese. The results showed as follows.The Free triiodothyronine (FT3) was significantly higher at6h and12h after acute cold stress than that of pre-cold stress. The Free thyroxine (FT4) was obviously higher at3h in12℃±1℃temperature, however, dropped gradually later, decreased at12h (P<0.05). The triiodothyronine (T3) was increased steadily during cold stress and significantly higher at6h after cold exposure and at0.5h,3h after end cold exposure. The Thyroxine (T4) got peak value at6h after cold exposure and6h after end cold exposure. The cortisone (Cor) retained in the higher level at more parts time of cold exposure and obviously higher at3h during cold exposure. The Adrenocorticotrophic hormone (ACTH) significant increased at6h after cold exposure and at3h after end cold exposure (P<0.01, P<0.05). The ACTH, Cor and FT3are sensitive to cold stimulus.They can probably be used as the supply indexes evaluating cold stress.
3. Analysis dynamic expression of gene mRNA correlated with hormone in HPA and HPT axis
TRH, TSH, CRH and ACTH gene in Wanxi white goose were coloned using RT-PCR. The sequences identity with that of chicken were94%,95%and95%, respectively. Dynamic expression of TRH, TSH, CRH and ACTH mRNA in HPA and HPT axis was analyzed by FQ-RT-PCR. Hypothalamic TRH mRNA transcription level went up significantly at0.5h,3h,6h,9h ad18h during cold stress; It increased again after cold exposure in1h,6h andl2h; It decreased to normal level in the other time. Pituitary TSH mRNA transcription level was increased significantly from1.5h to12h, moreover, it went up the highest level at12h; Hereafter it declined to normal level. It increased again after cold exposure from1h to6h and was decreased to normal level after6h. Hypothalamic CRH mRNA transcription level went up significantly at3h,9h,18h during cold stress and at1h,6h and12h after cold exposure; It was down-regulated to normal level at the other time. Adrenal gland ACTH gene mRNA transcription level went up significantly from1.5h to9h and at18h during cold stress; It increased again after cold exposure at0.5h and3h; It decreased to normal level in the other time.
Hypothalamic TRH mRNA transcription level had fluctuation trend and it's quanity was not over1.5, which indicated that Hypothalamic TRH was the down-regulation gene in cold stress reaction. The transcription quanity of TSH, CRH and ACTH was227-7.76、1.75-6.17、5.5-14.83and had ascend trend, which showed that they were the up-regulation gene in cold stress reaction. The results demonstrated that TRH, TSH, CRH and ACTH gene can be probably used as the molecular biomarker of animal cold stress.
4. HSP70gene cloning and bioinformatics
HSP70gene cDNA was cloned, whose size was2181bp in Wanxi white goose for the first time. It's1905bp of CDS and276bp of3'-UTR sequence, and encoded634amino acids. There were15nucleotides (in CDS) and only one amino acids difference, which were different between2populations. HSP70CDS sequence in goose had82-90%homology with mammals and fish, and up to99%with Chinese goose. Cluster analysis revealed that probably mammals, birds and fishes, each of these belonged to different categories separately.
Bioinformatics analysis indicated that goose HSP70protein contained34phosphorylation sites. The diversity of CDS and the structure of amino acids in HSP70genes resulted in the differences of protein secondary structure. HSP70protein secondary structure had more Alpha and Coil region, without signal peptide and transmembrane helix; The hydrophobicity structure prediction of goose HSP70protein indicated that it had major hydrophobicity region. The protein had abundant B cell antigen location. There were more overlapping region in flexibility, surfaceprobability and antigencity, which made antigen and antibody combine easily, and it was probably antigen enrichment region. It was predicted that HSP70protein probably played an important role in cell signal transfer and control mechanisms.
