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Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage
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Agricultural Sciences in China 2007, 6 ( 2 ) :249-254
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ScienceDirect
February 2007
Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th lnstar Stage Wei-chengl-5, GAO Qi-kang2, CHEN Jin-e1.5, YE Jianl, QIAN Yang-wen3,LI Jim-yingl, LU Hua-yun4,
MENG Zhi-qi5, NI Chun-xiao4 and ZHONG Bo-xiong’ 1
Laboratory of Molecular Biology of Silkworm and HoneybeeKollege of Animal Science, Zhejiang University, Hangzhou 310029, P.R.
China Institute of Applied Entomology, Zhejiang University, Hangzhou 310029, P.R. China 3 College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310029, P.R. China 4 Department of Speciality Good Seed Extension, Hangzhou 310002, P.R. China Laboratory of Moleculnr Biology of Insect, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R.China 2
J
Abstract The posterior silk gland (PSG) of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 7580% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis (2-DE) and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 (Dl) and day 4 (D4) in the 5th instar stage from five different strains of silkworm (Bombyx dori). While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.
Key words: silkworm (Bornbyx mori), silk gland, protein, 2-DE, image analysis
INTRODUCTION Silkworm is an economically important insect and a legitimate model for Lepidopteran genomics and genetics with 28 pairs of chromosomes. The silk gland of silkworm is a paired tubular gland that can be divided into anterior silk gland (ASG), middle silk gland (MSG), and posterior silk glands (PSG). Fibroin is synthesized and secreted in the PSG and constitutes 75-80% of the silk filament. When the silkworm reaches the 5th in-
star larval stage, fibroin was synthesized and secreted plentifully, and the metabolism and subcellular structure of the PSG undergoes considerable changes. The mechanism governing fibroin secretion, the quality and yield of cocoon can be elucidated by the study on the PSG. The completion of the draft sequence of silkworm genome has laid the foundation for the research on silkworm proteomics (Mita et al. 2004; Xia et al. 2004). The gene expression profile in the posterior silk gland cell of the 5th instar larva of silkworm was studied using EST analysis. Both the overlapped EST clus-
This paper is translated from its Chinese version in Scienria Agriculrura Sinica. Correspondence ZHONG Bo-xiong, Tel +X6-571-8697 1302, Fax: +86-571-86971302,E-mail: [email protected]
02007,CAAS. Ail rights resewed.Publishedby Elsevier Ltd.
WU Wei-cheng er al.
250
ter and single sequence numbers on D1 were twice as much as those on D5, whereas the fibroin H-chain gene, fibroin L-chain gene, and the fibroin P25 gene on D5 were, respectively, 18,9, and 8 times higher than those on D1. The results suggest that in addition to having an effect on the growth of silk gland cell, the gene expression on D1 in the 5th instar stage mainly prepares for the synthesis of fibroin, and the gene expression on D5 mostly contributes to the synthesis and secretion of fibroin (Zhong et al. 2004,2005a). The contrasting results of gene expression of PSG cell in the 5th instar stage silkworm show that there is no obvious variation in the following six enzymes: oxidoreductase, transferring enzyme, hydrolase, synthetase, lyase, and isomerase. Only cytochrome c oxidase subunit T , cytochrome c oxidase subunit 111, S-adenosine formylmethionine polymerase, and peptidyl proline cistrans-isomerase have expression frequency higher than 10 (Zhong et al. 2005b). The linear regression and derivation regression variance of the number of silk gland cells and cocoon property has attained a very significant level, which shows that the number of silk gland cell is one of the factors that determines fibroin yield and the synthesis and secretion capacity of each cell is an another factor (Zhong et al. 1993). In this experiment, we produced several silkworm strains and adopted the posterior silk gland from D1 and D4 in the 5th instar stage for use as study materials. The differences in protein composition among strains in various days were investigated using 2-DE and EST analysis. Fibroin has not yet been synthesized on D1 and is synthesized in large amounts on D4. Different characteristics of fibroin at the protein level and the gene level in the same stage were compared, and the molecular mechanism accounting for the differences in output of silk between strains was analyzed at the protein level.
Preparation of protein sample of posterior silk gland cell The PSG cells were prepared as described by Yan et al. (2003) and Shen et al. (2005). The silk gland on D1 and D4 in the 5th instar stage has been frozen using liquid nitrogen and was dissected in 0.7% NaCl solution at 4"C, cleaned using 0.7% NaCl solution, and soaked in precooled 60% ethanol for 2 min. The PSG cells were stripped and harvested, and their dry weight was determined. Preparation of protein sample was similar to the method described by Zhong et al. (2002, 2003).
