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猪HOXA10基因的表达调控研究及激素诱导型启动子转基因小鼠与猪的制备与鉴定
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摘要
产仔数性状的改良可以为养猪业带来巨大的经济效益。研究影响产仔数性状的重要候选基因的功能并应用于育种实践的工作已引起育种工作者们越来越多的重视。利用转基因动物技术进行母猪产仔数性状的改良具有广阔的发展前景。同源异形框基因HOXA10是近年来受到广泛关注的产仔数性状重要候选基因之一。转录因子HOXA10对子宫内膜的分化发育和胚胎附植的建立以及正常妊娠的维持的调控都起到必不可少的关键作用,并且受到雌激素(E2)和孕激素(P4)的调节而对产仔数产生影响。然而,关于猪HOXA10基因的转录调控研究很少。鉴于HOXA10基因在胚胎附植过程中的重要功效,我们对猪HOXA10基因的表达和调控机理进行了初步研究,并制备了转HOXA10基因动物模型。
     本研究主要结果如下:
     1.克隆获得了猪HOXA10基因包含完整CDS区域的cDNA序列(1363bp)并分析了其基因组的结构。HOXA10基因含有两个外显子和一个内含子;cDNA全长2628bp; CDS长1236bp编码411个氨基酸,其氨基酸序列与人类和小鼠同源性分别达90%和95%,发现氨基酸序列中存在一个同源结构域;
     2.发现了2个猪HOXA10基因的SNP位点,其中一个位于第一外显子上。该外显子MslI多态位点的等位基因频率在国内外不同品种猪中的存在显著差异;性状关联分析结果显示,HOXA10基因SNP位点与母猪总产仔数显著相关,与产活仔数和初生重呈极显著相关;
     3. RT-PCR结果显示猪HOXA10基因在子宫内膜组织中高表达,同时在膀胱和肾脏组织中表达量也很高,这一结果与小鼠HOXA10基因组织表达模式一致;定量PCR结果表明,HOXA10基因在不同妊娠时期的梅山猪和大白猪子宫内膜中呈差异表达,其中,在两个品种的各个时期中,妊娠15天的梅山猪子宫内膜中HOXA10基因的表达量最高,并且极显著的高于大白猪妊娠15天的子宫内膜(P<0.01);而在妊娠50天时,大白猪子宫内膜HOXA10基因的表达量极显著高于梅山猪(P<0.01);另外大白猪品种内比较,妊娠50天时子宫内膜中HOXA10基因的表达最高;
     4. QPCR检测结果显示,腹腔注射2mg孕激素/天,可以显著增加妊娠第5天小鼠子宫内膜中HOXA10基因的表达水平,同时平均胚胎着床数增加1只;
     5.利用系列删除载体构建和荧光素酶报告基因活性检测的方法首次鉴定了位于猪HOXA10基因5'上游的一个激素诱导型启动子区域,该启动子区域受到E2的刺激后活性增强;构建HOXA10基因激素诱导型重组载体pc3.1-Pro3-DNA,并且在Ishikawa细胞中验证该重组载体受到E2和P4的诱导后可以上调HOXA10基因的表达;
     6.将pc3.1-Pro3-DNA重组载体线性化,利用显微注射法制备了转基因小鼠和转基因猪,并进行了PCR和Southern blot检测,结果共获得5只阳性首建鼠(F0),Southern blot检测出其中2只为阳性。这2只F0代转基因小鼠与野生型小鼠交配用于建系。经PCR和Southern blot检测,获得9只F1代阳性小鼠,阳性率达40.9%。PCR检测获得21只F2代阳性小鼠,阳性率达61.8%。其中对F2代阳性个体RT-PCR检测发现子宫内膜、肾脏和膀胱中均有外源HOXA10基因的表达。经PCR检测F1代和F2代的PCR鉴定结果表明,F0代鼠的转基因可稳定遗传给后代。另外,QPCR检测发现,妊娠第5天的阳性转基因小鼠子宫内膜中外源HOXA10基因的表达量显著高于正常发情第5天的阳性转基因小鼠,同时胚胎附植平均数比非妊娠的转基因小鼠多1只左右。显微注射法第一批共获得33头仔猪,通过PCR与Southern blot检测,得到4头阳性HOXA10转基因猪,阳性率为12.1%。
Improvement of litter size can bring huge economic benefits to pig industry, identification of crucial candidate genes and new approached affecting litter size are very important in pig research..Transgenic technology have a very good prospect in porcine reproductive traits improvement. In a previous study, Homeobox A10(HOXA10) was shown to be one of the most promising candidate genes which play major roles in endometrial differentiation and development, establishing the conditions required for implantation and normal pregnancy maintenance. HOXA10is a well-known transcriptional factor that can be regulated by estrogen (E2) and progesterone (P4). However, little work has focused on the regulation of the porcine HOXA10gene. Considering the important effects of HOXA10on embryo implantation, we investigated its expression and regulation mechanism, also provide theoretical basis and animal model for the improvement of porcine litter size.
