Abstract
To elucidate the mechanism of biomineralization in Pinctada fucata, most of the researchers put their attention on the roles of matrix proteins in shell formation. Our group has identified and characterized many essential matrix proteins in the regulation of deposition of calcium carbonate crystals in both prism and nacre layers, as described in the former chapter. Meanwhile, the regulation of the transcription, translation and expression of matrix proteins and how these regulatory factors mediate the biomineralization have become a hot spot in recent years. In this chapter, we mainly assayed RACE to obtain the sequence of the members of signaling pathways and the transcriptional factors; real time-qPCR to analyze the expression pattern of these factors in different tissues and/or distinct time during shell repair and pearl sac; in situ hybridization to find out the expression location of specific genes in mantle tissue; luciferase assay and electrophoretic mobility assay (EMSA) to clarify the recruitment of transcriptional factors on promoters of matrix protein; yeast two hybridization to explore the interactions between different pathways. We demonstrate the function of NF-κB, TGFβ, Wnt signal pathway, G protein-mediated pathway and several transcriptional factors in mediating the biomineralization in Pinctada fucata. The mechanism of transcriptional regulation can give deep sight to the matrix protein expression pattern which enrich the theory of bionimeralization in Pinctada fucata.
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Supplementary Table 6.1
Supplementary Table 6.1
Chapter | Primer name | Primer sequence |
|---|---|---|
6.3.2 | 5′-GSP1 | TTATGGGGGTGGGGGTCGTATGGTGTGT |
5′-GSP2 | TCTTCTCATTTGTGCTGCTCTCC | |
3′-GSP | GCTAAAGGTGGAGATGAGGTGTT | |
sense | AACAACACTGGAAAGCCCAGACT | |
antisense | ATGCCATTGAGATGAGGGGGAAT | |
RT-FP | AGCAGCACAAATGAGAAGAA | |
RT-RP | TCCCAAATAGTATCACCAGAGC | |
6.3.3 | 5RP | CCGACAAAGGTGCGTAGGTGATGTGATT |
3RP | CCTGGAGAAGAAGATTGGGTGG | |
FLGSPF | GGCACAATTTAGGTGGAACGATCA | |
FLGSPR | GCTTTCTCTCTCTCTCTCTCTCATC | |
EPF | CCGGAATTCGGATGGCAGTCAATAGTAAAG | |
EPR | CCGCTCGAGCTATCAGGTATTGTCTAACGGTG | |
