抑制素对山羊繁殖力的影响
摘要
抑制素(Inhibin,INH)是雄性动物睾丸支持细胞(Sertoli cells)和雌性动物卵巢颗粒细胞(Granulosa cells)分泌的二聚体糖蛋白激素。活化素(Activin, ACT)又名激活素,是最早在性腺中发现的一种糖蛋白激素。在结构上,INH基因由α亚基和β亚基(pA或pB)通过二硫键连接形成INHA (α-βA)和INHB (α-βB)两种形式,ACT由pA和pB亚基相互通过二硫键连接后形成ACTA (βA-βA)、ACTB (βB-βB)和ACTAB (βA-βB)三种形式。在雌性动物中,INH和ACT分别起到抑制和促进卵泡刺激素(Follicle-stimulating hormone, FSH)的合成和分泌的作用。
本实验旨在研究抑制素在激素水平和基因水平上对不同繁殖力山羊品种(遗传资源)的影响情况,试图揭示抑制素对山羊繁殖力影响的遗传机制。1.山羊血浆中INHB、ACTA和FSH含量比较分析
采用酶联免疫法(ELISA)测定并分析了大足黑山羊(n=5)、南江黄羊(n=5)和萨能奶山羊(n=4)3个山羊品种(遗传资源)在一个发情周期内FSH、INHB和ACTA三种激素的分泌变化规律,得到以下结果：
(1)大足黑山羊发情周期为(20.1±0.5)d,南江黄羊的发情周期为(20.2±1.1)d,萨能奶山羊发情周期为(25.1±0.8)d。
(2)在整个发情周期中,3个山羊品种(遗传资源)FSH分泌曲线中高峰点出现的时间和INHB分泌曲线中较低点出现的时间基本一致,FSH与INHB在3个羊种中都呈现极显著负相关(P<0.01)。3个山羊品种(遗传资源)的ACTA分泌变化趋势与FSH分泌变化趋势也比较一致,ACTA与FSH相关系数为正值,但均未达到差异显著水平(P>0.05)
(3)大足黑山羊FSH在发情周期中出现的第1个分泌高峰点的水平显著高于南江黄羊和萨能奶山羊(P<0.05)；而大足黑山羊INHB在发情周期中出现的第1个分泌较低点的水平显著低于萨能奶山羊和南江黄羊(P<0.05)
2.山羊INHα、INHβA和INHβB亚基基因的克隆和序列分析
以大足黑山羊(n=3)、南江黄羊(n=3)和萨能奶山羊(n=3)为研究对象,采用RT-PCR技术克隆出INHα、INHβA和INHβB亚基基因cDNA序列,并已获得两个NCBI登录号(INHα:HQ699620和INHβA:HQ699621),与GenBank中其它物种的相同亚基相同区段的同源性均在95%以上。
(1)山羊INHα、INHβA和INHβB亚基基因cDNA序列分别为1124 bp、1364bp和1499 bp; CDS区分别为1083 bp、1278 bp和1227 bp;CDS区分别编码360、425和408个氨基酸,分别包含18、21和20个氨基酸信号肽,341、404和380个氨基酸成熟肽。CDS区编码的氨基酸与GenBank中其它物种相同亚基的相同区段编码的氨基酸同源性达96%以上。
(2) INHα、INHβA和INHβB多肽相对分子质量分别为94.655 KD、109.132KD和100.438 KD,等电点(pI)分别为4.95、4.92和4.94,疏水性最小值分别为-0.467、-0.633和-0.567,最大值分别为2.344、2.422和2.267。均无蛋白跨膜区；蛋白的二级结构中,α螺旋分别占17.22%、21.65%和23.04%,β转角分别占5.28%、3.53%和4.41%,延伸链分别占18.89%、19.53%和18.14%,无规卷曲分别占58.61%、55.29%和54.41%。INHβA和INHβB多肽的三级结构均与人的INHβA多肽有116个氨基酸残基相匹配。
(3)所测山羊INHα亚基基因cDNA序列与GenBank中绵羊(Ovis aries)外显子2比较,共发现19个变异位点,其中转换17个,颠换2个,氨基酸变异位点为8个；与NCBI上公布的波尔山羊(Capra hircus)外显子1和2部分序列相比,共发现3个变异位点,其中转换2个,颠换1个,氨基酸变异位点为2个,分别为第338位(外显子2第32位)C→G的突变导致氨基酸L→V,第345位(外显子2第39位)A→G的突变导致氮基酸Q→R。
(4)所测山羊INHβA亚基基因cDNA序列与GenBank中绵羊相同区段相比,发现有10个碱基变异位点,均为转换,无颠换、插入或后缺失,其中CDS区第198位(cDNA)序列的第211位)A→G的突变导致氨基酸R→K。
(5)所测山羊INHβB亚基基因cDNA序列与GenBank中绵羊序列相比,出现了12个变异位点,其中有5个转换,6个颠换,1个插入。引起氨基酸变异的位点有3个,860位G→T的变异导致氨基酸R→L,第871位插入碱基G使得氨基酸序列增加了一个G,第1372位G→T的突变使得氨基酸G→V。在大足黑山羊中却没有发现这3个位点的突变。
3.山羊INHα、INHβA和INHβB亚基基因的外显子遗传多态性分析
采用序列测定和PCR-SSCP技术,在大足黑山羊(n=28)和南江黄羊(n=27)中分析INHα、INHβA和INHβB亚基基因的外显子遗传多态性,结果发现：
