家蚕不同耐热型品系热休克蛋白70基因（Hsp70s）的克隆和定量表达
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摘要
热休克蛋白70(Heat shock proteins, Hsp70s)是从细菌到哺乳动物中均广泛存在的一类保守的蛋白质家族。Hsp70s不仅在应激条件(如:热应激、饥饿或者细菌感染)下高效表达,有些还可以在正常的生理条件下有组成型的表达。诱导型的热休克蛋白70家族成员(Heat shock proteins, Hsp70s)是应激时大量表达的诱导性产物;组成型的热休克蛋白70称为热休克相关蛋白(heat shock cognate proteins,Hsc70s),除了可以进行应激性表达外,也可以进行组成性表达,它们是参与热休克反应(Heat Shock Responds,HSR)的主要因子。
Hsp70s家族通常含有多个成员。对于人类,果蝇和酵母等物种的Hsp70s目前已经有了较详细的研究,人的基因组含有17个Hsp70s基因和30个假基因,诱导性表达的成员有Hsp70-1a、Hsp70-1b、Hsp70-1t、Hsp70-6等,组成型表达的成员有Hsp70-5、Hsp70-9等;果蝇Hsp70s家族至少有12个成员,诱导性的有Hsp68和Hsp70s,组成型成员有Hsc70-1、Hsc70-2、Hsc70-4、Hsc70-3等。2009年,Fabian M对位于果蝇内质网内的Hsc70-3做了定量表达分析,发现在热激过程中,Hsc70-3的表达量增加了。在不同的物种中,随着Hsp70s成员不同,其表达的数量和功能也可能不同,在热激过程中的表达情况也不尽相同。目前,家蚕Hsp7s0家族成员的报道尚属空白,各成员的热激表达也缺乏研究,所以有必要对家蚕Hsp70s家族的组成及基因成员的表达情况进行探索。
本研究首先利用电子克隆技术,在EST数据库中人工拼接家蚕Hsp70家族成员,然后克隆、测序Hsp70s家族的每一个成员,根据和其它物种的比对结果,参照其命名原则,对家蚕Hsp70s家族成员进行了命名,并对其进行了染色体定位分析。家蚕内至少有10个Hsp70s家族成员,其中,Hsp70A、Hsp70B、Hsp68和Hsp70-14在基因组上都没有内含子,为诱导型。Hsp70,Hsp70A和Hsp70B分布在家蚕第27号染色体上,在基因组上是串连的,有多个拷贝,但是方向相反。Hsp70-14是一个分子量只有52KD的蛋白质,没有C末端的基序,是一个不规范的Hsp70。Hsc70-4、Hsc70-3、Hsc70-5、Hsc70-2、Hsc70-cs和Hsc70Cb在基因组上都含有内含子,属于组成型表达的Hsp70,其中Hsc70-3是内质网内组成型表达的热休克蛋白,Hsc70-4是细胞质内组成型表达的一种热休克蛋白。此外,在家蚕的11号染色体上,有一个暂命名为Hsp70-w的假基因,它在基因组上存在,也有转录(CK517125),但成熟的mRNA不能翻译出正确的蛋白质,所以Hsp70-w是一个有转录的假基因。Hsc70s家族成员则分布在不同的染色体上,一般只有一个拷贝。
家蚕是鳞翅目昆虫的模式物种,也是我国养殖了几千年的经济昆虫,基因库里保存有大量不同性状的品系的基因信息,为本研究提供了较丰富的材料。本研究团队筛选了抗热家蚕品系cs-10,不抗热的温度敏感性品系cs-11和以抗性品系和感性品系为亲本构建的近等基因系cs-7。以这三种家蚕品系作为研究材料来分析家蚕Hsp70s家族成员(Hsp68、Hsp70B、Hsc70-3和Hsc70-4)的一级序列及在高温诱导下的表达模式。本实验首先对每个品系中Hsp70s成员的转录产物进行克隆、测序。用MegAlign软件分析每个成员的一级结构,结果显示Hsp70s成员在三个品系中的一级序列排布上相似度非常高,只有极个别氨基酸不同。其次将家蚕进行40℃热激,用qRT-PCR技术对其表达量上进行了研究。诱导型的Hsp70s包括Hsp68和Hsp70B与组成型的Hsc70s包括Hsc70-3和Hsc70-4在三个品系中的表达均上调,说明这4个Hsp70s成员在三个品系中均参与了抗热,但是在不同的品系中,每个Hsp70s成员上调的情况有很大的不同。说明抗性品系能够抗热与Hsp70s的表达量有密切关系。在不同的热激时间,每个成员的表达也有很大的变化,表明各个成员虽然是属于Hsp70s家族,对热的抵御能力不尽相同,也许各个成员并不能相互替代,有其各自特殊的功能。最后用Northern杂交技术对Hsp70B和Hsc70-3在三个品系中的表达情况作了验证分析。表达趋势和荧光定量结果是一致的。本研究可以为家蚕耐热机理方面的探索打下基础,也可以为Hsp70s家族各成员不同功能方面的研究提供数据。
Heat shock protein 70 (Heat shock proteins, Hsp70s) are stressed proteins which exist widely from bacteria to mammals in conditions such as heat stress, starvation and bacterial infection. Not only they highly expressed in stress conditions, but also they be present consititutively under normal conditions. It is widely accepted that the induceble Hsp70s are expressed highly against stressful environment to resist heat. and the constitutive Hsc70s are expressed for basic resistance when at high-temperature.