     The results are as follows:
     1. The full-length of coding region of porcine HOXA10was cloned (1363bp), and its genomic structure were defined. HOXA10contains1exons and1intron. The cDNA of porcine HOXA10was2628bp in length, with a CDS of1236bp encoding a peptide of411amino acid residues, which showed90%and95%sequence similarity to that of human and mouse homologues, respectively.
     2. Analysis of the DNA sequences of HOXA10revealed2SNPs, one of them is in exon1. Allele frequency of this HOXA10MslI polymorphism revealed great variation between6porcine breeds. Association analyses showed that the MslI polymorphism had significant (P<0.05) association with total number born (TNB), and extremely significant (P<0.01) association with number born alive (NBA) and birth weight (WB);
     3. RT-PCR confirmed that the porcine HOXA10gene is highly expressed in the endometrium, bladder and kidney. QPCR showed that the expression of HOXA10was significantly higher on day15of gestation (gd15) in comparison to gd26(P<0.01), gd50(P<0.01) and day15of the estrous cycle (ed15) in the endometrium of Meishan pigs. In contrast, the highest expression in the endometrium of Yorkshire pigs was on gd50. Moreover, the abundance of HOXA10mRNA was the highest on gd15in Meishan pigs than in any other stage tested in the two breeds;
     4. QPCR results showed that the endometrial expression of HOXA10is increased when the pregnant mice were injected progesterone (2mg/each per day), and the number of embryo is also increased, but the difference is not significant;
     5. Deletion analysis and reporter expression assays identified a promoter region (-1044bp to+18bp) that is responsible for E2and P4-induced HOXA10transcription. Moreover, this promoter region enhanced the E2/P4-induced HOXA10expression in Ishikawa cells.
     6. Lineralized pc3.1-Pro3-DNA plasmid was used to microinjection to establish transgenic mice and transgenic pigs. PCR and Southern blot analysis were used to identify the positive transgenic ones. Offspring carrying the foreign HOXA10were identified by PCR, and5of them were confirmed by Southern blot analysis. The two positive transgenic mice (F0) were mated to wild-type mice to establish transgenic lineages. PCR and Southern blot results showed there are9positive transgenic mice in F1, and21positive transgenic mice in F2. The positive transgenic rate was about40.9%and61.8%, respectively. RT-PCR analysis of F2showed that the foreign HOXA10were expressed in endometrium, kidney and bladder of all the ten female lineages. PCR analysis of F1and F2showed that the foreign HOXA10in the2positive Fo mice could be transmitted stably.In addition, QPCR results showed that the endometrial expression of HOXA10is increased significantly in pregnant transgenic mice on d5of gestation compared to the non-pregnant transgenic mice on d5of estrous cycle. Mean while the number of embryo is also increased.
     PCR and Southern blotting analysis identified4positive transgenic pigs which carried the foreign HOXA10gene, the positive rate is12.1%.
     In conclusion, we identified an E2/P4response element of the porcine HOXA10gene for the first time. Moreover, we assume that the porcine HOXA10gene may affect litter size by regulating implantation. The data from the present study contribute to the mechanism by which the porcine HOXA10gene regulates embryo implantation and prolificacy traits. We also established the hormone-inducible transgenic mice and transgenic pig which provide the animal model for the improvement of little size traits.
引文
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