籽鹅卵巢组织基因差异表达及产蛋相关基因定量的研究
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摘要
鹅产蛋性能较低是制约我国鹅产业发展的重要因素之一,鹅产蛋性能的改善和提高一直以来都是鹅遗传育种工作的重点。卵巢是雌性鹅的重要生殖器官,鹅产蛋前后卵巢组织中某些基因是否表达(或者表达量的差异)均可能影响鹅的产蛋性能。分子生物学技术的发展为提高鹅产蛋性能的研究提供了新的途径。抑制性消减杂交技术对于基因组信息较少的鹅来说,无疑是一种较好的筛选差异表达基因的技术。目前,已有报道应用抑制性消减杂交技术成功地筛选获得了产蛋前期和产蛋期鹅腺垂体、肝脏组织的差异表达基因。因此,本研究以黑龙江省籽鹅卵巢组织作为研究对象,利用抑制消减杂交技术构建产蛋前期和产蛋期籽鹅卵巢组织消减cDNA文库,采用反向斑点杂交技术对该文库进行了差异表达基因的筛选和鉴定,同时利用实时定量PCR技术检测了部分差异表达基因在产蛋前期和产蛋期卵巢组织中表达量的变化,另外,还对与生殖相关的激素受体ESR1、ESR2、FSHR和PRLR进行了表达模式的研究。从而获得了产蛋前期与产蛋期籽鹅卵巢组织基因表达变化的信息,为从分子水平上探讨鹅产蛋分子调控机理的研究以及今后采用遗传手段改良和选育高产蛋性能籽鹅的研究奠定基础。
The improvement of the laying performance in the poultry is always one of the important duties for the heredity and breeding workers, especially in the geese with the poor laying performance. The ovary is the key reproductive organ, where a lot of hormone receptors are expressed, and it is the target organ for the reproductive hormones. The changes of the expressed levels in the certain of genes from ovary may effect on the laying performance in the geese. So, it is an important meaning to identify the change of differentially expressed genes for elucidating the molecular mechanism of the laying process. The further functional research of the differentially expressed genes could provide a way for the study on the geese laying performance.
Suppression subtractive hybridization (SSH) needs no genomes information, specific, few false positive and simple. So, it has been ubiquitous employed to identify the differentially expressed genes in the life science fields. Furthermore SSH should be a very good technique to identify the differentially expressed genes in the geese which genome project has been not achieved. To date, the differentially expressed genes have been obtained in the anterior pituitary and liver from the laying geese compared with the prelaying geese using SSH.
Therefore, in the present study, the subtracted cDNA library of the geese ovary was conducted by the SSH. The reverse dot-blot hybridization was employed to screen the library, and the relative expression levels of the differentially expressed genes were measured using the real-time quantitative PCR (RTQ-PCR). Furthermore, the amount of the relative expression for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), follicle-stimulating hormone receptor (FSHR) and prolactin receptor (PRLR) were also determined. The results were showed as following:
1 Construction of subtracted cDNA library
In the present study, the subtracted cDNA library of the geese ovary was constructed for the first time using SSH. The subtraction efficiency was estimated by a housekeeping gene named GAPDH. The results showed that GAPDH was subtracted efficiently at 20 folds for the subtracted cDNA library which demonstrated that differentially expressed genes were also enriched at the same folds. The cDNA fragments from the library were mainly ranged from 300 bp to 750 bp, and it suggests that the library was constructed successfully.
2 Screening the library by reverse dot-blot hybridization
568 clones were identified by PCR, and 465 positive clones were selected and further identified by reverse dot-blot hybridization. Ninety-seven clones that were identified positive fragments were subjected to sequencing. Of which the 97 clones, 26 generated adequate sequencing results. Among 26 genes, 2 genes belonged to known gene of the geese, 16 genes were unknown genes of the geese, and 8 genes were novel. All the genes, except 2 known genes, were submitted to the dbEST database at NCBI.
3 Functional analysis of 18 known genes
To gain insight into the function of these ESTs, the functional categorizations were predicted by the gene ontology tree machine in search of the gene ontology database. Eighteen ESTs were categorized into 7 molecular function groups involving in binding, catalytic activity, enzyme regulator activity, signal transducer activity, structural molecule activity, transporter activity and other function.
4 Determining the expressed levels of FTH
The relative expressed levels of FTH in the prelaying and laying Zi geese ovary were measured using RTQ-PCR. The results showed that the amount of FTH relative expression in the laying Zi geese ovary was significantly higher 13.25 folds than the prelaying Zi geese ovary. It suggests that FTH should play an important role in the molecular mediation function of the laying process in the poultry.
