類別:

黑角舞蛾核多角體病毒融合蛋白基因之選殖及其在吉普賽舞蛾細胞之表現

Sat, 01 Jan 2005 00:00:00 GMT

標題: 黑角舞蛾核多角體病毒融合蛋白基因之選殖及其在吉普賽舞蛾細胞之表現; Cloning of a gene encoding Lymantria xylina nucleopolyhedrovirus fusion protein and its expression in LD cells 作者: Hsiu-Wen Pien; 潘秀雯摘要: 以黑角舞蛾核多角體病毒 (Lymantria xylina multiple nucleopolyhedrovirus; LyxyMNPV) 感染吉普賽舞蛾細胞株 (IPLB-LD652Y-5d) 後，發生細胞融合形成一大的多核細胞，此細胞融合現象可能與病毒套膜融合蛋白有關。核多角體病毒的套膜蛋白質包括 gp64 與 ld130 基因，此兩個蛋白質在低 pH 值的誘導下，會導致感染的細胞融合。第一群核多角體病毒的套膜融合蛋白為 GP64；第二群病毒則為 LD130。黑角舞蛾核多角體病毒之 ld130 基因全長為 2,025 個鹼基對。黑角舞蛾核多角體病毒與吉普賽舞蛾核多角體病毒的 ld130 之核苷酸與胺基酸序列，相似度分別為 93 與 96 %。電腦分析黑角舞蛾核多角體病毒的 LD130 蛋白質序列，發現具有一訊息肽 (signal peptide) 和穿膜區域 (transmembrane domain)，預測其為一膜蛋白。黑角舞蛾核多角體病毒 ld130 基因轉錄本 (transcript) 之分析顯示，其 5’ 端序列具有早期和晚期轉錄起始點。而在 3’ 端的聚腺嘌呤序列位於加聚腺嘌呤訊號 (ATTAAA, poly A signal) 下游第 17 個核苷酸的位置。利用 ld130 基因序列進行親緣關係的分析，黑角舞蛾核多角體病毒屬於第二群病毒。以西方墨漬法偵測 LyxyMNPV 的LD130 蛋白，發現 LD130 為一結構蛋白。以桿狀病毒表現系統 (baculovirus transient expression vector) 極早期基因啟動子所構築之重組 ld130 進行暫時性表現，顯示 LD130 蛋白質的表現和細胞融合有關，並且在酸的誘導下會促使細胞融合。此結果確定了黑角舞蛾核多角體病毒具有 LD130 之套膜融合蛋白，且功能上可能與核多角體病毒第二群之 LD130 相同，為酸誘導後促進細胞融合的重要蛋白質。; IPLB LD-652Y-5d cells infected with Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) were found that the infection cells were fused together and formed syncytial giant cells. This phenomenon is probably caused by the viral envelope fusion protein in the cell membrane of the infected cells. Recently, two viral genes encoding the envelope fusion proteins, GP64 and LD130, had been identified. Both GP64 and LD130 envelope fusion proteins mediate the cell fusion at low pH value. The envelope fusion protein of NPV Group I is GP64 while Group II is LD130. We cloned and sequenced ld130 from LyxyMNPV. The LyxyNPV ld130 consists of 2,025 bp. Comparison of the nucleotide and amino acid sequences of LyxyMNPV ld130 with those of LdMNPV ld130 showed 93 and 96% identities, respectively. The predicted peptide sequence of LyxyMNPV LD130 contains signal and transmembrane domains that are significant homology to membrane proteins. Analysis of LyxyNPV ld130 transcript revealed both early and late transcriptional initiation sites located at upstream of the 5’ ATG initiation codon. In addition, a poly A sequence located at 17 nt downstream of the 3’ polyadenylation signal (ATTAAA). Based on the sequences of ld130 gene, the NPV species can be divided into two Groups, I and II, in phylogenetic analysis, LyxyMNPV belongs to Group II. The LD130 of budded virus detected by Western blot revealed that LD130 is a viral structural protein. The LD130 were constructed with the promoter of immediately early gene and expressed by baculovirus transient expression vector in uninfected insect cells showed that LD130 could mediate the cell-to-cell fusion at low pH. These results suggest that a functional homolog of LD130 envelope fusion protein in group II NPVs was found in LyxyMNPV.