(1) INHα亚基基因外显子1内没有多态性位点,外显子2内都存在第32位(cDNA序列第338位)C→G和第39位(cDNA序列第345位)A→G的突变；INHβA亚基基因外显子部分序列中没有发现多态性位点；INHβB亚基基因两个外显子中没有发现多态性位点。
(2)在更大群体中测序发现山羊INHa亚基基因的外显子2的第57位(cDNA序列第363位)存在G→A的突变,并引起了氨基酸R→H。
(3) INHa亚基基因的外显子2 PCR-SSCP分析发现,在大足黑山羊和南江黄羊中出现了三种基因型(AA、AB和BB型),大足黑山羊AA、AB和BB基因型频率分别为0.071、0.429和0.500,而南江黄羊分别为0.391、0.435和0.174；大足黑山羊B等位基因频率(0.714)高于A等位基因频率(0.286),南江黄羊A等位基因频率(0.630)高于B等位基因频率(0.370)；经χ2检验发现,大足黑山羊和南江黄羊都处于Hardy-Weinberg平衡状态(P>0.05)。
(4) INHa亚基基因的外显子2的多态性与产羔数的最小二乘分析发现,就前三胎产羔数、前二胎产羔数、前一胎产羔数和平均产羔数,大足黑山羊的BB型个体分别比AB型个体多0.595、1.01、0.703和0.769只(P<0.05)；南江黄羊BB型个体比AB型个体产羔数分别多了0.787、1.375和0.754只(P<0.05),比AA型个体的产羔数分别多了0.849、1.486和1.131只(P<0.05),但AA与AB型个体之间的差异未达到显著水平(P>0.05)；南江黄羊BB和AB型个体的前一胎产羔数分别比AA型个体多1.01和0.983只(P<0.05),但AB型与BB型个体之间的差异未达到显著水平(P>0.05)
4.山羊INHa, INHβA和INHβB亚基基因定量表达分析
以大足黑山羊(n=7)、南江黄羊(n=7)和萨能奶山羊(n=7)为研究对象,采用实时荧光定量PCR技术测定了山羊INHa, INHβA和INHβB亚基基因定量表达情况,结果发现：
(1) INHa、INHβA和INHβB亚基基因在山羊的垂体、卵巢、心脏、肝、脾脏、肺和肾脏7种组织均有表达,卵巢组织表达水平高于垂体(P<0.05)；在卵巢和垂体组织中,3个山羊品种(遗传资源)中均表现出INHa表达水平最高、INHβA居中、INHPB表达水平最低。
(2)在3个山羊品种(遗传资源)中,大足黑山羊的INHa、INHPA和INHβB亚基基因表达水平的各个组织中最低,萨能山羊居中,南江黄羊最高(P<0.05)。
(3)相关性分析发现,卵巢组织INHa、INHβA和INHβB亚基基因表达水平与山羊采样时前一胎的产羔数呈负相关(P<0.10)。
5.结论
综上所述,得到如下结论：
(1)在一个发情周期中,3个山羊品种(遗传资源)FSH水平与1NHB水平呈负相关,FSH水平与ACTA水平呈正相关,激素变化趋势与山羊卵泡发育波的出现基本吻合。
(2)在一个发情周期中,伴随着最后一个卵泡发育波出现的较大量FSH分泌和较低水平的INHB分泌以及抑制素基因较低的表达水平可能与山羊的高繁殖力相关。
(3)在山羊INHa亚基基因外显子1、INHβA亚基基因外显子部分序列和INHβB亚基基因两个外显子中没有发现多态性位点,这些亚基基因外显子序列对山羊的产羔数影响可能较小。
(4)山羊INHa外显子2的第57位存在G→A的突变,并引起了氨基酸R→H,在不同个体中出现了三种基因型(AA、AB和BB型)。INHa外显子2的B等位基因可能是山羊高繁殖力的一个优势基因,INHa外显子2有可能作为山羊多胎性标记辅助选择的候选基因片段。
(5) INHa、INHβA和INHβB三个亚基基因在山羊卵巢组织表达水平高于垂体。在山羊卵巢和垂体组织中,INHa亚基基因表达水平最高、INHβA居中、INHβB表达水平最低。
Inhibin is one kind of dimeric glycoprotein hormone that is secreted by sertoli-like cells of testis and granulosa cells of ovaries. Activin is also a kind of glycoprotein hormone that was first found in the gonad. Inhibin and activin have intimate closely function in female. Inhibin has the capacity to suppress secretion of FSH, while activin has stimulative function on the FSH secretion. Inhibin gene is composed of one a subunit and one of twoβsubunits (PA orβB), and generates dimeric inhibin A(a-βA) and inhibin B(a-βB) by disulfide bond, while activin is composed ofβA andβB and forms activin A(PA-βA), activin B(PB-βB) and activin AB(βA-βB) by disulfide bond.
