CN111088281B - 中华蜜蜂耐热相关基因DnaJ1及其应用 - Google Patents
中华蜜蜂耐热相关基因DnaJ1及其应用 Download PDFInfo
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Abstract
本发明涉及生物化学与分子生物学技术领域,具体涉及一种中华蜜蜂耐热相关基因DnaJ1及其应用。所述DnaJ1基因在热激胁迫条件下被显著诱导表达,沉默DnaJ1基因可通过降低中华蜜蜂的总抗氧化能力、增加脂质过氧化、蛋白质和细胞氧化损伤的程度来降低蜜蜂耐热性;在蜜蜂中过表达DnaJ1基因有望提高蜜蜂在热激胁迫条件下的存活率。由此,DnaJ1可以作为培育耐热转基因蜜蜂品种的候选热激响应基因。鉴于DnaJ1在热应激中的作用,将来有望将该基因在蜜蜂中过表达或转基因到家蚕、鸡、羊和猪等经济动物中,以提高他们的耐热性,进而提高它们的品质及产量,具有重要的社会效益及经济效益。
Description
技术领域
本发明涉及生物化学与分子生物学技术领域,具体涉及一种中华蜜蜂耐热相关基因DnaJ1及其应用。
背景技术
当生物体所处的环境温度远超过其最佳生长环境温度时就容易引起热激胁迫。近年来,伴随着全球气候变暖,热激胁迫问题引起了研究者的高度重视。热激胁迫能够诱导活性氧的产生,造成氧化损伤,引起蛋白质变性。这些现象不仅会破坏细胞,影响细胞的正常分裂及生长进程,甚至导致细胞死亡。因此,筛选热激胁迫响应基因,研究生物体应对热激胁迫的具体分子机制显得尤为重要。
热激蛋白(Heat shock protein,HSP),也叫热应激蛋白或热休克蛋白,最早在1962年在果蝇中由于其受热激胁迫诱导表达而被发现并命名。HSP是生物体适应环境温度升高的一个重要蛋白,生物体对热激胁迫的耐受能力与HSP表达量的上调普遍成正相关,并且在热激胁迫消除后的一段时间内其表达量仍保持在一定水平。虽然HSP由于受热激胁迫诱导被发现及命名,但不是所有的HSP基因都受热激胁迫的诱导。由于亚细胞定位、所处的发育时期及所在的生长环境的不同,即使是同一家族的HSP基因在对热应激的反应能力上也有差异。
根据分子量(kDa)大小,HSP被分为小分子热激蛋白(small HSP,sHsp)、HSP40、HSP60、HSP70、HSP90、HSP100等6个家族。其中,HSP40家族的成员由于其蛋白结构中均含有J结构域,更常被命名为DnaJ。根据所含结构域类别及个数的不同,DnaJ又被分为DnaJA、DnaJB和DnaJC三类。虽然HSP在热激胁迫反应中发挥着重要的功能,但DnaJ基因家族中的多个成员的功能是未知的,需要进一步探究。
中华蜜蜂(Apis ceran cerana)属于亚洲蜜蜂的主要生态类群之一,是我国特有的蜜蜂种质资源。近年来,由于受包括热激胁迫在内的各种生物及非生物环境胁迫的影响,我国部分地区的中华蜜蜂的数量明显减少。热激胁迫问题在我国主要中华蜜蜂养殖区频繁发生,危害较大。至今中华蜜蜂对热激胁迫的适应性及中华蜜蜂热应激的具体分子机制尚未被详细探究,有许多问题需要进一步研究。HSP作为热应激的重要调控蛋白对阐明生物的热应激分子机制具有重要的意义。揭示中华蜜蜂DnaJ家族参与热应激的具体机理及所涉及的关键基因、信号通路,不仅可以丰富HSP热应激功能的理论体系,还可以为培育耐热转基因蜜蜂品种提供候选热激响应基因。
发明内容
针对上述现有技术,本发明的目的是提供一种中华蜜蜂耐热相关基因DnaJ1及其应用。所述DnaJ1基因在热激胁迫条件下被显著诱导表达,沉默DnaJ1基因可通过降低中华蜜蜂的总抗氧化能力、增加脂质过氧化、蛋白质和细胞氧化损伤的程度来降低蜜蜂耐热性;在蜜蜂中过表达DnaJ1基因有望提高蜜蜂在热激胁迫条件下的存活率。由此,DnaJ1可以作为培育耐热转基因蜜蜂品种的候选热激响应基因。
为实现上述目的,本发明采用如下技术方案:
本发明的第一方面,提供DnaJ1基因或者其表达产物作为靶标在如下1)或2)中的应用:
1)调控蜜蜂在热激胁迫条件下的抗热能力;
2)培育在热激胁迫条件下抗热能力增强的蜜蜂品种。
本发明的第二方面,提供特异性检测DnaJ1基因或者其表达产物的试剂在制备用于检测蜜蜂在热激胁迫下抗热能力的产品中的应用。
上述应用中,所述产品采用实时荧光定量PCR方法检测DnaJ1基因或者其表达产物。
上述应用中,所述产品包括:实时荧光定量PCR试剂盒。
上述应用中,所述实时荧光定量PCR试剂盒中含有:
特异性检测DnaJ1基因的引物对,其序列如SEQ ID NO.3和SEQ ID NO.4所示。
本发明的第三方面,提供沉默DnaJ1基因表达的试剂在构建热激胁迫下抗热能力降低的蜜蜂模型中的应用。
上述应用中,所述试剂中包含:用于扩增沉默DnaJ1的片段的引物,其序列分别如SEQ ID NO.7和SEQ ID NO.8所示。
上述应用中,所构建的蜜蜂模型通过沉默DnaJ1的表达来降低蜜蜂的总抗氧化能力、增加脂质过氧化、蛋白质和细胞氧化损伤的程度,从而降低蜜蜂在热激胁迫下抗热能力。
沉默DnaJ1基因降低了蜜蜂在热激胁迫条件下的生存能力,若在蜜蜂或其他经济动物(家蚕、猪和牛等)中超表达该基因有望提高它们在环境胁迫中的抗逆能力。
本发明的有益效果:
与现有的技术相比,本发明首次提供了在特定的热激胁迫条件下DnaJ家族表达量被诱导最明显的基因DnaJ1,并研究了DnaJ1的耐热性和参与热应激反应所涉及的分子机制和应用。鉴于DnaJ1在热应激中的作用,将来有望将该基因在蜜蜂中过表达或转基因到家蚕、鸡、羊和猪等经济动物中,以提高他们的耐热性,进而提高它们的品质及产量,具有重要的社会效益及经济效益。
附图说明
图1:通过热图呈现在热激胁迫条件下DnaJ家族所有基因表达量。基因的表达量是通过转录组测序测定的。
由图中可见,在34个DnaJ中,DnaJ1的表达量被增加的最多。
图2:梯度热应激反应中DnaJ1的转录水平的检测图。β-actin(GenBank注册号为HM640276.1)作为内参基因。
由图中可见与对照组相比,在3个梯度热温度处理条件下,DnaJ1的表达量均显著增加。
图3:沉默DnaJ1对中华蜜蜂耐热能力的影响的检测图。A:以β-actin作为内参基因鉴定DnaJ1的沉默效率,饲喂dsRNA-GFP的蜜蜂作为对照组;B:统计沉默DnaJ1后中华蜜蜂在热激胁迫条件下的存活率。
由图中可知DnaJ1在中华蜜蜂中被成功沉默。并且与对照组相比,沉默DnaJ1增加蜜蜂在热激胁迫条件下的死亡率,降低了蜜蜂的耐热能力。
图4:沉默DnaJ1对中华蜜蜂体内丙二醛(A)、总羰基(B)、维生素C含量(C)及总抗氧化能力(D)的影响,以饲喂dsRNA-GFP的蜜蜂作为对照。
由图中可见与对照组相比,沉默DnaJ1增加了中华蜜蜂体内丙二醛、蛋白质羰基的含量,降低了维生素含量及总抗氧化能力。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
正如背景技术部分所介绍的,热激胁迫问题在我国主要中华蜜蜂养殖区频繁发生,危害较大。至今中华蜜蜂对热激胁迫的适应性及中华蜜蜂热应激的具体分子机制尚未被详细探究,有许多问题需要进一步研究,HSP作为热应激的重要调控蛋白对阐明生物的热应激分子机制具有重要的意义。目前中华蜜蜂DnaJ基因家族中的多个成员的功能是未知的,而且由于亚细胞定位、所处的发育时期及所在的生长环境的不同,即使是同一家族的HSP基因在对热应激的反应能力上也有差异。
基于此,本发明旨在发现一种新的中华蜜蜂耐热相关基因,并研究其在中华蜜蜂热应激反应中的表达模式、耐热能力的大小和参与热应激反应所涉及的具体分子机理,以作为培育耐热转基因蜜蜂品种的候选热激响应基因。
