鸡卵泡发育相关基因和miRNA的鉴定及功能分析
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摘要
动物育种实践证明,性早熟性状可以遗传,可能存在控制性早熟的主效基因,因此利用性早熟地方鸡品种筛选性早熟相关基因,可用于动物培育性早熟高繁殖力的品种以及为人类性早熟疾病的控制和治疗提供参考。动物性成熟是一个受多基因调控的复杂性状,在哺乳动物进行了大量研究,但禽类相对较少。鸡的卵泡在发育成熟过程中有严格的等级性,卵泡一旦经过选择后就按等级发育直至排卵,且很少发生闭锁。提高进入优势化等级的卵泡数量对于提高鸡的产蛋性能具有重要的经济意义。目前对鸡卵泡优势化选择及等级化维持的分子机制大多仍不清楚。哺乳动物研究表明,有多种生长因子以自分泌/旁分泌的方式在促性腺激素协同下参与了卵泡发育调控。miRNA是近年来发现的具有许多调控功能的一类非编码小分子RNA,其在哺乳动物卵巢中的表达特征已有报道,但在禽类卵巢的表达及功能研究尚未见报道。本研究对鸡卵泡发育相关基因及miRNA进行了鉴定和功能分析。
一、鸡FSHR基因5′调控区-237和-868两个突变位点的多态性及功能分析
促卵泡素( FSH)是调控动物繁殖活动的重要激素之一,对动物卵巢卵泡的生长、发育、分化、成熟和排卵起着必不可少的作用,其生物学功能的发挥要通过位于靶细胞膜上的促卵泡素受体(FSHR)介导,研究FSHR 5′调控区突变与产蛋性能的关系,寻找繁殖相关的DNA分子标记,对于禽类育种实践将有着重要的现实意义。
本研究用5个品种分析了突变位点的基因型分布,用2个鸡品种共计943个个体进行了突变位点基因型和单倍型的检测以及与产蛋性状关联分析,用实时荧光定量PCR技术对突变位点TTG+G+、TTG+G-和TTG-G-三种不同基因型间FSHR mRNA的表达量进行分析,构建4种单倍型载体,体外转染鸡的卵泡颗粒细胞,利用双荧光素酶报告基因检测系统对其启动基因表达的效率进行了分析。5个品种比较的结果表明:-868位点200 bp缺失型(G-)频率在地方品种达90%以上;文昌鸡群体单位点多态与39 w产蛋数和开产日龄相关不显著,两位点双倍型分析结果与开产日龄相关密切,TTG+G-对应较小的开产日龄(123 d),TTG+G+对应较大的开产日龄(134 d),两者差异显著(P=0.016)。新杨褐群体在-237位点为TT型时-868位点与37 w产蛋数相关显著(P<0.05):G-型对应较早开产日龄,但G+型对应较高的后期产蛋数;荧光定量结果也表明,高峰期G+G+型FSHR mRNA的表达量最高(P<0.05)。荧光素酶的分析结果表明,AG-型的启动效率最高,显著高于TG+、TG-单倍型(P=0.015)。推测G-型可能与早开产有关,这与G-在开产较早的地方品种中的频率较高相一致,G+型与高的产蛋数有关,其在高产群体中频率较高,两位点间的作用机理还有待于进一步研究。
二、TNRC6A(又称GW182)参与RNAi(RNA干扰)和miRNA途径,与mRNA、Argonaute等蛋白聚集在细胞质小体(P body或GW-body),参与转录后调控。本研究分析了TNRC6A基因在下丘脑、输卵管、肝脏及卵巢和卵泡发育不同时期的表达规律。实时荧光定量结果显示,TNRC6A mRNA在17 w白来航鸡卵巢比23 w开产后卵巢的表达量呈现下调,而在下丘脑、肝脏及输卵管中则上调表达(P<0.05)。在直径4 mm的白卵泡中表达量最高,且跟其它几级卵泡差异显著(P<0.05),在直径6-8 mm的黄卵泡中表达量最低,之后随卵泡直径的增大而呈增高趋势,在排卵前最大的F1卵泡中表达量又有所下降,但从黄卵泡到F1卵泡TNRC6A mRNA表达量差异不显著。
三、cDNA-AFLP技术是一种新的研究基因差异表达的技术,具有可靠性高、重复性好、假阳性率低、不需要预先知道序列信息及所需仪器设备简单等优点,而被广泛用于生物基因表达特性的研究。本研究用cDNA-AFLP方法,以具有性早熟特性的济宁百日鸡为试验材料,筛选与性早熟有关的基因。使用EcoRI和MseI双酶切组合,用54对不同的引物组合扩增出403个差异显示片段,经差异带挖取,二次扩增,胶回收纯化及克隆测序后,得到27个与GenBank数据库高度同源的差异表达序列。
选择可能与繁殖有关的13个差异显示基因进行荧光定量验证,最终获得5个在表达量上有显著差异的基因,其中有4个差异基因在开产的卵巢中表达上调,功能分析发现CCT6A与孕酮的产生有关,ERCC8基因所编码的蛋白是miRNA的靶蛋白,ZNF183是一类具有锌指结构的重要转录因子,最初在爪蟾卵母细胞发现,Poly(A) polymeraseⅡ对于卵母细胞的成熟有重要作用。在开产的卵巢明显下调的LOC418883,其功能类似于酵母的vacuolar protein sorting 36,下一步将对这些在性成熟前后卵巢中存在明显表达差异基因的功能进行深入探讨。
四、采用Solexa测序技术分析了开产前后鸡卵巢差异表达的mRNA。采集3只42 d雏鸡和3只23 w产蛋白来航鸡的卵巢组织,混池测序,结果在开产的卵巢共筛选到差异表达的mRNA 3648个,其中有2088个呈下调表达,1560个呈上调表达,对测得的差异表达基因进行分析验证,为筛选性成熟相关基因奠定基础。
五、采用Solexa测序技术分析了开产前后鸡卵巢差异表达的miRNA。采集3只42 d雏鸡和3只23 w产蛋白来航鸡的卵巢组织,混池测序,结果共筛选到72个差异表达miRNA,其中34个上调表达,38个下调表达。在未开产鸡的卵巢中筛选到189个新miRNA,在开产鸡的卵巢中筛选到97个新miRNA。对获得的差异表达miRNA进行分析验证,为进一步研究miRNA在鸡卵泡发育中的作用奠定基础。
