Cloning and Functional Analysis of CncC and Keap1 Genes in

Subscriber access provided by Kaohsiung Medical University

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

Journal of Agricultural and Food Chemistry

1 2

Cloning and functional analysis of CncC and Keap1 genes in silkworm

3

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

4

†

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

5 6

†

7

Jiangsu 215123, P.R. China.

8

‡

9

Jiangsu 215123, P.R. China

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

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

10 11 12 13 14

*Correspondence should be addressed to:

15

B. Li, Ph.D.

16

Fax: +86 512 65880262

17

E-mail: [email protected]

18

ACS Paragon Plus Environment


19

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

20

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

21

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

22

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

23

mapped. Through phylogenetic analysis and sequence comparison among multiple

24

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

25

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

26

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

27

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

28

and expression analysis showed that both BmCncC and Bmkeap1 were highly

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

expression of downstream detoxifying enzymes and detoxification of phoxim in

39

silkworm.

40 41

KEYWORDS: silkworm, BmCncC, Bmkeap1, phoxim

42 43 44


Page 2 of 22

Page 3 of 22


45

INTRODUCTION

46

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

47

in relieving exogenous oxidative stress. Recent studies have shown that

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

of Keap17.

59 60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

14

70

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

71

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

72

cyp6BQ6, cyp6BQ7 and cyp6BQ9 in Tribolium castaneum16. The transcriptional

73

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

74

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

75

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

76

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

77

antioxidant genes, and regulates the resistance to pesticides.

. Interfering with the expression of CncC in Drosophila melanogaster can

78



79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90 91

MATERIALS AND METHODS

92

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

93

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

94

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

95

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

96

mg/L21.

97 98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109 110

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

111

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


Page 4 of 22

Page 5 of 22


112

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

113

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

114

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

115

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

116

experiment.

117

SOD(NM_001043619.1),

CAT(NM_001043447.1),

118

CarE(NM_001128312.1),

cyp4M5(NM_001110363.1),

119

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

120

shown in Table 1.

The

detected

target

genes

were

Akt(EU305740.1),

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

121 122

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

123

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

124

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

125

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

126

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

127

anchored

128

dT:CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC

oligo

dT

primers

(3 (T)

'race 18VN)

to

129

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

130

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

131

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

132

vector and recombinant plasmids were obtained. After the restriction digestion

133

confirmation, the plasmids were sequenced.

22

134 135

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

136

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

137

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

138

α-tubulin,

139

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

140

Bmkeap1 were purchased from GenScript Company.

Akt,

p-Akt

antibodies

against

Drosophila

melanogaster

from

141 142

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

143

commercial kits purchased from Nanjing Jiancheng Bioengineering Institute(China)

144

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

145

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


146 147

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

148

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

149

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

150

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

151

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

152 153

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

154

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

155

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

Cloning and Functional Analysis of CncC and Keap1 Genes in

Recommend Documents