Authors: Lu-Ping Zheng A,B Lin Hou B* Miao Yu B Xiang Li B Xiang-Yang Zouc

Title: Cloning and the Expression Pattern of Spätzle gene during Embryonic Development and Bacterial Challenge in Artemia sinica

Authors: Lu-ping Zheng a,b Lin Hou b* Miao Yu b Xiang Li b Xiang-yang Zouc**

Journal: Molecular Biology Reports

Affiliation: aResearch institute of Integrated Traditional and Western Medicine, Dalian Medical University, Dalian, 116044, China

bCollege of Life Sciences, Liaoning Normal University, Dalian, 116029, PR China

cDepartment of Biotechnology, Dalian Medical University, Dalian, 116040, China

E-mail address: * Corresponding author: Tel: 86-411-84258306. E-mail address:

**Co-corresponding author: Tel: 86-411-86110296. E-mail address:

Supplementary Figures S1

Supplementary Figures S1: The putative open reading frame and corresponding Amino-acids sequence of Spätzle. The signal peptide is underlined and the putative O-and N-linked glycosylation sites are showed in bold. ﹡denotes terminator. Conserved cysteines are shaded and numbered. The paired numbers (1–1, 2–2, 3–3) indicate the intrachain disulfide linkage. Cys4 forms an intermolecular disulfide bond with its counterpart in another subunit. The disulfide bond connections mentioned are all predictions. The proteolytic activation site is indicated with “‖”.

Supplementary Figures S2

Supplementary Figures S2: Phylogenetic analysis of Spätzle from A. sinica and other speices. Neighbour-joining phylogenetic tree was constructed based on the sequence as below utilizing the sequence analysis tool MEGA4.0: DmSPZ1, D. melanogaster Spätzle-1 (GenBank Accession Numeber NP_524526); DmSPZ2, D. melanogaster Spätzle-2 (ACZ94621); DmSPZ3, D. melanogaster Spätzle-3 (AAF52574); DmSPZ4, D. melanogaster Spätzle-4 (NP_609504); DmSPZ5, D. melanogaster Spätzle-5 (AAF47694); DmSPZ6, D. melanogaster Spätzle-6 (AAF47261); TcSPZ3, Tribolium castaneum Spätzle-3 (NP_001153625); TcSPZ4, T. castaneum Spätzle-4 (EFA09263); TcSPZ5, T. castaneum Spätzle-5 (EEZ97725); TcSPZ6, T. castaneum Spätzle-6 (NP_001164082); AsSPZ, A. sinica Spätzle; BmSPZ1, Bombyx mori Spätzle-1 (NP_001108066); ApSPZ1, Acyrthosiphon pisum Spätzle-1 (NP_001153590); ApSPZ3, A. pisum Spätzle-3 (NP_001153591); ApSPZ4, A. pisum Spätzle-4 (NP_001153592); MsSPZ1, Manduca sexta Spätzle-1 (ACU68554); AgSPZ, Anopheles gambiae Spätzle-like protein (XP_557166); NvSPZ1, Nasonia vitripennis Spätzle-1 (XP_001606529); AmSPZ3, Apis mellifera Spätzle-3 (XP_001121955). Pink box donates Spätzle-1 family; blue box donates Spätzle-4 family; yellow box donates Spätzle-1 family and purple box donates Spätzle-6 family.

Supplementary Figures S3

Supplementary Figures S3: Sequence alignment of Spätzle-4 members from several species. Identical amino acid residues are indicated by black box. Less conserved residues are indicated by grey box. Somewhat similar residues are indicated by pale grey box. The alignment is optimized by introducing gaps using the Clustal X1.8program.