综上所述,我们采用候选基因的方法对鸡FSHR基因启动子区两个位点的多态性及其与开产日龄及产蛋数的关系进行了分析,对其影响该基因表达的机制进行了探讨。对FSHR和TNRC6A的表达特征进行了定量分析。用cDNA-AFLP技术获得4个开产后上调的基因和1个下调的基因。用Solexa测序技术分析了开产前后卵巢中差异表达的基因和miRNA。这些研究为揭示鸡卵泡发育关键基因奠定了基础。
Animal breeding practice reveal that sexual precocity traits are inheritable, and it is likely that sexual maturity is controlled by some major genes. Therefore, by using an indigenous precocious chicken breeds, we aim to identify genes underlying early sexual maturity. To identification of these genes will be helpful for improving egg roduction as well as reproduction capability in chicken, and provide reference for the control and treatment of sexual precocity in human. Sexually maturity in animal is a complex trait, which is controlled by polygenes. Studies have been conducted in mammals, but relatively few was reported in birds.
In poultry, there are strictly hierarchical properties in the mature process of follicles. Once selected, the follicle will develop by hierarchy, until ovulation, and rarely atresia. To increase the follicular quantity entering preovulatory follicle hierarchy is important for improving egg-laying property. At present, the mechanisms of follicular hierarchy selection and hierarchy maintenance are largely unknown. Studies in mammals have shown that several growth factors are involved in the regulation of follicle development acting in autocrine/ paracrine pathway. MicroRNA (miRNA) is a large class of endogenous non-coding short RNAs that plays important roles in regulating gene expression. The expression of miRNA in mammalian ovaries have been reported, but studies concerning its function and expressions in chicken ovaries are unknown. In this study, genes and miRNAs associated with the chicken ovary follicle development were identified and their function was investigated.
PartⅠ: The polymorphism and functional analysis of mutations at -237 and -868 site of chicken FSHR promoter region.
Follicle-stimulating hormone (FSH) is one of the important hormones regulating animal breeding activities, it is essential for animal ovarian follicle growth, development, differentiation, maturation and ovulation. The biological function of FSH is mediated by the follicle-stimulating hormone receptor (FSHR). To understand the relationship between FSHR 5′promoter region mutation and laying performance, identify DNA markers associated with breeding, is prerequisite for the poultry molecular breeding.