In order to try to reveal the genetic mechanism of inhibin regulating goat's fecundity, the effects of inhibin on goat fecundity in gene level and hormone level were taken up in goat breeds of different fecundity ability in this study.
1. Plasma concentrations of INHB, ACTA and FSH
INHB, ACTA and FSH in an estrous cycle of goat (including 5 Dazu black goats,5 Nanjiang brown goats and 4 Saanen dairy goats) were measured and analyzed by ELISA. The resluts were as followings:
(1) An estrous cycle length was (20.1±0.5) days in Dazu black goat,(20.2=1.1) days in Nanjiang brown goat and (25.1±0.8) days in Saanen dairy goat.
(2) In a whole estrous cycle, the time of peak points of FSH and lower points of INHB were coincident, the relationship of FSH and INHB were significantly negative in three goat breeds(P< 0.01). In a whole estrous cycle,The secretion of ACTA and FSH were also coincident, the correlation coefficient of FSH and ACTA was positive (P>0.05).
(3) In the first peak point of FSH secretion in an estrous cycle, FSH level of Dazu black goat was significantly high than that of Nanjiang brown goat and Saanen dairy goat(P<0.05). While in the first lower point of INHB secretion, INHB level of Dazu black goat was significantly lower than that of Nanjiang brown goat and Saanen dairy goat(P<0.05).
2. Cloning and sequences analysis of inhibin alpha, beta A and beta B subunit genes in goat
Cloned cDNA sequences of inhibin alpha, beta A and beta B subunit genes in goats (including 3 Dazu black goats,3 Nanjiang brown goats and 3 Saanen dairy goats) by RT-PCR were submitted and received GenBank IDs (HQ699620 for INHa and HQ699621 for INHβA).The homologies of the sequences were higher compared with the sequences of other species in GenBank.
(1) The length of cDNA sequences of inhibin alpha, beta A and beta B subunit genes were 1124 bp,1364 bp and 1499 bp, respectively. Their CDS regions were at 1083 bp,1278 bp and 1227 bp, encoded a polypeptide of 360,425 and 408 amino acids, included 18,21 and 20 amino acids signal peptides and 341,404 and 380 amino acids mature peptides, respectively. Compared with other species in GenBank, the homology in codogenic amino acids of CDS regions was more than 96%.
(2) The relative molecular weight of the polypeptide of inhibin alpha, beta A and beta B subunit genes in goat were 94.655 KD,109.132 KD and 100.438 KD, respectively. The isoelectric points were 4.95,4.92 and 4.94, respectively; the minimum values of hydrophobicity were -0.467,-0.633 and -0.567, the maximum were 2.344,2.422 and 2.267, respectively. They had not protein transmembrane domains and had alpha helix (17.22%,21.65% and 23.04%), beta turn (5.28%, 3.53% and 4.41%), extended strand (18.89%,19.53% and 18.14%) and random coil (58.61%, 55.29% and 54.41%) in its secondary structure.116 amino acid residues of inhibin beta A and beta B subunit genes matched human inhibin beta A subunit gene in tertiary structure.
(3) Compared with the sequence of exon 2 for inhibin alpha subunit gene in sheep, the cDNA sequence in goat had 19 mutation sites (including 17 transitionsal sites and 2 transversional sites) and 8 amino acids mutation sites. Compared with the partial sequence of exon 1 and 2 in Boer goat in GenBank, the cDNA sequence in goat had 3 mutation sites (including two transitionsal sites and one transversional site) and two amino acids mutation sites.One of the amino acids mutation sites was 338 C→G (i.e.32 C→G of exon 2) that resulted in amino acid change (L→V). the other was 345 A→G(i.e.39 A→G of exon 2) that resulted in one amino acid change (Q→R).
