论文标题:鸡卵巢组织基因差异表达与繁殖性状杂种优势的关系研究
Studies on the Relationship between Differential Gene Expression in Ovary and Heterosis of Reproduction Traits in Chicken
论文作者 王慧
论文导师 张沅,论文学位 博士,论文专业 动物遗传育种与繁殖
论文单位 中国农业大学,点击次数 288,论文页数 79页File Size4991k
2004-05-01论文网 http://www.lw23.com/lunwen_2647382/ 杂种优势;mRNA差异显示;荧光定量反转录PCR;繁殖性状;鸡
heterosis,mRNA differential display,real time fluorescence quantify reverse transcriptional polymerase chain reaction,reproduction traits,chicken
为研究鸡繁殖性状杂种优势的分子遗传机理，选用白洛克肉鸡(E)、农大褐蛋鸡(D)、中国丝羽乌骨鸡(C)和白来航蛋鸡(A)四个品种，组建了4×4全双列杂交组合试验鸡群。采用mRNA差异显示(DDRT-PCR)技术和荧光定量反转录PCR(FQRT-PCR)技术，从随机mRNA差异显示和候选功能基因mRNA表达定量分析两个方面，研究了32周龄杂种鸡及其亲本纯种的基因差异表达及其与繁殖性状杂种优势的关系，探讨了鸡繁殖性状杂种优势形成的因素及其分子遗传机理。 (1) mRNA差异显示(DDRT-PCR)研究结果表明： 30对引物组合在16个组合试验鸡群中共显示出1161条能够重现的cDNA片段，其中331条为差异表达片段：杂种与亲本纯种间的基因表达差异表现为质和量两个方面；在杂种与亲本纯种间发现了七种基因差异表达模式：T1共显性、T2杂种减弱、T3杂种超亲、T4杂种沉默、T5单亲特异、T6单亲显性和T7杂种特异表达模式。相关性分析表明，32和42周龄产蛋数杂种优势率分别与T3早极显著止相关(p＜0.01)，并与T7呈极显著负相关(p＜0.01)；T6与蛋黄比色杂种优势率呈显著负相关(p＜0.05)；T2与蛋壳颜色杂种优势率呈显著正相关(p＜0.05)。七种基因差异表达模式与42周龄蛋重、蛋壳强度、蛋黄重、蛋壳重、肉斑、血斑利蛋型指数间均相关不显著(p＞0.05)。 Reverse Northern dot blot验证与克隆测序的18条差异表达片段中，发现其中的1条与人的组织型血纤维蛋白溶酶原激活因子(tissue type plasminogen activator, tPA)编码区的mRNA序列高度同源(e值为0)，推测为鸡的类tPA(CtPA-like)。CtPA-like在亲本纯种EE中仅微弱表达，而在杂种DE和ED中超强表达。tPA在人体内可诱发排卵，提示差异表达片段CtPA-like可能与32和42周龄产蛋数的杂种优势率有关。研究结果表明，基因的差异表达与繁殖性状的杂种优势有关。 (2) 荧光定量反转录PCR(FQRT-PCR)研究结果表明： 32周龄卵巢组织促卵泡素受体(Follicle stimulating Hormone Receptor, FSHR)基因和雌激素受体(Estrogen Receptor, ER)基因的mRNA表达量在杂种与亲本纯种之间差异显著(p＜0.05)；32周龄卵巢组织的FSHR基因在杂种中的mRNA表达量分别与32和42周龄产蛋数的杂种优势率呈显著止相关(p＜0.05)；32周龄卵巢组织ER基因在杂种中的mRNA表达量分别与32和42周龄产蛋数的杂种优势率呈显著正相关(p＜0.05)。研究结果提示，FSHR基因和ER基因的差异表达与产蛋数的杂种优势有关。
In order to understand the molecular basis of chicken heterosis in reproduction traits, mRNA differential display method and real time fluorescence quantify reverse transcriptional polymerase chain reaction (FQRT-PCR) were used to analyze the differential gene expression of ovary tissue between hybrids and their parental lines in a 4X4 diallel cross, involving 4 chicken breeds, which were White Plymouth Rock (E), CAU Brown (D), Silkies (C) and White Leghorn (A). (1) On the basis of DDRT-PCR technique we found that:Total of 331 differential displayed cDNA bands from 1161 were displayed in the 4x4 diallel cross combinations with 30 pairs of primers, which shows the differences of gene expression between hybrids and their parental lines were very obvious in quantity and quality. Seven types of differential expression patterns were found: Co-dominance expressed pattern (T1), Underexpression of parental fragements in hybrids (T2), Over-expression of parental fragements in hybrids (T3), Hybrid-absence expressed pattern (T4), Single parent-specific expressed pattern (T5), Dominant expression fragements of single parent in hybrids (T6), Hybrid-specific expressed pattern (T7).Correlation analysis indicated that there were significant correlation between the pattern of T3 and the heterosis percentage of egg number of 32- week and 42-week old (p<0.01), while there are negative significant correlation between the pattern of T7 and the heterosis percentage of egg number of 32-week and 42 week-old (P<0.01).Of 18 differential expressed sequences conirmed by rerverse Northern dot blot sequenced, one was in high homology with encoded mRNA sequences with known functions(e=0); Other 17 sequences, without homologous sequences by BLAST analysis, were new EST sequences. The sequence of differentially expressed CtPA-like had high similarity with human tissue type plasminogen activator (tPA) (e=0), which can induce ovulation. The differential expression of CtPA-like gene between hybrids (DE ED) and their parental pure breeds (EE> DD) was likely to the heterosis of egg number on 32-week-old and 42-week-old in DE and ED. These results showed the heterosis of reproduction traits were related to gene differential expression between chicken hybrids and their parents. ( 2 ) By using the technique of FQRT-PCR we found that:Differential expressed content of Follicle stimulating Hormone Receptor (FSHR) gene and Estrogen Receptor (ER) gene in 32-week-old ovary is significant between hybrids and their parental pure lines. The expressed content of FSHR gene in hybrids on 32-week-old of age positively correlated to their heterosis percentages of egg number on 32-week-old and 42-week-old respectively (p<0.05). The expressed content of ER gene in hybrids positively correlated with their heterosis percentages of 32-week old and 42-week-old egg number respectively (p<0.05). These results indicated the heterosis of egg number traits were related to the differential expression of FSHR gene and ER gene.
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