Protein electrophoresis According to the method of Zhong et al. (1997), we adopt the O'Farrell system (O'Farrell 1975) in protein two-dimensional electrophoresis. A nonlinear gradient of pH ranging from 3.5 to 10 was adopted in the first dimension. The gel length is 12.5 cm, the inner diameter is 3 mm, and the concentration of polyacrylamide gel is 4%. 30 pL of protein sample was loaded. The second dimension employed SDS-PAGE electrophoresis using 5% stacking gel and 15% resolving gel with size of 160 mm x 60 mm x 1 mm. The proteins separated were visualized with silver-staining.
Image analysis of protein 2-DE The 2-DE images were obtatined by scanning the gels into computer. The protein spots were detected and matched using the software Imagemaster 2D, ver. 2002. 01 (Amersham Biosciences, Sweden).
RESULTS MATERIALS AND METHODS Materials Five strains were used: E8, the bivoltine European strains with high yield; Sihai, the multivoltine Chinese strains with low yield; 301; LanlO; and sericin-cocoon, the mutant strain that can only synthesize pure scricin without fibroin.
The 2D-PAGE of the PSG proteins of D1 and D4 from five silkworm strains in the 5th instar stage was performed. The scanned and analyzed images are shown in Fig. Differences in protein composition were observed between days and strains, which were far less than those observed at the gene level using EST analysis. The change trends of protein composition from D1 to
02007,CWS. AII rights reserved. Published by Elsevier ud.
Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bornbyx mori)
PI-
25 I
lsoelectric focusing
1st
2nd
Fig. 2D-PAGE images of proteins extracted from the posterior silk gland (PSG) of silkworm in the 5th instar stage. A, D1 of European strain E8; B, D4 of European strain E8; C, D1 of Chinese polyvoltine strain Sihai; D, D4 of Chinese polyvoltine strain Sihai; E, D1 of Chinese polyvoltine strain LanlO; F, D4 of Chinese polyvoltine strain LanlO; G, D1 of Chinese polyvoltine strain 301; H, D4 of Chinese polyvoltine strain 301; I, D1 of sericin-cocoon; J, D4 of sericin-cocoon.
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WU Wei-cheng et al.
D4 were diverse in various strains (Zhong et al. 2004, 2005b). Table shows the normal volume, isoelectric point (PI), and molecular weight (MW) (kD)of the specific protein spots. It is observed from Table that, with comparison to D1, on D4, in European strain E8, two protein spots were newly-expressed, and the amount of five protein spots was significantly higher; in Chinese strain Sihai, two protein spots were newly-expressed, and the
amount of one protein spot was higher and one was lower; in Chinese strain 301, two protein spots were newly-expressed, and the amount of one protein spot was higher and eight was lower, and ten protein spots disappeared; in Chinese strain Lan 10, seven protein spots were newly-expressed, and the amount of nine protein spots was higher and one was lower, and twenty-five protein spots disappeared; in sericin-cocoon, the amount of thirteen protein spots was lower, and
Table Characteristics of protein spots from posterior silk gland of silkworm in the 5th instar stage ~~