(4) Compared with inhibin beta A subunit gene in sheep, the cDNA sequenceof goat had 10 mutation sites that were both transitionsal sites. Locus 198A→G of CDS region (i.e.211 A→G of cDNA sequence) resulted in amino acid change (P→K).
(5) Compared with inhibin beta B subunit gene in sheep, the cDNA sequence in goat had 12 mutation sites (with 5 transitionsal sites,6 transversional sites and 1 insertion site) and 3 amino acid mutation sites. Loci 860 (A→G) and 1372 (G→T) respectively resulted in amino acid change (P→K) and (G→V); inserted G in locus 871 resulted in amino acid increased.
3. Genetic polymorphism analysis for exons of inhibin alpha, beta A and beta B subunit genes in goat
After sequencing and PCR-SSCP in larger population including 28 Dazu black goats and 27 Nanjiang brown goats, the results were as followings:
(1) There were not polymorphic sites in exon 1 of inhibin alpha subunit gene in goat.32C→G and 39A→G in exon 2 of inhibin alpha subunit gene were found in all experimental goats. There were no polymorphic sites in partial sequence of exon of inhibin beta A subunit gene and two exons of inhibin beta B subunit gene in all goats.
(2) There was a mutation site 57G→A (i.e.363G→A of cDNA sequence) in exon 2 of inhibin alpha subunit gene was found by sequencing in larger population.The mutation site resulted in one amino acid change (R→H).
(3) Three genotypes (AA, AB and BB) were detected in two goat breeds after SSCP analysis. Genotype frequencies of AA, AB and BB were 0.071,0.429 and 0.500 in Dazu black goat, respectively, while three genotype frequencies were 0.391,0.435 and 0.174 in Nanjiang brown goat, respectively; Gene frequency of B allele (0.714) was higher than that of A (0.286) in Dazu black goat, while gene frequency of A allele (0.630) was higher than that of B (0.370) in Nanjiang brown goat. Theχ2 test showed that Dazu black goat and Nanjiang brown goat were in Hardy-Weinberg equilibrium(P>0.05).
(4) For the first three litter size, the first two litter size, the first one litter size and average litter size in Dazu black goat, the least square means (LSM) of BB genotype was 0.595,1.01,0.703 and 0.769 more than those of LSM of AB(P<0.05),respectively. For the first three litter size, the first two litter size and average litter size in Nanjiang brown goat,LSM of BB genotype was 0.787,1.375 and 0.754 than LSM of AB (P<0.05).respectively and 0.849,1.486 and 1.131 than LSM of AA (P< 0.05).respectively. but there was not significantly difference between AA and AB (P>0.05). For the first one litter size, LSM of BB and AB were 1.01 and 0.983 more than AA (P<0.05),respectively, but there was not significantly difference between the frequencies of BB and AB(P>0.05).
4. Quantitative expression analysis of inhibin alpha, beta A and beta B subunit genes in goat
Quantitative expression of inhibin alpha, beta A and beta B subunit genes in goats (including 7 Dazu black goats,7 Nanjiang brown goats and 7 Saanen dairy goats) were analyzed by real-time fluorescent quantitation PCR. The results were as followings:
(1) Expression of inhibin alpha, beta A and beta B subunit genes in pituitary gland, ovary, heart, liver, spleen, lung and kidney can be detected. The expression level of ovary was higher than that of pituitary gland(P<0.05). In ovaries and pituitary gland of three goat breeds, expression level of inhibin alpha subunit gene is the highest, inhibin beta A subunit gene is middle and inhibin beta A subunit gene is the lowest.
(2) Expression level of inhibin subunit genes in every tissue of Dazu black goat was the highest in three goat breeds. Saanen dairy goat was in middle level and Nanjiang brown goat was in the lowest level(P<0.05).
(3) The relationships between expression level of inhibin alpha, beta A and beta B subunit genes in ovary and the first one litter size of goat were in negative correlation (P<0.10).
5. Conclusions
(1) There was a negative correlation between FSH and INHB level and a positive correlation between FSH and ACTA level in an estrous cycle of the three goat breeds. The hormone variation tendency was basically coincided with the follicular development wave in goat.
(2) Higher fecundity of goat may be correlation with higher FSH level, lower INHB level and lower expression of inhibin subunit genes in the last follicular development wave.
(3) Exon 1 of inhibin alpha subunit gene, partial sequence of exon in inhibin beta A subunit and two exons of inhibin beta B subunit gene were not polymorphic sites in all goats and these sequences may faintly affect goat's fecundity.
(4) The mutation site (57G→A) in exon 2 of inhibin alpha subunit gene in goat resulted in amino acid change (R→H), three genotypes (AA, AB and BB) were detected in the different individual of two goat breeds. B allele of exon 2 in inhibin alpha subunit gene may be a predominant gene that controls high fecundity of goat,and inhibin alpha subunit gene may be a candidate gene.
