Cloning and Functional Analysis of CncC and Keap1 Genes in

Laboratory for Modern Silk, Soochow University, Suzhou, Jiangsu 215123, People's Republic of China. J. Agric. Food Chem. , Article ASAP. DOI: 10.1...
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Cloning and functional analysis of CncC and Keap1 genes in silkworm Jingsheng Hu, Jian Chen, Hui Wang, Tingting Mao, Jinxin Li, Xiaoyu Cheng, Jiahuan Hu, Bin Xue, and Bing Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05820 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Journal of Agricultural and Food Chemistry

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Cloning and functional analysis of CncC and Keap1 genes in silkworm

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Jingsheng Hu †, ‡, Jian Chen †, Hui Wang †, Tingting Mao †, Jinxin Li †, Xiaoyu Cheng

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, Jiahuan Hu †, Bin Xue†, B. Li †, ‡,*

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Jiangsu 215123, P.R. China.

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Jiangsu 215123, P.R. China

School of Basic Medicine and Biological Sciences, Soochow University, Suzhou,

National Engineering Laboratory for Modern Silk, Soochow University, Suzhou,

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*Correspondence should be addressed to:

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B. Li, Ph.D.

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Fax: +86 512 65880262

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E-mail: [email protected]

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Abstract: CncC/keap1-ARE is an important signaling pathway for detoxification and

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antioxidation in Diptera and Coleoptera insects. However, such signaling pathway has

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not been studied in Bombyx mori. In this study, BmCncC and Bmkeap1 genes were

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cloned, their amino acid sequences were analyzed, and each functional domain was

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mapped. Through phylogenetic analysis and sequence comparison among multiple

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species, we found that Neh1 motif of CncC was highly conserved and DLG motif was

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replaced by DMG motif in Neh2. Conformational analysis showed that Neh1 of

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BmCncC forms hairpin structure to bind DNA. DGR region of Bmkeap1 contained

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abundant β-sheets, which was involved in the recognition of Neh2. The transcription

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and expression analysis showed that both BmCncC and Bmkeap1 were highly

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expressed in the 1st instar larvae, and these two genes were expressed at high level in

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the reproductive gland, fat body and head. The transcriptional and expression levels of

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Akt and BmCncC in the fat body were significantly up-regulated and the expression

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of Bmkeap1 was down-regulated after the phoxim treatment in silkworm. The

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transcriptional levels of CncC-regulated detoxification enzymes GST, cyp4M5,

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cyp6AE2 and cyp9G3 were increased by 4.026, 5.246, 3.821 and 9.787 fold,

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respectively, while the activities of GST and CYP450 were increased by 1.521 and

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1.231 fold, respectively, after phoxim treatment. These results indicated that

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BmCncC/Bmkeap1 signaling pathway was activated by phoxim, leading to the

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expression of downstream detoxifying enzymes and detoxification of phoxim in

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silkworm.

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KEYWORDS: silkworm, BmCncC, Bmkeap1, phoxim

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INTRODUCTION

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Mammalian Nrf2 (nuclear factor erythroid 2 related factor 2) plays an important role

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in relieving exogenous oxidative stress. Recent studies have shown that

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Nrf2/keap1signaling pathway can be effectively targeted to treat diseases such as liver

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poisoning, cancer, and inflammation1. Nrf2 specifically recognizes the antioxidant

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regulatory element (ARE) that regulates the transcriptions of many detoxification

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genes and antioxidant genes2. Nrf2 is divided into 6 key functional domains, in which

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Neh1, Neh3, Neh4 and Neh5 are responsible for the Nrf2 recognition of ARE. Neh2

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and Neh6 are the key regions for the regulation of the ubiquitination of Nrf23,4. Neh2

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can recognize and bind the double glycine repeat region (DGR) of Keap15. Keap1

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contains three structural domains, which are intervention areas (IVR), BTB and DGR.

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The BTB domain binds Cullin3 through BTB-Cullin3 ligase6. The oxidative

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modification of the IVR cysteine plays an important role in the electrophilic reaction

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of Keap17.

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In insects, Nrf2 was renamed as CncC (cap 'n' collar isoform-C). The main function of

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the CncC/Keap1 signaling pathway in Drosophila melanogaster is to selectively bind

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ARE8. CncC has the function of regulating the homeostasis of redox, preventing aging,

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regulating cell proliferation, detoxification, and enhancing the resistance9-11.

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CncC/keap1-ARE signaling pathway can regulate the expression of CYP450 family

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genes, GST (glutathione S-transferase), HO-1(Heme oxygenas-1) and Superoxide

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Dismutase (SOD), and enhance their resistance to external stress12,13. The CncC level

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in the DDT resistant Drosophila strain was higher than that in the control group. The

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expression level of cyp6G1, cyp6A2 and cyp12D1 was higher than the control group

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significantly down-regulate the detoxification related genes, e.g., CYP6a8 and

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CYP6a215. CncC-Maf complex can enhance the transcriptional level of P450 family

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cyp6BQ6, cyp6BQ7 and cyp6BQ9 in Tribolium castaneum16. The transcriptional

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level of CncC is up-regulated when the expression of Keap1 is reduced or when acute

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exogenous pressure is present. When CncC is stimulated by heavy metal, ethyl ester,

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H2O2 and mitochondria-derived ROS, it regulates the transcription of the downstream

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genes17. CncC induces the expression of insect detoxifying enzyme genes and

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antioxidant genes, and regulates the resistance to pesticides.

