Supporting information

Table S1 Expression level of recombinant proteins in the silk gland of transgenic silkworm

Exogenous protein / Silkworm strain / Promoter / Expression level of procucts / Reference
antibacterial silk / GaoBai / Fib-L / null / Li et al. 2015
human acidic fibroblast growth factor (hFGF1) protein / Dazao / Ser1 / approximate 0.07% of the cocoon shell weight / Wang Fet al. 2014
Infectious bursal disease virus (IBDV) capsid protein VP2 / D9L / Ser1 / a yield of 3.33 mg (purity > 90%) purified from 30g cocoon / Xu Het al. 2014
Spider dragline silk / C515 / Fib-H / 0.37 to 0.61% (w/w) native silkworm fibroin / Kuwana et al. 2014
Single-chain variable fragment / pnd-w1 / Fib-L / 5%-10% of the total Fib-L / Sato et al. 2014
Human insulin-like growth factor / Dazao×Haoyue / Sericin-1 / approx 162.7 ng/g of cocoon shell weight / Song et al. 2014
DsRed / Dazao / Ser1 / 9.5 % (w/w) of cocoon shell weight / Wang et al. 2013
EGFP, DsRed &Monomeric Kusabira orange / pnd-w1 / Fib-H / null / Iizuka et al. 2013
Human granucyto-macrophage / GaoBai / Fib-H / approx 2.70 ng/g of freeze dried posterior silk gland / Xue et al. 2012
Partial spider silk fibroin sequence / pnd-w1 / Fib-H / approx 2%-5% (w/w)of cocoon shell weight / Teuléet al. 2012
Human insulin-like growth factor / GaoBai / Fhx / approx 150 ng/g of fresh posterior silk gland / Li et al. 2011
Ca binding sequence / pnd-w1 / Fib-H / Null / Nagano et al. 2011
Partial fibronectin sequence &Partial collagen sequence / pnd-w1 / Fib-L / Null / Nakazawa et al. 2010
EGFP / N4 / Fib-H / 15% (w/w) of silk fibroin / Zhao et al. 2010A
Non-triple helical collagen α chain / pnd-w1 / Ser1 / 8% of the dried cocoon silk / Adachi et al. 2010
EGFP / pnd-w1-Nd -Sd pnd-w1 / Ser1 / 500 μg per larva / Tatematsu et al. 2010
Partial spider silk fibroin sequence / pnd-w1 / Fib-H / 5% (w/w) of silk fibroin / Zhu et al. 2010
Mouse monoclonal antibody / pnd-w1 / Ser1 / 1.1% of the dried cocoon silk / Iizuka et al. 2009
Human μ-opioid receptor / pnd-w1 / Fib-L / 150-250 ng/g of fresh silk gland / Tateno et al. 2009
Human insulin-like growth factor-I / Haoyue / Ser1 / 2.44 ng/g of middle silk gland / Zhao et al. 2009
Feline interferon / pnd-w1 / Fib-H / 6% of the dried cocoon silk / Kurihara et al. 2007
Human serum albumin / pnd-w1 / Ser1 / 0.3% of the dried cocoon silk / Ogawa et al. 2007
Collagen & Fibronectin derived peptides / pnd-w1 / Fib-L / 0.2% & 0.6% of the dried cocoon silk / Yanagisawa et al. 2007
Mini human collagen & rolyl-hydroxylase α-subunits / pnd-w1 / Fib-L / Null / Adachi et al. 2006
Human basic fibroblast growth factor / pnd-w1 / Fib-L / 0.04% of the fibroin / Hino et al. 2006
Fib-L-GFP / C108 & Nd-Sd / Fib-L / 10% of the dried cocoon silk / Inoue et al. 2005
Human type III procollagen / pnd-w1 / Fib-L / 0.8% of the dried cocoon silk / Tomita et al. 2003

Table S2Primer sequences

Functions / Primers / Sequences
sqRT-PCR / Bm30Kc6 / S-AGACCGCTATCGCCAAATGC
A-CAACTTGAGGGCGTGATGGT
Bm30Kc12 / S-CAGCATGAGCCCTTCTAACC
A-GGGCCTTGAAGTACACTCTG
Bm30Kc19 / S-ACAGGACTCGACGCTCCAAAG
A-GCTGCTCATTATCATTGGTTCG
Bm30Kc21 / S-GCATACGGCGATGGTAAGGA
A-CGAAGCGTCCACAATCCTAC
Bm30Kc23 / S-GACTCATCATGGCCGGAAAC
A-TTTCGTACTTGGCGGGCTGGA
Rp49 / S-GCATCAATCGGATCGCTATG
A-GGACCTTACGGAATCCATTTG
qRT-PCR / BmAtg6 / S-GTTATACGGTTCGGGTGG
A-TGGAGTACGCATGTGGTG
BmAtg8 / S-AAGGCTAGGCTTGGAGAC
A-CAGATGTGGGTGGAATGA
BmDronc / S-TGTGGCTGTCTTCCTTC
A-ATCTAAGTCTGTGCCCTC
BmECR / S-CCACGATGCCTTTACCAATG
A-GTCGAGGTGCAGGACCTTTC
BmE74 / S-ACCGCGTTCGACAACTTCGATAC
A-CTGCAATCTTTGCTGCCCGTT
Bm18S rRNA / S-CGATCCGCCGACGTTACTACA
A-GTCCGGGCCTGGTGAGATTT

Identification of the transgenic mutation system and silk gland morphology

Fig.S1. Identification of thetransgenic mutationsystem (A) and silk gland morphology (B) at day 6 of 5th instar larvae.WT:wild-type of TBH; TBH: mutant (Hpl/Hpl);ASG: anterior silk gland;MSG:middle silkgland;PSG:posterior silk gland. Arrow in-frameindicates the larval eyes.Bar=2mm.

Internal structures of the middle silk gland during larval-pupal metamorphosis

Fig. S2. Internal structures of silkworm middle silk gland during larval-pupal metamorphosis. A cross-section of the silk glands stained by HE.WT:wild-type of TBH; TBH: mutant (Hpl/Hpl); V7:day 7 of the 5th instar larva; S2: hour 24 of the spinning stage; PP1: the pre-pupal stage (at 48 h after the spinning stage); P1: day 1 of the pupal stage. N:Nucleus; V: Vacuole; M: Membrane; A: Autophagosome. Bar=400 μm.

Detection of DNA ladder in posterior silk glands during the larval-pupal stage

Fig. S3. Detection of DNA ladder in silkwormposterior silk glands during the larval-pupal stage.M indicates DNA molecular weight marker.V7:day 7 of the 5th instar larva; W: the wandering stage; S2: hour 24 of the spinning stage; PP1: the pre-pupal stage (at 48 h after the spinning stage); P17: day 1 of the pupal stage.

Gene expression of 30Ks in silkwormposterior silk glands.

Fig.S4. Gene expression of 30Kc6, 30Kc12, 30Kc19 and 30Kc23 in silkwormposterior silk glands. qRT-PCR was used to determine transcript levels of these genes. Transcript levels ofBmRp49 gene were taken as an internal reference. W: the wandering stage; S1, S2 and S3 are hour 12, hour 24 and hour 36 of the spinning stage, respectively. PP1 and PP2: the pre-pupal stage at 48 h and 54 h after the spinning stage, respectively; P1: day 1 of the pupal stage.

Number of egg laid

Fig.S5.Number of eggs laidby WT and TBH. (A) A single moth laid eggsalone after 35h at 26–27°C and darkness. (B) Egg numberchart of picture A. WT:wild-type of TBH; TBH: mutant (Hpl/Hpl). **P0.01, n=3.

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