青岛文昌鱼肌肉生化分析及其功能基因的研究

IOCAS-IR > 海洋环流与波动重点实验室

	青岛文昌鱼肌肉生化分析及其功能基因的研究
其他题名	Biochemistry and functional genes analysis of musculature from Amphioxus (Branchiostoma belcheri tsingtauense)
	马俊凯
学位类型	博士
	2007-06-01
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	快、慢肌酶组织化学克隆表达青岛文昌鱼(Branchiostoma Belcheri Tsingtauense)
摘要	本论文主要对青岛文昌鱼(Branchiostoma belcheri tsingtauense)肌肉组织进行了酶组织化学实验和电镜观察，分离了青岛文昌鱼肌球蛋白重链基因异构体并将其重组表达；克隆了青岛文昌鱼新生肽链相关复合体α亚基基因，并检测了其时空表达情况。根据NADH酶组化实验可以将文昌鱼的肌细胞分成两类，即蓝紫色、有氧代谢型的浅层肌和基本不着色、无氧代谢型的深层肌。电镜观察进一步验证了酶组化的结果，并首次发现浅层肌和中间肌细胞肌丝密度明显大于深层肌。而mATPase组化实验没能对肌纤维进行分类，这也许是由于实验的pH条件不是文昌鱼的肌纤维ATP酶活的pH；或者其很容易失活；或者该酶的活化需要不同于其它动物的金属离子的缘故。克隆到三条对应于肌球蛋白重链尾部保守区的异构体，其中两条是首次报道。这三条异构体都属于II类肌球蛋白并和脊椎、无脊椎动物的多种类型的肌球蛋白分子有较高的同源性；利用原核表达系统对异构体进行重组表达，获得了可溶性的表达蛋白，为制备文昌鱼肌球蛋白特异性抗体打下了基础。从青岛文昌鱼成体中分离到了新生肽链相关复合体α亚基的全长cDNA，并检测了其时空表达情况。对其cDNA 5’端的基因组序列进行预测和比较分析，没有发现肌肉特异性外显子的存在。
其他摘要	This thesis mainly focus on the Enzyme histochemistry and TEM observation of trunk muscle from Branchiostoma belcheri tsingtauense (Bbt); isolation and recombinant expression of Bbt-myosin heavy chain isoforms; characterization and expression of Bbt-nascent-polypeptide-associated complex α subunit. In NADH Enzyme histochemistry, two types of muscle lamella can be distinguished .The oxidative superficial muscle was stained dark blue while the glycolytic deep one was almost unstained. TEM observation confirmed the results of Enzyme histochemistry. Furthermore, it was noticed that there was clearly higher myofilaments density in superficial and intermediate lamellae than the deep one. While in mATPase histochemistry, there is no difference among different lamellae. Maybe the lamellae were inactive or deactivation under those pHs, or additional metal ions being different from muscle fiber (lamellae) of other species should be added for this display. Three isoforms of myosin heavy chain fragments, two of which were the first time reported, were isolated. They belonged to class II myosin and had high identities with different kinds of muscle myosin of vertebrates and invertebrates. One of them was recombinant expressed in E.coli. A soluble fusion protein which would be a good resource for anti-Bbt-MyHC antibodies preparation was obtained. The full length cDNA of the nascent-polypeptide-associated complex α subunit gene was cloned and it was found to be expressed in all of the checked stages and tissues. The genomic sequence of 5’ end was obtained and analyzed; however, it seemed that there was no ortholog of the muscle-specific NAC in amphioxus.
页数	84
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1217
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	马俊凯. 青岛文昌鱼肌肉生化分析及其功能基因的研究[D]. 海洋研究所. 中国科学院海洋研究所,2007.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
10001_20042800681200（1188KB）			限制开放	--	浏览