The expression and functions of Hsp70s may indicate very diffrently in different species because of the multiple different mumbers in each specie. There are 17 Hsp70s and 30 pseudogene in human with induceble Hsp70s such as Hsp70-1a, Hsp70-1b, Hsp70-1t, Hsp70-6 and consitutitive Hsp70s including Hsp70-5, Hsp70-9 and so on. In fruitfly, there are at least 12 Hsp70s members such as the induceble Hsp68 and Hsp70s as well as the consitutitive Hsc70-1, Hsc70-2, Hsc70-4, Hsc70-3. In 2009, Fabian M found that the Hsc70-3 of endoplasmic reticulum expressed higher in fruitflies when at high temperature than that in normal environment. The function of the Hsp70s members have been studied much in humans, fruit flies and yeast. But the expression of each member of Hsp70s is relatively researched less, especially the expression of Hsp70s in silkworm is scarely studied and the report about functions is still blank. It is necessary to studied Hsp70s in silkworms.
In this thesis, the Hsp70s were cloned electronicly in silkworms and named according to the name of Hsp70s in other species. All the Hsp70s were also sited exactly on chromosomes.There are at least 10 Hsp70s. including 4 induceble Hsp70s and 6 constitutive Hsp70s. Hsp70A, Hsp70B, Hsp68 and Hsp70-14 are intronless belonging to induceble Hsp70s. Hsc70-4, Hsc70-3, Hsc70-5, Hsc70-2, Hsc70-cs and Hsc70Cb all contain introns which are expressed constitutively. Hsc70-3 is expressed constitutively in the endoplasmic reticulum and Hsc70-4 is expressed constitutively in the cytoplasm. Hsp70-14 is a protein of only 52KD and has a non-standard C motif end. In addition, there is a Hsp70-w named pseudogene on the chromosome 11 in silkworm, which is transcribed (CK517125), but can not be translated. The induced Hsp70B and Hsp70A are concentrated on chromosome 27, with multiple copies. Hsp70A and Hsp70B are chained on the genome, but in the opposite direction. Constitutive Hsp70s are located in different chromosomes, generally only one copy.
Silkworm is a specie of Lepidoptera insects, with thousands years of economic useness. There are a large number of strains in gene library with different traits providing lots of good research materials. Our experiments contain three different strains including the cs-10 of thermotolerance,cs-11 of thermosensitive and cs-7 of close isogenic strain which is descendant of cs-10 and cs-11. The expression of Hsp70s in silkworm when at high temprature is systematicly analysised. Hsp68 and Hsp70B are expressed induceblely . Hsc70-3 is a heat shock protein in the endoplasmic reticulum which is constitutived expressed. Hsc70-4 is a constitutive heat shock protein expressed in the cytoplasm. The four Hsp70s of the three strains were cloned and sequenced. The analysis of the sequence for each member in using MegAlign software showned that each member of Hsp70s in the three strains are highly similar with only a few amino acids are different. Quantitative real-time PCR (qRT-PCR) is used to test the level change of their expression when the three strains of silkworms are cultrued at 40℃. The induceble Hsp70s including Hsp68 and Hsp70B and the constitutives including Hsc70-3 and Hsc70-4 are all up-regulated. This means that the four members of Hsp70s are all involved in the heat resistant, but the amount of expression of each member is changed greatly which indicating that there are certain relationships between the amount of Hsp70s expression and the ability of resistanting heat. Furthermore, the four Hsp70s mumbers showned dramatically differently at different being heated shock time, these indicate that although they all belong to the same family of Hsp70, but the abilities of them in heat resisting are not similar and the mumber of Hsp70s may cannot be replaced by another one. At the same time, Northern is used for analysing the expression of Hsp70B and Hsc70-3 in the three strains. The reserch about the expression of Hsp70s in silkworms is the base of further study for the mechanism of heat-resistant and can offer data for the function research of Hsp70s.
