家蚕细胞色素P450基因及其定量方法的研究
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摘要
细胞色素P450酶系又称作多功能氧化酶(MFO),是广泛存在于所有需氧生物中的一类代谢酶系,细胞色素P450基因起源于35亿年前的一个共同祖先,是最古老和最庞大的超基因家族之一,在生命的过程中,它对内源物质的代谢与转化或外源化合物的活化与降解等进行催化和调控。在昆虫基因组中大约有100个左右的P450s,它们的作用涉及生长、发育、取食等过程,其对异生物质的代谢特性导致了昆虫对杀虫剂的抗药性和对植物有毒物质的耐受性,它还参与了昆虫体内保幼激素、蜕皮激素和脂肪酸等内源化合物的合成与代谢。
家蚕(Bombyx mori)是鳞翅目昆虫的模式生物,本研究以家蚕为研究对象,对家蚕P450基因CYP305B1基因组序列进行了分析;通过对目前家蚕实时定量PCR研究中常用的内源参照基因进行分析,提出了新的定量PCR方法,并应用此方法,对家蚕和野蚕(Bombyx mandarina)部分P450基因进行了定量研究。本论文的主要研究结果如下: 1.家蚕P450基因CYP305B1的克隆及其结构分析
为了研究家蚕P450基因CYP305B1的结构,采用了反向PCR(inverse PCR)技术,克隆了家蚕CYP305B1基因的基因组DNA,经序列测定,拼接得到家蚕CYP305B1全基因序列,并与野桑蚕P450基因CYP305B1V1进行了比较分析。结果表明,CYP305B1和CYP305B1V1在5’UTR上游-400至-770之间370 bp的序列同源性只有40.4%;在第1内含子中,与家蚕相比,野桑蚕在翻译起始密码ATG上游-340之前多了330 bp的插入序列,而在-110至-340 bp间两者同源性也只有46.9%。这些结果为深入研究该基因的功能提供了信息。
2.实时定量PCR方法的比较研究
为了比较目前常用的内源参照基因actin-3、GAPDH、28s rRNA,在实验样品中加入IFP2基因的mRNA和DNA混合物,作为跟踪标定基因,测定了家蚕各组织中的actin-3、GAPDH、28s rRNA基因的转录水平。同时测定了后部丝腺中的FibH、FibL基因,中部丝腺Ser-1基因的转录水平,并对两种归一化方法进行了比较。
结果表明,actin-3、GAPDH、28s rRNA在家蚕各组织中的差异极大,A3基因在后部丝腺中转录水平最高(1,098),中肠最低(9.9);GAPDH基因的转录水平以脂肪体最高(10,475),中肠最低(6.57)。28S rRNA基因的转录水平以脂肪体最高(97,321),中肠最低(2,178)。
如用与actin-3、GAPDH、28s rRNA相比的相对表达结果,则后部丝腺中的FibH ,FibL基因,中部丝腺Ser-1基因的转录水平,相互之间差距较大。而应用加入IFP2基因的mRNA和DNA混合物作为跟踪标定基因测到的是实际转录水平,基因FibL和FibH在后部丝腺中的转录水平分别为106,453和192,029,Ser-1基因在中部丝腺中的转录水平为196,539。我们把同时加入mRNA和DNA混合物,作为跟踪标定基因的测定方法,称为双跟踪标定定量PCR,所得结果称为基因的表观转录活性。
3.杀虫剂诱导的P450基因的表观转录活性
应用双跟踪标定定量PCR方法,测定了野蚕和不同品种家蚕P450基因的表观转录活性。同时,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P450基因的表观转录活性。
结果表明,家蚕品种菁松中P450基因主要在中肠中有较高的转录,其次是脂肪体;家蚕品种皓月P450基因在丝腺中有较高转录;野桑蚕的P450基因的表观转录水平,以丝腺最高,其次是中肠。
在敌敌畏和溴氰菊酯诱导下,L11中肠的P450基因的转录水平上调最显著,其次是脂肪体。L11的P450基因以CYP9A19、CYP9A22显著上调。野桑蚕在溴氰菊酯诱导下,CYP9A19、CYP9A22均有显著上调,且达到较高转录活性,而在敌敌畏诱导下野桑蚕被测P450基因未检测到较高的转录水平。
4.氟化物诱导下家蚕P450基因的表观转录水平
应用双跟踪标定定量PCR方法,测定了家蚕在NaF诱导下的P450基因的表观转录活性。结果表明,CYP9A19,CYP9A22在中肠中具有较高表观转录活性,而CYP305B1,CYP4M5则在脂肪体中有较高表观转录活性。
实验表明,NaF诱导下的时间曲线有组织特异性,我们把中肠、脂肪体的诱导称为正向诱导,这反映了家蚕对NaF的抵抗作用,把马氏管中的诱导称为反向诱导,这反映了NaF对家蚕的毒害作用。并由此提出了在今后家蚕抗性研究中应综合考虑这两方面因素的新观点。
Cytochrome P450 monooxygenases, known as multi-functional oxidase(MFO),are a very important enzymatic system, which has been found in all living organisms systems examined. Cytochrome P450 gene originated from a common ancestor 35 million years ago,is one of the oldest and the largest super-gene families. In the process of life, they catalyse and regulate the metabolism and transform of endogenous compounds or the activation and degradation of exogenous compounds. There are around 100 or so P450s in insect genome. Their functions involve growth, development, feeding, etc., which not merely involves a series of endogenous materials such as juvenile hormone(JH) and its analogue, ecdysone, biologic pheromone, but also involve the supersession to exogenous many kinds of compounds such as insecticide, plant secondary materials, environmental pollutant.
