泥蚶(Tegillarca granosa)个体发育过程中同工酶基因表达与调控的研究

EXPRESSIONS AND REGULATION OF ISOZYME GENES DURING ONTOGENY OF BLOODY CLAM TEGILLARCA GRANOSA

采用不连续垂直聚丙烯酰胺凝胶电泳法,对泥蚶(TegillarcagranosaLinnaeus)19个发育阶段的ADH、LDH、EST、IDH、ALP、MDH、GDH、POD、ATPase和ME等10种同工酶的基因表达进行检测分析。结果表明,泥蚶在个体发育过程中随着机体分化和生理活动的改变,同工酶基因的表达呈现出显著的时序变化。在10种酶中,LDH和ATPase两种酶的所有酶带及EST、MDH和ME等的部分酶带在各个发育阶段持续表达,其他酶带只出现在发育阶段的某一或某些阶段,并且表现出了与泥蚶营养方式、代谢形式等生理活动的关联性。在泥蚶的个体发育阶段过程中,IDH、MDH、GDH、ME、ALP等5种酶在32细胞期表达的酶带数剧增,因此32细胞期可能是合子基因表达启动的时间;ADH、EST、LDH、MDH、GDH、ME等6种酶在泥蚶的附着变态期酶带大幅减少或产生新酶带,可能与泥蚶附着期运动方式、代谢强度及生活环境的改变有关。

Bloody clam Tegillarca granosa, distributes along the south and west Pacific Ocean, belonging to Mollusca, Bivalvia, Arcoida, Arcidae, is one of four traditional culture species of shellfish in China. Containing abundant protein and vitamin B12, also being tasty and delicious, it has been farmed in a large scale along the south coast of China. In recent years, expending culture in area and scale, some diseases in T.granosa population are causing heavy harm in some farms. Except for the deterioration of the ocean environment, degradation of the genetic resource of T. granosa is thought to be responsible for the problem, demanding better breeding and seeding technology for T. granosa, which is also, to study in its bioenergetics. The analysis of isozyme gene expression is not only important for the discovery of certain functional gene and its regulation mechanism but also useful for exiting some special physiological phenomenon happened during early life stage of an organism. Many previous cases reported that certain gene expression pattern in ontogeny is related to some important physiological events. T. granosa is an important economical shellfish in China seas. Although many researches have been done on this species, but yet there is no study reported on isozyme gene expression pattern in its ontogeny. In this study, mechanism of the isozyme gene expression change in ontogeny of T. granosa. 10 isozymes were chosen to carry out the experiment by vertical polyacrylamide gel electrophoresis. They are lactate dehydrogenase (LDH), malic enzyme (ME), malate dehydrogenase (MDH), ester (EST), alcohol dehydrogenase (ADH), isocitrate dehydrogenase (IDH), alkaline phosphatase (ALP), glutamate dehydrogenase (GDH), adenosine triphosphatase (ATPase) and peroxidase (POD). Samples from 19 different life stages of T. granosa were collected from the Institute of Zhejiang Mariculture. In laboratory, the samples were homogenized in 0.05% mercaptoethanol buffer at ratio of 1∶3 (W/V) with ice-bath immediately and centrifuged by 12000r/min for 30 minutes at 4℃. The supernatant was collected and used for the experiment. Like other organisms, the expression of isozyme genes in T. granosa showed significant changes in different life stages and physiological activity. In general, the enzymes could be divided into two types. The first type was found in all stages of individual development, which may be coded by “house keeping genes”; the second type was found only in some special stages, whose expression showed certain connection with special physiological activities. Our analysis on the changes of all zymograms revealed that there are two basic changes in 19 stages of embryo development. One appears in the stage of 32-cell-period, in which the amount of enzyme bands increased rapidly, suggesting that the 32-cell-period is the expression start of zygote genes, the other happened in the stage of metamorphosis period, in which the amount of enzyme bands decreased rapidly, it may be related with changes of moving fashion, intensity of metabolism and living environment of T. granosa larva. The regulative mechanism of isozyme gene of T. granosa was also discussed in this paper, which may give a reference to further research of gene regulations in other organisms.