5. Analysis dynamic expression of HSP70mRNA in HPA and HPT axis during cold stress
The dynamic expression of HSP70mRNA in HPA and HPT axis of7d Wanxi White goose was got utilizing FQ-RT-PCR. Hypothalamic HSP70mRNA transcription level went up significantly atl.5h,6h and9h; It decreased to normal level in the other time; It increased again after cold exposed in6hours, moreover, it went up the highest level at6h;Hereafter it declined to normal level. Pituitary HSP70mRNA transcription level descended significantly at0.5h,1.5h,6h and12h. It went up again gradually to normal level in the next12hours. It decreased again significantly at0.5h,3h,6h after cold exposure, hereafter, gradually increased to normal level. Thyroid HSP70mRNA transcription level went up quickly at0.5h, hereafter decreased at3h,6h,12h significantly under cold stress. It went up significantly at0.5h and3h after cold exposure and it was down-regulated gradually to normal level in the other time. Adrenal gland HSP70mRNA transcription level was decreased at1.5h,6h,9h during cold stress and at3h,6h after end cold exposure. It was up-regulated gradually to normal level in the other time. The Adrenal gland HSP70mRNA transcription quanity was0.02-0.57, which was predicted that it was down-regulation gene in cold stress reaction.
The expression rule of HSP70mRNA was different in Hypothalamic, Pituitary, Thyroid and Adrenal gland. Hypothalamic HSP70mRNA transcription level was the highest which showed going up trend as a whole. HSP70was the up-regulation gene of Hypothalamic in cold stress reaction. Pituitary, thyroid and adrenal gland HSP70mRNA transcription level was repressed, which showed that it was the down-regulation gene of pituitary, thyroid and adrenal gland in cold stress reaction. The results demonstrated that it play a part in protective cell function, on the other hand, denoted that histiocyte function was placed in the urgent condition. The lymphocyte HSP70can probably be used as the molecular biomarker of animal cold stress.
1.雏鹅受到冷应激时血相指标变化的测定结果
以7日龄皖西白鹅为试验对象,研究于12℃±1℃条件下进行急性冷应激对皖西白鹅雏鹅某些血相指标变化的影响。研究结果表明,全血中红细胞(RBC)、红细胞压积(HCT)、血红蛋白(HGB)、红细胞分布宽度(RDW)总体呈减少趋势,且RBC在冷暴露3h、6h和冷暴露结束后0.5h和3h极显著低于冷应激前的水平；HCT和HGB在冷暴露3h、6h和冷暴露结束后3h时显著低于冷应激前的水平；RDW在冷暴露6h后至结束冷暴露后12h期间均极显著低于应激前水平(P<0.01)。白细胞(WBC)在冷暴露12h和24h时显著升高。淋巴细胞绝对值(LYM)在冷暴露6h时显著低于应激前的水平(P<0.05),淋巴细胞百分比(LYM%)在冷暴露后3h、6h和12h时显著升高(P<0.01),但冷应激24h时淋巴细胞百分比已经恢复正常,雏鹅已经开始适应。首次获得了皖西白鹅血液生理指标参考值,为兽医临床诊断与治疗提供了重要依据之一
2.雏鹅冷应激时HPA、HPT轴应激激素测定的结果
研究于12℃±1℃条件下进行急性冷应激处理时对雏鹅下丘脑-垂体-肾上腺皮质(HPA)轴和下丘脑-垂体-甲状腺(HPT)轴的影响。结果表明,FT3在冷暴露6h和12h时显著升高。FT4在冷暴露3h显著升高(P<0.01),随后逐渐下降,至结束冷暴露后12h显著降低(P<0.05)。T3在冷暴露过程中逐渐升高,在冷暴露6h和结束冷暴露后0.5h和3h时明显高于应激前水平。T4在冷暴露6h和结束冷暴露后6h时达到峰值。Cor在冷暴露3h时显著升高(P<0.05),并在冷暴露期间保持高水平。血清中ACTH随冷暴露时间延长而逐渐升高,在冷应激6h时显著高于应激前的水平(P<0.01),至冷暴露结束后3h时,ACTH再次明显升高(P<0.05)。ACTH、Cor和FT3反应敏感,初步认为它们可以作为评估冷应激程度的辅助指标。