Bombyx mori is one of model organisms in Lepidoptera. This research take the Bombyx mori as the object of study, and mark the genomic sequence analysis of CPY305B1 gene in the Bombyx mori. Through the current real-time quantitative PCR study of silkworm commonly used endogenous reference gene analysis, it has proposed the new quantitative PCR method, and using this method, has conducted the quantitative research to the Bombyx mori and the Bombyx mandarina part of P450 genes. The main results are as followed.
1. Cloning and sequence analysis of cytochrome CPY305B1 gene in the Bombyx mori
In order to study the gene structure of CYP305B1, the genomic sequence of this P450 gene was cloned by reverse PCR method. The complete genomic sequence of CYP305B1 was obatined after sequencing and assembling. Sequence analysis showed the first intron is located in the 5′UTR of this gene. Comparison of the genomic sequence of CYP305B1 with that of CYP305B1V1 from Bombyx mandarina demonstrated that the 370 bp sequence between 770 bp and 400 bp upstream of the 5′UTR of these two P450 genes shared only 40.4% identity to each other. The biggest differences between the two genes lies in the first and sixth introns. In the first intron, there is an additional insertion sequence of 330 bp before the 340 bp sequence upstream of the translation initiation codon ATG of CYP305B1V1 compared with CYP305B1, and the 230 bp sequences between 340 bp and 110 bp upstream of the translation initiation codon of the two genes showed only 46.9% identity to each other. In the sixth intron, there is an extra insertion sequence of about 300 bp in CYP305B1 compared with CYP305B1V1. This research will help us to research the mechanism of transcriptional regulation of this P450 gene.
2. The comparative study of real-time quantitative PCR methods
In order to compare the current commonly used endogenous reference genes actin-3, GAPDH, 28s rRNA, We determined the gene transcription levels of actin-3, GAPDH, 28s rRNA in different tissues of Bombyx mori with IFP2 mRNA and DNA mixed in the experiment type variety as the spike-in gene.
Simultaneously it has determined the transcription level of FibH,the FibL gene in middle silk gland, Ser-1 gene in the the posterior silk gland, and comparison with the consequences determined with actin-3,GAPDH,28s rRNA.
As the results shown , actin-3, GAPDH and 28s rRNA are very different in each tissue of Bombyx mori.A3 gene in the posterior silk gland transcription level is in the highest (1,098),the lowest in the mid-gut(9.9);GAPDH gene transcription level in the fat body is the highest(10,475), the lowest in the mid-gut (6.57). 28S rRNA gene transcription level of the highest is in the fat body (97,321), the lowest in the mid-gut (2,178).
With actin-3, GAPDH, 28s rRNA measured , FibH, FibL genes in the middle silk gland,the Ser-1 gene transcription level in the posterior silk gland,there is a large gap between any two.
