Cloning and Activity Analysis of a Midgut-specific Promoter in Silkworm (<i>Bombyx mori</i>)

doi:10.13523/j.cb.20180203

China Biotechnology

2018, Vol. 38

Issue (2): 13-17 DOI: 10.13523/j.cb.20180203

Orginal Article

Cloning and Activity Analysis of a Midgut-specific Promoter in Silkworm (Bombyx mori)

Jia-zhen WANG¹,Lun-guang YAO¹,Feng WANG²,Yun-chao KAN¹,Jin-ping LUO¹,Qian-qian HUANG¹,Jian-ping DUAN^1*(

)

1 Henan Provincial Key Laboratory of Funiu Mountain Insect Biology, Nanyang Normal University, Nanyang 473061, China
2 State Key Laboratory of Silkworm Genome Biology, Chongqing 400716, China

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Abstract

The midgut is not only a digestive organ, but also a physiological barrier against invasive pathogens in silkworm, Bombyx mori. To clone and identify a novel midgut-specific promoter in silkworm, firstly detected the expression characteristics of a candidate gene BmP56 of tissue-specific expression by RT-PCR, and found that it midgut-specifically expressed. Furthermore, cloned its upstream regulatory region P56, and constructed a transgenic vector pBac[P56DsRedSV40,3×P3EGFP] of the expression of red fluorescence protein gene DsRed drived by the upstream regulatory region P56. By the way of microinjection and fluorescence screening, finally obtained the transgenic silkworm. The expression detection showed that the reporter gene DsRed only expressed in the midgut of transgenic silkworm, in consistent with the expression mode of BmP56, indicating that the upstream regulatory region P56 is an active, midgut-specifcally expressed promoter in silkworm.

Key words： Bombyx mori Midgut Specific promoter Transgene

Received: 05 September 2017 Published: 21 March 2018

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	Jia-zhen WANG
	Lun-guang YAO
	Feng WANG
	Yun-chao KAN
	Jin-ping LUO
	Qian-qian HUANG
	Jian-ping DUAN

Cite this article:

Jia-zhen WANG,Lun-guang YAO,Feng WANG,Yun-chao KAN,Jin-ping LUO,Qian-qian HUANG,Jian-ping DUAN. Cloning and Activity Analysis of a Midgut-specific Promoter in Silkworm (Bombyx mori). China Biotechnology, 2018, 38(2): 13-17.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180203 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I2/13

Table 1 Primers used in this study

Fig.1 Schematic diagram of the transgenic vector pBac[P56DsRedSV40,3×P3EGFP]

Fig.2 The expression profile of three midgut-specifically expressed candidate genes in silkworm gene microarray
T: Testis; O: Ovary; H-F: Female head; H-M: Male head; F-F: Female fat body; F-M: Male fat body; I-F: Female integument; I-M: Male integument; Mi-F: Female midgut; Mi-M: Male midgut; He-F: Female hemocyte; He-M: Male hemocyte; Ma-F: Female malpighian tube; Ma-M: Male malpighian tube; A/M-F: Female anterior and middle silkgland; A/M-M: Male anterior and middle silkgland; P-F: Female posterior silkgland; P-M: Male posterior silkgland

Fig.3 Expression profile of BmP56 in different tissues
M: DNA marker; 1: Testis; 2: Ovary; 3: Fat body; 4: Integument; 5: Midgut; 6: Hemocyte; 7: Malpighian tube; 8: Anterior silkgland; 9:Middle silkgland; 10: Posterior silkgland; 11: Trachea; 12:Head

Fig.4 The digested map of T-P56 by restriction enzyme
M: Trans2K plus II DNA marker; 1: The digested result of T-P56; 2: The control of plasmid T-P56

Fig.5 The fluorescent observation of transgenic silkworm

Fig.6 The expression profile of DsRed in different tissues
M: Trans2K plus II DNA marker; 1: Midgut tissue of transgenic silkworm; 2: Mixed tissues of transgenic silkworm except of midgut; 3: Midgut tissue of non-transgenic silkworm; 4: Mixed tissue of non-transgenic silkworm except of midgut


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