机构地区:[1]广东海洋大学农学院动物科学系 [2]广东海洋大学家禽育种中心,广东湛江524008
出 处:《中国农业科学》2015年第4期821-830,共10页Scientia Agricultura Sinica
基 金:科技部农转资金项目(2012GB2E000341);广东省科技攻关项目(2012B020305008);广东省省创新校项目(DOU201305022);国家特种经济动物种质资源共享平台子课题(201402-09);湛江市2012年科技攻关项目(湛财工【2012】196号);广东海洋大学校选课题(1212339;0712130)
摘 要:【目的】研究卷羽鸡卷羽弯曲过程、结构特点、卷羽毛囊发育规律和卷羽候选基因KRT75序列特征及其在卷羽毛囊发育过程中的表达规律。【方法】通过对360只卷羽鸡从1日龄到300日龄卷羽发生情况进行观察,了解其羽毛出壳后弯卷过程;制作成年卷羽和片羽羽毛切片,了解卷羽羽轴、羽枝、羽小枝、羽小钩显微结构特点;对卷羽鸡和片羽鸡胚胎期8—19 d皮肤毛囊制作石蜡切片,阐明卷羽毛囊发生过程,分析卷羽和片羽毛囊发育异同;分别提取13胚龄卷羽和片羽鸡皮肤RNA,反转录为cDNA,连接pMD18-T载体,将连接产物加入DH5α感受态细胞中,转化涂板,挑取白色菌落摇菌后PCR鉴定,阳性菌液送测序,同时随机抽取15只300日龄片羽鸡和15只卷羽鸡,翅下静脉采血,血液基因组柱式小量提取试剂盒提取DNA,PCR产物纯化后测序,采用DNAMAN软件比对分析KRT75基因的遗传特征;分别提取卷羽和片羽胚胎期E 8—E 17d皮肤RNA,反转录成cDNA,根据SYBR试剂盒操作说明荧光定量KRT75基因在卷羽和片羽整个毛囊发育过程中的表达变化规律。【结果】卷羽鸡初生绒羽弯曲明显,以后逐渐弯曲,在第一次换羽结束时(约6周龄左右)全身羽毛显著弯曲;卷羽羽轴向外翻卷,羽小枝上着生羽钩,但羽小枝之间不能相互勾连形成闭合羽片;卷羽毛囊发育从胚胎期第8天开始到第16天结束,整个过程类似于片羽毛囊发育,卷羽毛囊在胚胎期第8—16天完成,整个过程和片羽毛囊发育过程基本相似,但在12—15胚龄卷羽毛囊髓质面积、羽小枝大小与片羽毛囊存在明显差异,其中在第12—14天,卷羽毛囊髓质面积要明显大于片羽,羽小枝板要明显小于片羽;卷羽鸡和片羽鸡KRT75基因CDS全长均为1 569bp,编码522个氨基酸,全序列没有发生69 bp的缺失突变,但在CDS区的3个SNP(954bp:T>C;967bp:T>C;978bp:C>T)可能是卷羽、片羽区别的分子标记;KRT75基因在片羽和卷�【Objective】The Objective of this study is to analyzes the features and curve process of frizzled feather,the development of frizzled feather follice,sequence features and expression rules of candidate gene KRT75 in frizzled feather follicle development.【Method】 The frizzled feathers of 360 frizzled chicks from 1 to 300 days were observedto know the curve process,and the microstructure of rachis,barbules,anf hooks between frizzled and contour feather were compared.The development of feather follicle between frizzled feather and contour feather at 8th in embryonic period was compared by Microscopic section technology.The total RNA from the embryonic 13 day skin of frizzled feather and contour feather was extracted,respectively,reverse transcripted cDNA,connected with the PMD-18 T vector,the products were transformed into DH5α competent cells,coated plates,monoclonal colony was selected for PCR,and the positive plasmid was chosen for sequencing.In addition,15 frizzled and contour feather chicks at 300 days were randomly selected to collect vein blood under the wing,blood genome pillar extraction kit was used to extract DNA,the PCR products were also sequenced after purification,and the DNAMAN software was used to analyze genetic characteristics of KRT75 gene.The total RNA was also extracted from embryonic 8 to 17 day skin of frizzled feather and contour feather; respectively,reverse transcripted cDNA,and the SYBR kit was used to analyze KRT75 gene expression during the formation of feather follicle.【Result】 The down feather appeared disorder and curved in newborn frizzle chicks,the largest curvature of feather was observed at 6 weeks.The frizzled feather rachis curved outward,barbules could not hooked together to form a closed feather; the development of frizzled feather follicle and contour feather follicle was both from E8 to E16,there were some differences in the medulla and barb ridge between frizzled feather and normal one from E12 to E15,in the E12 to E14 day,the medulla of frizzled feather were b
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