| In the wool type animal, its wool is an important economic trait. Scanning the candidate gene relative to animal wool quality had been the target of many studies. Most of current researches focused on human and sheep, and it was few studies of rabbits that restricted the rabbit breeding greatly.Keratin associated proteins (KAPs) were the basic component of hair. In hair cortex, intermediate filaments (IFs) composed of keratin embedded in the matrix formed by the KAPs, they connected together by a disulfide bond of cysteine (Cys) residues, and then formed the hair shaft. In mammals, keratin and keratin associated protein which is composed of wool fiber skeleton and accounted65-95%of the total fibers weight. FGF5gene of mice and rats had important biological functions, it was important regulatory factor of the hair follicles growth cyclical, and was the only gene which had been found concerned with the length of hair in recently study.Therefore, KAPs and FGF5genes were significance for wool traits breeding of rabbit. This study, we scanned genetic diversity of KAP3.1, KAP3.3, KAP6.1and FGF5gene in Minxinan Black rabbit, Beaver Rex rabbit, White Rex rabbit, Jiuyishan rabbit, Wanxi Angora rabbit by PCR-SSCP and PCR-RFLP; analyzed the growth pattern of wool by measure the fine and coarse wool length of neck, back, buttocks and abdomen of Fujian yellow rabbit, Germany Angora rabbit, American Rex rabbit (2weeks of age,4weeks of age,6weeks of age,8weeks of age,12weeks of age,16weeks of age,20weeks of age); analyzed the hair follicles histological changes at different stages by observe the hair follicles appearance of back and buttocks skin tissue in Fujian yellow rabbit, Germany Angora rabbit, American Rex rabbit (0weeks of age,2weeks of age,4weeks of age,6weeks of age,8weeks of age,12weeks of age,16weeks of age,20weeks of age); analyzed KAP3.1, KAP3.3, KAP6.1and FGF5expression pattern of back and buttocks skin tissues in Fujian yellow rabbit, Germany Angora rabbit, American Rex rabbit (0weeks of age,2weeks of age,4weeks of age,6weeks of age,8weeks of age,12weeks of age,16weeks of age,20weeks of age) by real-time quantitative PCR. The main results of this study as follow:1. In this study, we obtained the complete CDS of rabbit KAP3.1, KAP3.3gene for the first time. KAP3.1and KAP6.1sequences had been submitted to the GenBank database (HM147283, HM147284, HM068868and HM068869). KAP3.1, KAP3.3, KAP6.1and FGF5gene sequences of rabbit compared with mouse, chimpanzee, human sheep and goat, both their homology were more than80%, indicating that the genes of different species kept high conservation in evolution process.The polymorphisms of KAP3.1, KAP3.3, KAP6.1and FGF5gene full-length CDS region in different rabbit populations were detected. According to the sequencing and sequence alignment, we found that C91T was a silent mutation (serine) in coding region at KAP3.1-1locus. Four mutations (A358G, CA373-374TG, A435G, G467T) which belonged to3’untranslated region of KAP3.3gene and2alleles were found at KAP3.3-2locus. The basic components of KAP6.1gene (CAAT box, enhancer, CACCC box, TATA box) at5’untranslated region were obtained, there were5insertion mutations at KAP6.1-1locus and6basic radical deletion at position1036-1041(CTATGG) at KAP6.1-2locus compared to the rabbit sequence in GenBank (M95718). An insertion mutation (694(G)), a transition mutation (G744C), a transversion mutation (A755G) were found between C1and C2allele in KAP6.1-1locus, and A755G mutation was belonged to the enhancer region. In FGF5gene, the TCT (serine) deletion mutation was found at220-222position at FGF5-1A locus, T58C (leucine- serine) mutation was detected at FGF5-3B locus, both of2mutations lead to amino acid sequence changed. There were not polymorphism been detected at other loci.The distribution of these genotypes in all populations were all in the Hardy-Weinberg equilibrium (P>0.05).5rabbit populations were presented as low diversity at KAP3.1-1locus, their polymorphic information content were uniformity. Minxinan Black rabbit, Beaver Rex rabbit and Jiuyishan rabbit showed intermediate diversity at KAP3.3-2loci, indicating that the natural and artificial selection pressure were considerable. The frequency of C1allele was higher than C2at KAP6.1-1locus, the polymorphic information content of meat-type rabbit (Minxinan Black rabbit and Jiuyishan rabbit) were higher than Rex and Angora rabbit while Wanxi Angora rabbit was0. The frequency of El allele was higher than E2at FGF5-1A locus, there was not E2allele been found in Wanxi Angora rabbit. The result probably due to that Angora and Rex rabbit were selcted in the meat-type rabbit, long-term selection process may result in decreased frequency of C2and E2alleles.2. In this study, hair follicle development of different rabbit breeds (from birth to adulthood) were analyzed, measured the length of wool in different periods and different rabbit breeds to analysis growth regularity of rabbit wool.The average growth speed of primary follicles of