低温胁迫对鲤红细胞膜表面结构的影响及相关基因分析

Erythrocyte membrane surface structure of different vitality of Cyprinus carpio under low temperature conditions and analysis of related genes

以荷包红鲤(Cyprinus carpio var.wananensis)耐寒品系(♂,耐低温)与云南大头鲤(Cyprinus pellegnini pellegnini Tchang)(♀,不耐低温)的杂交F2为研究对象,研究抑制消减杂交技术构建的鲤耐低温差异表达基因cDNA文库中2个具有多态性的新差异表达基因[克隆号为CC005(GenBank_Accn为FF339846)和CC062(GenBank_Accn为FF677502)]与低温下鲤红细胞膜形态之间关联性,结果显示,活力正常的实验鱼红细胞膜边缘有数个棘状凸起,直径在500nm左右,高度为300nm左右;两个未知新基因CC005和CC062在膜表面结构凸起组和正常组(相对正常)之间基因型组合分布存在极显著差异(χ2=21.345,P<0.01),推测这2个与低温相关的基因对低温下鲤红细胞膜表面出现的凸起结构有协同效应,这一实验结果将为鲤的耐低温机理研究和标记辅助选择育种提供理论依据。

Cold tolerance is one of the major economic characteristics in fish. Although lots of research has been conducted by fish biologists, the molecular mechanism about cold tolerance is still not available. Experiments were conducted to study： 1） the polymorphism of the new CC005 （Genbank Accession Number was FF339846）and CC062 （Genbank Accession Number was FF677502）differentially expressed genes and 2）the relationship between the carp erythrocyte structures under the temperature of（4.0±2.0） ℃. The F2 of purse red carp cold-tolerant strains （Cyprinus carpio vat. wananensis）and Boshi carp （C. pellegrini）were used as experimental objects in this study. Erythrocyte membrane surface structure with different vitality experimental fish was compared under atomic force microscope （AFM）. The results showed that： the length of erythrocytes from normal vitality fish is （11.990±1.457） μm and the short axis is （7.225 ± 1.158） μm; the length of erythrocytes from slower fish is （11.938 ± 0.969） μm and the short axis is （7.394±0.864） μm; the length of erythrocytes from the sick fish group is （12.860± 1.080） μm and the short axis is （8.635±0.864） μm. The variance analysis showed that there is no significant difference in both the length （F=0.853, P〉0.05） and the short axis of the erythrocytes （F=3.081,P〉0.05）. Under 3 μm ×3 μm scanning reach, different size particles on the membrane surface were observed and found to be the exposed part of the erythrocyte membrane protein molecules. The erythrocytes from the normal vitality fish had several spine-uplifts on the edge. It was also found that all erythrocytes had same spin-uplifts. Being measured, these uplifts are about 500 nm in diameter and 300 nm high. The erythrocyte structures from sick fish with white spots differed from those from normal and slow vitality fish. In addition, Analysis showed that the two unknown genes CC005 and CC062 have significant differences in their genotypes between the membrane surface structure with uplifts group and the normal surface structure group （χ2= 21.345, P〈0.01）. Thus, we can assume that the carp erythrocyte membrane structure is controlled by the genes related to the low-temperature. The result of this study would provide theoretical basis for the study of carp cold tolerance and breeding using marker-assisted selection.