RTGSPF | AAGCAGTAGTAATCCTGGTAG | |
RTGSPR | GTGTATTCGTCTTGTTCCCT | |
6.3.4 | Nacrein-F | GGGGTACCTGAGTATCGACGAGAAACGCTTA |
Nacrein-R | CCCAAGCTTCAGTCAAATGAAGATACATCACC | |
Rel-exp-F | GCGAGCGAGCGGTGACTTA | |
Rel-exp-R | GCACAGGCGCACACATACG | |
Sense-Rel | CATCCCCGGGGAGAGCAGCAC | |
Antisense-Rel | GTGCTGCTCTCCCCGGGATG | |
Nacrein-RT-F | GGCTTTGGCGACGAACCGGA | |
Nacrein-RT-R | ACACGGGGGAGTGGTCAGGG | |
Rel-RT-F | CTCGAGTGAAAGCTTCAACA | |
Rel-RT-R | CGAGCTATACGAGCACGCAGC | |
actin-RT-F | CTCCTCACTGAAGCCCCCCTCA | |
actin-RT-R | ATGGCTGGAATAGGGATTCTGG | |
KBWTL-5 | GATCACTGCAACAGGGTCTGGTTGGAGATCCCCTCCCTTCTTGTAAGA | |
AGTTGAGGGGACTTTCCCAGGC | ||
6.4.2.1 | AR1 | GGSACDGGVTCCTGGACNCAG |
AR2 | GAGCCATGTAACGTTTGGTTCC | |
AR5R2 | CTTTGGAGATAATCATATAATGACCCG | |
AR3R1 | TTGTTACGTCACGACTCGATCCTGGG | |
AR3R2 | CGGAAAGTGGAAAAATAGATTACGG | |
ALR-cf1 | TATCAACGCAGAGTACGCGGGGAG | |
ALR-cf2 | CTGAGCTTCAAATTCAGTACCGTGG | |
AR3 | CGAGGTGTAGGGTGTTTTGTGTTC | |
AR4 | TAGGTTTTCCTGGCTCTCTTGTCG | |
SB1 | TATTGAAGCACCAGTGTTGCCTCC | |
SB2 | TCTAAAGGTGGGCTTGGTGAAACG | |
RTREL1 | AGCAGCACAAATGAGAAGAA | |
RTREL2 | TCCCAAATAGTATCACCAGAGC | |
RTIPF | AAGCAGTAGTAATCCTGGTAG | |
RTIPR | GTGTATTCGTCTTGTTCCCT | |
6.4.2.2 | BMPR1B-GSP1 | TACAGTAATCCTCGTCATCACAGCAC |
BMPR1B-NGSP1 | GAAGTTGTTGTATTGCTGATCTGGGATTAGC | |
BAMBI-GSP1 | GCAATAGGGCCTCCATAAACTGTCAACC | |
BAMBI-NGSP1 | TCCATAAACTGTCAACCCCCTCGTCCC | |
RT-PfACTIN-F | TACCGCCGCGTCATCATCAT | |
RT-PfACTIN-R | TGCCTCGGGACATCTGAACC | |
RT-PfBAMBI-F | ACTGTACAAGGAAGGCGTGTCAC | |
RT-PfBAMBI-R | GAGCTGGCATTTGTTTGGACGTG | |
RT-PfBMPR1B-F | GCACATAACACACGGCAAGGAAC | |
RT-PfBMPR1B-R | TCTGCTTGTCGGTAGGCTTCAAA | |
RT-KRMP-F | GAATGAAGTTCGCCGCTGTT | |
RT-KRMP-R | TTCCAATCCCARGGRTGACA | |
RT-PRISMALIN14-F | AAAGAAATACTTAACTGGTGCTA | |
RT-PRISMALIN14-R | CATGAGCAGCCCGGGTC | |
RT-PIF-F | TGCTGCCATCACGTGAGTATG | |
RT-PIF-R | GACTTCCCTTTCTCACACTTCCA | |
RT-MSI60-F | GAACAATGACTGGAATGACA | |
RT-MSI60-R | GGAAAGGTATCCAATAACAAC | |
ISH-BAMBI-F | GAGAAGAGGTGCTACTGC | |
ISH-BAMBI-R | GCTGGATTCTGCTTCTCG | |
ISH-BMPR1B-F | GAGTGCTGTGATGACGAGGATTAC | |
ISH-BMPR1B-R | TTGATTCACTTATATATCGCACTGC | |
RNAi-BAMBI-F | GCGTAATACGACTCACTATAGGGAGATGCGATGTTGTAAAGAGGA | |