Protein characteristic Specific spots on D1
Protein spots 8 22 24 26 28 30 32 34 36 38 40 42 44 65 68 70 72 74 76 78 80 92 Specific spots on D4 6 9 11 13 15 20 49 52 57 59 63 85 Spots of higher amount on D1 than on D4 46 81 87 89 91 94 97 99 101 Spots of lower amount on D1 than on D4 1 3 4
16 50 54 61 64 84
Norm. Vol. 276044 85 734 109 643 60314 132 944 55 364 40 832 102311 102 563 89 156 172 521 167 333 43 087 105 864 124111 50 132 47 364 214456 67 442 115 377 241 573 202 225 22317 84 792 30 814 20958 286 309 81 732 64 626 26 891 167 825 149 498 68 035 324 835 422 527 423 177 119217 164 292 67 597 103 867 120588 132 582 160 037 113 393 59 168 101 510 238 362 1284 924 186 237 625 762 673 694 584852
PI 5.22 5.75 5.79 5.75 5.82 5.51 5.38 5.10 5.39 5.93 5.84 5.47 5.40 5.53 4.92 5.14 4.73 5.63 5.99 5.51 5.59 4.72 4.61 5.44 5.13 5.04 5.96 4.94 5.72 4.95 4.91 5.58 5.90 6.19 6.30 5.91 4.68 4.57 4.62 4.91 4.63 4.76 4.98 4.48 4.47 4.52 5.63 6.47 6.21 5.86 5.50 6.06
-
MW (kD) A"
LI.1
61.5 33.5 32.3 31.1 27.6 26.3 25.7 23.4 22.1 20.9 22.3 21.2 43.8 31.3 31.5 23.0 27.1 26.3 23.3 23.0 26.2 27.0 102.2 105.1 105.1 90.9 25.1 97.1 111.5 47.0 38.9 20.4 25.1 16.8 18.0 30.0 25.2 22.8 22.5 21.7 20.4 20.5 35.8 30.4 27.7 83.8 71.0 75.3 26.1 22.0
23 25 27 29 31 33 35 37 39 41 43 45 66 69 71 73 75 77 79 86 95 7 10 12 14 17 47 51 56 58 60 83
Norm.Vol. 108 637 73 070 119 359 41 616 123 367 49817 68 038 80 484 103 170 49 105 53 579 74 55.8 315 139 63 237 87 523 87 156 174 232 103 622 80 580 127 630 236 143 174 642 22 934 60 678 13438 58 597 70 157 290 909 54 224 46 122 48 987 171 236 122 604
PI 6.24 5.09 5.11 5.80 5.61 5.45 5.35 5.34 5.75 5.89 5.55 5.43 5.89 5.13 4.89 4.71 5.21 5.96 5.61 5.95 4.42 4.72 4.68 5.36 5.07 5.62 6.29 4.74 4.92 4.96 5.58 6.07 5.97
MW (W 55.0 38.4 35.9 32.4 28.9 27.2 26.3 25.0 22.4 22.6 21.9 20.6 17.1 44.0 29.5 24.3 26.3 32.0 26.1 23.4 30.0 26.7 29.1 102.1 105.0 92.5 41.8 146.3 111.7 47.7 38.9 32.3 92.6
67 a2 88 90 93 96 98 100
140 985 427 072 142 969 161 993 82 984 102 532 85 829 104 624
5.15 6.32 4.64 4.60 4.85 4.73 4.77 4.84
35.5 17.6 26.8 23.9 23.1 21.9 22.5 20.5
2 5 18 19 53 55 62 48
212232 69 162 199 907 176981 441 400 222 173 635 678 233 814
4.43 4.49 5.69 5.04 5.73 6.25 5.99 5.81
32.1 29.3 50.4 45.7 93.1 69.6 26.0 102.1
Protein spots
-. LI
25.1 ~~
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Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori)
three protein spots disappeared. The above results indicated that the protein composition in PSG cell of various strains changed over time, but each strain had its unique change trend. The expression level of protein on 04 is significantly higher than that on D1 in European strain E8. Only a few protein spots express specifically between D1 and D4 in Chinese strain Sihai; the protein expression level on D1 was significantly higher than that on D4 in Chinese strain 301; there were relatively more proteins with differences between D1 and D4 in Chinese strain LanlO; the protein expression level on D1 was much higher than that on D4 in sericin-cocoon strain. On the whole, these protein spots do not overlap and vary with strains.