在本发明中,我们首先用热激胁迫处理蜜蜂,然后提取其RNA,未处理的蜜蜂作为对照,做转录组测序。通过对转录组数据的分析,有34个基因被聚类属于DnaJ家族。在这34个DnaJ中,DnaJ1的表达量在热激胁迫条件下被诱导的最明显(图1)。通过氨基酸多序列比对及保守结构域预测,我们把DnaJ1归类于DnaJA家族,DnaJ1基因的核苷酸序列如SEQ IDNO.1所示,DnaJ1蛋白的氨基酸序列如SEQ ID NO.2所示。
本发明进一步提供了DnaJ1在热应激中的具体表达模式。鉴于相对其他时期的蜜蜂,采集蜂遭受环境胁迫的概率大,本发明选择采集蜂为实验材料探究DnaJ1的热应激能力。本发明发现,与对照组相比,在3种梯度热激胁迫条件下DnaJ1均被显著诱导表达(图2)。
本发明还提供了可以在中华蜜蜂中沉默DnaJ1的编码区的部分核苷酸序列。该核苷酸序列被合成双链RNA(Double-stranded RNA,dsRNA)后用来饲喂中华蜜蜂即可成功沉默DnaJ1的转录水平(图3A)。用热激胁迫处理DnaJ1被沉默的中华蜜蜂,本发明发现,与对照组相比,沉默DnaJ1增加中华蜜蜂在热应激中的死亡率(图3B)。
本发明还发现沉默DnaJ1加剧了中华蜜蜂体内丙二醛及总羰基的含量(图4A和4B),降低了中华蜜蜂体内维生素C含量(图4C)及总抗氧化能力(图4D)。因此本发明指出沉默DnaJ1可通过降低中华蜜蜂的总抗氧化能力、增加脂质过氧化、蛋白质和细胞氧化损伤的程度来降低蜜蜂耐热性,在蜜蜂中过表达DnaJ1有望提高蜜蜂在热激胁迫条件下的存活率。
与现有的技术相比,本发明首次提供了在特定的热激胁迫条件下DnaJ家族表达量被诱导最明显的基因DnaJ1及DnaJ1的耐热性和参与热应激反应所涉及的分子机制和应用。鉴于DnaJ1在热应激中的作用,将来有望将该基因在蜜蜂中过表达或转基因到家蚕、鸡、羊和猪等经济动物中,以提高他们的耐热性,进而提高它们的品质及产量,具有重要的社会效益及经济效益。
综上所述,本发明证实了在特定热激胁迫条件下DnaJ1是DnaJ家族表达量被增加最多的一个基因,并且热激胁迫诱导DnaJ1的转录水平的增加有利于蜜蜂在热激胁迫条件下的存活。本发明提供的内容为研究蜜蜂的抗逆分子生物学机制、培育耐热蜜蜂新品种及蜜蜂的遗传改良具有重要的指导意义。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案,以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例中所用的试验材料均为本领域常规的试验材料,均可通过商业渠道购买得到。未注明详细条件的实验方法是按照常规试验方法或按照供应商所建议的操作说明书进行的。
本发明实施例中所处理的蜜蜂均为中华蜜蜂。
实施例1:通过转录组测序检测热激胁迫条件下蜜蜂DnaJ家族所有基因表达量
1.热激处理蜜蜂
选取采集蜂60只,分成两组,每组30只。第一组蜜蜂为对照组,放在温度被设置在33℃的环境中,第二组蜜蜂为实验组,被放置在47℃的环境中。两组蜜蜂所处环境的相对湿度均被设置为70%,光照度设置为0。在处理2h后取样,并迅速在液氮中冷冻后放-80℃冰箱保存。
2.RNA的提取
利用TaKaRa公司的RNAiso Plus试剂提取蜜蜂的总RNA,具体步骤如下所述。
(a)在陶瓷研钵中用液氮把蜜蜂研成粉末,称取0.07g粉末放入预先盛有1mL的RNAiso Plus的1.5mL离心管中,用振荡器使其混匀,然后室温静置5min;
(b)4℃,12000g离心10min;
(c)取步骤(b)离心后的上清到一个新的1.5mL的离心管中,加入500μL氯仿,剧烈震荡混匀,室温静止3min,然后4℃,12000g离心15min;
(d)取步骤(c)离心后的上清到一个新的1.5mL离心管中,加入等体积的异丙醇,轻轻颠倒混匀,室温放置10min,4℃,12000g离心10min;
(e)用真空泵把上清抽去,加入1mL的75%的乙醇洗沉淀,4℃,7500g离心5min;
(f)用真空泵把上清抽干,加入100μL的无RNase的水溶解沉淀。
3.RNA被送往百迈克生物公司进行转录组测序。
4.试验结果