RNAi-BAMBI-R | GCGTAATACGACTCACTATAGGGAGATTTCACGACGGATTTGTAT | |
RNAi-BMPR1B-F | GCGTAATACGACTCACTATAGGGAGACTGGCGACTCAATGTCTCAA | |
RNAi-BMPR1B-R | GCGTAATACGACTCACTATAGGGAGACTGGCATTGGTGTTGTTGAC | |
BAMBI-EcoRI | CGGAATTCATGGAGGCCCTATTGCTTC | |
BAMBI-PstI | AACTGCAGCTATACAGAAGCCACCAAGTC | |
BMPR1B-EcoRI | CGGAATTCATGGCAGACCTCTGCTGG | |
BMPR1B-PstI | AACTGCAGCTAGCTTTCTCCGGGTTTTATGAC | |
BMPR1B-BamHI | CGGGATCCCTAGCTTTCTCCGGGTTTTATGAC | |
SMAD1/5/8-ClaI | CCATCGATATGAGTTCACCCATCTCC | |
SMAD1/5/8-BamHI | CGGGATCCTCATGATACAGATGAAATTGGG | |
BMP2-ClaI | CCATCGATATGATTTACGGATTTGGACATTACC | |
BMP2-BamHI | CGGGATCCCTACCGACATCCGCATCC | |
SMAD4-EcoRI | CGGAATTCATGTTTCGGTCTAAAAGATCTACCCTC | |
SMAD4-BamHI | CGGGATCCTCACCTGTGGACGTTCAATAAAATTTC | |
6.4.4.1 | SD5-1 | GCAAACTCCTGATTGTTAAATATC |
SD5-2 | GCATGAAATGGTTCTCCAACACG | |
SD3-1 | GGTGCTCGATAGCTTACTATGAGC | |
SD3-2 | CAGTCTGCAAAATACCCCCAGG | |
Smad-cf1 | AATCGTCGTTTTGTTCTCTTCTAG | |
Smad-cf2 | CGGTATCAGTCAATATCACACAG | |
SB1 | TATTGAAGCACCAGTGTTGCCTCC | |
SB2 | TCTAAAGGTGGGCTTGGTGAAACG | |
SI1 | TTCCCCATTTACTCCACCCATC | |
SI2 | GCAACACTGGTGCTTCAATACGA | |
Pf-engrailed-ES1 | CGGCATTCTCTAACGATCAGC | |
Pf-engrailed-ES2 | CCATGAGATGTAACGCTAACAAG | |
Pf-Smad3-SB1 | TTCCCCATTTACTCCACCCATC | |
Pf-Smad3-SB2 | TCGTATTGAAGCACCAGTGTTGC | |
Pf-BMP2-BMP2F | GCGGTCGAAGAACTAAAA | |
Pf-BMP2-BMP2R | ATCCGCATCCTTCAACAA | |
6.5 | BCAT-3F1 | CGAGGAATGAAGGGGTTGC |
BCAT-3F2 | TCACGGTAGTCAGGGAAGCAT | |
BCAT-5R1 | GGGTGGAGGGGATGGGTAC | |
BCAT-5R2 | CGGCGTGTATGCCTGTTCT | |
BCAT-5 | ACAAATAAGGAACCACCAAGATG | |
BCAT-3 | CATGGTTACAGAAGTGGAATATCC | |
DVL-3F1 | CCCCTCAGCCTGGGTAGCA | |
DVL-3F2 | GAAAGACTCCGCTGAAACTCC | |
DVL-5R1 | CCAGCACTTGGCAACGACT | |
DVL-5R2 | TGATGTGCGGTTTGTGGTGT | |
DVL-5 | AATCGGACGCCATTTTACATCA | |
DVL-3 | TTGCGGTACTGCGTTGAGGT | |
TCF-3F1 | GGGAATCCACCAGAAGACAAA | |
TCF-3F2 | AAAGAAGTGCGAGCCCAAGT | |
TCF-5R1 | TGGGCTCGCACTTCTTTCAT | |
TCF-5R2 | ACATCAAGCCTGGGTGGG | |
TCF-5 | TTGTTTTTAACTTTGACAATGCCG | |
TCF-3 | AATGTCTACTACCGTTGCCATGTTC | |
BCAT-F | AACCATTCTACAAGCAGGCGATA | |
BCAT-R | TCCCTCAGTGGTGCGTGGTTAG | |
DVL-F | TATTAGCAGGGCTTCGTCATTCA | |
DVL-R | CCCGTCTCCTCCTTTGTTACTCT | |
TCF-F | TGAAGGCGAGGAAGAGCAGA | |