3.雏鹅冷应激反应中HPA、HPT轴应激激素相关基因表达调控规律
采用RT-PCR技术对HPA、HPT轴应激激素相关基因TRH、TSH、CRH和ACTH克隆、测序,同源性比较,鹅TSH基因测序序列与GenBank上鹅TSH序列同源性为100%。鹅TRH、CRH、ACTH基因序列与GenBank上鸡TRH、CRH、ACTH基因序列同源性分别为94%、95%和95%。利用实时荧光定量逆转录PCR技术分析这些基因的表达调控规律。下丘脑TRH基因mRNA转录量在冷应激0.5h、3h、6h、9h、18h与应激结束后1h、6h和12h显著升高,其他时间内均下降到室温时的转录水平。垂体TSH基因nRNA转录量在冷应激1.5h到12h期间显著升高,应激12h时转录水平达到最高点,而其他应激时间均降低到应激前的水平,应激结束后1h到6h期间垂体TSH基因mRNA转录水平也被显著升高,冷处理结束后9h-24h已恢复到应激前的正常水平。下丘脑CRH基因nRNA转录量在冷应激3h、9h、18h时与应激结束后0.5h、1h和9h显著升高,其他时间点降到应激前的正常水平。肾上腺ACTH基因mRNA转录水平在冷应激1.5h-9h、18h时显著升高,其他时间点均迅速下降到应激前水平；冷处理结束后0.5h和3h,由于又突然受到热的室温应激,ACTH基因mRNA转录量又显著升高,其他时间点降到应激前的正常水平。
下丘脑TRH基因mRNA表达呈现波动性变化趋势,但表达量均不超过1.5,说明下丘脑TRH基因是负调控的。下丘脑CRH、垂体TSH和肾上腺ACTH基因1nRNA的表达量分别在2.27-7.76、1.75-6.17、5.5-14.83范围内,总体呈上升趋势,说明TSH、CRH和ACTH基因均是冷应激反应时的正调控基因。
4.鹅HSP70基因的克隆和生物信息学分析
首次克隆了皖西白鹅HSP70基因cDNA序列2181bp,包括CDS全序列1905bp和3’UTR序列276bp,编码634个氨基酸。与GenBank上乌棕鹅HSP70基因cDNA序列进行比对分析,皖西白鹅HSP70基因共存在15个核苷酸变异(均在CDS区),只有1个蛋白质位点的差异,编码区同源性序列比对,与乌棕鹅同源性达99%,与家鸭、鸡、日本鹌鹑HSP70基因编码区同源性序列同源性分别达97%、92%和92%,与哺乳动物同源性均高于82%,进一步证实了HSP70在不同生物间的高度保守性。
利用生物信息学预测鹅HSP70蛋白质的结构与功能,结果表明,HSP70蛋白质以亲水性区域为主；该蛋白具有丰富的B细胞抗原位点；抗原表位区域、柔韧性区域和表面可能性区域出现了较多的重叠区,这些区域相对易于形变,便于抗原、抗体的自由结合,可能是抗原位点的富集区；该蛋白二级结构以α-螺旋和无规则卷曲为主,无明显的跨膜螺旋,含有34个磷酸化位点,无糖基化位点,推测其可能在细胞信号转导与调控中发挥重要作用。
5.雏鹅冷应激反应中HPT、HPTA轴HSP70mRNA的动态表达规律
实时荧光定量逆转录PCR法检测结果表明,下丘脑HSP70mRNA转录量在冷应激(12℃±1℃)1.5h、6h和9h显著升高,其他时间点均恢复到正常水平；在冷处理结束后6h内HSP70mRNA转录水平均显著升高,应激结束后6h转录水平最高,随后又迅速下降到室温下的转录水平。垂体HSP70mRNA转录量在冷应激0.5h、1.5h、6h和12h时显著降低,其他应激时间均升高到应激前的水平,应激结束后0.5h、3h和6h转录水平也被显著降低,随后逐渐升高到应激前的正常水平。甲状腺HSP70mRNA转录量在冷应激0.5h时显著升高,随后迅速下降到应激前的水平,应激3h、6h和12h时显著降低。应激结束后0.5h和3h转录量显著升高,其他时间点逐渐恢复到正常水平。肾上腺HSP70基因mRNA转录水平在冷应激1.5h、6h、9h时和冷处理结束后3h到6h期间表达水平被显著降低,其他应激时间恢复到应激前的水平。冷应激期间及结束冷处理后一段时间,肾上腺HSP70基因mRNA表达水平都很低,在0.02-0.57范围内,HSP70基因mRNA表达量仍然不超过1,推测HSP70基因也是肾上腺组织细胞表达的下调基因,是负调控的。雏鹅冷应激反应中,HSP70基因在下丘脑、垂体、甲状腺和肾上腺中的表达规律是不同的,在不同的组织中有不同的调控规律。下丘脑HSP70mRNA转录水平最高,表达量总体呈现明显上升趋势,是冷应激反应时的正调控基因：而它在垂体、甲状腺和肾上腺中的表达总体是被抑制的,是垂体、甲状腺和肾上腺冷应激反应时的负调控基因。初步认为可以将HSP70作为动物冷应激的分子生物标志。
Stress is a nonspecific response to various kinds of stimulates in organism which can cause performance descends, induce various diseases, even result in death in livestock and poultries. The cold is one of the most important factor to restrict livestock farming development owing to the direct and indirect economic loss. Studying'the animal cold stress mechanism and investigating scientific stress monitor method have important meaning to elevate preventative level of stress and ensure livestock farming development. Cold stress has important effect on the HPA and HPT axis of nerve internal system, however, there was no correlated study in young goose. In this study,7d Wanxi White geese were used to study the effects on hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-thyroid (HPT) axis under cold stress (12℃±1℃), using the radioimmunity, fluorescence quantitative RT-PCR etc.
