Sex-linked Red Ants (sch) Silkworm Embryonic Temperature Sensitivity Of Ddrt-pcr Study

Differential display reverse-transcriptase PCR (DDRT-PCR), reported first by Peng.Liang cf at in 1992. has the characteristics of convenience, high sensitivity and is suitable to many samples. DDRT-PCR effectively overcomes the drawbacks of the other similar methods, such as high cost, laboriousness and only two comparative samples can be analyzed etc. So DDRT-PCR is a powerful protocol to identify and c lone differentially expressed genes. The ch~ractet of sc/i of temperature-sensitive lethality during embryonic period suggests that temperature-sensitive lethality of sc/i is related with differential expression of genes controlled by environment. So an effective method of DDRT-PCR for silkworm eggs was developed in this investigation and then eggs of Xia sc/i incubated under 60% RI-I at 35~C for 36h (Abbreviate as:Xia sch/high) ,eggs of Xia sc/i treated in normal temperature and humidity (Abbreviate as:Xia sch /normal), eggs of Xia fang under 60% RH at 35扖 for 36h (Abbreviate as :Xia fang/high) eggs of Xia fang treated in normal temperature and humidity (Abbreviate as :Xia fang/normal) were used to research. The gene expression of them 憊ere analyzed by DDRT-PCR.The results are as the following: I Total RNA extracted by one-step-method and treated with DNase free of RNase was found to be applicable to DDRT-PCR. Its absorbance ratio A260/A280 and results of the electrophosis showed that the RNA samples thus-obtained were in good integrity?and free of degradation of RNA and contamination of DNA. 2.An effective method of DDRT-PCR for silkworm eggs was developed in this investigation. Reagents in RNA image kit \vere replaced by those purchased seperately and primers synthesized commercially; autoradiography was replaced by silver staining, so its cost was considerably declined and safety and convenience were improved. This method overcomes the general defects of less, small and weak bands when RNA image kit and silver staining are used together. Using this method, one pair of primers can obtain 60 clear hands range from 200bp to 600hp. Although there were some non-specifically amplified bands in differentially expressed bands, false-positive was obviously controlled and its ratio was down to VI 50% because only differentially expressed bands that could be replicated were further analyzed. 3. The results of DDRT-PCR on Xia sch/high and Xia sc/i/normal, Xia fang/high, Xia fang/normal indicated that the great majority of bands amplified by every pair of primers were the same among the four samples, the small minority of differentially expressed bands included 6 types as the following: (a) bands specific to Xia fang; (b) bands specific to Xia sch; (c) bands sepecific to high temperature treatment; (d)deftciency bands in Xia sc/i/normal; (e) bands specific to Xia sch/normal, Xia fang/high, Xia fang/normal; (f)bands specific to Xia sc/i/high. Type (e) and type (f) were found to be in good accordance with temperature sensitivity of sc/i. 4. 24 differentially expressed bands of type(e) and (0 were obtained in the triplication. But only HA P8280 and I-1AP5200 which could complicate steadily were used as the bands of interest for we study the machanism of temperature sensitivity of sc/i. 5.The results of Norhern blot indicated that only HAP8280, the band specific to Xia schlhigh, was true.