In this study, two chicken breeds (Wenchang and Xinyang), totally 943 individuals were used to analyze the genotype frequency, hapolytypes and dipolytypes and the associations with egg production traits. By using real-time quantitative RT-PCR technology, we analyzed FSHR mRNA expression of the three different genotypes (TTG+G+, TTG+G-,TTG-G) in mutation site, construted four haplotype vector, transfected chicken follicular granulosa cell in vitro, and compared expression efficiency of the haplotype.
The results revealed that, 200 bp deletion (G-) at position -868 upstream of the translation initiation codon is a predominant allele in Chinese indigenous chicken breeds, the frequency of which was up to 90%. Single site geneotypes were not significantly associated with laying egg number at 39 w and AFE in Wenchang breed, however, their diplotypes were significantly associated with AFE: TTG+G- with the low AFE(123 d), TTG+G+ with the high AFE(134 d), the difference was significant (P=0.016). In Xinyang chicken, all individuals were genotype TT at -237 site upstream of translation initiation codon. At -868 site, genotypes were significantly associated with egg number at 37 w (P<0.05): allele G- was associated with early AFE, while G+ with high laying-egg number at later period.
Real-time quantitative RT-PCR results also showed that chicken FSHR mRNA expression levels of G+G+ was the highest (P<0.05) in the follicles of 4 mm. Luciferase reporter assays, using pGL3 vector constructs, showed that haplotype AG- has significantly higher promoter activity than other three haplotypes (P=0.015). We speculated that G- may affect sexual maturity, which is consistent with the extremely higher frequency in indigenous chicken breeds, whereas G+ affect high laying-egg number, and there were some synergy between the two sites, the mechanism of which needs to be further investigated.
PartⅡ: TNRC6A (also known as GW182), a protein involved in RNAi and miRNA pathway, is a component of cytoplasmic compartments referred to as processing bodies (P-bodies or GW bodies) in which mRNAs, Argonaute proteins were found and involved in post-transcriptional regulation. In this study, we analyzed TNRC6A expression pattern at different periods in the developing process of hypothalamus, oviduct, liver and ovarian follicle. Real-time quantitative RT-PCR results indicated that the TNRC6A mRNA expression level was higher in ovary of 17 w single-comb white leghorn hens than 23 w, but the expression in hypothalamus, liver and oviduct were up-regulated. Among these follicles, the highest mRNA level was found in white follicles of 4 mm in diameter and the lowest in yellow follicles of 6-8 mm in diameter. After this stage, it increases following follicle growth, showing a trend towards rising, however, decreased to its lowest level in the F1 follicle of 33-34 mm in diameter. The expression level of TNRC6A mRNA has no significant difference from yellow to F1 follicles.
PartⅢ: cDNA-AFLP was a nwe differential display technique with the advantage of high reproducibility, specificity and sensitivity. It was a reliable method for monitoring differential expression gene. Jining Bairi chicken comes from Shandong Province of China and is well-known for early sexual maturity. In the present study, cDNA-AFLP differential display was used to isolate genes differentially expressed in ovary of chicken with low AFE compared with high AFE. In cDNA-AFLP analysis, 403 differentially expressed fragments were identified by using 54 different combinations of EcoRI and MseI primers. After isolation, cloning and sequencing of the re-amplified bands and BLAST in GenBank we found 27 differentially expressed genes.
Thirteen clones of them were confirmed by real-time qRT-PCR. The results showed that there were five genes which were significantly different in mRNA level. In which four genes were up-regulated in laying ovary. The CCT6A have a coordinating role with progesterone and ERCC8 protein was modulated by miRNA. ZNF183 zinc finger protein is one of the important transcription factors with zinc finger domain that regulates gene expression. It was found first in Xenopus oocytes and most of the zinc finger protein function are remain unknown. The poly(A) polymeraseⅡplays an important role in oocyte maturation. The Loc418883 was a down-regulated fragments in laying ovary whose function similar to vacuolar protein sorting 36 (yeast).The function of these differentially expressed genes will be studied further.
PartⅣ: Solexa was a new high-throughput sequencing technology. The differentially expressed mRNA in the ovaries of 42 d and 23 w chickens were screened by this approach. The ovary tissue of three individuals were pooled and used for Solexa sequencing. The results showed 2088 mRNAs were down-regulated and 1560 mRNAs were up-regulated in laying ovary.
PartⅤ: The differentially expressed miRNA in ovary of 42 d and 23 w chicken were screened by using Solexa sequencing method. The ovary tissue of three individuals were pooled and used. By this approach we obtained 72 known chicken miRNA in which 34 miRNAs were up-regulated and 38 miRNAs were down-regulated. Our study is a basis for clarification the relationship between miRNA and follicle development.