Hsp70s家族通常含有多个成员。对于人类,果蝇和酵母等物种的Hsp70s目前已经有了较详细的研究,人的基因组含有17个Hsp70s基因和30个假基因,诱导性表达的成员有Hsp70-1a、Hsp70-1b、Hsp70-1t、Hsp70-6等,组成型表达的成员有Hsp70-5、Hsp70-9等;果蝇Hsp70s家族至少有12个成员,诱导性的有Hsp68和Hsp70s,组成型成员有Hsc70-1、Hsc70-2、Hsc70-4、Hsc70-3等。2009年,Fabian M对位于果蝇内质网内的Hsc70-3做了定量表达分析,发现在热激过程中,Hsc70-3的表达量增加了。在不同的物种中,随着Hsp70s成员不同,其表达的数量和功能也可能不同,在热激过程中的表达情况也不尽相同。目前,家蚕Hsp7s0家族成员的报道尚属空白,各成员的热激表达也缺乏研究,所以有必要对家蚕Hsp70s家族的组成及基因成员的表达情况进行探索。
本研究首先利用电子克隆技术,在EST数据库中人工拼接家蚕Hsp70家族成员,然后克隆、测序Hsp70s家族的每一个成员,根据和其它物种的比对结果,参照其命名原则,对家蚕Hsp70s家族成员进行了命名,并对其进行了染色体定位分析。家蚕内至少有10个Hsp70s家族成员,其中,Hsp70A、Hsp70B、Hsp68和Hsp70-14在基因组上都没有内含子,为诱导型。Hsp70,Hsp70A和Hsp70B分布在家蚕第27号染色体上,在基因组上是串连的,有多个拷贝,但是方向相反。Hsp70-14是一个分子量只有52KD的蛋白质,没有C末端的基序,是一个不规范的Hsp70。Hsc70-4、Hsc70-3、Hsc70-5、Hsc70-2、Hsc70-cs和Hsc70Cb在基因组上都含有内含子,属于组成型表达的Hsp70,其中Hsc70-3是内质网内组成型表达的热休克蛋白,Hsc70-4是细胞质内组成型表达的一种热休克蛋白。此外,在家蚕的11号染色体上,有一个暂命名为Hsp70-w的假基因,它在基因组上存在,也有转录(CK517125),但成熟的mRNA不能翻译出正确的蛋白质,所以Hsp70-w是一个有转录的假基因。Hsc70s家族成员则分布在不同的染色体上,一般只有一个拷贝。
家蚕是鳞翅目昆虫的模式物种,也是我国养殖了几千年的经济昆虫,基因库里保存有大量不同性状的品系的基因信息,为本研究提供了较丰富的材料。本研究团队筛选了抗热家蚕品系cs-10,不抗热的温度敏感性品系cs-11和以抗性品系和感性品系为亲本构建的近等基因系cs-7。以这三种家蚕品系作为研究材料来分析家蚕Hsp70s家族成员(Hsp68、Hsp70B、Hsc70-3和Hsc70-4)的一级序列及在高温诱导下的表达模式。本实验首先对每个品系中Hsp70s成员的转录产物进行克隆、测序。用MegAlign软件分析每个成员的一级结构,结果显示Hsp70s成员在三个品系中的一级序列排布上相似度非常高,只有极个别氨基酸不同。其次将家蚕进行40℃热激,用qRT-PCR技术对其表达量上进行了研究。诱导型的Hsp70s包括Hsp68和Hsp70B与组成型的Hsc70s包括Hsc70-3和Hsc70-4在三个品系中的表达均上调,说明这4个Hsp70s成员在三个品系中均参与了抗热,但是在不同的品系中,每个Hsp70s成员上调的情况有很大的不同。说明抗性品系能够抗热与Hsp70s的表达量有密切关系。在不同的热激时间,每个成员的表达也有很大的变化,表明各个成员虽然是属于Hsp70s家族,对热的抵御能力不尽相同,也许各个成员并不能相互替代,有其各自特殊的功能。最后用Northern杂交技术对Hsp70B和Hsc70-3在三个品系中的表达情况作了验证分析。表达趋势和荧光定量结果是一致的。本研究可以为家蚕耐热机理方面的探索打下基础,也可以为Hsp70s家族各成员不同功能方面的研究提供数据。
Heat shock protein 70 (Heat shock proteins, Hsp70s) are stressed proteins which exist widely from bacteria to mammals in conditions such as heat stress, starvation and bacterial infection. Not only they highly expressed in stress conditions, but also they be present consititutively under normal conditions. It is widely accepted that the induceble Hsp70s are expressed highly against stressful environment to resist heat. and the constitutive Hsc70s are expressed for basic resistance when at high-temperature.