(5) Expression levels of inhibin alpha, beta A and beta B subunit genes in ovaries were higher than that in pituitary gland. In goat's ovaries and pituitary gland,expression level of inhibin alpha subunit gene is the highest, inhibin beta A subunit gene is middle and inhibin beta A subunit gene is the lowest.
本实验旨在研究抑制素在激素水平和基因水平上对不同繁殖力山羊品种(遗传资源)的影响情况,试图揭示抑制素对山羊繁殖力影响的遗传机制。1.山羊血浆中INHB、ACTA和FSH含量比较分析
采用酶联免疫法(ELISA)测定并分析了大足黑山羊(n=5)、南江黄羊(n=5)和萨能奶山羊(n=4)3个山羊品种(遗传资源)在一个发情周期内FSH、INHB和ACTA三种激素的分泌变化规律,得到以下结果：
(1)大足黑山羊发情周期为(20.1±0.5)d,南江黄羊的发情周期为(20.2±1.1)d,萨能奶山羊发情周期为(25.1±0.8)d。
(2)在整个发情周期中,3个山羊品种(遗传资源)FSH分泌曲线中高峰点出现的时间和INHB分泌曲线中较低点出现的时间基本一致,FSH与INHB在3个羊种中都呈现极显著负相关(P<0.01)。3个山羊品种(遗传资源)的ACTA分泌变化趋势与FSH分泌变化趋势也比较一致,ACTA与FSH相关系数为正值,但均未达到差异显著水平(P>0.05)
(3)大足黑山羊FSH在发情周期中出现的第1个分泌高峰点的水平显著高于南江黄羊和萨能奶山羊(P<0.05)；而大足黑山羊INHB在发情周期中出现的第1个分泌较低点的水平显著低于萨能奶山羊和南江黄羊(P<0.05)
2.山羊INHα、INHβA和INHβB亚基基因的克隆和序列分析
以大足黑山羊(n=3)、南江黄羊(n=3)和萨能奶山羊(n=3)为研究对象,采用RT-PCR技术克隆出INHα、INHβA和INHβB亚基基因cDNA序列,并已获得两个NCBI登录号(INHα:HQ699620和INHβA:HQ699621),与GenBank中其它物种的相同亚基相同区段的同源性均在95%以上。
(1)山羊INHα、INHβA和INHβB亚基基因cDNA序列分别为1124 bp、1364bp和1499 bp; CDS区分别为1083 bp、1278 bp和1227 bp;CDS区分别编码360、425和408个氨基酸,分别包含18、21和20个氨基酸信号肽,341、404和380个氨基酸成熟肽。CDS区编码的氨基酸与GenBank中其它物种相同亚基的相同区段编码的氨基酸同源性达96%以上。
(2) INHα、INHβA和INHβB多肽相对分子质量分别为94.655 KD、109.132KD和100.438 KD,等电点(pI)分别为4.95、4.92和4.94,疏水性最小值分别为-0.467、-0.633和-0.567,最大值分别为2.344、2.422和2.267。均无蛋白跨膜区；蛋白的二级结构中,α螺旋分别占17.22%、21.65%和23.04%,β转角分别占5.28%、3.53%和4.41%,延伸链分别占18.89%、19.53%和18.14%,无规卷曲分别占58.61%、55.29%和54.41%。INHβA和INHβB多肽的三级结构均与人的INHβA多肽有116个氨基酸残基相匹配。
(3)所测山羊INHα亚基基因cDNA序列与GenBank中绵羊(Ovis aries)外显子2比较,共发现19个变异位点,其中转换17个,颠换2个,氨基酸变异位点为8个；与NCBI上公布的波尔山羊(Capra hircus)外显子1和2部分序列相比,共发现3个变异位点,其中转换2个,颠换1个,氨基酸变异位点为2个,分别为第338位(外显子2第32位)C→G的突变导致氨基酸L→V,第345位(外显子2第39位)A→G的突变导致氮基酸Q→R。
(4)所测山羊INHβA亚基基因cDNA序列与GenBank中绵羊相同区段相比,发现有10个碱基变异位点,均为转换,无颠换、插入或后缺失,其中CDS区第198位(cDNA)序列的第211位)A→G的突变导致氨基酸R→K。
(5)所测山羊INHβB亚基基因cDNA序列与GenBank中绵羊序列相比,出现了12个变异位点,其中有5个转换,6个颠换,1个插入。引起氨基酸变异的位点有3个,860位G→T的变异导致氨基酸R→L,第871位插入碱基G使得氨基酸序列增加了一个G,第1372位G→T的突变使得氨基酸G→V。在大足黑山羊中却没有发现这3个位点的突变。
3.山羊INHα、INHβA和INHβB亚基基因的外显子遗传多态性分析
采用序列测定和PCR-SSCP技术,在大足黑山羊(n=28)和南江黄羊(n=27)中分析INHα、INHβA和INHβB亚基基因的外显子遗传多态性,结果发现：
(1) INHα亚基基因外显子1内没有多态性位点,外显子2内都存在第32位(cDNA序列第338位)C→G和第39位(cDNA序列第345位)A→G的突变；INHβA亚基基因外显子部分序列中没有发现多态性位点；INHβB亚基基因两个外显子中没有发现多态性位点。