黑角舞蛾核多角體病毒抑制細胞凋亡基因iap2 及iap3 之選殖、定序、表現及比較

Sun, 01 Jan 2006 00:00:00 GMT

標題: 黑角舞蛾核多角體病毒抑制細胞凋亡基因iap2 及iap3 之選殖、定序、表現及比較; The study of anti-apoptotic genes iap2 and iap3 of Lymantria xylina multiple nucleopolyhedrovirus, (LyxyMNPV): cloning, sequencing, expression and comparison 作者: Yu-Shin Nai; 乃育昕摘要: 昆蟲桿狀病毒感染非寄主昆蟲細胞，即可以觀察到細胞凋亡的現象。昆蟲桿狀病毒為能夠順利在感染昆蟲細胞達到增殖的目的，其基因組內主要含有兩種抑制細胞凋亡之基因，即為p35 和iap 基因群(inhibitor of apoptosis gene family, iap1~iap5)，其中iap 基因較p35 基因廣泛地分布於桿狀病毒中。經PCR 增幅再利用南氏墨點法證實，黑角舞蛾核多角體病毒(Lymantria xylina multiple nucleopolyhedrovirus, LyxyMNPV) 之基因組內含有兩個iap 基因，分別為ly-iap2 (687 bp) 與ly-iap3 (450 bp)，而不具有加州苜蓿夜蛾核多角體病毒(Autographa californica MNPV, AcMNPV) 之iap1 及p35 同源基因。此外黑角舞蛾核多角體病毒之iap 基因與吉普賽舞蛾核多角體病毒(L.dispar MNPV) 之iap 基因皆呈高相似度，iap2 及iap3 分別為81% 及86%。利用北方雜合法證實黑角舞蛾核多角體病毒ly-iap2 在細胞感染72 小時後，即可偵測到一1.95-kb 的轉錄區；而ly-iap3 在感染48 小時後，可偵測到兩個轉錄區，分別為1.04-kb 及2.09-kb。黑角舞蛾核多角體病毒的兩個iap 基因和榕樹透翅毒蛾核多角體病毒(Perina nuda MNPV, PenuMNPV) 的相同iap 基因均已解序，據此序列與其他桿狀病毒之相同基因序列分析親緣關係，結果顯示LyxyMNPV 之iap 基因與LdMNPV 關係密切，而與PenuMNPV 和 AcMNPV 皆處於不同群中。LY-IAP3 經表現後，並成功製造出抗血清，利用間接螢光免疫偵測法(indirect fluorescen immunoassay, IFIA)，LY-IAP3 蛋白質在LyxyMNPV 感染之不接受細胞株(NTU-SL7B) 以及可接受細胞株(IPLB-LD 652Y-7) 後之表現，發現LY-IAP3 皆可在SL7B 或LD7 細胞中表現。因此LY-IAP3 在病毒感染過程中所扮演的角色，需更進一步釐清。; The anti-apoptosis genes of baculoviruses are essential genes for viral propagation in their susceptible host cells. There are two main groups of antiapoptotic genes in baculovirus genome, p35 and iap gene family (iap1~iap5), the latter is more wildly distributed in baculovirus. According to the results of PCR amplification and Southern blot hybridization, Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) contains only two iap genes ly-iap2 (687 bp) and ly-iap3 (450 bp). The sequences of iap2 and iap3 of LyxyMNPV had high identities with those of L. dispar MNPV (LdMNPV), about 81% and 86%, respectively. The results of Northern blot hybridization revealed that, a 1.95-kb transcript of ly-iap2 was first detected at 72 h postinfection (pi) and 1.04- and 2.09-kb transcripts of ly-iap3 were first detected at 48 h pi. The iap genes of Perina nuda MNPV (PenuMNPV) had been also cloned and sequenced. LyxyMNPV and PenuMNPV were compared with other known baculovirus iap genes for phylogenetic analysis. The trees showed that LyxyMNPV was closely related to LdMNPV, in addition, LyxyMNPV, PenuMNPV and Autographa californica MNPV (AcMNPV) were located seperately in three different clusters. LY-IAP3 polyclonal antibody had been prepared and used to detect the expression of LY-IAP3 in LyxyMNPV-infected IPLB-LD 652Y-7 cells (LyxyMNPV permissive cells) and NTU-SL7B cells (LyxyMNPV non-permissive cells) by indirdct fluorescence immunoassay (IFIA). LY-IAP3 could be expressed in the cytoplasm of both cells; therefore, the role of LY-IAP3 needs to be further elucidated.