The expression and functions of Hsp70s may indicate very diffrently in different species because of the multiple different mumbers in each specie. There are 17 Hsp70s and 30 pseudogene in human with induceble Hsp70s such as Hsp70-1a, Hsp70-1b, Hsp70-1t, Hsp70-6 and consitutitive Hsp70s including Hsp70-5, Hsp70-9 and so on. In fruitfly, there are at least 12 Hsp70s members such as the induceble Hsp68 and Hsp70s as well as the consitutitive Hsc70-1, Hsc70-2, Hsc70-4, Hsc70-3. In 2009, Fabian M found that the Hsc70-3 of endoplasmic reticulum expressed higher in fruitflies when at high temperature than that in normal environment. The function of the Hsp70s members have been studied much in humans, fruit flies and yeast. But the expression of each member of Hsp70s is relatively researched less, especially the expression of Hsp70s in silkworm is scarely studied and the report about functions is still blank. It is necessary to studied Hsp70s in silkworms.
In this thesis, the Hsp70s were cloned electronicly in silkworms and named according to the name of Hsp70s in other species. All the Hsp70s were also sited exactly on chromosomes.There are at least 10 Hsp70s. including 4 induceble Hsp70s and 6 constitutive Hsp70s. Hsp70A, Hsp70B, Hsp68 and Hsp70-14 are intronless belonging to induceble Hsp70s. Hsc70-4, Hsc70-3, Hsc70-5, Hsc70-2, Hsc70-cs and Hsc70Cb all contain introns which are expressed constitutively. Hsc70-3 is expressed constitutively in the endoplasmic reticulum and Hsc70-4 is expressed constitutively in the cytoplasm. Hsp70-14 is a protein of only 52KD and has a non-standard C motif end. In addition, there is a Hsp70-w named pseudogene on the chromosome 11 in silkworm, which is transcribed (CK517125), but can not be translated. The induced Hsp70B and Hsp70A are concentrated on chromosome 27, with multiple copies. Hsp70A and Hsp70B are chained on the genome, but in the opposite direction. Constitutive Hsp70s are located in different chromosomes, generally only one copy.
Silkworm is a specie of Lepidoptera insects, with thousands years of economic useness. There are a large number of strains in gene library with different traits providing lots of good research materials. Our experiments contain three different strains including the cs-10 of thermotolerance,cs-11 of thermosensitive and cs-7 of close isogenic strain which is descendant of cs-10 and cs-11. The expression of Hsp70s in silkworm when at high temprature is systematicly analysised. Hsp68 and Hsp70B are expressed induceblely . Hsc70-3 is a heat shock protein in the endoplasmic reticulum which is constitutived expressed. Hsc70-4 is a constitutive heat shock protein expressed in the cytoplasm. The four Hsp70s of the three strains were cloned and sequenced. The analysis of the sequence for each member in using MegAlign software showned that each member of Hsp70s in the three strains are highly similar with only a few amino acids are different. Quantitative real-time PCR (qRT-PCR) is used to test the level change of their expression when the three strains of silkworms are cultrued at 40℃. The induceble Hsp70s including Hsp68 and Hsp70B and the constitutives including Hsc70-3 and Hsc70-4 are all up-regulated. This means that the four members of Hsp70s are all involved in the heat resistant, but the amount of expression of each member is changed greatly which indicating that there are certain relationships between the amount of Hsp70s expression and the ability of resistanting heat. Furthermore, the four Hsp70s mumbers showned dramatically differently at different being heated shock time, these indicate that although they all belong to the same family of Hsp70, but the abilities of them in heat resisting are not similar and the mumber of Hsp70s may cannot be replaced by another one. At the same time, Northern is used for analysing the expression of Hsp70B and Hsc70-3 in the three strains. The reserch about the expression of Hsp70s in silkworms is the base of further study for the mechanism of heat-resistant and can offer data for the function research of Hsp70s.
引文
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