本文主要研究内容
作者刘深贺（2019）在《水牛耐热性和抗热应激方法及耐热基因鉴定研究》一文中研究指出：热应激影响动物的生长发育和泌乳,降低生产性能。水牛主要生活在高温高湿气候地区,对高温高湿气候条件具有一定的适应性,但当其暴露在太阳下或在高温天气下劳作时,也会表现不适反应,进而影响生理和生产表现,如呼吸加快,体温升高,繁殖和泌乳性能紊乱等。目前,预防热应激的措施主要包括营养调控,环境改善和遗传改良,其中耐热品种选育是最经济有效的措施。耐热品种的传统选育方法进展缓慢,新兴的高通量测序技术为研究耐热性状提供了新的方向。因此,本研究首先评估水牛抗热应激能力,其次利用高通量测序技术对影响耐热性能的候选基因进行筛选和鉴定,最后进行喷淋水牛的抗热应激效果评估,旨在为提高水牛抗热应激能力,加快高产耐热品种培育提供理论依据和技术支撑。1.水牛耐热性及其影响因素研究(1)水牛耐热性研究分析90头杂交水牛在冬、春和夏季的生理指标、生产性能和血液参数,发现在夏季水牛直肠温度(39.24±0.07°C)、呼吸频率(69.74±3.82次/分钟)、体表温度(38.01±0.09°C)及血浆HSP70(421.28±53.11pg/mL)和HSP90(3348.48±306.44pg/mL)水平显著性高于春季(38.34±0.03°C、15.76±0.62次/分钟、35.44±0.35°C、263.65±31.36 pg/mL和1311.08±222.51pg/mL)和冬季(38.13±0.05°C、10.49±0.16次/分钟、24.51±0.24°C、174.11±53.13pg/mL和947.21±230.00pg/mL);在夏季的受胎率(28.27%)、排卵卵泡直径(10.36±0.26mm)、大卵泡直径(10.22±0.24mm)、排卵卵泡个数(0.71±0.07)和大卵泡个数(0.79±0.06)显著性低于春季(32.35%、11.77±0.30mm、11.59±0.29mm、0.76±0.09和0.85±0.07)和冬季(35.48%、12.58±0.32mm、12.46±0.31mm、0.84±0.08和0.90±0.07)。在夏季,水牛的呼吸频率(r=0.84)和直肠温度(r=0.81)与温湿度指数(THI)显著性正相关,当THI高于78时,水牛呼吸频率和直肠温度呈显著上升趋势,说明水牛的耐热性较奶牛强。(2)纯种和杂交水牛耐热性比较当THI大于78时,比较夏季14头纯种地中海水牛和30头杂交水牛的生理指标和血液参数,发现杂交水牛的直肠温度(39.12±0.09°C)、呼吸频率(60.82±5.45次/分钟)和体表温度(37.81±0.12°C)显著性低于纯种水牛(39.38±0.09°C、76.84±4.31次/分钟和38.23±0.12°C);血浆HSP90(2938±520.73pg/mL)、HSP70(375.12±64.47pg/mL)和皮质醇(214.06±22.83ng/mL)水平显著性高于纯种水牛(1381.61±339.00pg/mL、202.11±39.45pg/mL和145.45±19.96ng/mL)。说明杂交水牛的耐热性强于纯种地中海水牛。(3)犊牛和成年水牛耐热性比较当THI大于78时,比较夏季15头犊牛(<3月龄)和30头成年水牛(>12月龄)的生理指标和血液参数,发现成年水牛的直肠温度(39.12±0.09°C)、呼吸频率(60.82±5.45次/分钟)和体表温度(37.81±0.12°C)显著性低于犊牛(39.46±0.09°C、98.72±7.21次/分钟和38.92±0.12°C);血浆HSP90(2938±520.73pg/mL)、HSP70(375.12±64.47pg/mL)和皮质醇(214.06±22.83ng/mL)水平显著性高于犊牛(1098.81±391.23pg/mL、172.11±27.15pg/mL和128.25±21.36ng/mL)。说明成年水牛的耐热性强于犊牛。(4)泌乳高产和低产水牛耐热性比较当THI大于78时,比较夏季16头高产泌乳水牛(>8~12Kg)和12头低产泌乳水牛(<4Kg)的生理指标和血液参数,发现低产水牛的直肠温度(38.86±0.07°C)和呼吸频率(56.52±7.36次/分钟)显著性低于高产水牛(39.21±0.07°C和84.72±3.61次/分钟);血浆HSP90(2723±413.33pg/mL)和HSP70(357.14±34.53pg/mL)水平显著性高于高产水牛(1278.21±278.00pg/mL和176.17±29.13pg/mL)。说明低产水牛的耐热性强于高产水牛。(5)妊娠晚期与空怀水牛耐热性比较当THI大于78时,比较夏季8头妊娠晚期水牛和17头空怀水牛的生理指标和血液参数,发现空怀水牛的直肠温度(39.03±0.05°C)、呼吸频率(56.62±7.35次/分钟)和体表温度(37.91±0.14°C)显著性低于妊娠晚期水牛(39.46±0.05°C、106.23±6.47次/分钟和38.77±0.14°C);血浆HSP90(3458±478.23pg/mL)和HSP70(359.17±58.39pg/mL)水平显著性高于妊娠晚期水牛(1419.27±279.00pg/mL和237.15±25.47pg/mL)。说明空怀水牛的耐热性强于妊娠晚期水牛。2.水牛耐热基因发掘与鉴定(1)耐热和热敏感杂交水牛和奶牛的筛选在夏季(THI>78)连续5天检测30头杂交水牛的生理指标和血液参数,获得耐热和热敏感水牛各4头。