In conclusion, the two polymorphisms in the promoter region of chicken FSHR gene and their relationship with age at first egg and egg production as well as their effect on gene expression were investigated. The expression profile of tissues and dynamics in a range of follicles was quantitatively characterized. Four up-regulated genes and one dow-regulated gene were identified using cDNA-AFLP approach. By Solexa sequencing, differentially expressed genes or miRNAs were revealed. These data may provide a basis for the identification of critical gene(s) underlying follicle growth in chicken.
一、鸡FSHR基因5′调控区-237和-868两个突变位点的多态性及功能分析
促卵泡素( FSH)是调控动物繁殖活动的重要激素之一,对动物卵巢卵泡的生长、发育、分化、成熟和排卵起着必不可少的作用,其生物学功能的发挥要通过位于靶细胞膜上的促卵泡素受体(FSHR)介导,研究FSHR 5′调控区突变与产蛋性能的关系,寻找繁殖相关的DNA分子标记,对于禽类育种实践将有着重要的现实意义。
本研究用5个品种分析了突变位点的基因型分布,用2个鸡品种共计943个个体进行了突变位点基因型和单倍型的检测以及与产蛋性状关联分析,用实时荧光定量PCR技术对突变位点TTG+G+、TTG+G-和TTG-G-三种不同基因型间FSHR mRNA的表达量进行分析,构建4种单倍型载体,体外转染鸡的卵泡颗粒细胞,利用双荧光素酶报告基因检测系统对其启动基因表达的效率进行了分析。5个品种比较的结果表明:-868位点200 bp缺失型(G-)频率在地方品种达90%以上;文昌鸡群体单位点多态与39 w产蛋数和开产日龄相关不显著,两位点双倍型分析结果与开产日龄相关密切,TTG+G-对应较小的开产日龄(123 d),TTG+G+对应较大的开产日龄(134 d),两者差异显著(P=0.016)。新杨褐群体在-237位点为TT型时-868位点与37 w产蛋数相关显著(P<0.05):G-型对应较早开产日龄,但G+型对应较高的后期产蛋数;荧光定量结果也表明,高峰期G+G+型FSHR mRNA的表达量最高(P<0.05)。荧光素酶的分析结果表明,AG-型的启动效率最高,显著高于TG+、TG-单倍型(P=0.015)。推测G-型可能与早开产有关,这与G-在开产较早的地方品种中的频率较高相一致,G+型与高的产蛋数有关,其在高产群体中频率较高,两位点间的作用机理还有待于进一步研究。
二、TNRC6A(又称GW182)参与RNAi(RNA干扰)和miRNA途径,与mRNA、Argonaute等蛋白聚集在细胞质小体(P body或GW-body),参与转录后调控。本研究分析了TNRC6A基因在下丘脑、输卵管、肝脏及卵巢和卵泡发育不同时期的表达规律。实时荧光定量结果显示,TNRC6A mRNA在17 w白来航鸡卵巢比23 w开产后卵巢的表达量呈现下调,而在下丘脑、肝脏及输卵管中则上调表达(P<0.05)。在直径4 mm的白卵泡中表达量最高,且跟其它几级卵泡差异显著(P<0.05),在直径6-8 mm的黄卵泡中表达量最低,之后随卵泡直径的增大而呈增高趋势,在排卵前最大的F1卵泡中表达量又有所下降,但从黄卵泡到F1卵泡TNRC6A mRNA表达量差异不显著。
三、cDNA-AFLP技术是一种新的研究基因差异表达的技术,具有可靠性高、重复性好、假阳性率低、不需要预先知道序列信息及所需仪器设备简单等优点,而被广泛用于生物基因表达特性的研究。本研究用cDNA-AFLP方法,以具有性早熟特性的济宁百日鸡为试验材料,筛选与性早熟有关的基因。使用EcoRI和MseI双酶切组合,用54对不同的引物组合扩增出403个差异显示片段,经差异带挖取,二次扩增,胶回收纯化及克隆测序后,得到27个与GenBank数据库高度同源的差异表达序列。
选择可能与繁殖有关的13个差异显示基因进行荧光定量验证,最终获得5个在表达量上有显著差异的基因,其中有4个差异基因在开产的卵巢中表达上调,功能分析发现CCT6A与孕酮的产生有关,ERCC8基因所编码的蛋白是miRNA的靶蛋白,ZNF183是一类具有锌指结构的重要转录因子,最初在爪蟾卵母细胞发现,Poly(A) polymeraseⅡ对于卵母细胞的成熟有重要作用。在开产的卵巢明显下调的LOC418883,其功能类似于酵母的vacuolar protein sorting 36,下一步将对这些在性成熟前后卵巢中存在明显表达差异基因的功能进行深入探讨。
四、采用Solexa测序技术分析了开产前后鸡卵巢差异表达的mRNA。采集3只42 d雏鸡和3只23 w产蛋白来航鸡的卵巢组织,混池测序,结果在开产的卵巢共筛选到差异表达的mRNA 3648个,其中有2088个呈下调表达,1560个呈上调表达,对测得的差异表达基因进行分析验证,为筛选性成熟相关基因奠定基础。
五、采用Solexa测序技术分析了开产前后鸡卵巢差异表达的miRNA。采集3只42 d雏鸡和3只23 w产蛋白来航鸡的卵巢组织,混池测序,结果共筛选到72个差异表达miRNA,其中34个上调表达,38个下调表达。在未开产鸡的卵巢中筛选到189个新miRNA,在开产鸡的卵巢中筛选到97个新miRNA。对获得的差异表达miRNA进行分析验证,为进一步研究miRNA在鸡卵泡发育中的作用奠定基础。
综上所述,我们采用候选基因的方法对鸡FSHR基因启动子区两个位点的多态性及其与开产日龄及产蛋数的关系进行了分析,对其影响该基因表达的机制进行了探讨。对FSHR和TNRC6A的表达特征进行了定量分析。用cDNA-AFLP技术获得4个开产后上调的基因和1个下调的基因。用Solexa测序技术分析了开产前后卵巢中差异表达的基因和miRNA。这些研究为揭示鸡卵泡发育关键基因奠定了基础。
