【作者】 俸艳萍；

【导师】 龚炎长；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 鸡的性别控制在现代养鸡生产中具有重要意义，然而目前仍没有较好的方法实现这一目标。现有的性别控制方法有通过影响控制鸡性比的因素使性比偏向生产所需及通过干扰鸡的性腺分化产生性反转鸡等，本研究围绕这两个主题，一方面利用早期鸡胚性别鉴定方法全面分析正常生产条件下鸡的各种性比及产生性比偏向的可能原因；另一方面利用抑制消减杂交（SSH）技术分离性分化早期雌、雄鸡胚性腺的差异表达基因，并对这些基因进行进一步研究，为探讨性别决定与性分化的分子机理奠定基础。具体研究如下：组合扩增W染色体上360 bp的EE0.6特异片段和Z染色体上970 bp的OVR基因片段的两对引物，应用双重PCR技术建立了鸡性别鉴定分子方法，经验证其准确率达100％；利用该方法成功判定了早期鸡胚的性别，其中早期死亡鸡胚的性别判出率达92.34％。结合分子鉴定鸡胚性别及剖检鉴定性别的方法分析了人工饲养条件下140只母鸡在30天内的子代性比，结果表明“所有母鸡”组和“高繁殖力母鸡”组（76只）的出壳雏鸡和总体子女（包括孵化过程中的死胚）的性比均不显著偏离理论性比值1∶1，但孵化过程中死亡的雌性胚胎数量显著超过雄性（P＜0.05）；高繁殖力母鸡中，大部分个体的亲本性比不同程度地偏离理论性比，其中有7只（占9.21％）子代性比显著偏离理论值（P＜0.05），另有37只产生至少连续4个为同一性别的种蛋。以孵化3.5—6天的鸡胚性腺组织为材料，应用SSH技术构建了性分化早期雌、雄鸡胚间正反向消减cDNA文库，以持家基因GAPDH为参照指标检测消减文库的消减效率，结果表明两个文库的消减效率均高达2⁵倍。从每个文库中随机挑选96个克隆进行PCR鉴定，发现cDNA插入片段的长度主要分布于250—750 bp之间，F-M（以雌性鸡胚为tester、雄性鸡胚为driver）和M-F（以雄性鸡胚为tester、雌性鸡胚为driver）消减cDNA文库中有效阳性克隆率分别达93.8％和90.6％。利用斑点杂交技术，以正向消减cDNA和反向未消减cDNA作为探针对F-M文库中1200个克隆和M-F文库中900个克隆进行筛选，结果表明两个文库中差异明显（相差至少3倍以上）的阳性克隆率分别为31％和26％。将已测序的152个差异表达克隆的cDNA序列在NCBI数据库中进行比对，结果表明F-M文库的86个克隆代表了41个基因，其中39个为已鉴定基因、2个具有同源EST序列；M-F文库的66个克隆代表了47个基因，其中16个为已鉴定基因，12个有EST同源序列，25个没有任何同源序列。根据哺乳动物中同源基因的功能推测41个已知功能的差异表达基因（F-M中31个，M-F中10个）分属于DNA水平相关因子、RNA水平相关因子、结构蛋白、酶类、癌症相关基因及信号转导因子等。分析88个差异表达基因在鸡基因组中的分布，结果发现这些基因主要分布于鸡1—5号染色体上，其中F-M文库中有1个基因位于W染色体上，F-M文库和M-F文库中分别有2个和3个基因位于Z染色体上。将差异表达cDNA片段在GenBank的鸡EST数据库中进行比对，结果表明分别有80.49％和34.04％的雌、雄性差异表达基因在两个已报道鸡胚性腺相关文库（gonad cDNA library and gPGC cDNA library）中有同源序列；通过比较cDNA片段在消减文库及两个已报道性腺相关文库中的重复率，初步分析了这些基因在鸡胚性腺中的基本表达特征。用RT-PCR方法分析了7个功能候选基因及46个新发现的差异表达基因（F-M文库34个，M-F文库12个）在鸡胚早期性腺发育过程中（第4—12天）的表达谱，结果表明：功能候选基因中除cEki2和cFog2在两性中表达水平相似外，cBrd3、cVnn1、cPpt1、cLhx9和cGATA4五个基因在鸡性腺分化过程中呈两性差异性表达；另外从表达模式上看，除cVnn1的表达模式及cGATA4／cFog2的相互作用模式与哺乳动物相似外其它都不相同；新发现的差异表达基因大部分（F-M，85.29％；M-F，91.67％）为真实差异表达，相对表达水平相差达2倍以上的基因在F-M、M-F文库中分别有13个和6个，其中于性分化前期（第4—5天）两性中表达差异明显的分别有10个和1个；而表达差异小于2倍的基因在F-M、M-F文库中分别有16及5个。此外，还通过WISH定位分析基因表达的方法初步分析了SMARCE1在5.5天和8.5天鸡胚泌尿生殖系统的表达，结果表明该基因在5.5天两性鸡胚的泌尿生殖系统中均有表达且表达量基本相同，在性腺形态已分化的8.5天雄性鸡胚的性腺和肾脏外侧呈明显阳性表达。应用SMART技术成功克隆了HMG-14A基因的一个新转录本（F071）和Z染色体上一个非编码RNA（A041）的全长cDNA序列，GenBank登录号分别为EF422210、EF422212。F071基因全长cDNA序列为1026 bp，ORF Finder预测其最大开放阅读框为318 bp，编码105个氨基酸：该基因的核苷酸和氨基酸序列比对结果表明F071与鸡HMG-14A基因（NM₀01079480.1）的同源性最高，两者核苷酸同源区段覆盖率为85％，但两者所编码的氨基酸相似性为100％。用Signal P3.0、ProtParam、Prosite等软件对基因所编码的蛋白质结构、功能等特征进行预测和分析，结果表明F071蛋白为富含赖氨酸的非分泌性蛋白，它含有HMG14和HMG17信号、一个依赖于cAMP和cGMP的蛋白激酶磷酸化位点、一个酰胺化位点、一个酪蛋白激酶Ⅱ磷酸化位点、两个蛋白激酶C磷酸化位点和一个N-糖基化位点；根据CLUSTALW结果，用MEGA3.1软件构建了HMG14（HMGN1）基因家族在多个物种间的系统进化树，结果表明鸡F071（HMG-14A）与其它物种HMGN1的进化关系相距较远。A041分子有两个长度分别为1249 bp和790 bp、仅在5’UTR区长度不同的转录本，ORF Finder预测其最大开放阅读框为144 bp，编码47个氨基酸，在NCBI数据库进行比对未发现与该分子具有同源核苷酸序列或同源蛋白序列的基因，根据该分子特征推测其为非编码RNA。更多还原

【Abstract】 Sex control of chicken is signality in modern commercial poultry production, but unfortunately, there is no authorized way yet to realize. Current approaches to control the sex of chicken include manipulating the factors which have effect on chicken sex ratio to make it deflect according to the requirement of production, producing sex reversal chicken by interfere with the sex differentiation etc. In present research, two topics are focused on: firstly, the sex ratios under normal condition of chicken production would be studied by an approach being able to identify the sex of embryo during the early development stage and the causes to lead the deflection of sex ratios would be simply attributed, and secondly, sexual differentially expressed genes in chicken embryos during early sexual differentiation would be isolated by using