耐热水牛在夏季的直肠温度和呼吸频率分别为38.72±0.10°C和41.67±4.43次/分钟,显著低于热敏感水牛(39.67±0.06°C、100.12±3.48次/分钟);血浆HSP70、HSP90和皮质醇水平分别为454.26±91.15pg/mL、3972.53±668.49pg/mL和251.64±10.68ng/mL,显著高于热敏感水牛(142.86±41.35pg/mL、845.42±236.96pg/mL和121.46±24.20ng/mL)。在夏季连续7天检测42头高产荷斯坦奶牛的生理指标、血液参数和产奶量变化,获得耐热和热敏感奶牛各3头。耐热奶牛在夏季的直肠温度和呼吸频率分别为38.7±0.02°C和57±1次/分钟,显著低于热敏感奶牛(39.7±0.05°C、100±6次/分钟);血浆HSP70、HSP90和皮质醇水平分别为391±59pg/mL、2747±196pg/mL和247±19 ng/mL,显著高于热敏感奶牛(183±12pg/mL、1075±84pg/mL和144±11 ng/mL)。(2)耐热和热敏感水牛血液转录组分析应用mRNA-Seq和miRNA-Seq方法分析耐热和热敏感水牛血液转录组,共鉴定出753个差异基因和16个差异miRNA。应用加权基因共表达网络分析(WGCNA)方法,将差异基因分为5个模块,其中4个模块与热应激指标相关。在turquoise模块中,发现6个与耐热性相关的基因(IL18RAP、IL6R、CCR1、PPBP、IL1B和IL1R1),且差异miRNA bta-miR-1246靶向结合ABCC4基因与枢纽基因构建mRNA-miRNA网络图,进而调控水牛热应激反应。应用mRNA-Seq方法分析耐热和热敏感奶牛血液转录组,共鉴定出200个差异表达基因。其中,14个基因参与到蛋白互作(PPI)网络,4个与耐热性相关的基因(OAS2、MX2、IFIT5和TGFB2)富集到免疫效应过程。整合分析水牛和奶牛耐热性相关的差异基因,发现7个(BMP4、DIRAS2、CD72、DOCK5、MOV10L1、NAT14和TSPAN15)共有的且表达趋势一致的基因。以上结果为进一步探讨水牛的耐热机制奠定了基础。(3)耐热基因BMP4的解析取奶牛乳腺上皮细胞,用RNAi技术敲低水牛和奶牛共有的耐热基因BMP4的表达,然后进行热应激(42℃,3小时)处理,检测细胞的增殖和凋亡率及HSP70、HSP27、Bax和Bcl-2基因的表达。结果表明,敲低组的细胞早期凋亡率明显增加,但基因HSP70、HSP27、Bax和Bcl-2的表达水平没有显著差异。推测该基因的上调可能是通过其他途径来增强水牛耐热性。3.喷淋水牛的抗热应激效果当THI大于78时,比较不同喷淋方法对30头泌乳水牛的体温和体表温度及乳成分的影响,发现从喷淋后水牛乳品质、体温和体表温度的变化及节水减污的角度考虑,实际生产中建议采用间隔14分钟喷淋时长1分钟的方案。
Abstract
re ying ji ying xiang dong wu de sheng chang fa yo he bi ru ,jiang di sheng chan xing neng 。shui niu zhu yao sheng huo zai gao wen gao shi qi hou de ou ,dui gao wen gao shi qi hou tiao jian ju you yi ding de kuo ying xing ,dan dang ji bao lou zai tai yang xia huo zai gao wen tian qi xia lao zuo shi ,ye hui biao xian bu kuo fan ying ,jin er ying xiang sheng li he sheng chan biao xian ,ru hu xi jia kuai ,ti wen sheng gao ,fan shi he bi ru xing neng wen luan deng 。mu qian ,yu fang re ying ji de cuo shi zhu yao bao gua ying yang diao kong ,huan jing gai shan he wei chuan gai liang ,ji zhong nai re pin chong shua yo shi zui jing ji you xiao de cuo shi 。nai re pin chong de chuan tong shua yo fang fa jin zhan huan man ,xin xing de gao tong liang ce xu ji shu wei yan jiu nai re xing zhuang di gong le xin de fang xiang 。yin ci ,ben yan jiu shou xian ping gu shui niu kang re ying ji neng li ,ji ci li yong gao tong liang ce xu ji shu dui ying xiang nai re xing neng de hou shua ji yin jin hang shai shua he jian ding ,zui hou jin hang pen lin shui niu de kang re ying ji xiao guo ping gu ,zhi zai wei di gao shui niu kang re ying ji neng li ,jia kuai gao chan nai re pin chong pei yo di gong li lun yi ju he ji shu zhi cheng 。