Animal breeding practice reveal that sexual precocity traits are inheritable, and it is likely that sexual maturity is controlled by some major genes. Therefore, by using an indigenous precocious chicken breeds, we aim to identify genes underlying early sexual maturity. To identification of these genes will be helpful for improving egg roduction as well as reproduction capability in chicken, and provide reference for the control and treatment of sexual precocity in human. Sexually maturity in animal is a complex trait, which is controlled by polygenes. Studies have been conducted in mammals, but relatively few was reported in birds.
In poultry, there are strictly hierarchical properties in the mature process of follicles. Once selected, the follicle will develop by hierarchy, until ovulation, and rarely atresia. To increase the follicular quantity entering preovulatory follicle hierarchy is important for improving egg-laying property. At present, the mechanisms of follicular hierarchy selection and hierarchy maintenance are largely unknown. Studies in mammals have shown that several growth factors are involved in the regulation of follicle development acting in autocrine/ paracrine pathway. MicroRNA (miRNA) is a large class of endogenous non-coding short RNAs that plays important roles in regulating gene expression. The expression of miRNA in mammalian ovaries have been reported, but studies concerning its function and expressions in chicken ovaries are unknown. In this study, genes and miRNAs associated with the chicken ovary follicle development were identified and their function was investigated.
PartⅠ: The polymorphism and functional analysis of mutations at -237 and -868 site of chicken FSHR promoter region.
Follicle-stimulating hormone (FSH) is one of the important hormones regulating animal breeding activities, it is essential for animal ovarian follicle growth, development, differentiation, maturation and ovulation. The biological function of FSH is mediated by the follicle-stimulating hormone receptor (FSHR). To understand the relationship between FSHR 5′promoter region mutation and laying performance, identify DNA markers associated with breeding, is prerequisite for the poultry molecular breeding.
In this study, two chicken breeds (Wenchang and Xinyang), totally 943 individuals were used to analyze the genotype frequency, hapolytypes and dipolytypes and the associations with egg production traits. By using real-time quantitative RT-PCR technology, we analyzed FSHR mRNA expression of the three different genotypes (TTG+G+, TTG+G-,TTG-G) in mutation site, construted four haplotype vector, transfected chicken follicular granulosa cell in vitro, and compared expression efficiency of the haplotype.