(2)在更大群体中测序发现山羊INHa亚基基因的外显子2的第57位(cDNA序列第363位)存在G→A的突变,并引起了氨基酸R→H。
(3) INHa亚基基因的外显子2 PCR-SSCP分析发现,在大足黑山羊和南江黄羊中出现了三种基因型(AA、AB和BB型),大足黑山羊AA、AB和BB基因型频率分别为0.071、0.429和0.500,而南江黄羊分别为0.391、0.435和0.174；大足黑山羊B等位基因频率(0.714)高于A等位基因频率(0.286),南江黄羊A等位基因频率(0.630)高于B等位基因频率(0.370)；经χ2检验发现,大足黑山羊和南江黄羊都处于Hardy-Weinberg平衡状态(P>0.05)。
(4) INHa亚基基因的外显子2的多态性与产羔数的最小二乘分析发现,就前三胎产羔数、前二胎产羔数、前一胎产羔数和平均产羔数,大足黑山羊的BB型个体分别比AB型个体多0.595、1.01、0.703和0.769只(P<0.05)；南江黄羊BB型个体比AB型个体产羔数分别多了0.787、1.375和0.754只(P<0.05),比AA型个体的产羔数分别多了0.849、1.486和1.131只(P<0.05),但AA与AB型个体之间的差异未达到显著水平(P>0.05)；南江黄羊BB和AB型个体的前一胎产羔数分别比AA型个体多1.01和0.983只(P<0.05),但AB型与BB型个体之间的差异未达到显著水平(P>0.05)
4.山羊INHa, INHβA和INHβB亚基基因定量表达分析
以大足黑山羊(n=7)、南江黄羊(n=7)和萨能奶山羊(n=7)为研究对象,采用实时荧光定量PCR技术测定了山羊INHa, INHβA和INHβB亚基基因定量表达情况,结果发现：
(1) INHa、INHβA和INHβB亚基基因在山羊的垂体、卵巢、心脏、肝、脾脏、肺和肾脏7种组织均有表达,卵巢组织表达水平高于垂体(P<0.05)；在卵巢和垂体组织中,3个山羊品种(遗传资源)中均表现出INHa表达水平最高、INHβA居中、INHPB表达水平最低。
(2)在3个山羊品种(遗传资源)中,大足黑山羊的INHa、INHPA和INHβB亚基基因表达水平的各个组织中最低,萨能山羊居中,南江黄羊最高(P<0.05)。
(3)相关性分析发现,卵巢组织INHa、INHβA和INHβB亚基基因表达水平与山羊采样时前一胎的产羔数呈负相关(P<0.10)。
5.结论
综上所述,得到如下结论：
(1)在一个发情周期中,3个山羊品种(遗传资源)FSH水平与1NHB水平呈负相关,FSH水平与ACTA水平呈正相关,激素变化趋势与山羊卵泡发育波的出现基本吻合。
(2)在一个发情周期中,伴随着最后一个卵泡发育波出现的较大量FSH分泌和较低水平的INHB分泌以及抑制素基因较低的表达水平可能与山羊的高繁殖力相关。
(3)在山羊INHa亚基基因外显子1、INHβA亚基基因外显子部分序列和INHβB亚基基因两个外显子中没有发现多态性位点,这些亚基基因外显子序列对山羊的产羔数影响可能较小。
(4)山羊INHa外显子2的第57位存在G→A的突变,并引起了氨基酸R→H,在不同个体中出现了三种基因型(AA、AB和BB型)。INHa外显子2的B等位基因可能是山羊高繁殖力的一个优势基因,INHa外显子2有可能作为山羊多胎性标记辅助选择的候选基因片段。
(5) INHa、INHβA和INHβB三个亚基基因在山羊卵巢组织表达水平高于垂体。在山羊卵巢和垂体组织中,INHa亚基基因表达水平最高、INHβA居中、INHβB表达水平最低。
Inhibin is one kind of dimeric glycoprotein hormone that is secreted by sertoli-like cells of testis and granulosa cells of ovaries. Activin is also a kind of glycoprotein hormone that was first found in the gonad. Inhibin and activin have intimate closely function in female. Inhibin has the capacity to suppress secretion of FSH, while activin has stimulative function on the FSH secretion. Inhibin gene is composed of one a subunit and one of twoβsubunits (PA orβB), and generates dimeric inhibin A(a-βA) and inhibin B(a-βB) by disulfide bond, while activin is composed ofβA andβB and forms activin A(PA-βA), activin B(PB-βB) and activin AB(βA-βB) by disulfide bond.