黑角舞蛾核多角體病毒基因體解序及一種類吉普賽舞蛾核多角體病毒特性之分析

Fri, 01 Jan 2010 00:00:00 GMT

標題: 黑角舞蛾核多角體病毒基因體解序及一種類吉普賽舞蛾核多角體病毒特性之分析; Genomic Anlyses of LyxyMNPV and the Characterization of an LdMNPV-like Virus 作者: Yu-Shin Nai; 乃育昕摘要: 黑角舞蛾 (Lymantria xylina Swinhoe) 為台灣林木及果樹類重要害蟲，本實驗室由野外罹病蟲體分離出兩種病毒病原體，皆屬桿狀病毒科，Alphabaculovirus 屬。在野外黑角舞蛾族群中以 LyxyMNPV 感染為主要，將 LyxyMNPV 基因體解序，並進行深入研究。LyxyMNPV 基因體大小為 156,344 bp，GC 百分比為 53.47%，其中包含 157 個開放譯讀區 (open reading frame, ORF)，13 個同源區域 (homologus regsions, hrs) 及 14 個桿狀病毒重複開放譯讀區 (baculovirus repeat orf, bro)。親緣關係分析上顯示 LyxyMNPV 屬於 group II NPV 與 LdMNPV 最為接近，唯基因排列上有所差異。LyxyMNPV 157 個 ORFs 中，包括 151 個 ORFs 與 LdMNPV 互為同源基因；2 個 ORFs 與其他桿狀病毒之基因同源；4 個 ORFs為特有基因，而其中一個為桿狀病毒中首次發現之特有基因，為 gag-like 的同源基因。LyxyMNPV 具有兩個非經由複製產生之 odv-e27 同源基因。LyxyMNPV 缺乏 host range factor-1 (hrf-1)，但仍可感染 NTU-LY-1 (LY) 及 IPLB-LD-652Y (LD) 細胞；進一步比較三種可感染 LD 細胞之病毒基因體 (LyxyMNPV、LdMNPV 及 OpMNPV)，結果顯示其共同擁有三個基因：inhibitor of apoptosis-3 (iap-3)、ribonucleotide reductase 2b (rr2b) 及 dutpase，其中 iap 基因在病毒感染細胞初期扮演非常重要的角色。 LyxyMNPV 具有兩個 iap 基因：iap-2 (687 bp) 與 iap-3 (450 bp)，經轉錄本分析結果顯示，iap-2 之轉譯起始點 (ATG) 上游 (upstream) 第15個鹼基對具有一個晚期及非常晚期基因轉錄起始位置 (TAAG)，而 iap-2 之轉錄起始點位於 ATG 上游第 16 個鹼基對；iap-3 之轉錄起始點位於 ATG 上游第 44 個鹼基對上之 CTTGT 保守區域，而保守區域下游第 25 個鹼基對接有 TCGT 序列；利用反轉錄-聚合酶鏈鎖反應得知此二基因 mRNA 表現量在 LyxyMNPV 感染寄主 LD 細胞後 6 小時開始上升至 72 小時，但於 72 小時後便開始下降直至 120 小時；而在 SF 細胞中，iap-2 之mRNA 表現量至感染後 120 小時仍相當低，而 iap-3 之mRNA 表現量在感染 48 小時後開始表現，且持續上升至感染後 120 小時。進一步在 SF 細胞株中利用共表現實驗 (co-expression) 進行蛋白質功能截斷分析，結果顯示 IAP-2、IAP-3 及 IAP-2-BIR 均可抑制由果蠅 RPR 蛋白所誘導之細胞凋亡反應 (P<0.05) 而 IAP-3-BIR、IAP-2-RZF 及 IAP-3-RZF在抑制由果蠅 RPR 蛋白所誘導之細胞凋亡反應則無顯著功效，推測 IAP-3 需要全長表現才具抑制由果蠅 RPR 蛋白所誘導之細胞凋亡效果。另一株由黑角舞蛾分離出的病毒，經由 polyhedrin，lef-8 及 lef-9 基因序列分析，証實此病毒係異於 LyxyMNPV 而近於 LdMNPV的病毒，因此命名為 LdMNPV-like virus，其感染 LD 細胞呈現典型寡多角體 (Few polyhedra, FP) 病徵，經由選殖定發現其 fp25k 基因缺失 44 bp。此病毒是首次野外所發現呈現 FP 的病毒。; The casuarina moth, Lymantria xylina Swinhoe (Lepidoptera: Lymantriidae), is an important forest pest in Taiwan. Two nucleopolyhedrovirus (NPV) strains were isolated from L. xylina larvae with an epizootic nucleopolyhedrosis. In the field, LyxyMNPV is the most prevalent vius strain, therefore, the nucleotide sequence was determined and analysed. The genome of LyxyMNPV consists of 156,344 bases with a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The LyxyMNPV genome encoded 14 bro genes. 