Dynamic change rule of blood physiological and biochemical indicators, stress hormone, TRH, TSH, CRH, ACTH, HSP70mRNA by different times during cold exposure0,0.5,3,6,9,12,18,24hour and after terminate cold exposed0,0.5,3,6,9,12,24hour using radioimmunity, fluorescence quantitative PCR. Moreover, HSP70gene was cloned from Wanxi white goose by RT-PCR, the protein structure and function were further predicted by bioinformatics. The studies can provide experimental evidence for investigating reaction mechanism and earlier monitoring of animal cold stress. The main results were showed as following:
1. Analysis of blood routines
7d Wanxi White geese were used to study the influence on some parameters of blood under cold stress (12℃±1℃). The results were as follows. The Red blood cell (RBC), Haematocrit (HCT), Hemoglobin (HGB), Red blood cell distribution width (RDW) were decreased collectively. RBC was significant lower at3h,6h and at0.5h,3h after terminating cold stress than pre-cold stress. HCT and HGB were obviously decreased at3h,6h during cold stress and at3h after terminating cold stress. RDW was lower at6h during cold stress and at12h after end cold stress than pre-cold stress significantly (P<0.01). WBC was increased at12h,24h during cold stress. The absolute value of Lymphoid cell (LYM) was lower at6h during cold stress than pre-cold stress significantly (P<0.05). LYM%was increased at3h,6h and12h after cold stress (P<0.01). however, at normal level at24h after cold stress. Collecting detected the hematological results from all test groups during cold stress, obtaining Wanxi white goose blood physiological and biochemical reference values, which can provide important proof for veterinary clinical diagnosis and therapy.
2. Analysis of stress hormone in HPA and HPT anxis
The effect on HPA axis and HPT axis was analyzed under cold stress (12℃±1℃) in Wanxi White geese. The results showed as follows.The Free triiodothyronine (FT3) was significantly higher at6h and12h after acute cold stress than that of pre-cold stress. The Free thyroxine (FT4) was obviously higher at3h in12℃±1℃temperature, however, dropped gradually later, decreased at12h (P<0.05). The triiodothyronine (T3) was increased steadily during cold stress and significantly higher at6h after cold exposure and at0.5h,3h after end cold exposure. The Thyroxine (T4) got peak value at6h after cold exposure and6h after end cold exposure. The cortisone (Cor) retained in the higher level at more parts time of cold exposure and obviously higher at3h during cold exposure. The Adrenocorticotrophic hormone (ACTH) significant increased at6h after cold exposure and at3h after end cold exposure (P<0.01, P<0.05). The ACTH, Cor and FT3are sensitive to cold stimulus.They can probably be used as the supply indexes evaluating cold stress.