In order to try to reveal the genetic mechanism of inhibin regulating goat's fecundity, the effects of inhibin on goat fecundity in gene level and hormone level were taken up in goat breeds of different fecundity ability in this study.
1. Plasma concentrations of INHB, ACTA and FSH
INHB, ACTA and FSH in an estrous cycle of goat (including 5 Dazu black goats,5 Nanjiang brown goats and 4 Saanen dairy goats) were measured and analyzed by ELISA. The resluts were as followings:
(1) An estrous cycle length was (20.1±0.5) days in Dazu black goat,(20.2=1.1) days in Nanjiang brown goat and (25.1±0.8) days in Saanen dairy goat.
(2) In a whole estrous cycle, the time of peak points of FSH and lower points of INHB were coincident, the relationship of FSH and INHB were significantly negative in three goat breeds(P< 0.01). In a whole estrous cycle,The secretion of ACTA and FSH were also coincident, the correlation coefficient of FSH and ACTA was positive (P>0.05).
(3) In the first peak point of FSH secretion in an estrous cycle, FSH level of Dazu black goat was significantly high than that of Nanjiang brown goat and Saanen dairy goat(P<0.05). While in the first lower point of INHB secretion, INHB level of Dazu black goat was significantly lower than that of Nanjiang brown goat and Saanen dairy goat(P<0.05).
2. Cloning and sequences analysis of inhibin alpha, beta A and beta B subunit genes in goat
Cloned cDNA sequences of inhibin alpha, beta A and beta B subunit genes in goats (including 3 Dazu black goats,3 Nanjiang brown goats and 3 Saanen dairy goats) by RT-PCR were submitted and received GenBank IDs (HQ699620 for INHa and HQ699621 for INHβA).The homologies of the sequences were higher compared with the sequences of other species in GenBank.
(1) The length of cDNA sequences of inhibin alpha, beta A and beta B subunit genes were 1124 bp,1364 bp and 1499 bp, respectively. Their CDS regions were at 1083 bp,1278 bp and 1227 bp, encoded a polypeptide of 360,425 and 408 amino acids, included 18,21 and 20 amino acids signal peptides and 341,404 and 380 amino acids mature peptides, respectively. Compared with other species in GenBank, the homology in codogenic amino acids of CDS regions was more than 96%.
(2) The relative molecular weight of the polypeptide of inhibin alpha, beta A and beta B subunit genes in goat were 94.655 KD,109.132 KD and 100.438 KD, respectively. The isoelectric points were 4.95,4.92 and 4.94, respectively; the minimum values of hydrophobicity were -0.467,-0.633 and -0.567, the maximum were 2.344,2.422 and 2.267, respectively. They had not protein transmembrane domains and had alpha helix (17.22%,21.65% and 23.04%), beta turn (5.28%, 3.53% and 4.41%), extended strand (18.89%,19.53% and 18.14%) and random coil (58.61%, 55.29% and 54.41%) in its secondary structure.116 amino acid residues of inhibin beta A and beta B subunit genes matched human inhibin beta A subunit gene in tertiary structure.
(3) Compared with the sequence of exon 2 for inhibin alpha subunit gene in sheep, the cDNA sequence in goat had 19 mutation sites (including 17 transitionsal sites and 2 transversional sites) and 8 amino acids mutation sites. Compared with the partial sequence of exon 1 and 2 in Boer goat in GenBank, the cDNA sequence in goat had 3 mutation sites (including two transitionsal sites and one transversional site) and two amino acids mutation sites.One of the amino acids mutation sites was 338 C→G (i.e.32 C→G of exon 2) that resulted in amino acid change (L→V). the other was 345 A→G(i.e.39 A→G of exon 2) that resulted in one amino acid change (Q→R).
(4) Compared with inhibin beta A subunit gene in sheep, the cDNA sequenceof goat had 10 mutation sites that were both transitionsal sites. Locus 198A→G of CDS region (i.e.211 A→G of cDNA sequence) resulted in amino acid change (P→K).
(5) Compared with inhibin beta B subunit gene in sheep, the cDNA sequence in goat had 12 mutation sites (with 5 transitionsal sites,6 transversional sites and 1 insertion site) and 3 amino acid mutation sites. Loci 860 (A→G) and 1372 (G→T) respectively resulted in amino acid change (P→K) and (G→V); inserted G in locus 871 resulted in amino acid increased.