5 Determining the expressed levels of 8 novel genes
The relative expressed levels of 8 novel genes (Accession No. GE616653~GE616660) in the prelaying and laying Zi geese ovary were measured using RTQ-PCR. The results showed that the amount of 8 novel genes (Accession No. GE616653~GE616660) relative expression in the laying Zi geese ovary was significantly higher 7.68, 11.82, 14.23, 8.25, 15.14, 9.78, 6.48 and 14.21 folds than the prelaying Zi geese ovary, respectively. It suggests that these genes should play an important role in the geese ovary function. It is significant for elucidating the molecular mechanism of the laying process to study on the sequence and the function of these genes.
6 Expression profiling of ESR (ESR1 and ESR2) in the ovary
The dynamic regulation of ESR1 and ESR2 mRNA expression in the ovary of geese during the developmental and egg laying stage were determined using RTQ-PCR with SYBRⅠdye. Relative quantitation of gene expression was normalized to GAPDH. The results showed that ESR1 mRNA expression levels increased since the geese was aged 1 day (P<0.05), and decreased from 2 month. The amount of ESR1 mRNA was lowest in the ovary of the geese aged 3 month, and it is increased from 4 month. The levels of ESR1 mRNA in the ovary among the geese on 1, 2, 3 and 4 month were not significant difference (P>0.05). ESR1 mRNA expression were significantly greater at the age of 5 and 8 month compared with at 1 day (P<0.05), and the amount of ESR1 mRNA expression in the ovary of geese aged 8 month was the highest during the development and laying performance.
ESR2 mRNA expression levels increased since the geese was aged 1 day (P<0.05), and decreased from 4 month. The levels of ESR2 mRNA in the ovary among the geese on 4 month were not significant difference compared with the levels of 1, 2 and 3 month (P>0.05). It is increased when geese were 5 month. ESR2 mRNA expression were significantly greater at the age of 5 and 8 month compared with at 1 day (P<0.05), and the amount of the ESR2 mRNA expression in the ovary of geese aged 8 month was the highest during the development and laying performance. These suggest that ESR1 and ESR2 interact to mediate the ovarian function and the process of egg laying in the geese, and ESR2 may play a more important role in mediating the response of the ovary to estrogen during the developmental and egg laying stage.
7 Expression profiling of FSHR in the ovary The change of the FSHR mRNA expression level was similar to ESR1. FSHR mRNA expression levels increased since the geese was aged 1 day (P<0.05), and decreased from 2 month. The amount of FSHR mRNA was lowest in the ovary of the geese aged 3 month, and it is increased from 4 month. FSHR mRNA expression were significantly greater at the age of 5 and 8 month compared with at 1 day (P<0.05), and the amount of the FSHR mRNA expression was the highest during the development and laying performance and was higher 5.11 folds than the geese aged 1 day.
8 Expression profiling of PRLR in the ovary
The change of the mRNA expression level was significantly difference between PRLR and ESR. PRLR mRNA expression levels decreased since the geese was aged 1 day (P<0.05), and increased from 5 month. PRLR mRNA expression was not significantly difference between 1 and 8 month geese ovary, and it was also not significantly difference among the 2, 3, 4 and 5 month geese ovary. They were significantly lower than the PRLR mRNA expression level of the ovary in the 1 day geese.
In conclusion, in the paper, the differentially expressed genes between laying and prelaying geese ovary were identified, and the expression profiling was also determined. The information suggests that this work will be helpful for facilitating future study of the reproductive biology of the geese, acquiring new insights into the molecular mechanism involved, and improving the egg production of the geese.
The improvement of the laying performance in the poultry is always one of the important duties for the heredity and breeding workers, especially in the geese with the poor laying performance. The ovary is the key reproductive organ, where a lot of hormone receptors are expressed, and it is the target organ for the reproductive hormones. The changes of the expressed levels in the certain of genes from ovary may effect on the laying performance in the geese. So, it is an important meaning to identify the change of differentially expressed genes for elucidating the molecular mechanism of the laying process. The further functional research of the differentially expressed genes could provide a way for the study on the geese laying performance.
Suppression subtractive hybridization (SSH) needs no genomes information, specific, few false positive and simple. So, it has been ubiquitous employed to identify the differentially expressed genes in the life science fields. Furthermore SSH should be a very good technique to identify the differentially expressed genes in the geese which genome project has been not achieved. To date, the differentially expressed genes have been obtained in the anterior pituitary and liver from the laying geese compared with the prelaying geese using SSH.
Therefore, in the present study, the subtracted cDNA library of the geese ovary was conducted by the SSH. The reverse dot-blot hybridization was employed to screen the library, and the relative expression levels of the differentially expressed genes were measured using the real-time quantitative PCR (RTQ-PCR). Furthermore, the amount of the relative expression for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), follicle-stimulating hormone receptor (FSHR) and prolactin receptor (PRLR) were also determined. The results were showed as following:
1 Construction of subtracted cDNA library
In the present study, the subtracted cDNA library of the geese ovary was constructed for the first time using SSH. The subtraction efficiency was estimated by a housekeeping gene named GAPDH. The results showed that GAPDH was subtracted efficiently at 20 folds for the subtracted cDNA library which demonstrated that differentially expressed genes were also enriched at the same folds. The cDNA fragments from the library were mainly ranged from 300 bp to 750 bp, and it suggests that the library was constructed successfully.
