Acta Entomologica Sinica ›› 2019, Vol. 62 ›› Issue (7): 779-786.doi: 10.16380/j.kcxb.2019.07.001
• RESEARCH PAPERS •
LU Yi-Ping1, HUO Chun-Yue2, MA Tian-Tian2, YANG Zong-Lin2, SHEN Ya-Wen1, KAN Yun-Chao2, LI Dan-Dan2,*
【Aim】 To investigate the convenient method for functional study of non-coding RNAs (ncRNAs) in the silkworm Bombyx mori, via injection of the overexpression vector of ncRNAs into the coelom of B. mori larva and detection of the expression changes of ncRNAs in different tissues of the larvae after injection. 【Methods】 The overexpression vectors piggyBac［A3-EGFP-A3-Bm-15］ and piggyBac［A3-EGFP-A3-Bm-152］ of ncRNA Bm-15 and Bm-152, respectively, were injected into the junction of the 7th－8th abdominal segments of the day-3 5th instar larvae of B. mori by microinjector (glass microinjection needle). The total RNA was extracted from larval head, thorax, caudal end, abdominal tergum, abdominal sternum and silk gland after 48 h of injection, and the expression levels of Bm-15 and Bm-152 in different larval tissues were detected by qPCR. 【Results】 The wound surface caused by glass microinjection needle was small, and the infection and mortality rates of B. mori larvae were less than 10%. Bm-15 was significantly overexpressed in the larval head and caudal end after injection of the overexpression vector, with the expression level increased by 17-fold in the head as compared to that in the injection group of the blank vector piggyBac［A3-EGFP］. However, the expression levels of Bm-15 in the other four tissues of B. mori larvae after injection of overexpression vector showed little change as compared to those of the uninjected control group. Bm-152 was also significantly overexpressed in larval head and caudal end of the silkworm after injection of the overexpression vector, with the expression level being 6.6-fold higher than that in the injection group of the blank vector piggyBac ［A3-EGFP］ in larval head. Bm-152 also showed obvious overexpression in larval abdominal tergum and silk gland, with the expression level being 3.4 and 4.5 times as high as that in the injection group of the blank vector piggyBac ［A3-EGFP］, respectively. 【Conclusion】 By coelomic microinjection of overexpression vectors of ncRNAs, ncRNAs can be stably overexpressed in the larval head of B. mori. This study provides a good strategy and method for the subsequent functional study of neuro-related ncRNAs.
LU Yi-Ping, HUO Chun-Yue, MA Tian-Tian, YANG Zong-Lin, SHEN Ya-Wen, KAN Yun-Chao, LI Dan-Dan. Stable overexpression of non-coding RNA in the head of Bombyx mori via coelomic microinjection[J].Acta Entomologica Sinica, 2019, 62(7): 779-786.
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