1.shui niu nai re xing ji ji ying xiang yin su yan jiu (1)shui niu nai re xing yan jiu fen xi 90tou za jiao shui niu zai dong 、chun he xia ji de sheng li zhi biao 、sheng chan xing neng he xie ye can shu ,fa xian zai xia ji shui niu zhi chang wen du (39.24±0.07°C)、hu xi pin lv (69.74±3.82ci /fen zhong )、ti biao wen du (38.01±0.09°C)ji xie jiang HSP70(421.28±53.11pg/mL)he HSP90(3348.48±306.44pg/mL)shui ping xian zhe xing gao yu chun ji (38.34±0.03°C、15.76±0.62ci /fen zhong 、35.44±0.35°C、263.65±31.36 pg/mLhe 1311.08±222.51pg/mL)he dong ji (38.13±0.05°C、10.49±0.16ci /fen zhong 、24.51±0.24°C、174.11±53.13pg/mLhe 947.21±230.00pg/mL);zai xia ji de shou tai lv (28.27%)、pai luan luan pao zhi jing (10.36±0.26mm)、da luan pao zhi jing (10.22±0.24mm)、pai luan luan pao ge shu (0.71±0.07)he da luan pao ge shu (0.79±0.06)xian zhe xing di yu chun ji (32.35%、11.77±0.30mm、11.59±0.29mm、0.76±0.09he 0.85±0.07)he dong ji (35.48%、12.58±0.32mm、12.46±0.31mm、0.84±0.08he 0.90±0.07)。zai xia ji ,shui niu de hu xi pin lv (r=0.84)he zhi chang wen du (r=0.81)yu wen shi du zhi shu (THI)xian zhe xing zheng xiang guan ,dang THIgao yu 78shi ,shui niu hu xi pin lv he zhi chang wen du cheng xian zhe shang sheng qu shi ,shui ming shui niu de nai re xing jiao nai niu jiang 。(2)chun chong he za jiao shui niu nai re xing bi jiao dang THIda yu 78shi ,bi jiao xia ji 14tou chun chong de zhong hai shui niu he 30tou za jiao shui niu de sheng li zhi biao he xie ye can shu ,fa xian za jiao shui niu de zhi chang wen du (39.12±0.09°C)、hu xi pin lv (60.82±5.45ci /fen zhong )he ti biao wen du (37.81±0.12°C)xian zhe xing di yu chun chong shui niu (39.38±0.09°C、76.84±4.31ci /fen zhong he 38.23±0.12°C);xie jiang HSP90(2938±520.73pg/mL)、HSP70(375.12±64.47pg/mL)he pi zhi chun (214.06±22.83ng/mL)shui ping xian zhe xing gao yu chun chong shui niu (1381.61±339.00pg/mL、202.11±39.45pg/mLhe 145.45±19.96ng/mL)。shui ming za jiao shui niu de nai re xing jiang yu chun chong de zhong hai shui niu 。(3)du niu he cheng nian shui niu nai re xing bi jiao dang THIda yu 78shi ,bi jiao xia ji 15tou du niu (<3yue ling )he 30tou cheng nian shui niu (>12yue ling )de sheng li zhi biao he xie ye can shu ,fa xian cheng nian shui niu de zhi chang wen du (39.12±0.09°C)、hu xi pin lv (60.82±5.45ci /fen zhong )he ti biao wen du (37.81±0.12°C)xian zhe xing di yu du niu (39.46±0.09°C、98.72±7.21ci /fen zhong he 38.92±0.12°C);xie jiang HSP90(2938±520.73pg/mL)、HSP70(375.12±64.47pg/mL)he pi zhi chun (214.06±22.83ng/mL)shui ping xian zhe xing gao yu du niu (1098.81±391.23pg/mL、172.11±27.15pg/mLhe 128.25±21.36ng/mL)。