13 homologous regions (hrs) were identified. In a phylogenetic analysis, LyxyMNPV is a member of Group II NPV and closely related to LdMNPV but with highly distinct genomic organization. The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified homologous to those reported in the LdMNPV genome. Two genes were homologous to other baculoviruses and four unique ORFs were identified in LyxyMNPV genome, including a gag-like gene which was not reported in baculoviruses. Two putative odv-e27 homologues were identified in LyxyMNPV, each of which has been acquired independently and not by gene duplication. LyxyMNPV lacks host range factor-1 (hrf-1). However, in vitro host range assay indicated that LyxyMNPV could infect both NTU-LY and IPLB-LD-652Y cell lines with the absence of hrf-1. Therefore, we compared LyxyMNPV gene content to those of LdMNPV and OpMNPV, and found that these three NPVs shared three genes, iap-3, rr2b, and dutpase, that are absent in AcMNPV genome, of these three genes, iap-3 is one of the baculovirus genes that affect viral host range and prevent apoptosis in baculovirus-infected cells. Two groups of anti-apoptotic genes, p35 and iap (inhibitor of apoptosis) family, have been identified in baculovirus. There are two iap genes, iap-2 (687 bp) and iap-3 (450 bp), in LyxyMNPV genome. The 5’UTR of iap-2 contains a transcriptional initiation site, TAAG, of late and very late gene motif, but 5’UTR of iap-3 contains CTTGT promoter motif plus TCGT in 25 bp spacing context. The mRNA expression profiles of these two genes in permissive cell line LD were evaluated using RT-PCR. The expression levels of iap-2 and iap-3 in the LD cells infected with LyxyMNPV raised from 3 to 72 hours post-infection (p.i.), but declined after 72 to 120 hours. Interestingly, the expression level of iap-2 and iap-3 of the infected SF cells showed significant difference. The mRNA of iap-2 was not detected; in contrary, iap-3 was detectable. During the process of infecting SF cells with LyxyMNPV, iap-3 presented a delayed expression pattern, which was detected at 48 hours p.i. and continued to rise for 120 hours. Functional assay of two iap genes was performed using over-expression method. In SF cells, full length IAP-2, IAP-3 and IAP-2-BIR domain inhibited the apoptosis induced by Drosophila RPR protein (P<0.05), however, IAP-3-BIR domain could not rescue the apoptosis induced by D-RPR. LdMNPV-like virus was nominaded base on the sequences analyses of polyhedrin, lef-8 and lef-9. This virus was closely related to LdMNPV but far from LyxyMNPV. LdMNPV-like virus showed a few polyhedra (occlusion bodies) CPE in the infected LD cells. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. This is the first FP virus isolated from the field-collected larvae infected with NPV.