3. Analysis dynamic expression of gene mRNA correlated with hormone in HPA and HPT axis
TRH, TSH, CRH and ACTH gene in Wanxi white goose were coloned using RT-PCR. The sequences identity with that of chicken were94%,95%and95%, respectively. Dynamic expression of TRH, TSH, CRH and ACTH mRNA in HPA and HPT axis was analyzed by FQ-RT-PCR. Hypothalamic TRH mRNA transcription level went up significantly at0.5h,3h,6h,9h ad18h during cold stress; It increased again after cold exposure in1h,6h andl2h; It decreased to normal level in the other time. Pituitary TSH mRNA transcription level was increased significantly from1.5h to12h, moreover, it went up the highest level at12h; Hereafter it declined to normal level. It increased again after cold exposure from1h to6h and was decreased to normal level after6h. Hypothalamic CRH mRNA transcription level went up significantly at3h,9h,18h during cold stress and at1h,6h and12h after cold exposure; It was down-regulated to normal level at the other time. Adrenal gland ACTH gene mRNA transcription level went up significantly from1.5h to9h and at18h during cold stress; It increased again after cold exposure at0.5h and3h; It decreased to normal level in the other time.
Hypothalamic TRH mRNA transcription level had fluctuation trend and it's quanity was not over1.5, which indicated that Hypothalamic TRH was the down-regulation gene in cold stress reaction. The transcription quanity of TSH, CRH and ACTH was227-7.76、1.75-6.17、5.5-14.83and had ascend trend, which showed that they were the up-regulation gene in cold stress reaction. The results demonstrated that TRH, TSH, CRH and ACTH gene can be probably used as the molecular biomarker of animal cold stress.
4. HSP70gene cloning and bioinformatics
HSP70gene cDNA was cloned, whose size was2181bp in Wanxi white goose for the first time. It's1905bp of CDS and276bp of3'-UTR sequence, and encoded634amino acids. There were15nucleotides (in CDS) and only one amino acids difference, which were different between2populations. HSP70CDS sequence in goose had82-90%homology with mammals and fish, and up to99%with Chinese goose. Cluster analysis revealed that probably mammals, birds and fishes, each of these belonged to different categories separately.
Bioinformatics analysis indicated that goose HSP70protein contained34phosphorylation sites. The diversity of CDS and the structure of amino acids in HSP70genes resulted in the differences of protein secondary structure. HSP70protein secondary structure had more Alpha and Coil region, without signal peptide and transmembrane helix; The hydrophobicity structure prediction of goose HSP70protein indicated that it had major hydrophobicity region. The protein had abundant B cell antigen location. There were more overlapping region in flexibility, surfaceprobability and antigencity, which made antigen and antibody combine easily, and it was probably antigen enrichment region. It was predicted that HSP70protein probably played an important role in cell signal transfer and control mechanisms.
5. Analysis dynamic expression of HSP70mRNA in HPA and HPT axis during cold stress
The dynamic expression of HSP70mRNA in HPA and HPT axis of7d Wanxi White goose was got utilizing FQ-RT-PCR. Hypothalamic HSP70mRNA transcription level went up significantly atl.5h,6h and9h; It decreased to normal level in the other time; It increased again after cold exposed in6hours, moreover, it went up the highest level at6h;Hereafter it declined to normal level. Pituitary HSP70mRNA transcription level descended significantly at0.5h,1.5h,6h and12h. It went up again gradually to normal level in the next12hours. It decreased again significantly at0.5h,3h,6h after cold exposure, hereafter, gradually increased to normal level. Thyroid HSP70mRNA transcription level went up quickly at0.5h, hereafter decreased at3h,6h,12h significantly under cold stress. It went up significantly at0.5h and3h after cold exposure and it was down-regulated gradually to normal level in the other time. Adrenal gland HSP70mRNA transcription level was decreased at1.5h,6h,9h during cold stress and at3h,6h after end cold exposure. It was up-regulated gradually to normal level in the other time. The Adrenal gland HSP70mRNA transcription quanity was0.02-0.57, which was predicted that it was down-regulation gene in cold stress reaction.
The expression rule of HSP70mRNA was different in Hypothalamic, Pituitary, Thyroid and Adrenal gland. Hypothalamic HSP70mRNA transcription level was the highest which showed going up trend as a whole. HSP70was the up-regulation gene of Hypothalamic in cold stress reaction. Pituitary, thyroid and adrenal gland HSP70mRNA transcription level was repressed, which showed that it was the down-regulation gene of pituitary, thyroid and adrenal gland in cold stress reaction. The results demonstrated that it play a part in protective cell function, on the other hand, denoted that histiocyte function was placed in the urgent condition. The lymphocyte HSP70can probably be used as the molecular biomarker of animal cold stress.
引文
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