shui ming cheng nian shui niu de nai re xing jiang yu du niu 。(4)bi ru gao chan he di chan shui niu nai re xing bi jiao dang THIda yu 78shi ,bi jiao xia ji 16tou gao chan bi ru shui niu (>8~12Kg)he 12tou di chan bi ru shui niu (<4Kg)de sheng li zhi biao he xie ye can shu ,fa xian di chan shui niu de zhi chang wen du (38.86±0.07°C)he hu xi pin lv (56.52±7.36ci /fen zhong )xian zhe xing di yu gao chan shui niu (39.21±0.07°Che 84.72±3.61ci /fen zhong );xie jiang HSP90(2723±413.33pg/mL)he HSP70(357.14±34.53pg/mL)shui ping xian zhe xing gao yu gao chan shui niu (1278.21±278.00pg/mLhe 176.17±29.13pg/mL)。shui ming di chan shui niu de nai re xing jiang yu gao chan shui niu 。(5)ren shen wan ji yu kong huai shui niu nai re xing bi jiao dang THIda yu 78shi ,bi jiao xia ji 8tou ren shen wan ji shui niu he 17tou kong huai shui niu de sheng li zhi biao he xie ye can shu ,fa xian kong huai shui niu de zhi chang wen du (39.03±0.05°C)、hu xi pin lv (56.62±7.35ci /fen zhong )he ti biao wen du (37.91±0.14°C)xian zhe xing di yu ren shen wan ji shui niu (39.46±0.05°C、106.23±6.47ci /fen zhong he 38.77±0.14°C);xie jiang HSP90(3458±478.23pg/mL)he HSP70(359.17±58.39pg/mL)shui ping xian zhe xing gao yu ren shen wan ji shui niu (1419.27±279.00pg/mLhe 237.15±25.47pg/mL)。shui ming kong huai shui niu de nai re xing jiang yu ren shen wan ji shui niu 。2.shui niu nai re ji yin fa jue yu jian ding (1)nai re he re min gan za jiao shui niu he nai niu de shai shua zai xia ji (THI>78)lian xu 5tian jian ce 30tou za jiao shui niu de sheng li zhi biao he xie ye can shu ,huo de nai re he re min gan shui niu ge 4tou 。nai re shui niu zai xia ji de zhi chang wen du he hu xi pin lv fen bie wei 38.72±0.10°Che 41.67±4.43ci /fen zhong ,xian zhe di yu re min gan shui niu (39.67±0.06°C、100.12±3.48ci /fen zhong );xie jiang HSP70、HSP90he pi zhi chun shui ping fen bie wei 454.26±91.15pg/mL、3972.53±668.49pg/mLhe 251.64±10.68ng/mL,xian zhe gao yu re min gan shui niu (142.86±41.35pg/mL、845.42±236.96pg/mLhe 121.46±24.20ng/mL)。zai xia ji lian xu 7tian jian ce 42tou gao chan he si tan nai niu de sheng li zhi biao 、xie ye can shu he chan nai liang bian hua ,huo de nai re he re min gan nai niu ge 3tou 。