由於蝦類的防禦系統大部分是藉由血淋巴細胞所引發，S�丼erh�纜l研究發現淡水螯蝦astakine基因所產生之蛋白質具有血淋巴細胞增生和增加存活時間的功能，鑑於astakine基因對於引發蝦類防禦系統的血淋巴細胞具有上述功能，本研究對於目前為台灣經濟海水蝦種的白蝦(Litopenaeus vannamei)進行astakine基因選殖並探討其於細菌性病原之哈維弧菌(Vibrio harveyi)進行攻毒試驗，觀察攻毒後不同時間點之血淋巴細胞astakine基因表現的情�捸A進而了解此類內因性細胞激素astakine產生之蛋白質產物參與白蝦的病原菌防禦機制中的角色。藉由3端和5端rapid amplification of cDNA ends (RACE) PCR進行基因選殖，獲得白蝦astakine基因全長為1488 鹼基對，其中372鹼基對轉譯成124個胺基酸，經由蛋白質結構預測發現第1~21個胺基酸具有signal peptide，另外在第22~107個胺基酸具有類似前人研究功能蛋白Prokineticin 結構，同時藉由染色體基因序列(genomic DNA)與訊息表現基因互補序列比對(mRNA complementary DNA, cDNA)獲此基因開放讀序框(open reading frame, ORF)由三個Exon和兩個 Intron組成。所轉譯出的蛋白質序列與草蝦(Penaeus monodon，black tiger shrimp)的astakine具有87% 相似度(87% Identity)，還有在淡水螯蝦發現的astakine (Pacifastacus leniusculus，signal crayfish) 具有56% 相似度(56% Identity)。注射滅菌海水和哈維弧菌(Vibrio harveyi)後不同時間所採集的血淋巴細胞萃取其RNA進行astakine基因的RT-PCR 分析，其中一對專一性引子分析結果發現在電泳膠體分析中此基因具有三個大小不同片段，經過片段序列分析，顯示此基因在經過注射後可能藉由選擇性接合(alternative splicing)方式來調控產生不同的表現型態；在收集不同的組織中基因表現經過哈維弧菌注射後顯示astakine基因在不同組織所表現型態的情形產生改變，在經由不同位置設計專一性引子進行RT-PCR分析後，推斷此基因在蝦子體內可能藉由選擇性接合基因調控方式產生至少有四種不同的基因形態存在。

Astakine has been reported to involve in hematopoietic function that plays an important role in shrimp hematopoiesis, however, its molecular mechanism has not been clearly elucidated. An astakine was isolated by cloning from hemocytes of white shrimp Litopenaeus vannamei by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The cDNA of astakine consists of 1488 base pairs with a 372 base pairs open reading frame (ORF) able to code a 124 amino acids peptide. Nucleotide sequence analysis reveals that the nucleotide sequence of L. vannamei astakine has homology with that of Penaeus mondom and freshwater crayfish (Pacifastacus leniusculus) respectively. The putative protein domain contains a signal peptide start from amino acid 1 to 21, and a functional structure similar to prokineticin domain, locate from amino acid 22 to 107. The genomic DNA of astakine in ORF codes three exons and two introns. The gene expression of astakine from hemocytes and various tissues and the time course of astakine gene expression after sterile sea water or V. harveyi injection were analyzed with RT-PCR and sequence analysis. We suppose that expression of astakine may be regulated by alternative splicing and predicate that expression patterns of astakine show at last four types after injection.

Catalogue

中文摘要．．．．．．．．．．．І
Abstract ．．．．．．．．．ІІI
Catalogue．．．．．．．．．．．．ІV
Catalogues of Figure and Table ．．．．．V

Study
1. Introduction ．．．．．．．．．．．．．．．．1
2. Materials and Methods．．．．．．．．．．．．6
3. Results．．．．．．．．．．．．．．．．．14
4. Discussion ．．．．．．．．．．．．．19
5. Reference ．．．．．．．．．．．．．．．23
6. Figure and Table．．．．．．．．．．．．．31
7. Appendix．．．．．．．．．．．．．．．．．53

Catalogues of Figure and Table

Figure 1. The innate defense mechanism of crustacean modified by Pikul et al. ．．．．．．．．．．．．．．．．31
Figure 2. The flow chart of experiment ．．．．32
Figure 3. The full-length cDNA sequences of astakine from L. vannamei. ．．．．．．．．．33
Figure 4. Putative amino acid sequence of astakine from L. vannamei and protein features. ．．．．．．．．．．．．34
Figure 5. Astakine amino acid sequence alignment with different species. ．．．．．．．．．．．35
Figure 6. The phylogenetic tree analysis of astakine form L. vannamei.．．．．．．．．．．．．．．．37
Figure 7. The tissue distribution of astakine gene by RT-PCR analysis. ．．．．．．．．．．．．．．．38
Figure 8. The relationships of Lvastakine F2 and F3. ．．39
Figure 9. Contaminative analysis of Genomic DNA.．．40
Figure 10. The relationship of Lvastakine F1, F2, and F3.．．．．．．41
Figure 11. The quality and contaminative analysis of haemocyte cDNA in time course study post sterile sea water and V. harveyi injection by RT-PCR analysis．．．．．．．．．．．．42
Figure 12. The haemocyte expression pattern of astakine gene in time course study after V. harveyi challenge by RT-PCR analysis. ．．．．44
Figure 13. Hypothesis of astakine gene alternative splicing. ．．．．49
Table 1. The cellular immune response of crustacean. ．．．．．．．50
Table 2. Primer sequence used for amplification of astakine gene. ．．51
Appendix 1. pGEM��-T Easy Vectors. (Promega, Cat. No.A1360) ．．53
Appendix 2. The solutions of experiment. ．．．．．．．．．．．54

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