家蚕谷胱甘肽-S-转移酶基因转录水平定量研究
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摘要
谷胱甘肽-S-转移酶(glutathione S-transferases, GSTs)是一个多功能超基因家族,在昆虫抗性方面起到很重要的作用。为探究家蚕(Bombyx mori)谷胱甘肽-S-转移酶基因(BmGSTs)的功能,本研究采用双跟踪标定实时荧光定量RT-PCR(dual spike-in qPCR)方法,对家蚕和野蚕(Bombyx mandarina)部分重要的BmGSTs基因转录水平进行了定量研究,主要研究结果如下:
1、BmGSTd1基因诱导表达定量分析
采用实时荧光定量RT-PCR方法,对BmGSTd1基因在正常饲养及添食NaF的家蚕5龄幼虫不同组织中的转录水平进行检测,并采用家蚕Actin3、GAPDH、28S rRNA 3种內源参照基因对检测结果进行归一化处理?峁砻鳎築mGSTd1基因在家蚕5龄幼虫各组织的转录水平存在差异,且采用不同内参照基因归一化的结果也有较大差异。研究认为,检测基因在不同组织中的转录水平,采用合适的内参照基因对保证检测结果的可靠性非常重要。
2、不同蚕品种BmGSTs基因转录水平的测定
采用dual spike-in qPCR方法,对野蚕和不同品种家蚕各组织中BmGSTs基因的转录水平进行检测,结果表明:不同蚕品种之间被测BmGSTs基因的转录水平存在差异,菁松和皓月BmGSTs基因的转录水平较高,老挝L11种的转录水平次之,大造和野桑蚕BmGSTs基因的转录水平相对较低;不同组织之间BmGSTs基因的转录水平也存在一定的差异。
3、杀虫剂诱导下BmGSTs基因转录水平的测定
采用dual spike-in qPCR方法,测定了在杀虫剂敌敌畏和溴氰菊酯诱导下,家蚕L11和野蚕不同BmGSTs基因的表观转录水平。结果表明:在敌敌畏和溴氰菊酯诱导下,老挝L11 BmGSTs基因在中肠的转录水平上调最显著,其次是脂肪体。野桑蚕在溴氰菊酯诱导下,被测BmGSTs基因转录水平有显著上调,而在敌敌畏诱导下,未检测到较高的转录水平。所选基因以BmGSTe5在中肠和BmGSTs1基因在脂肪体中被诱导上调为主,预测这两个基因与家蚕抗性的关系比较密切。
4、氟化物诱导下BmGSTs基因转录水平的测定
采用dual spike-in qPCR方法,测定了家蚕皓月在NaF诱导下,5龄幼虫各组织BmGSTs基因的转录水平。结果表明:NaF对家蚕体内某些组织BmGSTs基因的表达具有诱导作用,比如:中肠中的BmGSTe5和BmGSTs1基因,脂肪体中的BmGSTo3和BmGSTs1基因;而在另外一些组织中则具有抑制作用,例如BmGSTs基因在马氏管中的表达。
本研究阐明了BmGSTs基因在家蚕和野蚕体内各组织表达的基本信息,为研究该基因家族在家蚕抗性中的作用提供数据参考,并为培育家蚕抗逆性新品种提供重要的理论依据。
Glutathione S-transferases (GSTs) are a multifunctional supergene family and some play an important role in insecticide resistance. In order to explore the roles of Bombyx mori glutathione S-transferase genes (BmGSTs), we investigated the transcription levels of some BmGST genes in Bombyx mori and Bombyx mandarina with the dual spike-in qPCR method. The main results are as follows:
1. Induced expression and quantitative analysis of BmGSTd1
We used a real-time fluorescent quantitative PCR method to detect the transcription levels of BmGSTd1 in different tissues of the 5th instar larvae feeding on both normal mulberry leaves and sodium fluoride treated mulberry leaves. The detection results were normalized by using 3 Bombyx mori internal reference genes namely Actin3, GAPDH and 28S rRNA. The results indicated that the transcription levels of BmGSTd1 were different in various tissues of the 5th instar larvae. They were also varied while different internal reference genes were used. It is thus concluded that, for detection of gene transcription levels, it is very important to choose adequate internal reference genes so as to ensure the reliability of the detection results.