Fujian Yellow rabbit in different skin part was fastest, slower in German Angora rabbit and slowest in Rex rabbit, but the difference of average secondary follicles diameter was small among different rabbit breeds. The result were conform to the growth regularity of rabbit wool diameter:coarse wool was difficult to find in all varieties rabbit growth prophase (after birth), but increased significantly in Fujian Yellow rabbit and German Angora rabbit breeds at growth anaphase. The difference of average secondary follicles diameter was small among different rabbit breeds, but there were significant deviation in morphology among different growth periods. Indicated that the development of rabbit hair follicle was same to sheep with a periodicity and the cycle conform to molt cycle of rabbit.The difference of average thickness of the epidermis was small among German Angora rabbit, Fujian Yellow rabbit and American Rex rabbit. The total skin thickness (epidermis and dermis layer) and dermis layer thickness of American Rex rabbit was the thickest, and thinnest in German Angora rabbit. The skin thickness of meat-type and Rex rabbit were thicker than Angora rabbit.The determination result of rabbit wool length as followed:the wool length of same breed but different skin parts was different, the wool of back and buttocks were longer than neck and abdomen parts; the coarse wool was longer than fine wool in the same parts. The strength and duration of rabbit wool growth wave were different among different rabbit breeds, meat-type and Rex rabbit wool growth wave duration were shorter but stronger than angora rabbit at earlier period.3. KAP3.1, KAP3.3, KAP6.1and FGF5the expression pattern in different skin tissues of German Angora rabbit, Fujian Yellow rabbit and American Rex rabbit were analyzed by real-time fluorescent quantitative PCR technology for the first time. The expression patterns of these genes were not identical among different species, stages and skin parts. KAP3.1gene expression showed higher level in0weeks of age in all breeds and the expression level of Fujian Yellow rabbit and American Rex rabbit were higher than German Angora rabbit. The expression patterns of German Angora rabbit were similar between different sex and parts which showed a gender rising trend in general. But it presented greater fluctuation in Fujian Yellow rabbit and American Rex rabbit, most tissues expression descend greatly of in4weeks and12weeks of age excepted for females Fujian Yellow rabbit back tissue appear modest rise.The expression trends of KAP3.3gene were similar to KAP3.1gene.All the expression patterns of KAP6.1gene presented greater fluctuation in3rabbit breeds at different stages. In Fujian Yellow rabbit, the expressions of back and buttocks tissue in different sex were minimum:appearing the highest expression in16weeks of age except female rabbit buttocks tissue was in12weeks of age. The expression differences of different tissue among0-6weeks in German Angora rabbit breed were significant. In American Rex rabbit, all of the tissues and sex showed similar expression trend overall, The expression difference of different tissue in0weeks of age were significant, then they decreased to a low level in4weeks of age and then raised gently. FGF5gene of3rabbit breeds were widely expressed in0weeks of age, the expression level of Fujian Yellow rabbit was the highest and German Angora rabbit was the lowest. In Fujian Yellow rabbit, although the expression of different skin parts, stages and sex was different, the overall trend was a fundamental consistent. The expression patterns of American Rex rabbit were similar to Fujian Yellow rabbit but the expression of American Rex rabbit decreased more obvious than Fujian Yellow rabbit in12weeks of age. In German Angora rabbit, the expression were all at lowest level in0weeks of age and then increased continuously.In this study, all the genes had certain expression levels in0weeks of age in all rabbit breeds indicating that hair follicle development and wool growth had been conducted in the rabbit embryonic period. The expression patterns of4genes in different rabbit breeds trend were not entirely consistent. In German Angora rabbit breed, KAP3.1, KAP3.3, FGF5gene expression showed a gentle upward trend but KAP6.1genes were irregular fluctuations. In American Rex rabbit and Fujian Yellow rabbit, all the genes expression in4and12weeks of age showed great fluctuations may relate to the molt cycle of rabbit. Meanwhile,4genes expression pattern of Fujian Yellow rabbit and American Rex rabbit were similar to each other, the result was consistent with the commercial use of different rabbit breeds. The expression pattern of these genes were not identical among different species, stages and skin parts which conform to the wool length of different species, stages and skin parts was different. The expression trends of KAP3.3gene were similar to KAP3.1gene, so we speculated that perhaps the expression mechanism of these genes existed synergistic effect. |