'
DISCUSSION no-dimensional electrophoresis is currently the most widely used method for protein analysis in proteome research, by which high-throughput identification of proteins in cells and organs may be accomplished. The PSG of silkworm is the organ where fibroin is synthesized and secreted. The cocoon shell weight is related with the secretive ability of PSG, which depends on the metabolic ability of cells. Proteins are the main excutors on various metabolism and regulation responsible for life process. The metabolic status of cells in a certain stage can be found out through the study of protein composition. The metabolism and structure of PSG cells undergoes considerable changes after the 5th instar stage is reached. And the study of the changes in protein composition of different strains during this stage will help better understand the metabolic ability of PSG cell in the 5th instar stage of various strains. It is believed that discovering the protein influencing fibroin synthesis and secretion and revealing the mechanism of production of silk will lay the foundation for screening of higher yield strains. The change of protein composition in PSG cell of the five strains in the 5th instar stage shows that the cellular metabolism varies with time, which is in compliance with its physiological function during the stage. The gene expression analysis indicate that besides having an influence on the growth of silk gland cell, the gene expression on D1 in the 5th instar stage mainly
253
prepares for fibroin synthesis. The gene expression on D5 in the 5th instar stage is primarily related to fibroin synthesis. According to the analysis at the cell level, the function of PSG cells in prophase of the 5th instar stage is mainly the replication of DNA as a preparation for transcription and translation of fibroin gene, and the establishment of fibroin secretion system. The fibroin is synthesized in large amount on D4 in the 5th instar stage. The results showed that in European strain E8, the amount of protein secreted by the silk gland cells on D4 is obviously higher than that on D 1. In Sijiao silkworm, the amount of protein on D1 is obviously higher than that on D4, which implies that the protein type and amount secreted by silk gland cells on D4 may have a close relationship with the synthesis and secretion of fibroin. It reveals that the protein composition in PSG cells from D1 to D4 are diverse, not only among strains with different cocoon shell weight but also among strains with similar cocoon shell weight (Chinese polyvoltine strains Sihai, LanlO, and 301 having similar cocoon shell weight), and the changes have different trends. Thus, it is speculated that the synthesis of fibroin may be regulated by more than one type protein in a strain, and that different strains may not have all the same kind of regulation proteins. It is also possible that the function of some same proteins may be different among different strains. The final cocoon shell weight is the sum of all regulative factors via vector addition. Therefore, the regulative factors of the various strains with similar cocoon shell weight may be different, and the mechanism responsible for the production of large quantities of silk may differ with strains. The sequence and functional analysis of the concerned proteins will help understand their role in physiological processes involving secretion as well as the relationship between differences in metabolism and secretive ability, so as to provide the theoretical basis for breeding and screening of the strain of interest with considerable output.
CONCLUSIONS Differences were observed at protein level between days and strains and these were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse
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among various strains, which suggests that fibroin secretion may be regulated by various proteins and regulating sites, and these distinctions result in the differences in the secretive ability for fibroin among different strains.
Acknowledgements The program was supported by the National Basic Research Program (973 Program), China (2005CB121003), Science and Technology Program of Zhejiang Province, China (2004(332048),and Key Science and Technology Program of Hangzhou City, China (200432241).
References Mita K, Kasahara M, Sasaki S, Nagayasu Y, Yamada T, Kanamori H, Namiki N, Kitagawa M, Yamashita H, Yasukochi, KadonoOkuda K, Yamamoto K, Ajimura M, Ravikumar G, Shimomura M, Nagamura Y, Shin-i T, Abe H, Shimada T, Morishita S, Sasaki T. 2004. The genome sequence of silkworm, Bombyx mori. DNA Research, 11,27-35. O’Farrell P H. 1975. High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemical, 250,4007-4021. Shen F Y, Zhong B X, Lou C F, Su S K, Xu H S, YanX P, Yao G H, Lu Y C. 2005. A comparison of the protein composition of the middle silk gland cells of the fifth instar larvae of silkworm. Scientia Agriculturu Sinica, 38, 1052-1058. (in Chinese) Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, Zhao P, Zha X, Cheng T, Chai C, Pan G, Xu J, Liu C, Lin Y, Qian J, Hou Y, Wu Z, Li G, Pan M, Li C, Shen Y, Lan X, Yuan L, Li T, Xu H, Yang G, Wan Y, Zhu Y, Yu M, Shen W, Wu D, Xiang Z, Yu J, Wang J, Li R, Shi J, Li H, Li G, Su J, Wang X, Li G, Zhang Z, Wu Q, Li J, Zhang Q, Wei N, Xu J, Sun H, Dong L, Liu D, Zhao S, Zhao X, Meng Q, Lan F, Huang X, Li Y, Fang L, Li C, Li D, Sun Y, Zhang Z, Yang Z, Huang Y, Xi Y, Qi Q, He D, Huang H, Zhang X, Wang Z, Li W, Cao Y, Yu Y, Yu H, Li J, Ye J, Chen H, Zhou Y, Liu B, Wang J, Ye J, Ji H, Li S, Ni