TCF-R | CCCAAGGGAGAAGATCCATTAGG | |
actin-F | CTCCTCACTGAAGCCCCCCTCA | |
actin-R | ATGGCTGGAATAGGGATTCTGG | |
18S-F | AGGACCTCGGTTCTATTTTGTTGG | |
18S-R | TTTCACCTCTAACACCGCAATACC | |
6.6.2 | 5AGSP1 | GTACA GGGGGATTCAGTGTGCTCATGG |
3AGSP2 | CATGAGCACACTGAATCCCCCTGTACC | |
AeF | CGCGGATCCATGGGATGTTTTAAGCCTAAGGGAG | |
AeR | CCGCTCGAGCTATCACAGAAGCTCGTATTGTCGCAG | |
6.6.3 | GSP3 | CCTGTAAATGCTGTCGTCTGCTGACCT |
6.7.2 | POU3-3F1 | GCCATCCGCCCAAGAAAT |
POU3-3F2 | ACCAAATGGAGAACTGCTGATG | |
POU3-5R1 | CCTGTGGTCGAGTCCGCTTCTTCAA | |
POU3-5R2 | CCAGGAATCACCGCCCGTAATCG | |
POU3F4-5 | AATAAGCCAGGACCAAGTAAAGG | |
POU3F4-3 | CATTCCCAACACCTTACTTTCTG | |
AS-1F | GGGATAGCCATTCTTATGTGTCT | |
AS-1R1 | AGCATCACTGGGCTCCGATA | |
AS-1R2 | ACTACAGCACCAGCGGCAG | |
P14-1F | GGGACACAGTCCCAAAGAAATAC | |
P14-1R1 | AACAAACATGAGCAGCCCG | |
P14-1R2 | GATTCGCTGGATTCCGCTA | |
AS-GWR1 | TGACATCAGAAACGAATAAACAGGA | |
AS-GWR2 | TATCAGCATTTCCTTGAGCAGCCAT | |
AS-GWR3 | ACAATACCAAACAAACGAAATCACT | |
AS-GWR4 | GTCCGTTACCGAAAGCGAAAGA | |
AS-GWR5 | TAACTGACAAGGATCGGGCTAG | |
AS-GWR6 | CTAGAGCGTGCTTTGTGGAACT | |
ASP-F | TCGGATGCCCTGTAGAGT | |
ASP-R | AATCACTTACAAGTAACGCTTA | |
P14-GWR1 | CACAAGCAGCTAAGGCAAGGA | |
P14-GWR2 | GAATGGAAATGTAGCACCTGTTGAT | |
P14-GWR3 | GCTTGCCGAAAATCTTTTACGA | |
P14P-F | AAGGCTTCTGCGGGGTTT | |
P14P-R | TCGAACTCCGGTGCCAGAG | |
POU2F1-F | CTCCGATTTCCGCCCCTC | |
POU2F1-R | GCTAGAATGATTTGTCCGTTAGTCG | |
POU3F4-F | AGATGTGGGACTTGCTTTGGG | |
POU3F4-R | TTCTTTCGCTTTCTACCTTGTGC | |
AS-F | GAAGGGGATAGCCATTCTTATGTG | |
AS-R | GCATCACTGGGCTCCGATACTA | |
P14-F | CTATTTCCCGCGTTTCTCCTATC | |
P14-R | TCCTCCGTAACCACCGTTAAATC | |
actin-F | CTCCTCACTGAAGCCCCCCTCA | |
actin-R | ATGGCTGGAATAGGGATTCTGG | |
18S-F | AGGACCTCGGTTCTATTTTGTTGG | |
18S-R | TTTCACCTCTAACACCGCAATACC | |
dsPOU3F4- 1F | GGATCCTAATACGACTCACTATAGGTTGGCGGA CCCTACTCA | |
dsPOU3F4- 1R | CTTCTTTCGCTTTCTACCTT | |
dsPOU3F4- 2F | TTGGCGGACCCTACTCA | |
dsPOU3F4- 2R | GGATCCTAATACGACTCACTATAGGCTTCTTTCG CTTTCTACCTT | |
dsGFP-1F | GGATCCTAATACGACTCACTATAGGATGGTGAG CAAGGGCGA | |
dsGFP-1R | ACTTGTACAGCTCGTCCATG | |
dsGFP-2F | ATGGTGAGCAAGGGCGA | |
dsGFP-2R | GGATCCTAATACGACTCACTATAGGACTTGTAC AGCTCGTCCATG | |
P14B1F | GTCAAAATTGCAAATTTAATTAGATTCAAAAAC GCACGG | |