suppressive subtraction hybridization （SSH）, and the developmental expression profiles of the isolated genes and some candidates which involve in the sex determination and differentiation of other species would be studied to form the basis of molecular mechanism of chicken sex determination and sex differentiation. Details as follows:A molecular approach to sex the chicken and the embryo was established by using duplex PCR which simultaneously amplifys a 360 bp EE0.6 fragment on W chromosome and 970 bp OVR fragment on Z chromosome. It has been validated to be accurate completely （100 %） and is used successfully to identify the sexes of chicken embryos at early stage, and the sexes of 92.34 % embryos died during early developmental stage can be identified by using this way. Offspring sex ratios of 140 hens during 30 days under normal condition are studied, it shows that the sex ratios of hatching and all offspring （including embryos died） respectively in "all hens" group and "high fecundity" group, i. e. 76 hens among all hens, do not deviate significantly from expected 1:1, but the number of female embryos died is higher significantly than that of males （P＜0.05） which has validated through the study of a large number of dead embryos. Parental sex ratio of most "high fecundity" hens appear bias in different degree, of which the offspring sex raio of 7 hens （account for 9.21%） deviate significantly from 1:1 （P＜0.05）, and 37 hens produce successively more than 4 eggs with the same sex.Forward and reverse subtracted cDNA libraries between female and male are constructed from chicken embryonic gonads at days 3.5-6 post-incubation by SSH. The subtractive efficiency of 2⁵ folds is obtained in the two libraries by using the housekeeping gene encoding for glyceraldehyde-6-phosphate dehydrogenase （GAPDH） as the reference. PCR of 96 recombined clones picked randomly from each library shows that most of them contained inserts of 250-750 bp, and 93.8 % clone in the F-M library, in which female embryos as tester, male embryos as driver, is recombinants and corresponding, 90.6 % in the M-F. Dot Blot hybridization is carried out to screen differentially expressed genes by using forward subtracted cDNA and reverse unsubtracted cDNA as probes, results show that, respectively in F-M and M-F library, there are 31% and 26 % clones having obviously different expression （three times difference at least） among 1200 clones detected in F-M and 900 in M-F. BLAST of sequences of 152 sequenced clones reveals that 86 clones from F-M library represent 41 genes, including 39 being identified and 2 having homologous EST sequence, and 66 clones from M-F library represent 47 genes, 16 being identified, 12 having homologous EST sequence and another 25 having no homolog in GenBank database. Based on the function of the homologs in mammals, all 41 genes identified （31 in F-M, 10 in M-F） are predicted to be functional molecular belonging to DNA association, RNA association, structural protein, enzyme, cancer relation and signing transport etc. It is demonstrated that most of 88 differentially expressed genes locate on macrochromosomes （1-5）, one from F-M library locate on W and 2 from F-M library, 3 from M-F library on Z. The cDNA sequences of differentially expressed genes are blasted with EST sequences in two reported chicken gonad related libraries （gonad cDNA library and gPGC cDNA library） from GenBank, it reveals that there are 80.49 % and 34.04 % differentially expressed genes respectively