And adding IFP2 into a mixture of mRNA and DNA at the same time, as a tracking method of spike-in gene,we have measured the transcription level of gene FibH, FibL genes in the MSG and the Ser-1 gene in the PSG.The transcription level (106,453 and 192,029) of gene FibL and FibH in PSG, and the transcription level (196,539) of Ser-1 gene in MSG compare the level closer to the actual transcription.Adding a mixture of mRNA and DNA at the same time,as a tracking method of spike-in gene,known as the dual-sipke-in quantitative PCR. The obtained result is called the gene apparent transcription activity.
3. Pesticides-induced apparent transcription levels of P450 gene
It has applied of dual-spike-in method of quantitative PCR to measure the apparent transcription levels of P450 gene in the Bombyx mandarina and Bombyx mori different varieties. At the same time, it has determined Bombyx mori L11 and Bombyx mandarina different P450 gene apparent transcription activity under the induction of the pesticide DDVP(dimethyl dichloro vinyl phosphate) and DKDD (Decamethrin Kothrin Decis Deltamethrin)
The results show that P450 gene mainly has the higher transcription level in the middle gut, followed by the fat body; Bombyx mori Haoyue P450 genes also have a higher transcription in the silk gland.The apparent transcription level of P450 gene in the Bombyx mandarina is the highest in silk gland, followed by the mid-gut.
Induced by DDVP and DKDD,the transcription level of P450 gene in the mid-gut of Bombyx mori L11 increases significantly, followed by that of the fat body.CYP9A19 and CYP9A22 in Bombyx mori L11 are significantly raised.CYP9A19 and CYP9A22 in Bombyx mandarina are significantly raised, and reach a higher transcriptional activity under the induction of DDKD.However,the transcription of the DDKP-induced P450 gene measured in the Bombyx mandarina is not detected in a higher level.
4. Fluoride-induced apparent transcription levels of P450 gene in Bombyx mori Using the dual-sipke-in quantitative PCR, the apparent transcription levels of P450 gene under the induction of NaF are measured.The results indicated that CYP9A19 and CYP9A22 have the higher apparent transcriptional levels in the middle gut, but CYP305B1 and CYP4M5 have the higher apparent transcriptional levels in the fat body(FB).
In this study,As shown in the NaF-induced time curve,there is tissue speciality. The induction in the Mid-gut and fat body is called the positive induction,and it reflects the resistance of Bombyx mori to NaF,and that in the malpighian tubules is called the negative induction, it reflects the poison effect of NaF to Bombyx mori. In this study,we proposed a new opinion that we should consider these two aspects in study of resistance of Bombyx mori in the future.
家蚕(Bombyx mori)是鳞翅目昆虫的模式生物,本研究以家蚕为研究对象,对家蚕P450基因CYP305B1基因组序列进行了分析;通过对目前家蚕实时定量PCR研究中常用的内源参照基因进行分析,提出了新的定量PCR方法,并应用此方法,对家蚕和野蚕(Bombyx mandarina)部分P450基因进行了定量研究。本论文的主要研究结果如下: 1.家蚕P450基因CYP305B1的克隆及其结构分析
为了研究家蚕P450基因CYP305B1的结构,采用了反向PCR(inverse PCR)技术,克隆了家蚕CYP305B1基因的基因组DNA,经序列测定,拼接得到家蚕CYP305B1全基因序列,并与野桑蚕P450基因CYP305B1V1进行了比较分析。结果表明,CYP305B1和CYP305B1V1在5’UTR上游-400至-770之间370 bp的序列同源性只有40.4%;在第1内含子中,与家蚕相比,野桑蚕在翻译起始密码ATG上游-340之前多了330 bp的插入序列,而在-110至-340 bp间两者同源性也只有46.9%。这些结果为深入研究该基因的功能提供了信息。
2.实时定量PCR方法的比较研究
为了比较目前常用的内源参照基因actin-3、GAPDH、28s rRNA,在实验样品中加入IFP2基因的mRNA和DNA混合物,作为跟踪标定基因,测定了家蚕各组织中的actin-3、GAPDH、28s rRNA基因的转录水平。同时测定了后部丝腺中的FibH、FibL基因,中部丝腺Ser-1基因的转录水平,并对两种归一化方法进行了比较。
结果表明,actin-3、GAPDH、28s rRNA在家蚕各组织中的差异极大,A3基因在后部丝腺中转录水平最高(1,098),中肠最低(9.9);GAPDH基因的转录水平以脂肪体最高(10,475),中肠最低(6.57)。28S rRNA基因的转录水平以脂肪体最高(97,321),中肠最低(2,178)。