. Interfering with the expression of CncC in Drosophila melanogaster can

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In China, silkworm is an important economic insect which produced more than 78%

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of the worldwide raw silk18. Moreover, silkworm is also an important model insect of

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Lepidoptera. In recent years, due to the widespread use of pesticides, pesticide

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residues on mulberry leaves can cause silkworm poisoning, resulting in reduced or no

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silk production19. Phoxim is one of the most widely used organophosphorus pesticides

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at present. Trace intoxication can cause mitochondrial damage and produce excessive

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ROS, which leads to oxidative stress20. Currently, there are no reports on the role of

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CncC/keap1-ARE signaling pathways in regulating the expression of detoxifying

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enzyme CYP450 and antioxidant enzyme HO-1 in Lepidoptera insects. Studying the

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function of the signal pathway of CncC/keap1 in silkworm is beneficial to elucidate

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the regulation mechanism of Lepidoptera metabolism and anti-oxidation response.

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MATERIALS AND METHODS

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Chemicals. Phoxim[O,O-diethyl O-(alpha-cyanobenzylideneamino) phosphorothioate]

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(Sigma) had a purity of 98.1%. Acetone was added to Phoxim to make the stock

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solution (100 g/L). Work solution at the concentration of 10g/L was made with double

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distilled water. Finally, distilled water was added to obtain the final concentration of 4

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mg/L21.

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Insects and Treatments. The silkworm variety “Jingsong × Haoyue” was stocked in

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the laboratory and cultivated with mulberry leaf at the condition of 26±1℃ and

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humidity of 60%~75%. The 5th instar larvae were divided into 2 groups (control

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group and treatment group) with 100 larvae in each group. Both groups were fed with

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clean water-treated mulberry leaves, 3 times per day. On the fourth day, the control

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group was fed with water-treated mulberry leaves, and the treatment group was fed

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with Phoxim-treated mulberry leaves. After 24h of treatment, silkworm was dissected

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and fat body was obtained and stored at -80℃. The leaves were soaked in the liquor

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and dry out in lab with 25℃ before feeding. The larvae were cryopreserved at each

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instar. 5th instar and third day larvae was dissected. The fat body, midgut, head,

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epidermis, hemolymph, malpighian tube, gonad and silk gland were saved at -80℃.

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RNA extraction, reverse transcription and qRT-PCR. RNA was extracted using

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the RNAiso Plus (Takara). The reverse transcription was conducted using Reverse

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Transcriptase M-MLV (RNase H-) to synthesize the first chain of cDNA. The

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qRT-PCR experiment was carried out using the SYBR Premix Ex Taq II (Tli RNaseH

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Plus). All experiments were carried out in strict accordance with the instructions. The

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relative expressions were compared with actin 3 of silkworm in the qRT-PCR

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experiment.

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SOD(NM_001043619.1),

CAT(NM_001043447.1),

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CarE(NM_001128312.1),

cyp4M5(NM_001110363.1),

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cyp9G3(EU435134.1) and GSTd(AB176691.2) in Bombyx mori. All primers are

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shown in Table 1.

The

detected

target

genes

were

Akt(EU305740.1),

HO-1(NM_001046896.1), cyp6AE2(EF415296.1),

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Gene cloning. The CncC and keap1 primers were designed according to the

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sequences in other species. CncC and keap1 were renamed by BmCncC and Bmkeap1

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in the silkworm. The genes were cloned, and the results were analyzed by the NCBI

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database Blast. After the sequence was correctly cloned, 3 'RACE and 5' RACE

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primers were designed for RACE amplification. 3 'RACE was performed using 3' end

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anchored

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dT:CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC

oligo

dT

primers

(3 (T)

'race 18VN)

to

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synthesize cDNA first chain . Nested PCR was performed using specific primers and

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3' race M primers for nested PCR. 5 'RACE was performed using the 5' -Full RACE

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Kit according to the instructions. All the PCR products were ligated to the pMD-18T

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vector and recombinant plasmids were obtained. After the restriction digestion

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confirmation, the plasmids were sequenced.

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Western blot analysis. PMSF was added to the lysate, and the fatty tissues were

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homogenized. After the samples were placed in room temperature for 10 min, they

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were centrifuged and the supernatant was saved. Western blot was performed using

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α-tubulin,

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univ(ShangHai) Bioscience Co., Inc(China). Polyclonal antibodies for BmCncC and

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Bmkeap1 were purchased from GenScript Company.

Akt,

p-Akt

antibodies

against

Drosophila

melanogaster

from

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Enzyme activity determination. The GST enzyme activity was measured using the

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commercial kits purchased from Nanjing Jiancheng Bioengineering Institute(China)

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according to the instructions. The P450 enzyme activity test kit was provided by

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Jiangsu KeJing Biological Technology Co., Ltd.(China), according to the instruction. ACS Paragon Plus Environment

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Sequence analysis. All sequences were processed by DNAMAN 8. The evolutionary

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tree was generated by MEGA 6. Comparison of sequences from different species was

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analyzed by Align software. The three-dimensional conformation was made by the

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following website: https://swissmodel.expasy.org23. The selected sequences were all

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extracted from the NCBI (https://www.ncbi.nlm.nih.gov/) database.

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Statistical analysis. All presented data were the mean values of three independent

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measurements for each sample. Data were analyzed by one-way ANOVA followed by

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Dunnett’s t-test for comparisons among groups. A difference with * p