关键词: 家蚕, 马氏管, 无机磷酸盐协同转运蛋白, 启动子, 转基因, 组织特异表达

Abstract:  【Aim】 To identify the specifically expressed genes and promoters in the Malpighian tubules of Bombyx mori, providing new tools and support for the functional study and utilization of genes related to metabolic excretion, detoxification and immunity in B. mori.【Methods】 The Malpighian tubule-specifically expressed genes were screened based on the whole-tissue proteomics data of B. mori and their expression in the head, cuticle, midgut, fat body, Malpighian tubules, silk glands, testes, ovaries, hemocytes and tracheae of the 5th instar larvae of B. mori was validated via RT-PCR. qRT-PCR was used to detect the expression levels of the above specifically expressed genes in the Malpighian tubules of different developmental stages of B. mori (egg, 3rd instar molting, 4th instar larva, 5th instar larva, wandering stage, pupa and adult) and in the head, cuticle, midgut, fat body, Malpighian tubules, silk glands, testes, ovaries, hemocytes and tracheae of the 5th instar larvae. The upstream promoter sequence of Malpighian tubule-specifically expressed gene BmPiT4175 of B. mori was cloned by PCR. A transgenic vector with EGFP as the reporter gene was constructed to obtain transgenic B. mori, and the activity and tissue specificity of the BmPiT4175 upstream promoter were verified at the individual level.【Results】 BmPiT4175, that was specifically expressed in the Malpighian tubules of B. mori, was selected and obtained. The cloned promoter of BmPiT4175 was 2 955 bp in length, which could drive EGFP to be just specifically expressed in the Malpighian tubules of transgenic B. mori, and the expression level of EGFP was comparable to that of the endogenous BmPiT4175.【Conclusion】 Both the expression pattern of BmPiT4175 and the activity of its promoter are Malpighian tubule-specific in B. mori.

Key words: Bombyx mori, Malpighian tubules, inorganic phosphate cotransporter, promoter, transgene, tissue-specific expression