P14B1R | CCGTGCGTTTTTGAATCTAATTAAATTTGCAATT TTGAC | |
P14B1WTF | GTCAAAATTGCAAATGGAAGGAGAGGCAAAA ACGCACGG | |
P14B1WTR | CCGTGCGTTTTTGCCTCTCCTTCCATTTGCAATT TTGAC | |
P14B2F | GATTAGACTATAATTAATTTTAATTATTCTGTGCA ATAT | |
P14B2R | ATATTGCACAGAATAATTAAAATTAATTATAGTC TAATC | |
P14B2WTF | GATTAGACTATGGTTGGTTTTAATGGTTCTGTGC AATAT | |
P14B2WTR | ATATTGCACAGAACCATTAAAACCAACCATAGT CTAATC | |
ASB1F | TTGTCAGTTATTAAAATACCTATAACGTATTTTA ATCACAATA | |
ASB1R | TATTGTGATTAAAATACGTTATAGGTATTTTAATA ACTGACAA | |
ASB2F | TTAATCACAATATGTATGCATGATTGTTCAAAAA ATTGTGATG | |
ASB2R | CATCACAATTTTTTGAACAATCATGCATACATAT TGTGATTAA | |
ASB3F | AAAATTGTGATGTTTCCATAAATTATAGGTACTG AACCTCAAT | |
ASB3R | ATTGAGGTTCAGTACCTATAATTTATGGAAACAT CACAATTTT | |
P14B1F | GTCAAAATTGCAAATTTAATTAGATTCAAAAAC GCACGG | |
P14B1R | CCGTGCGTTTTTGAATCTAATTAAATTTGCAATT TTGAC | |
P14B1WTF | GTCAAAATTGCAAATGGAAGGAGAGGCAAAA ACGCACGG | |
P14B1WTR | CCGTGCGTTTTTGCCTCTCCTTCCATTTGCAATT TTGAC | |
P14B2F | GATTAGACTATAATTAATTTTAATTATTCTGTGCA ATAT | |
P14B2R | ATATTGCACAGAATAATTAAAATTAATTATAGTC TAATC | |
P14B2WTF | GATTAGACTATGGTTGGTTTTAATGGTTCTGTGC AATAT | |
P14B2WTR | ATATTGCACAGAACCATTAAAACCAACCATAGT CTAATC | |
ASB1F | TTGTCAGTTATTAAAATACCTATAACGTATTTTA ATCACAATA | |
ASB1R | TATTGTGATTAAAATACGTTATAGGTATTTTAATA ACTGACAA | |
ASB2F | TTAATCACAATATGTATGCATGATTGTTCAAAAA ATTGTGATG | |
ASB2R | CATCACAATTTTTTGAACAATCATGCATACATAT TGTGATTAA | |
ASB3F | AAAATTGTGATGTTTCCATAAATTATAGGTACTG AACCTCAAT | |
ASB3R | ATTGAGGTTCAGTACCTATAATTTATGGAAACAT CACAATTTT | |
6.7.3 | c-Jun186-f | TCTCCKCACGTKGGKCTKCTCAARC |
c-Jun873-f | CAAGTAGCWGAGCTCAARCARAAAG | |
c-Jun35-r | TCATCGTAGAAAGTAGTTTCCAT | |
c-Jun489-r | GAGTGATGAAATGACGAGTTAGAATCATC | |
c-Jun1152-f | AATGGCAATATTATGTCTCTTGTTAGCAC | |
cjun-qf | CGGTGGCAAAACAAAACAGC | |
cjun-qr | TGGGGCACGGGTAAAACTT | |
6.7.4 | yy10-f | GGCGACACCCTCTACATTGCC |
yy95-f | CGACAGAAATCAAACATGGCGTC | |
yy1391-r | CCTCGTATGTGTATCCTATTAACATGGC | |
yy833-3f | TAGCAGAATTTGCTAGTCTCCAGCC | |
yy1098-3f | TTTTCAGTGTACGTTTGAGGGATGC | |
yy1251-3f | TATACTGACACATGCAAAAGCAAATAGC | |
yy776-5r | AATTCCCCCTGGGGGAAGTTTTTTCC | |
yy545-5r | CTCCCAAAGTTCCAGCCCCGAGAAG | |
yy238-5r | TGGAAGCGGTTGTAACGCTATCATAGGTTG | |