The results revealed that, 200 bp deletion (G-) at position -868 upstream of the translation initiation codon is a predominant allele in Chinese indigenous chicken breeds, the frequency of which was up to 90%. Single site geneotypes were not significantly associated with laying egg number at 39 w and AFE in Wenchang breed, however, their diplotypes were significantly associated with AFE: TTG+G- with the low AFE(123 d), TTG+G+ with the high AFE(134 d), the difference was significant (P=0.016). In Xinyang chicken, all individuals were genotype TT at -237 site upstream of translation initiation codon. At -868 site, genotypes were significantly associated with egg number at 37 w (P<0.05): allele G- was associated with early AFE, while G+ with high laying-egg number at later period.
Real-time quantitative RT-PCR results also showed that chicken FSHR mRNA expression levels of G+G+ was the highest (P<0.05) in the follicles of 4 mm. Luciferase reporter assays, using pGL3 vector constructs, showed that haplotype AG- has significantly higher promoter activity than other three haplotypes (P=0.015). We speculated that G- may affect sexual maturity, which is consistent with the extremely higher frequency in indigenous chicken breeds, whereas G+ affect high laying-egg number, and there were some synergy between the two sites, the mechanism of which needs to be further investigated.
PartⅡ: TNRC6A (also known as GW182), a protein involved in RNAi and miRNA pathway, is a component of cytoplasmic compartments referred to as processing bodies (P-bodies or GW bodies) in which mRNAs, Argonaute proteins were found and involved in post-transcriptional regulation. In this study, we analyzed TNRC6A expression pattern at different periods in the developing process of hypothalamus, oviduct, liver and ovarian follicle. Real-time quantitative RT-PCR results indicated that the TNRC6A mRNA expression level was higher in ovary of 17 w single-comb white leghorn hens than 23 w, but the expression in hypothalamus, liver and oviduct were up-regulated. Among these follicles, the highest mRNA level was found in white follicles of 4 mm in diameter and the lowest in yellow follicles of 6-8 mm in diameter. After this stage, it increases following follicle growth, showing a trend towards rising, however, decreased to its lowest level in the F1 follicle of 33-34 mm in diameter. The expression level of TNRC6A mRNA has no significant difference from yellow to F1 follicles.
PartⅢ: cDNA-AFLP was a nwe differential display technique with the advantage of high reproducibility, specificity and sensitivity. It was a reliable method for monitoring differential expression gene. Jining Bairi chicken comes from Shandong Province of China and is well-known for early sexual maturity. In the present study, cDNA-AFLP differential display was used to isolate genes differentially expressed in ovary of chicken with low AFE compared with high AFE. In cDNA-AFLP analysis, 403 differentially expressed fragments were identified by using 54 different combinations of EcoRI and MseI primers. After isolation, cloning and sequencing of the re-amplified bands and BLAST in GenBank we found 27 differentially expressed genes.
Thirteen clones of them were confirmed by real-time qRT-PCR. The results showed that there were five genes which were significantly different in mRNA level. In which four genes were up-regulated in laying ovary. The CCT6A have a coordinating role with progesterone and ERCC8 protein was modulated by miRNA. ZNF183 zinc finger protein is one of the important transcription factors with zinc finger domain that regulates gene expression. It was found first in Xenopus oocytes and most of the zinc finger protein function are remain unknown. The poly(A) polymeraseⅡplays an important role in oocyte maturation. The Loc418883 was a down-regulated fragments in laying ovary whose function similar to vacuolar protein sorting 36 (yeast).The function of these differentially expressed genes will be studied further.
PartⅣ: Solexa was a new high-throughput sequencing technology. The differentially expressed mRNA in the ovaries of 42 d and 23 w chickens were screened by this approach. The ovary tissue of three individuals were pooled and used for Solexa sequencing. The results showed 2088 mRNAs were down-regulated and 1560 mRNAs were up-regulated in laying ovary.
PartⅤ: The differentially expressed miRNA in ovary of 42 d and 23 w chicken were screened by using Solexa sequencing method. The ovary tissue of three individuals were pooled and used. By this approach we obtained 72 known chicken miRNA in which 34 miRNAs were up-regulated and 38 miRNAs were down-regulated. Our study is a basis for clarification the relationship between miRNA and follicle development.
In conclusion, the two polymorphisms in the promoter region of chicken FSHR gene and their relationship with age at first egg and egg production as well as their effect on gene expression were investigated. The expression profile of tissues and dynamics in a range of follicles was quantitatively characterized. Four up-regulated genes and one dow-regulated gene were identified using cDNA-AFLP approach. By Solexa sequencing, differentially expressed genes or miRNAs were revealed. These data may provide a basis for the identification of critical gene(s) underlying follicle growth in chicken.
引文
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