3. Genetic polymorphism analysis for exons of inhibin alpha, beta A and beta B subunit genes in goat
After sequencing and PCR-SSCP in larger population including 28 Dazu black goats and 27 Nanjiang brown goats, the results were as followings:
(1) There were not polymorphic sites in exon 1 of inhibin alpha subunit gene in goat.32C→G and 39A→G in exon 2 of inhibin alpha subunit gene were found in all experimental goats. There were no polymorphic sites in partial sequence of exon of inhibin beta A subunit gene and two exons of inhibin beta B subunit gene in all goats.
(2) There was a mutation site 57G→A (i.e.363G→A of cDNA sequence) in exon 2 of inhibin alpha subunit gene was found by sequencing in larger population.The mutation site resulted in one amino acid change (R→H).
(3) Three genotypes (AA, AB and BB) were detected in two goat breeds after SSCP analysis. Genotype frequencies of AA, AB and BB were 0.071,0.429 and 0.500 in Dazu black goat, respectively, while three genotype frequencies were 0.391,0.435 and 0.174 in Nanjiang brown goat, respectively; Gene frequency of B allele (0.714) was higher than that of A (0.286) in Dazu black goat, while gene frequency of A allele (0.630) was higher than that of B (0.370) in Nanjiang brown goat. Theχ2 test showed that Dazu black goat and Nanjiang brown goat were in Hardy-Weinberg equilibrium(P>0.05).
(4) For the first three litter size, the first two litter size, the first one litter size and average litter size in Dazu black goat, the least square means (LSM) of BB genotype was 0.595,1.01,0.703 and 0.769 more than those of LSM of AB(P<0.05),respectively. For the first three litter size, the first two litter size and average litter size in Nanjiang brown goat,LSM of BB genotype was 0.787,1.375 and 0.754 than LSM of AB (P<0.05).respectively and 0.849,1.486 and 1.131 than LSM of AA (P< 0.05).respectively. but there was not significantly difference between AA and AB (P>0.05). For the first one litter size, LSM of BB and AB were 1.01 and 0.983 more than AA (P<0.05),respectively, but there was not significantly difference between the frequencies of BB and AB(P>0.05).
4. Quantitative expression analysis of inhibin alpha, beta A and beta B subunit genes in goat
Quantitative expression of inhibin alpha, beta A and beta B subunit genes in goats (including 7 Dazu black goats,7 Nanjiang brown goats and 7 Saanen dairy goats) were analyzed by real-time fluorescent quantitation PCR. The results were as followings:
(1) Expression of inhibin alpha, beta A and beta B subunit genes in pituitary gland, ovary, heart, liver, spleen, lung and kidney can be detected. The expression level of ovary was higher than that of pituitary gland(P<0.05). In ovaries and pituitary gland of three goat breeds, expression level of inhibin alpha subunit gene is the highest, inhibin beta A subunit gene is middle and inhibin beta A subunit gene is the lowest.
(2) Expression level of inhibin subunit genes in every tissue of Dazu black goat was the highest in three goat breeds. Saanen dairy goat was in middle level and Nanjiang brown goat was in the lowest level(P<0.05).
(3) The relationships between expression level of inhibin alpha, beta A and beta B subunit genes in ovary and the first one litter size of goat were in negative correlation (P<0.10).
5. Conclusions
(1) There was a negative correlation between FSH and INHB level and a positive correlation between FSH and ACTA level in an estrous cycle of the three goat breeds. The hormone variation tendency was basically coincided with the follicular development wave in goat.
(2) Higher fecundity of goat may be correlation with higher FSH level, lower INHB level and lower expression of inhibin subunit genes in the last follicular development wave.
(3) Exon 1 of inhibin alpha subunit gene, partial sequence of exon in inhibin beta A subunit and two exons of inhibin beta B subunit gene were not polymorphic sites in all goats and these sequences may faintly affect goat's fecundity.
(4) The mutation site (57G→A) in exon 2 of inhibin alpha subunit gene in goat resulted in amino acid change (R→H), three genotypes (AA, AB and BB) were detected in the different individual of two goat breeds. B allele of exon 2 in inhibin alpha subunit gene may be a predominant gene that controls high fecundity of goat,and inhibin alpha subunit gene may be a candidate gene.
(5) Expression levels of inhibin alpha, beta A and beta B subunit genes in ovaries were higher than that in pituitary gland. In goat's ovaries and pituitary gland,expression level of inhibin alpha subunit gene is the highest, inhibin beta A subunit gene is middle and inhibin beta A subunit gene is the lowest.
引文
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