| 932 strain of silkworm was used as research material in the paper. To further explore the molecular regulation mechanism of diapauses occurring and releasing, diapauses-related mi RNA and its target genes were screened and different expression and regulation function in diapause-related eggs, instant pickling eggs, red bean color pickling eggs were analyzed by modern biotechnologies such as PCR, q RT-PCR, DLR, bioinformatics and the ELISA method. The researches got the following results:(1) The significantly up- or down-regulated miRNAs in post-picking silkworm eggs were analyzed by bioinformatics tools(eg. RNAhybrid et.al). Two significantly down-regulated mi RNA named bmo-mi R-3384-3p and bmo-mi R-2761-3p were screened. Target genes predications showed that bmo-mi R-3384-3p have binding sites at BmNLK 3’UTR 11-64 position bases and bmo-mi R-2761-3p have binding sites at BmSDH 3’UTR 396-423 position bases.(2) By the PCR amplification, comparisons of the transcriptional expression of candidate target genes BmNLK and BmSDH in diapauses-related eggs and red bean color pickled eggs showed that dipping treatment with hydrochloric acid, BmSDH and BmNLK significantly increased, while in the non-pickled eggs was at the low level of expression status. The results indicated that the two genes significantly differentiate expressed in diapauses-related eggs and pickled eggs.(3) By the q RT-PCR technique, the quantitative expressions of bmo-mi R-2761-3p, bmo-mi R-3384-3p, BmSDH and BmNLK in the postpartum 3-7d diapauses-related eggs and red bean color pickled eggs were calculated. The bmo-mi R-3384-3p and bmo-mi R-2761-3p showed relative lower expression after acid treatment. Compared to the samples of before acid treatment, it fell by 76% and 78% in pickled eggs. However, the BmNLK and BmSDH showed relative higher expression than that of the previous pickle, it raised by 217% and 4287%. Statistical analysis confirmed that the relationship was negatively correlated(R12 = 0.94, R22 = 0.92)between the expression bmo-mi R-2761-3p vs BmSDH, bmo-mi R-3384-3p vs BmNLK. This indicates that BmSDH, BmNLK expressed at low levels when the silkworm eggs in diapause state, while silkworm eggs pickling causes significantly upregulate.(4) To verify target relation of bmo-mi R-2761-3p and BmSDH, bmo-mi R-3384-3p and BmNLK, vectors containing BmSDH 3’UTR, BmNLK 3’UTR were constructed respectively. The DLR assay confirmed that bmo-mi R-2761-3p inhibits the expression of BmSDH, but bmo-mi R-3384-3p inhibits the expression of BmNLK.(5) By means of ELISA method for the enzyme activity determinationof BmSDH and BmNLK in the instant pickled eggs, red bean color pickled eggs(until 1d before hatch) and 5 ℃ cold storage eggs(storage passed through 0,10,20,30,40,60,80 d), the results showed that once pickling, these two enzymes activities rapid increased in instant pickled eggs and red bean color pickled eggs, BmNLK activity reached the peak 2d later pickled. The amounts are 2972.24 m U/g, 2800.45 m U/g, respectively. Compared with the control, it increased by 13.26 times and 12.43 times. BmSDH activity reached the peak 3d later pickled. Instant pickled egg is 1003.36 m IU/g, while red bean color pickled egg is 662.29 m IU/g. Compared with the control, it increased by 4.50 times and 2.63 times. Diapause eggs in cold storage at 5 ℃, the BmNLK and BmSDH activity decreased in initial, when passed through 30d/20 d respectively the activity began to increase until the 80 d, BmNLK 、 BmSDH activity reaches maximum. This indicated presence of linkage between BmNLK, BmSDH enzymes activities and silkworm eggs diapause. |
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