2. Transcription levels of BmGST genes in varieties of silkworm
We have determined the transcription levels of BmGSTs in five varieties of Bombyx mori and Bombyx mandarina with the dual-spike-in qPCR method. The results indicated that the transcription levels of BmGSTs shows differences between different varieties. To sum up, the transcription levels of BmGSTs in Jingsong and Haoyue are highest, those of L11 are following, and the transcription levels of BmGSTs in Dazao and Bombyx mandarina are lowest. And the transcription levels of BmGSTs in different tissues are also different.
3. Insecticides-induced transcription levels of BmGST genes We have determined the transcription levels of BmGSTs induced by Dichlorvos and Deltamethrin in silkworm L11 and Bombyx mandarina with the dual-spike-in qPCR method. The results indicated that the transcription levels of BmGSTs induced by Dichlorvos and Deltamethrin in midgut of L11 are up-regulated most, the following are fat body. In Bombyx mandarina, the transcription levels of BmGSTs induced by Deltamethrin are significantly up-regulated, but those induced by Dichlorvos can not be detected significantly. The transcription levels of BmGSTe5 in midgut and BmGSTs1 in fatbody are up-regulated most, so we predict that these two genes are related with resistance of silkworm.
4. NaF-induced transcription levels of BmGST genes
We have determined the transcription levels of BmGSTs induced by NaF in silkworm Haoyue with dual-spike-in qPCR method. The results indicated that the transcription of some BmGSTs such as BmGSTe5 and BmGSTs1 in the midgut as well as BmGSTo3 and BmGSTs1 in the fat body can be induced by NaF, and they can be inhibited by NaF, such as the BmGSTs in the Malpighian tubule.
The production of this research illuminated the basic information of distributing of BmGSTs in various tissues of Bombyx mori, and it can provide data for studying the function of BmGSTs in the development of resistance and developing new pesticide resistant silkworm varieties.
1、BmGSTd1基因诱导表达定量分析
采用实时荧光定量RT-PCR方法,对BmGSTd1基因在正常饲养及添食NaF的家蚕5龄幼虫不同组织中的转录水平进行检测,并采用家蚕Actin3、GAPDH、28S rRNA 3种內源参照基因对检测结果进行归一化处理?峁砻鳎築mGSTd1基因在家蚕5龄幼虫各组织的转录水平存在差异,且采用不同内参照基因归一化的结果也有较大差异。研究认为,检测基因在不同组织中的转录水平,采用合适的内参照基因对保证检测结果的可靠性非常重要。
2、不同蚕品种BmGSTs基因转录水平的测定
采用dual spike-in qPCR方法,对野蚕和不同品种家蚕各组织中BmGSTs基因的转录水平进行检测,结果表明:不同蚕品种之间被测BmGSTs基因的转录水平存在差异,菁松和皓月BmGSTs基因的转录水平较高,老挝L11种的转录水平次之,大造和野桑蚕BmGSTs基因的转录水平相对较低;不同组织之间BmGSTs基因的转录水平也存在一定的差异。
3、杀虫剂诱导下BmGSTs基因转录水平的测定
采用dual spike-in qPCR方法,测定了在杀虫剂敌敌畏和溴氰菊酯诱导下,家蚕L11和野蚕不同BmGSTs基因的表观转录水平。结果表明:在敌敌畏和溴氰菊酯诱导下,老挝L11 BmGSTs基因在中肠的转录水平上调最显著,其次是脂肪体。野桑蚕在溴氰菊酯诱导下,被测BmGSTs基因转录水平有显著上调,而在敌敌畏诱导下,未检测到较高的转录水平。所选基因以BmGSTe5在中肠和BmGSTs1基因在脂肪体中被诱导上调为主,预测这两个基因与家蚕抗性的关系比较密切。
4、氟化物诱导下BmGSTs基因转录水平的测定
采用dual spike-in qPCR方法,测定了家蚕皓月在NaF诱导下,5龄幼虫各组织BmGSTs基因的转录水平。结果表明:NaF对家蚕体内某些组织BmGSTs基因的表达具有诱导作用,比如:中肠中的BmGSTe5和BmGSTs1基因,脂肪体中的BmGSTo3和BmGSTs1基因;而在另外一些组织中则具有抑制作用,例如BmGSTs基因在马氏管中的表达。
本研究阐明了BmGSTs基因在家蚕和野蚕体内各组织表达的基本信息,为研究该基因家族在家蚕抗性中的作用提供数据参考,并为培育家蚕抗逆性新品种提供重要的理论依据。