如用与actin-3、GAPDH、28s rRNA相比的相对表达结果,则后部丝腺中的FibH ,FibL基因,中部丝腺Ser-1基因的转录水平,相互之间差距较大。而应用加入IFP2基因的mRNA和DNA混合物作为跟踪标定基因测到的是实际转录水平,基因FibL和FibH在后部丝腺中的转录水平分别为106,453和192,029,Ser-1基因在中部丝腺中的转录水平为196,539。我们把同时加入mRNA和DNA混合物,作为跟踪标定基因的测定方法,称为双跟踪标定定量PCR,所得结果称为基因的表观转录活性。
3.杀虫剂诱导的P450基因的表观转录活性
应用双跟踪标定定量PCR方法,测定了野蚕和不同品种家蚕P450基因的表观转录活性。同时,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P450基因的表观转录活性。
结果表明,家蚕品种菁松中P450基因主要在中肠中有较高的转录,其次是脂肪体;家蚕品种皓月P450基因在丝腺中有较高转录;野桑蚕的P450基因的表观转录水平,以丝腺最高,其次是中肠。
在敌敌畏和溴氰菊酯诱导下,L11中肠的P450基因的转录水平上调最显著,其次是脂肪体。L11的P450基因以CYP9A19、CYP9A22显著上调。野桑蚕在溴氰菊酯诱导下,CYP9A19、CYP9A22均有显著上调,且达到较高转录活性,而在敌敌畏诱导下野桑蚕被测P450基因未检测到较高的转录水平。
4.氟化物诱导下家蚕P450基因的表观转录水平
应用双跟踪标定定量PCR方法,测定了家蚕在NaF诱导下的P450基因的表观转录活性。结果表明,CYP9A19,CYP9A22在中肠中具有较高表观转录活性,而CYP305B1,CYP4M5则在脂肪体中有较高表观转录活性。
实验表明,NaF诱导下的时间曲线有组织特异性,我们把中肠、脂肪体的诱导称为正向诱导,这反映了家蚕对NaF的抵抗作用,把马氏管中的诱导称为反向诱导,这反映了NaF对家蚕的毒害作用。并由此提出了在今后家蚕抗性研究中应综合考虑这两方面因素的新观点。
Cytochrome P450 monooxygenases, known as multi-functional oxidase(MFO),are a very important enzymatic system, which has been found in all living organisms systems examined. Cytochrome P450 gene originated from a common ancestor 35 million years ago,is one of the oldest and the largest super-gene families. In the process of life, they catalyse and regulate the metabolism and transform of endogenous compounds or the activation and degradation of exogenous compounds. There are around 100 or so P450s in insect genome. Their functions involve growth, development, feeding, etc., which not merely involves a series of endogenous materials such as juvenile hormone(JH) and its analogue, ecdysone, biologic pheromone, but also involve the supersession to exogenous many kinds of compounds such as insecticide, plant secondary materials, environmental pollutant.
Bombyx mori is one of model organisms in Lepidoptera. This research take the Bombyx mori as the object of study, and mark the genomic sequence analysis of CPY305B1 gene in the Bombyx mori. Through the current real-time quantitative PCR study of silkworm commonly used endogenous reference gene analysis, it has proposed the new quantitative PCR method, and using this method, has conducted the quantitative research to the Bombyx mori and the Bombyx mandarina part of P450 genes. The main results are as followed.
1. Cloning and sequence analysis of cytochrome CPY305B1 gene in the Bombyx mori
In order to study the gene structure of CYP305B1, the genomic sequence of this P450 gene was cloned by reverse PCR method. The complete genomic sequence of CYP305B1 was obatined after sequencing and assembling. Sequence analysis showed the first intron is located in the 5′UTR of this gene. Comparison of the genomic sequence of CYP305B1 with that of CYP305B1V1 from Bombyx mandarina demonstrated that the 370 bp sequence between 770 bp and 400 bp upstream of the 5′UTR of these two P450 genes shared only 40.4% identity to each other. The biggest differences between the two genes lies in the first and sixth introns. In the first intron, there is an additional insertion sequence of 330 bp before the 340 bp sequence upstream of the translation initiation codon ATG of CYP305B1V1 compared with CYP305B1, and the 230 bp sequences between 340 bp and 110 bp upstream of the translation initiation codon of the two genes showed only 46.9% identity to each other. In the sixth intron, there is an extra insertion sequence of about 300 bp in CYP305B1 compared with CYP305B1V1. This research will help us to research the mechanism of transcriptional regulation of this P450 gene.
