SPECIAL ECONOMIC ANIMAL NUTRITION

Study on 30K Protein Phosphorylation of Silkworm Haemolymph under Heat Shock

  • LI Na ,
  • JIA Manli ,
  • YANG Guiming ,
  • LI Jisheng
Expand
  • 1. Sericulture Institute of Chengde Medical University, Hebei Universities R & D Centre for Sericulture and Specialty Enabling Technologies, Chengde 067000, China;
    2. College of Animal Sciences, Zhejiang University, Hangzhou 310029, China

Received date: 2015-12-18

  Online published: 2016-06-27

Abstract

This experiment was conducted to investigate the expression changes of phosphorylated protein in silkworm haemolymph under heat shock, and provided a reference for further study of its function. Two groups were designed, and 30 fifth-instar silkworms (half male and half female) were randomly divided in each group, and treated with heat shock under 40℃ for 0 (control group) and 10 min (heat shock group), respectively. The differentially expressed phosphorylation proteins were studied utilizing two-dimensional electrophoresis, Pro-Q Diamond dye and mass spectrometry. The results showed as follows: 1) amount of proteins were accumulated in the 30 ku area of silver staining map and revealed as phosphorylated proteins by Pro-Q Diamond dyeing. 2) Three 30K proteins were identified as PBMHPC-6, PBMHPC-19 and PBMHPC-12 by multiple sampling and mass spectrometry identification. 3) Compared with 0 min heat shock, the expression levels of three 30K proteins were significantly up-regulated after 10 min heat shock (P<0.05). It is concluded that fifth-instar silkworm haemolymph are rich in 30K proteins. These proteins not only play important roles in lipid transport, but also may involve in heat shock response.

Cite this article

LI Na , JIA Manli , YANG Guiming , LI Jisheng . Study on 30K Protein Phosphorylation of Silkworm Haemolymph under Heat Shock[J]. Chinese Journal of Animal Nutrition, 2016 , 28(6) : 1720 -1725 . DOI: 10.3969/j.issn.1006-267x.2016.06.012

References

[1] RAGGIASCHI R,GOTTA S,TERSTAPPEN G C.Phosphoproteome analysis[J].Bioscience Reports,2005,25(1/2):33-44.
[2] 杨策,王正国,朱佩芳.蛋白质组中蛋白质磷酸化研究进展[J].生理科学进展,2004,35(2):119-124.
[3] KIM E J,PARK H J,PARK T H.Inhibition of apoptosis by recombinant 30K protein originating from silkworm hemolymph[J].Biochemical and Biophysical Research Communications,2003,308(3):523-528.  
[4] SAKAI N,MORI S,IZUMI S,et al.Structures and expression of mRNAs coding for major plasma proteins of Bombyx mori[J].Biochimica et Biophysica Acta:Gene Structure and Expression,1988,949(2):224-232.  
[5] UJITA M,KIMURA A,NISHINO D,et al.Specific binding of silkworm Bombyx mori 30-kDa lipoproteins to carbohydrates containing glucose[J].Bioscience,Biochnology,and Biochemistry,2002,66(10):2264-2266.  
[6] KIM E J,RHEE W J,PARK T H.Isolation and characterization of an apoptosis-inhibiting component from the hemolymph of Bombyx mori[J].Biochemical and Biophysical Research Communications,2001,285(2):224-228.  
[7] PIETRZYK A J,BUJACZ A,ŁOCHYÑSKA M,et al.Isolation,purification,crystallization and preliminary X-ray studies of two 30 kDa proteins from silkworm haemolymph[J].Acta Crystallographica Section F,2011,67(3):372-376.  
[8] GOLDSMITH M R,SHIMADA T,ABE H.The genetics and genomics of the silkworm,Bombyx mori[J].Annual Review of Entomology,2005,50:71-100.
[9] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1/2):248-254.
[10] ZHOU Z H,YANG H J,CHEN M,et al.Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet[J].Journal of Proteome Research,2008,7(12):5103-5111.  
[11] GHARAHDAGHI F,WEINBERG C R,MEAGHER D A,et al.Mass spectrometric identification of proteins from silver-stained polyacrylamide gel:a method for the removal of silver ions to enhance sensitivity[J].Electrophoresis,1999,20(3):601-605.  
[12] LI J S,YE L P,LAN T Y,et al.Comparative proteomic and phosphoproteomic analysis of the silkworm (Bombyx mori) posterior silk gland under high temperature treatment[J].Molecular Biology Reports,2012,39(8):8447-8456.  
[13] HUANG H D,LEE T Y,TSENG S W,et al.KinasePhos:a web tool for identifying protein kinase-specific phosphorylation sites[J].Nucleic Acids Research,2005,33(Suppl.2):W226-W229.
[14] SUN Q,ZHAO P,LIN Y,et al.Analysis of the structure and expression of the 30K protein genes in silkworm,Bombyx mori[J].Insect Science,2007,14(1):5-14.
[15] YANG J P,MA X X,HE Y X,et al.Crystal structure of the 30 K protein from the silkworm Bombyx mori reveals a new member of the β-trefoil superfamily[J].Journal of Structural Biology,2011,175(1):97-103.  
[16] 吴能表,朱利泉,王小佳.温度胁迫对甘蓝蛋白质磷酸化的影响[J].西南农业大学学报:自然科学版,2004,26(2):198-200.
[17] WU H Y,TSENG V S M,LIAO P C.Mining phosphopep tide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis[J].Journal of Proteome Research,2007,6(5):1812-1821.  
Outlines

/