Glutathione S-transferases (GSTs) are a multifunctional supergene family and some play an important role in insecticide resistance. In order to explore the roles of Bombyx mori glutathione S-transferase genes (BmGSTs), we investigated the transcription levels of some BmGST genes in Bombyx mori and Bombyx mandarina with the dual spike-in qPCR method. The main results are as follows:
1. Induced expression and quantitative analysis of BmGSTd1
We used a real-time fluorescent quantitative PCR method to detect the transcription levels of BmGSTd1 in different tissues of the 5th instar larvae feeding on both normal mulberry leaves and sodium fluoride treated mulberry leaves. The detection results were normalized by using 3 Bombyx mori internal reference genes namely Actin3, GAPDH and 28S rRNA. The results indicated that the transcription levels of BmGSTd1 were different in various tissues of the 5th instar larvae. They were also varied while different internal reference genes were used. It is thus concluded that, for detection of gene transcription levels, it is very important to choose adequate internal reference genes so as to ensure the reliability of the detection results.
2. Transcription levels of BmGST genes in varieties of silkworm
We have determined the transcription levels of BmGSTs in five varieties of Bombyx mori and Bombyx mandarina with the dual-spike-in qPCR method. The results indicated that the transcription levels of BmGSTs shows differences between different varieties. To sum up, the transcription levels of BmGSTs in Jingsong and Haoyue are highest, those of L11 are following, and the transcription levels of BmGSTs in Dazao and Bombyx mandarina are lowest. And the transcription levels of BmGSTs in different tissues are also different.
3. Insecticides-induced transcription levels of BmGST genes We have determined the transcription levels of BmGSTs induced by Dichlorvos and Deltamethrin in silkworm L11 and Bombyx mandarina with the dual-spike-in qPCR method. The results indicated that the transcription levels of BmGSTs induced by Dichlorvos and Deltamethrin in midgut of L11 are up-regulated most, the following are fat body. In Bombyx mandarina, the transcription levels of BmGSTs induced by Deltamethrin are significantly up-regulated, but those induced by Dichlorvos can not be detected significantly. The transcription levels of BmGSTe5 in midgut and BmGSTs1 in fatbody are up-regulated most, so we predict that these two genes are related with resistance of silkworm.
4. NaF-induced transcription levels of BmGST genes
We have determined the transcription levels of BmGSTs induced by NaF in silkworm Haoyue with dual-spike-in qPCR method. The results indicated that the transcription of some BmGSTs such as BmGSTe5 and BmGSTs1 in the midgut as well as BmGSTo3 and BmGSTs1 in the fat body can be induced by NaF, and they can be inhibited by NaF, such as the BmGSTs in the Malpighian tubule.
The production of this research illuminated the basic information of distributing of BmGSTs in various tissues of Bombyx mori, and it can provide data for studying the function of BmGSTs in the development of resistance and developing new pesticide resistant silkworm varieties.
引文
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