2. The comparative study of real-time quantitative PCR methods
In order to compare the current commonly used endogenous reference genes actin-3, GAPDH, 28s rRNA, We determined the gene transcription levels of actin-3, GAPDH, 28s rRNA in different tissues of Bombyx mori with IFP2 mRNA and DNA mixed in the experiment type variety as the spike-in gene.
Simultaneously it has determined the transcription level of FibH,the FibL gene in middle silk gland, Ser-1 gene in the the posterior silk gland, and comparison with the consequences determined with actin-3,GAPDH,28s rRNA.
As the results shown , actin-3, GAPDH and 28s rRNA are very different in each tissue of Bombyx mori.A3 gene in the posterior silk gland transcription level is in the highest (1,098),the lowest in the mid-gut(9.9);GAPDH gene transcription level in the fat body is the highest(10,475), the lowest in the mid-gut (6.57). 28S rRNA gene transcription level of the highest is in the fat body (97,321), the lowest in the mid-gut (2,178).
With actin-3, GAPDH, 28s rRNA measured , FibH, FibL genes in the middle silk gland,the Ser-1 gene transcription level in the posterior silk gland,there is a large gap between any two.
And adding IFP2 into a mixture of mRNA and DNA at the same time, as a tracking method of spike-in gene,we have measured the transcription level of gene FibH, FibL genes in the MSG and the Ser-1 gene in the PSG.The transcription level (106,453 and 192,029) of gene FibL and FibH in PSG, and the transcription level (196,539) of Ser-1 gene in MSG compare the level closer to the actual transcription.Adding a mixture of mRNA and DNA at the same time,as a tracking method of spike-in gene,known as the dual-sipke-in quantitative PCR. The obtained result is called the gene apparent transcription activity.
3. Pesticides-induced apparent transcription levels of P450 gene
It has applied of dual-spike-in method of quantitative PCR to measure the apparent transcription levels of P450 gene in the Bombyx mandarina and Bombyx mori different varieties. At the same time, it has determined Bombyx mori L11 and Bombyx mandarina different P450 gene apparent transcription activity under the induction of the pesticide DDVP(dimethyl dichloro vinyl phosphate) and DKDD (Decamethrin Kothrin Decis Deltamethrin)
The results show that P450 gene mainly has the higher transcription level in the middle gut, followed by the fat body; Bombyx mori Haoyue P450 genes also have a higher transcription in the silk gland.The apparent transcription level of P450 gene in the Bombyx mandarina is the highest in silk gland, followed by the mid-gut.
Induced by DDVP and DKDD,the transcription level of P450 gene in the mid-gut of Bombyx mori L11 increases significantly, followed by that of the fat body.CYP9A19 and CYP9A22 in Bombyx mori L11 are significantly raised.CYP9A19 and CYP9A22 in Bombyx mandarina are significantly raised, and reach a higher transcriptional activity under the induction of DDKD.However,the transcription of the DDKP-induced P450 gene measured in the Bombyx mandarina is not detected in a higher level.
4. Fluoride-induced apparent transcription levels of P450 gene in Bombyx mori Using the dual-sipke-in quantitative PCR, the apparent transcription levels of P450 gene under the induction of NaF are measured.The results indicated that CYP9A19 and CYP9A22 have the higher apparent transcriptional levels in the middle gut, but CYP305B1 and CYP4M5 have the higher apparent transcriptional levels in the fat body(FB).
In this study,As shown in the NaF-induced time curve,there is tissue speciality. The induction in the Mid-gut and fat body is called the positive induction,and it reflects the resistance of Bombyx mori to NaF,and that in the malpighian tubules is called the negative induction, it reflects the poison effect of NaF to Bombyx mori. In this study,we proposed a new opinion that we should consider these two aspects in study of resistance of Bombyx mori in the future.
引文
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