通过热图呈现在热激胁迫条件下DnaJ家族所有基因的表达量(基因的表达量是通过转录组测序测定的),结果见图1。由图中可见,在34个DnaJ中,DnaJ1的表达量被增加的最多。
实施例2:DnaJ1在热应激中转录水平的分析
1.试验方法
(a)选取采集蜂240只,分成四组,每组60只。第一组蜜蜂放在温度被设置在33℃的培养箱中(对照组),第二、三和四组蜜蜂分别放在温度被设置在40℃、43℃和46℃的培养箱中(实验组)。四组培养箱的相对湿度均被设置为70%,光照度设置为0。在处理0h、1h、2h、3h、4h和5h后取样,并迅速在液氮中冷冻后放-80℃冰箱保存;
(b)利用TaKaRa公司生产的的RNAiso Plus试剂提取蜜蜂的总RNA;
(c)采用Thermo ScientificTM NanoDropTM One超微量紫外-可见光分光光度计检测上一步提取的RNA的浓度及质量。然后利用TaKaRa公司生产的的PrimeScriptTM RTreagent Kit with gDNA Eraser合成cDNA第一链;
(d)以上一步合成的cDNA为模版,采用TaKaRa公司生产的TB GreenTM Premix ExTaqTM(Tli RNaseH Plus)及Bio-Rad公司制造的CFX96TM Real-Time System做qRT-PCR检测在热应激胁迫条件下DnaJ1的转录水平。以β-actin(GenBank注册号为HM640276.1)作为内参基因。做qRT-PCR所用的引物序列如下:
DnaJ1的qRT-PCR引物:
正向引物:GTGGCAATGTATTCTCTTCA;(SEQ ID NO.3)
反向引物:GTGGCAATGTATTCTCTTCA。(SEQ ID NO.4)
β-actin的qRT-PCR引物:
正向引物:TTATATGCCAACACTGTCCTTT;(SEQ ID NO.5)
反向引物:AGAATTGATCCACCAATCCA。(SEQ ID NO.6)
2.试验结果
试验结果见图2,由图2可见,与对照组相比,在3个梯度热温度处理条件下,DnaJ1的表达量均被显著增加。
实施例3:沉默DnaJ1对中华蜜蜂耐热性的影响
1.试验方法
(a)利用PCR技术扩增用于沉默DnaJ1的片段,该片段在DnaJ1的编码区核苷酸序列所处的位置为236-730bp。同时扩增500bp的GFP(GenBank上的注册号为:U87974)的序列。
扩增DnaJ1的引物为:
正向引物:GGATCCTAATACGACTCACTATAGGCAGGAGGAGGCGGTGGTGGC;(SEQ ID NO.7)
反向引物:GGATCCTAATACGACTCACTATAGGCTTTCCCACGGCCTTGATCAC。(SEQ ID NO.8)
扩增GFP用的引物为:
正向引物:GGATCCTAATACGACTCACTATAGGAGTGGAGAGGGTGAAGGTGA;(SEQ ID NO.9)
反向引物:GGATCCTAATACGACTCACTATAGGGGTAAAAGGACAGGGCCATC。(SEQ ID NO.10)
以上4条引物的划线部分的序列均为T7 RNA聚合酶启动子序列。
(b)把上述扩增的PCR产物进行胶回收,并以胶回收产物为模版,借助Promega公司生产的T7 RiboMAXTM Express RNAi System合成dsRNA-DnaJ1和dsRNA-GFP。
(c)选取采集蜂60只,分成2组,每组30只蜜蜂。第1组的每只蜜蜂饲喂5μg的dsRNA-DnaJ1(实验组),第2组的每只饲喂5μg的dsRNA-GFP(对照组)。两组蜜蜂放在培养箱中正常培养。两天后提取两组蜜蜂的RNA,并反转录成cDNA,最后用qRT-PCR的方法验证DnaJ1在中华蜜蜂中沉默的效率;