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P,Zhang J, Zhang Y, Zheng H, Mao B, Wang W, Ye C, Li S, Wang J, Wong K-S G, Yang H. 2004. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science, 306, 1937-1940. Yan X P, Zhong B X, Xu M K, Guo D G, Yao G H. 2003. The relation between the protein composition from posterior silkgland in the fifth instar larva of silkworm and cocoon shell weight. Acta Sericologica Sinica, 29, 344-348. (in Chinese) Zhong B X, Karibe H, Komatsu S, Ichimura H, Nagamura Y, Sasaki T, Hiraro H. 1997. Screening of rice genes from a cDNA catalog based on the sequence data-file of proteins separated by two-dimensional electrophoresis. Straining Science, 47,245-251. Zhong B X, Weng H B, Fang W H. 2002. Preparation of protein samples for gel electrophoresis by sequential extraction. Journal of Zhejiang University SCIENCE, 3,606-610. Zhong B X, Wu Y C, Zhu F, Zhao Q M, Wang X L. 1993. Studies on relation between cell numbers of posterior silkgland and cocoon quality characters of silkworm, Bombyx mori. Acta Sericologica Sinica, 19, 149-155.(in Chinese) Zhong B X, Yan H Y, Shen F Y, Li J K, Zhou L. 2003. Preparation of silkworm protein using two dimensional polyacryamide gel electrophoresis. Acta Sericologica Sinica, 29,427-432. (in Chinese) Zhong B X, Yu D L, Liang J S, Miao Y G, Su S K, Xu H S. 2005a. Comparative analysis of gene expression of enzymes in posterior silkgland cells of silkworm Bombyx mori fifth instar larva. Acta Sericologica Sinica, 31, 121-128. (in Chinese) ZhongB X, Yu Y P, Xu Y S, Yu H, Lu X M , Mia0 Y G, Yang J, Xu H, Hu S N, Lou C F. 2004. Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L. Science in China (C), 34,436-443. (in Chinese) B o n g B X, Yu Y P, Xu Y S, Yu H, Lu X M, Miao Y G, Yang J, Xu H, Hu S N, Lou C F. 2005b. Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L. Science in China (C), 48,25-33. (Edited by WANG Lu-han)
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Agricultural Sciences in China 2007, 6 ( 2 ) :249-254
-@
‘%
ScienceDirect
February 2007
Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th lnstar Stage Wei-chengl-5, GAO Qi-kang2, CHEN Jin-e1.5, YE Jianl, QIAN Yang-wen3,LI Jim-yingl, LU Hua-yun4,
MENG Zhi-qi5, NI Chun-xiao4 and ZHONG Bo-xiong’ 1
Laboratory of Molecular Biology of Silkworm and HoneybeeKollege of Animal Science, Zhejiang University, Hangzhou 310029, P.R.
China Institute of Applied Entomology, Zhejiang University, Hangzhou 310029, P.R. China 3 College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310029, P.R. China 4 Department of Speciality Good Seed Extension, Hangzhou 310002, P.R. China Laboratory of Moleculnr Biology of Insect, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R.China 2
J
Abstract The posterior silk gland (PSG) of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 7580% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis (2-DE) and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 (Dl) and day 4 (D4) in the 5th instar stage from five different strains of silkworm (Bombyx dori). While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.
Key words: silkworm (Bornbyx mori), silk gland, protein, 2-DE, image analysis
INTRODUCTION Silkworm is an economically important insect and a legitimate model for Lepidopteran genomics and genetics with 28 pairs of chromosomes. The silk gland of silkworm is a paired tubular gland that can be divided into anterior silk gland (ASG), middle silk gland (MSG), and posterior silk glands (PSG). Fibroin is synthesized and secreted in the PSG and constitutes 75-80% of the silk filament. When the silkworm reaches the 5th in-
star larval stage, fibroin was synthesized and secreted plentifully, and the metabolism and subcellular structure of the PSG undergoes considerable changes. The mechanism governing fibroin secretion, the quality and yield of cocoon can be elucidated by the study on the PSG. The completion of the draft sequence of silkworm genome has laid the foundation for the research on silkworm proteomics (Mita et al. 2004; Xia et al. 2004). The gene expression profile in the posterior silk gland cell of the 5th instar larva of silkworm was studied using EST analysis. Both the overlapped EST clus-
This paper is translated from its Chinese version in Scienria Agriculrura Sinica. Correspondence ZHONG Bo-xiong, Tel +X6-571-8697 1302, Fax: +86-571-86971302,E-mail: [email protected]
02007,CAAS. Ail rights resewed.Publishedby Elsevier Ltd.
WU Wei-cheng er al.