nai re nai niu zai xia ji de zhi chang wen du he hu xi pin lv fen bie wei 38.7±0.02°Che 57±1ci /fen zhong ,xian zhe di yu re min gan nai niu (39.7±0.05°C、100±6ci /fen zhong );xie jiang HSP70、HSP90he pi zhi chun shui ping fen bie wei 391±59pg/mL、2747±196pg/mLhe 247±19 ng/mL,xian zhe gao yu re min gan nai niu (183±12pg/mL、1075±84pg/mLhe 144±11 ng/mL)。(2)nai re he re min gan shui niu xie ye zhuai lu zu fen xi ying yong mRNA-Seqhe miRNA-Seqfang fa fen xi nai re he re min gan shui niu xie ye zhuai lu zu ,gong jian ding chu 753ge cha yi ji yin he 16ge cha yi miRNA。ying yong jia quan ji yin gong biao da wang lao fen xi (WGCNA)fang fa ,jiang cha yi ji yin fen wei 5ge mo kuai ,ji zhong 4ge mo kuai yu re ying ji zhi biao xiang guan 。zai turquoisemo kuai zhong ,fa xian 6ge yu nai re xing xiang guan de ji yin (IL18RAP、IL6R、CCR1、PPBP、IL1Bhe IL1R1),ju cha yi miRNA bta-miR-1246ba xiang jie ge ABCC4ji yin yu shu niu ji yin gou jian mRNA-miRNAwang lao tu ,jin er diao kong shui niu re ying ji fan ying 。ying yong mRNA-Seqfang fa fen xi nai re he re min gan nai niu xie ye zhuai lu zu ,gong jian ding chu 200ge cha yi biao da ji yin 。ji zhong ,14ge ji yin can yu dao dan bai hu zuo (PPI)wang lao ,4ge yu nai re xing xiang guan de ji yin (OAS2、MX2、IFIT5he TGFB2)fu ji dao mian yi xiao ying guo cheng 。zheng ge fen xi shui niu he nai niu nai re xing xiang guan de cha yi ji yin ,fa xian 7ge (BMP4、DIRAS2、CD72、DOCK5、MOV10L1、NAT14he TSPAN15)gong you de ju biao da qu shi yi zhi de ji yin 。yi shang jie guo wei jin yi bu tan tao shui niu de nai re ji zhi dian ding le ji chu 。(3)nai re ji yin BMP4de jie xi qu nai niu ru xian shang pi xi bao ,yong RNAiji shu qiao di shui niu he nai niu gong you de nai re ji yin BMP4de biao da ,ran hou jin hang re ying ji (42℃,3xiao shi )chu li ,jian ce xi bao de zeng shi he diao wang lv ji HSP70、HSP27、Baxhe Bcl-2ji yin de biao da 。jie guo biao ming ,qiao di zu de xi bao zao ji diao wang lv ming xian zeng jia ,dan ji yin HSP70、HSP27、Baxhe Bcl-2de biao da shui ping mei you xian zhe cha yi 。tui ce gai ji yin de shang diao ke neng shi tong guo ji ta tu jing lai zeng jiang shui niu nai re xing 。3.pen lin shui niu de kang re ying ji xiao guo dang THIda yu 78shi ,bi jiao bu tong pen lin fang fa dui 30tou bi ru shui niu de ti wen he ti biao wen du ji ru cheng fen de ying xiang ,fa xian cong pen lin hou shui niu ru pin zhi 、ti wen he ti biao wen du de bian hua ji jie shui jian wu de jiao du kao lv ,shi ji sheng chan zhong jian yi cai yong jian ge 14fen zhong pen lin shi chang 1fen zhong de fang an 。
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