(d)在确定可以在中华蜜蜂中成功沉默DnaJ1后,选取采集蜂60只,分成2组,第1组的蜜蜂饲喂dsRNA-DnaJ1(5μg/只),第2组的蜜蜂饲喂dsRNA-GFP(5μg/只),常规培养2天后,2组蜜蜂均用44℃处理,然后每隔4h统计一次蜜蜂的死亡率。每组处理至少做3个生物学重复。
2.试验结果
沉默DnaJ1对中华蜜蜂耐热能力的影响的检测结果见图3。图3A是以β-actin作为内参基因鉴定DnaJ1的沉默效率,饲喂dsRNA-GFP的蜜蜂作为对照组;图3B是统计沉默DnaJ1后中华蜜蜂在热激胁迫条件下的存活率。
由图3可知DnaJ1在中华蜜蜂中被成功沉默。并且与对照组相比,沉默DnaJ1增加蜜蜂在热激胁迫条件下的死亡率,降低了蜜蜂的耐热能力。
实施例4:沉默DnaJ1对中华蜜蜂体内丙二醛、蛋白质总羰基、维生素C的含量及总抗氧化能力的影响
1.试验方法
(a)把采集蜂分成2组(20只/组),第1组(实验组)的每只蜜蜂饲喂5μg的dsRNA-DnaJ1,第2组(对照组)的每只蜜蜂饲喂5μg的dsRNA-GFP。正常条件下培养2天后存样,把存好的样品放到-80℃冰箱保存;
(b)把上一步存的样本用液氮研磨成粉末后,再用生理盐水制备成10%的组织匀浆;
(c)把部分样本稀释成1%的组织匀浆,用南京建成公司生产的蛋白定量测试盒(考马斯亮蓝法)测组织匀浆的蛋白浓度;
(d)利用南京建成公司提供的总抗氧化能力测定试剂盒(比色法)测定每份组织匀浆的总抗氧化能力;
(e)借助南京建成公司生产的丙二醛测试盒(TBA法)检测上述步骤中提取的组织匀浆的丙二醛含量;
(f)采用南京建成公司制造的总羰基试剂盒,通过分光光度法来测定上述提取的组织匀浆的总羰基的含量;
(g)使用南京建成公司生产的维生素C含量测定试剂盒检测上面提及的组织匀浆的维生素C含量;
以上每个测定组至少做3个生物学重复。
2.试验结果
沉默DnaJ1对中华蜜蜂体内丙二醛、总羰基、维生素C含量及总抗氧化能力的影响结果见图4。由图4中可见,与对照组相比,沉默DnaJ1增加了中华蜜蜂体内丙二醛、蛋白质羰基的含量,降低了维生素含量及总抗氧化能力。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 山东农业大学
<120> 中华蜜蜂耐热相关基因DnaJ1及其应用
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Claims (6)
1.DnaJ1基因或者其表达产物作为靶标在如下1)或2)中的应用:
1)调控蜜蜂在热激胁迫条件下的抗热能力;
2)培育在热激胁迫条件下抗热能力增强的蜜蜂品种;
所述DnaJ1基因的核苷酸序列如SEQ ID NO.1所示;DnaJ1基因表达产物的氨基酸序列如SEQ ID NO.2所示。
2.特异性检测DnaJ1基因或者其表达产物的试剂在制备用于检测蜜蜂在热激胁迫下抗热能力的产品中的应用;
所述DnaJ1基因的核苷酸序列如SEQ ID NO.1所示;DnaJ1基因表达产物的氨基酸序列如SEQ ID NO.2所示;
所述产品采用实时荧光定量PCR方法检测DnaJ1基因或者其表达产物。
3.根据权利要求2所述的应用,其特征在于,所述产品包括:实时荧光定量PCR试剂盒。
4.根据权利要求3所述的应用,其特征在于,所述实时荧光定量PCR试剂盒中含有:特异性检测DnaJ1基因的引物对,其序列如SEQ ID NO.3和SEQ ID NO.4所示。
5.沉默DnaJ1基因表达的试剂在构建热激胁迫下抗热能力降低的蜜蜂模型中的应用;
所述DnaJ1基因的核苷酸序列如SEQ ID NO.1所示;
所述试剂中包含:用于扩增沉默DnaJ1的片段的引物,其序列分别如SEQ ID NO.7和SEQID NO.8所示。
6.根据权利要求5所述的应用,其特征在于,所构建的蜜蜂模型通过沉默DnaJ1的表达来降低蜜蜂的总抗氧化能力、增加脂质过氧化、蛋白质和细胞氧化损伤的程度,从而降低蜜蜂在热激胁迫下抗热能力。
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