yy92-5r | CGACGCCATGTTTGATTTCTGTCGAAG | |
yy-confirmf | AGCAGTGGTATCAACGCAGAG | |
yy-confirmr | CCGATTCCCAAAACAGAGTAA | |
yy-ef | CCATCGATATGGCGTCGGTTATCGGTTC | |
yy-er | AACCTCGAGCTATGACTCCTCGTATGTGTATCCTATTAAC | |
yy-sf | ATCCCAAACAGTTAGCAGA | |
yy-sr | CTTACCGCATCCCTCAA | |
β-actin-qf | CTCCTCACTGAAGCCCCCCTCA | |
β-actin-qr | ATGGCTGGAATAGGGATTCTGG | |
yyqf | AGAGGAAGATGCGTCAGTTTCAG | |
yyqr | GTTTGGGATCACTAAGGTCAAGG | |
P39-qf | CTGGAATGAGAGGATATG | |
P39-qr | TGCTGCTGTAATAACTATA | |
ACCBP-qf | GACATGGAACAAAGATGGTGGA | |
ACCBP-qr | CTGTGGCTGGAATGGTTGG | |
Pif-qf | TGCTGCCATCACGTGAGTATG | |
Pif-qr | GACTTCCCTTTCTCACACTTCCA | |
Aspein-qf | TGATAGTGAAGACGATGA | |
Aspein-qr | TGTCATCATCATCATCATC | |
6.7.5 | sp157-3f | CTAGCCGCTCAATGTAACAAGATC |
sp1311-3f | TACSTGGCAGACCATCCAGCT | |
sp1324-3r | TGGATGGTCTGCCASGTAACTTG | |
sp1318-3r | CASGTAACTTGGCCATTWGGRCC | |
sp180-5r | GATCTTGTTACATTGAGCGGCTAGCATTG | |
sp1705-5r | CATTGAGCGGCTAGCATTGCTAATGG | |
sp-85r | AAGGATGGAACCCCTTCCCTACA | |
sp-43r | GCGGGCTTTTGTTTGTGATCTTG | |
spqf | CTCACGGCATCAAGTCTGAAAT | |
spqr | CGGACTGCTGAACGGGTAAT | |
Pif-qf | TGCTGCCATCACGTGAGTATG | |
Pif-qr | GACTTCCCTTTCTCACACTTCCA | |
aspein-qf | TGATAGTGAAGACGATGA | |
aspein-qr | TGTCATCATCATCATCATC | |
Pearlin-qf | TACTCATACTGCTGGATA | |
Pearlin-qr | TATCATCATCGGTGTAAC | |
KRMP-ef1 | AAAGGTACCGTACATTTTTGACATATTTTTCACACTCTTTG | |
KRMP-er1 | AACCTCGAGTCTGAACCTGTAGAAAAATATTATTCATTAGATTC | |
pearlin-ef | AAAGGTACCAAAATTACTGTGTCCAAAGG | |
pearlin-er | AAACTCGAGAGACAAAAGATATTTCTCTTTAACC | |
p39-ef | AAAGGTACCCCGTACTTCATCATTGTAGCTCCG | |
p39-er | AACCTCGAGGACAATTTTGTAACCTGAAACAAAG | |
spef | AAAGGTACCATGCTAGCCGCTCAATGTAACAAGA | |
sper | GGATCGATTCACTTCATAGCACGGTTATCCCTG |
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Zhang, R., Xie, L., Yan, Z. (2019). Molecular Regulation Mechanism of Biomineralization of Pinctada fucata . In: Biomineralization Mechanism of the Pearl Oyster, Pinctada fucata. Springer, Singapore. https://doi.org/10.1007/978-981-13-1459-9_6
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DOI: https://doi.org/10.1007/978-981-13-1459-9_6
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