250
ter and single sequence numbers on D1 were twice as much as those on D5, whereas the fibroin H-chain gene, fibroin L-chain gene, and the fibroin P25 gene on D5 were, respectively, 18,9, and 8 times higher than those on D1. The results suggest that in addition to having an effect on the growth of silk gland cell, the gene expression on D1 in the 5th instar stage mainly prepares for the synthesis of fibroin, and the gene expression on D5 mostly contributes to the synthesis and secretion of fibroin (Zhong et al. 2004,2005a). The contrasting results of gene expression of PSG cell in the 5th instar stage silkworm show that there is no obvious variation in the following six enzymes: oxidoreductase, transferring enzyme, hydrolase, synthetase, lyase, and isomerase. Only cytochrome c oxidase subunit T , cytochrome c oxidase subunit 111, S-adenosine formylmethionine polymerase, and peptidyl proline cistrans-isomerase have expression frequency higher than 10 (Zhong et al. 2005b). The linear regression and derivation regression variance of the number of silk gland cells and cocoon property has attained a very significant level, which shows that the number of silk gland cell is one of the factors that determines fibroin yield and the synthesis and secretion capacity of each cell is an another factor (Zhong et al. 1993). In this experiment, we produced several silkworm strains and adopted the posterior silk gland from D1 and D4 in the 5th instar stage for use as study materials. The differences in protein composition among strains in various days were investigated using 2-DE and EST analysis. Fibroin has not yet been synthesized on D1 and is synthesized in large amounts on D4. Different characteristics of fibroin at the protein level and the gene level in the same stage were compared, and the molecular mechanism accounting for the differences in output of silk between strains was analyzed at the protein level.
Preparation of protein sample of posterior silk gland cell The PSG cells were prepared as described by Yan et al. (2003) and Shen et al. (2005). The silk gland on D1 and D4 in the 5th instar stage has been frozen using liquid nitrogen and was dissected in 0.7% NaCl solution at 4"C, cleaned using 0.7% NaCl solution, and soaked in precooled 60% ethanol for 2 min. The PSG cells were stripped and harvested, and their dry weight was determined. Preparation of protein sample was similar to the method described by Zhong et al. (2002, 2003).
Protein electrophoresis According to the method of Zhong et al. (1997), we adopt the O'Farrell system (O'Farrell 1975) in protein two-dimensional electrophoresis. A nonlinear gradient of pH ranging from 3.5 to 10 was adopted in the first dimension. The gel length is 12.5 cm, the inner diameter is 3 mm, and the concentration of polyacrylamide gel is 4%. 30 pL of protein sample was loaded. The second dimension employed SDS-PAGE electrophoresis using 5% stacking gel and 15% resolving gel with size of 160 mm x 60 mm x 1 mm. The proteins separated were visualized with silver-staining.
Image analysis of protein 2-DE The 2-DE images were obtatined by scanning the gels into computer. The protein spots were detected and matched using the software Imagemaster 2D, ver. 2002. 01 (Amersham Biosciences, Sweden).
RESULTS MATERIALS AND METHODS Materials Five strains were used: E8, the bivoltine European strains with high yield; Sihai, the multivoltine Chinese strains with low yield; 301; LanlO; and sericin-cocoon, the mutant strain that can only synthesize pure scricin without fibroin.
The 2D-PAGE of the PSG proteins of D1 and D4 from five silkworm strains in the 5th instar stage was performed. The scanned and analyzed images are shown in Fig. Differences in protein composition were observed between days and strains, which were far less than those observed at the gene level using EST analysis. The change trends of protein composition from D1 to
02007,CWS. AII rights reserved. Published by Elsevier ud.
Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bornbyx mori)
PI-
25 I
lsoelectric focusing
1st
2nd
Fig. 2D-PAGE images of proteins extracted from the posterior silk gland (PSG) of silkworm in the 5th instar stage. A, D1 of European strain E8; B, D4 of European strain E8; C, D1 of Chinese polyvoltine strain Sihai; D, D4 of Chinese polyvoltine strain Sihai; E, D1 of Chinese polyvoltine strain LanlO; F, D4 of Chinese polyvoltine strain LanlO; G, D1 of Chinese polyvoltine strain 301; H, D4 of Chinese polyvoltine strain 301; I, D1 of sericin-cocoon; J, D4 of sericin-cocoon.
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WU Wei-cheng et al.