from F-M and M-F library having homologous sequences in two reported libraries, and accordingly, expression feature in chicken embryonic gonad of differentially expressed genes are predicted by comparing the redundancy of differentially expressed cDNA in subtracted library with that in two reported gonad libraries.Expression profiles of seven candidate genes putatively involved in chicken gonadal development and 46 differentially expressed genes newly isolated from subtracted cDNA libraries （34 in F-M, 12 in M-F） during chicken early gonadal development are studied by RT-PCR. Results show that the cBrd3, cVnn1, cPpt1, cLhx9 and cGATA4 are sexually dimorphic expression with the exception of cEki and cFog2 having similar expression pattern in both sexes, and comparative expression analysis between mammals and chickens show both conserved and divergent elements: expression patterns of cVnn1 and the interaction between cGATA4/cFog2 are similar to those in mammals, while others appearing differences. Expression profiles of genes newly isolated reveals that most of them （respectively 85.29 % in F-M and 91.67 % in M-F） present really different expression. There are 13 and 6 genes, respectively in F-M and M-F, presenting the difference of more than 2 fold relative expression and among them, 10 genes in F-M and 1 in M-F showing obvious difference before sex differentiation （i. e. at 4-5 days）. In addition, there are 16 and 5 genes respectively in F-M and M-F library presenting difference of less than 2 times relative expression. The expression profile of SMARCE1 in urogenital system of chicken embryos at days of 5.5 and 8.5 are studied by WISH, a method for positional gene expression, it indicates that the expression of SMARCE1 is similar in both sexes in the urogenital system including embryonic mesonephros and gonad at 5.5-day, and positive expression of this gene has been found in the gonad and the lateral of mesonephros of embryo at 8.5-day.Full-length cDNA sequences of a new transcript of chicken HMG-14A （Clone F071） and a non-coding molecular （Clone A041） on Z chromosome are obtained by using SMART technology, and these two sequences are submitted to GenBank with accession number EF422210 and EF422212. The cDNA sequence of F071 is 1026 bp, with largest ORF of 318 bp, encoding 105 amino acids predicted by ORF Finder. BLAST of nucleotide and amino acid sequences of F071 show that the coverage rate of homologous nucleotide sequence of F071 with its homolog HMG-14A （NM₀01079480.1） is 85 %, and for the similarity of amino acid, 100 %. The structure and function of the protein encoded by F071 gene are predicted by using Signal P3.0, ProtParam and Prosite software, results show that this protein is Lysine-rich but non-secretory, having HMG14 and HMG17 signature, a cAMP-and cGMP-dependent protein kinase phosphorylation site, a protein kinase C phosphorylation site, an amidation site, a casein kinaseⅡphosphorylation site and a N-glycosylation site. Phylogenetic tree of HMG14 （HMGN1） among several species are constructed by using MEGA3.1 based on CLUSTALW, it shows that chicken HMG-14A （F071） is far away from HMGN1 of other compared species. A041 is found having two transcripts with difference length in 5’ UTR and the full length cDNA sequence are 1249 bp and 790 bp respectively, its largest ORF predicted by ORF Finder is 144 bp, encoding possibly 47 amino acids. No any homolog of A041 is found in NCBI and it is tentatively recognized as a non-coding RNA （ncRNA）.更多还原