2 Screening the library by reverse dot-blot hybridization
568 clones were identified by PCR, and 465 positive clones were selected and further identified by reverse dot-blot hybridization. Ninety-seven clones that were identified positive fragments were subjected to sequencing. Of which the 97 clones, 26 generated adequate sequencing results. Among 26 genes, 2 genes belonged to known gene of the geese, 16 genes were unknown genes of the geese, and 8 genes were novel. All the genes, except 2 known genes, were submitted to the dbEST database at NCBI.
3 Functional analysis of 18 known genes
To gain insight into the function of these ESTs, the functional categorizations were predicted by the gene ontology tree machine in search of the gene ontology database. Eighteen ESTs were categorized into 7 molecular function groups involving in binding, catalytic activity, enzyme regulator activity, signal transducer activity, structural molecule activity, transporter activity and other function.
4 Determining the expressed levels of FTH
The relative expressed levels of FTH in the prelaying and laying Zi geese ovary were measured using RTQ-PCR. The results showed that the amount of FTH relative expression in the laying Zi geese ovary was significantly higher 13.25 folds than the prelaying Zi geese ovary. It suggests that FTH should play an important role in the molecular mediation function of the laying process in the poultry.
5 Determining the expressed levels of 8 novel genes
The relative expressed levels of 8 novel genes (Accession No. GE616653~GE616660) in the prelaying and laying Zi geese ovary were measured using RTQ-PCR. The results showed that the amount of 8 novel genes (Accession No. GE616653~GE616660) relative expression in the laying Zi geese ovary was significantly higher 7.68, 11.82, 14.23, 8.25, 15.14, 9.78, 6.48 and 14.21 folds than the prelaying Zi geese ovary, respectively. It suggests that these genes should play an important role in the geese ovary function. It is significant for elucidating the molecular mechanism of the laying process to study on the sequence and the function of these genes.
6 Expression profiling of ESR (ESR1 and ESR2) in the ovary
The dynamic regulation of ESR1 and ESR2 mRNA expression in the ovary of geese during the developmental and egg laying stage were determined using RTQ-PCR with SYBRⅠdye. Relative quantitation of gene expression was normalized to GAPDH. The results showed that ESR1 mRNA expression levels increased since the geese was aged 1 day (P<0.05), and decreased from 2 month. The amount of ESR1 mRNA was lowest in the ovary of the geese aged 3 month, and it is increased from 4 month. The levels of ESR1 mRNA in the ovary among the geese on 1, 2, 3 and 4 month were not significant difference (P>0.05). ESR1 mRNA expression were significantly greater at the age of 5 and 8 month compared with at 1 day (P<0.05), and the amount of ESR1 mRNA expression in the ovary of geese aged 8 month was the highest during the development and laying performance.
ESR2 mRNA expression levels increased since the geese was aged 1 day (P<0.05), and decreased from 4 month. The levels of ESR2 mRNA in the ovary among the geese on 4 month were not significant difference compared with the levels of 1, 2 and 3 month (P>0.05). It is increased when geese were 5 month. ESR2 mRNA expression were significantly greater at the age of 5 and 8 month compared with at 1 day (P<0.05), and the amount of the ESR2 mRNA expression in the ovary of geese aged 8 month was the highest during the development and laying performance. These suggest that ESR1 and ESR2 interact to mediate the ovarian function and the process of egg laying in the geese, and ESR2 may play a more important role in mediating the response of the ovary to estrogen during the developmental and egg laying stage.
7 Expression profiling of FSHR in the ovary The change of the FSHR mRNA expression level was similar to ESR1. FSHR mRNA expression levels increased since the geese was aged 1 day (P<0.05), and decreased from 2 month. The amount of FSHR mRNA was lowest in the ovary of the geese aged 3 month, and it is increased from 4 month. FSHR mRNA expression were significantly greater at the age of 5 and 8 month compared with at 1 day (P<0.05), and the amount of the FSHR mRNA expression was the highest during the development and laying performance and was higher 5.11 folds than the geese aged 1 day.
8 Expression profiling of PRLR in the ovary
The change of the mRNA expression level was significantly difference between PRLR and ESR. PRLR mRNA expression levels decreased since the geese was aged 1 day (P<0.05), and increased from 5 month. PRLR mRNA expression was not significantly difference between 1 and 8 month geese ovary, and it was also not significantly difference among the 2, 3, 4 and 5 month geese ovary. They were significantly lower than the PRLR mRNA expression level of the ovary in the 1 day geese.
In conclusion, in the paper, the differentially expressed genes between laying and prelaying geese ovary were identified, and the expression profiling was also determined. The information suggests that this work will be helpful for facilitating future study of the reproductive biology of the geese, acquiring new insights into the molecular mechanism involved, and improving the egg production of the geese.
引文
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