D4 were diverse in various strains (Zhong et al. 2004, 2005b). Table shows the normal volume, isoelectric point (PI), and molecular weight (MW) (kD)of the specific protein spots. It is observed from Table that, with comparison to D1, on D4, in European strain E8, two protein spots were newly-expressed, and the amount of five protein spots was significantly higher; in Chinese strain Sihai, two protein spots were newly-expressed, and the
amount of one protein spot was higher and one was lower; in Chinese strain 301, two protein spots were newly-expressed, and the amount of one protein spot was higher and eight was lower, and ten protein spots disappeared; in Chinese strain Lan 10, seven protein spots were newly-expressed, and the amount of nine protein spots was higher and one was lower, and twenty-five protein spots disappeared; in sericin-cocoon, the amount of thirteen protein spots was lower, and
Table Characteristics of protein spots from posterior silk gland of silkworm in the 5th instar stage ~~
Protein characteristic Specific spots on D1
Protein spots 8 22 24 26 28 30 32 34 36 38 40 42 44 65 68 70 72 74 76 78 80 92 Specific spots on D4 6 9 11 13 15 20 49 52 57 59 63 85 Spots of higher amount on D1 than on D4 46 81 87 89 91 94 97 99 101 Spots of lower amount on D1 than on D4 1 3 4
16 50 54 61 64 84
Norm. Vol. 276044 85 734 109 643 60314 132 944 55 364 40 832 102311 102 563 89 156 172 521 167 333 43 087 105 864 124111 50 132 47 364 214456 67 442 115 377 241 573 202 225 22317 84 792 30 814 20958 286 309 81 732 64 626 26 891 167 825 149 498 68 035 324 835 422 527 423 177 119217 164 292 67 597 103 867 120588 132 582 160 037 113 393 59 168 101 510 238 362 1284 924 186 237 625 762 673 694 584852
PI 5.22 5.75 5.79 5.75 5.82 5.51 5.38 5.10 5.39 5.93 5.84 5.47 5.40 5.53 4.92 5.14 4.73 5.63 5.99 5.51 5.59 4.72 4.61 5.44 5.13 5.04 5.96 4.94 5.72 4.95 4.91 5.58 5.90 6.19 6.30 5.91 4.68 4.57 4.62 4.91 4.63 4.76 4.98 4.48 4.47 4.52 5.63 6.47 6.21 5.86 5.50 6.06
-
MW (kD) A"
LI.1
61.5 33.5 32.3 31.1 27.6 26.3 25.7 23.4 22.1 20.9 22.3 21.2 43.8 31.3 31.5 23.0 27.1 26.3 23.3 23.0 26.2 27.0 102.2 105.1 105.1 90.9 25.1 97.1 111.5 47.0 38.9 20.4 25.1 16.8 18.0 30.0 25.2 22.8 22.5 21.7 20.4 20.5 35.8 30.4 27.7 83.8 71.0 75.3 26.1 22.0
23 25 27 29 31 33 35 37 39 41 43 45 66 69 71 73 75 77 79 86 95 7 10 12 14 17 47 51 56 58 60 83
Norm.Vol. 108 637 73 070 119 359 41 616 123 367 49817 68 038 80 484 103 170 49 105 53 579 74 55.8 315 139 63 237 87 523 87 156 174 232 103 622 80 580 127 630 236 143 174 642 22 934 60 678 13438 58 597 70 157 290 909 54 224 46 122 48 987 171 236 122 604
PI 6.24 5.09 5.11 5.80 5.61 5.45 5.35 5.34 5.75 5.89 5.55 5.43 5.89 5.13 4.89 4.71 5.21 5.96 5.61 5.95 4.42 4.72 4.68 5.36 5.07 5.62 6.29 4.74 4.92 4.96 5.58 6.07 5.97
MW (W 55.0 38.4 35.9 32.4 28.9 27.2 26.3 25.0 22.4 22.6 21.9 20.6 17.1 44.0 29.5 24.3 26.3 32.0 26.1 23.4 30.0 26.7 29.1 102.1 105.0 92.5 41.8 146.3 111.7 47.7 38.9 32.3 92.6
67 a2 88 90 93 96 98 100
140 985 427 072 142 969 161 993 82 984 102 532 85 829 104 624
5.15 6.32 4.64 4.60 4.85 4.73 4.77 4.84
35.5 17.6 26.8 23.9 23.1 21.9 22.5 20.5
2 5 18 19 53 55 62 48
212232 69 162 199 907 176981 441 400 222 173 635 678 233 814
4.43 4.49 5.69 5.04 5.73 6.25 5.99 5.81
32.1 29.3 50.4 45.7 93.1 69.6 26.0 102.1
Protein spots
-. LI
25.1 ~~
02007, CAAS.All rights resenred. Publishedby ElsevierLtd.
Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori)
three protein spots disappeared. The above results indicated that the protein composition in PSG cell of various strains changed over time, but each strain had its unique change trend. The expression level of protein on 04 is significantly higher than that on D1 in European strain E8. Only a few protein spots express specifically between D1 and D4 in Chinese strain Sihai; the protein expression level on D1 was significantly higher than that on D4 in Chinese strain 301; there were relatively more proteins with differences between D1 and D4 in Chinese strain LanlO; the protein expression level on D1 was much higher than that on D4 in sericin-cocoon strain. On the whole, these protein spots do not overlap and vary with strains.
'
DISCUSSION no-dimensional electrophoresis is currently the most widely used method for protein analysis in proteome research, by which high-throughput identification of proteins in cells and organs may be accomplished. The PSG of silkworm is the organ where fibroin is synthesized and secreted. The cocoon shell weight is related with the secretive ability of PSG, which depends on the metabolic ability of cells. Proteins are the main excutors on various metabolism and regulation responsible for life process. The metabolic status of cells in a certain stage can be found out through the study of protein composition. The metabolism and structure of PSG cells undergoes considerable changes after the 5th instar stage is reached. And the study of the changes in protein composition of different strains during this stage will help better understand the metabolic ability of PSG cell in the 5th instar stage of various strains. It is believed that discovering the protein influencing fibroin synthesis and secretion and revealing the mechanism of production of silk will lay the foundation for screening of higher yield strains. The change of protein composition in PSG cell of the five strains in the 5th instar stage shows that the cellular metabolism varies with time, which is in compliance with its physiological function during the stage. The gene expression analysis indicate that besides having an influence on the growth of silk gland cell, the gene expression on D1 in the 5th instar stage mainly
253
prepares for fibroin synthesis. The gene expression on D5 in the 5th instar stage is primarily related to fibroin synthesis. According to the analysis at the cell level, the function of PSG cells in prophase of the 5th instar stage is mainly the replication of DNA as a preparation for transcription and translation of fibroin gene, and the establishment of fibroin secretion system. The fibroin is synthesized in large amount on D4 in the 5th instar stage. The results showed that in European strain E8, the amount of protein secreted by the silk gland cells on D4 is obviously higher than that on D 1. In Sijiao silkworm, the amount of protein on D1 is obviously higher than that on D4, which implies that the protein type and amount secreted by silk gland cells on D4 may have a close relationship with the synthesis and secretion of fibroin. It reveals that the protein composition in PSG cells from D1 to D4 are diverse, not only among strains with different cocoon shell weight but also among strains with similar cocoon shell weight (Chinese polyvoltine strains Sihai, LanlO, and 301 having similar cocoon shell weight), and the changes have different trends. Thus, it is speculated that the synthesis of fibroin may be regulated by more than one type protein in a strain, and that different strains may not have all the same kind of regulation proteins. It is also possible that the function of some same proteins may be different among different strains. The final cocoon shell weight is the sum of all regulative factors via vector addition. Therefore, the regulative factors of the various strains with similar cocoon shell weight may be different, and the mechanism responsible for the production of large quantities of silk may differ with strains. The sequence and functional analysis of the concerned proteins will help understand their role in physiological processes involving secretion as well as the relationship between differences in metabolism and secretive ability, so as to provide the theoretical basis for breeding and screening of the strain of interest with considerable output.
CONCLUSIONS Differences were observed at protein level between days and strains and these were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse
Q2007, CAAS.All rightsreserved.Publishedby Dsevier ud.
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among various strains, which suggests that fibroin secretion may be regulated by various proteins and regulating sites, and these distinctions result in the differences in the secretive ability for fibroin among different strains.
Acknowledgements The program was supported by the National Basic Research Program (973 Program), China (2005CB121003), Science and Technology Program of Zhejiang Province, China (2004(332048),and Key Science and Technology Program of Hangzhou City, China (200432241).
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Q2007, CAAS.All rightsreSeNSd. PuMished by ElsevierLtd.