Primary Culture Of Four Tissues From Small Brown Leaf Hopper(Laodelphax Striatellus) And Their CDNA RAPD Analysis

In last several years, the epidemiology of rice stripe disease caused by Rice stripe virus (RSV) had a rise trend in China and other countries of Southeast Asia. This disease had broken out in Jiangsu and Henan provinces, and also caused serious damage to the rice production in Yunnan, Shandong, Liaoning, Fujian, Zhejiang provinces and Beijing, Shanghai cities. RSV is spread by small brown leaf hopper (Laodelphax striatellus Fallen) only, which can persistently spread RSV through its eggs, and the symtom of Rice stripe disease straightly relates with distribution, reproduction and tansference of this insect. Thus, the study on mechanism of the interaction between RSV and small brown leaf hopper will help further understanding of the regulation of Rice stripe disease, and will provide new ways for prevention and cure of Rice stripe disease. To avoid researching this mechanism only by insect pathology, this experiment tried to establish a cell line of small brown leaf hopper, then built up a new pattern to research this mechanism, which would facilitate researching this mechanism on the level of cell biology and molecular biology. So, the most content of this experiment was the primary culture of tissues and cells of small brown leaf hopper.On the basis of cell culture experiences of predecessors, expectly of insect culture experiences, we had developed the primary culture of tissues and cells of small brown leaf hopper, which was first in China.The first step of cell primary culture is to isolate tissues from eggs or insect bodies, we compared the effects of two methods of isolation, the microscope glass-needle isolation and microscope silver-needle dissection, which showed that the microscope silver-needle dissection could dissect tissues more easy, without sybiosis yeast of small brown leaf hopper .The difficults of isolation, such as sybiosis yeast stain, time waste and other troubles, were settled.To dissect single cells promising to culture from tissues, we researched the time of hydrolyzation of the tissues by tryposin, the result showed that the optimum time of hydrolyzation of the tissues was 5~10min when pretreated with 0.25% tryposin under the optimum conditions of pH8.0 at 37 C.We cultured four tissues: embryo at mediat-anaphase, head of high-age nymphae, spermary and ovary of adult. And on the basis of Grace medium, different mixes of Fetal Bovine Serum (FBS), MM medium, Hydrolysate lactalbumin (HL) and Yeast extract (YE) was developed on different levels to culture these tissues and cells. The result showed that it is more easier to culture tissues and cells from embryo at mediat-anaphase than the other three, for it isolated more tissues and cells adhered to the bottom of bottle and which could survive more longer, moreover, the combination made up of a copy of Grace, a copy of MM medium and 20%FBS would maitain the time of growth of tissues and cells adhered to bottom live for longer time than other conbinations, especially the time of growth of tissues and cells from embryo at mediat-anaphase adhered to bottom could maitain above two monthes. Thus, we had optimized culturing conditions (pH, osmoc pressure and others) for tissues and cells from small brown leaf hopper.The different result of culture of these four tissues from small brown leaf hopper and the different form of cells indicated that the expression of genes was different in these four tissues. In order to understand these differences on gene transcript level, even to get the genes and their expression related to cell culture, we researched the differences of total mRNA of these tissues by RAPD-PCR, the result showed that the difference between embryo and head of nymphae was much closer than embyo with others, which was accorded to the time order of insect upgrown, and the tissue which is more different from embryo on transcript level ismore diiBcult to be cultured in vitro.