黑翅螢之生物學與保育研究

Fri, 01 Jan 2010 00:00:00 GMT

標題: 黑翅螢之生物學與保育研究; The biology and conservation of the black-winged firefly, Luciola cerata Olivier (Coleoptera: Lampyridae) 作者: Chia-Hsiung Wu; 吳加雄摘要: 保育生物學為一整合性科學，涵蓋自然、社會及人文科學之研究成果，以應用於保育策略之制訂與執行。黑翅螢(Luciola cerata Olivier)為臺灣特有種，在臺灣除台東、墾丁地區外，廣泛分佈於海拔1500公尺以下無光害污染之平地與山區，為臺灣賞螢季節之主要觀賞物種之一。本研究包含三個生物學研究，黑翅螢之形態、不同地區黑翅螢閃光模式之差異、黑翅螢雄蟲之二形性對於性擇之影響及一項社會科學研究—阿里山地區螢火蟲生態產業產值調查，共四個不同研究主題已做為日後制定黑翅螢之保育策略之基礎。黑翅螢屬中型螢火蟲，其溝通系統為HP系統，即雌蟲回應雄蟲閃光之閃光持續時間極為固定，約0.07~0.09秒。在臺灣中部之4月份，雄蟲夜間之閃光活動時間可區分為三階段，第一階段從18:40~19:01，為暖身期；第二階段為活動期，由19:02~21:39；最後階段為休息期，由21:40~23:39；各階段雄蟲之閃光持續時間、閃光間隔時間及雄蟲數量皆有不同，而雄蟲飛行及爬行之閃光模式，可能為雄蟲之間相互競爭的方式。黑翅螢雄蟲具二形性，可依第二發光節之外形，區分成五角形及半橢圓形雄蟲，在不同性比之室內試驗中，在雄蟲比例較高時，雌蟲偏好與五角形雄蟲交尾；相較於與半橢圓形雄蟲交尾之雌蟲，與五角形雄蟲交尾者，能產下更多的卵，但當雌蟲比例較高時，雌蟲雖仍偏好與五角型雄蟲交尾，但對於半橢圓形雄蟲之雌蟲閃光回應時間則減少。本研究另於 2007 年 6 月至 2008 年 2 月止，以問卷成功訪問了 31 家賞螢活動興盛之阿里山地區民宿業者，結果得知業者經營賞螢活動平均 7.5 年，以春、夏季為主；導覽解說賞螢時段在18:00~21:00間，每晚一場；並推估阿里山地區民宿活動年營收約為 9 億 3 仟 1 百萬元。賞螢季 (4 月至 6 月) 業者總營收約 52,654,560 元，約占全年營收之 28.67%；非賞螢季 (7 月至次年 3 月)業者總營收 131,011,920 元，約占全年營收之 71.33%；賞螢季的月營收約 17,551,520 元 (54%)，顯著高於非賞螢季之月營收 14,556,880 元 (46%)，證實賞螢活動對於民宿及旅行業者所帶來的收益甚高。本研究最後建議：良好規劃之賞螢活動及持續的螢火蟲生態教育，是保育黑翅螢之較佳方式；但賞螢活動需有足夠之解說人員，以免少數遊客捕捉螢火蟲造成干擾；而賞螢活動所創造之利益亦需部分回歸社區，方能促使當地社區居民有意願持續投入螢火蟲之保育工作。; Conservation biology is an integrated science, the studying results of nature, applying, social and humane science could be used in the management of conservation strategies. The black winged firefly, Luciola cetata Olivier is endemic to Taiwan, and also the main watching species in firefly-watching season. It is wide-separated firefly species under altitude of 1500 meter beside Taitung and Kenting area. It is the most abundant in the unlit mountain area. The topics of this study are three biological studying and one social science studying; these are the studying of morphology, the bioluminescence of L. cerata within a night, the dimorphism and sexual selection of male L.cerata and the out value of firefly eco-industry in Mt. Ali area. The results of these studies could become the basic of the conservation strategy for L. cerata., L. cerata is middle firefly, the communication system are HP system, it means that the female response delay time was fix, about 0.07~0.09 sec. The flashing behavior of male L. cerata could devided to three stages. The first stage is warm-up stage, 18: 40~ 19:01, then the active stage, 19:02~21:39; the last one is resting stage, 21:40~23:39. The time of flashing duration, interval and male numbers in each stage are different. The male L. cerata has dimorphism, pentagonal and semi-oval male upon the sharp of the 2nd light segment. In the laboratory experiment with different sex ratios, when the male ratio were high, female preferred mating with pentagonal male and laid more eggs than the female matted with semi-oval male. Female still preferred mating with pentagonal male in relative high female ratio, the female response delay to semi-oval male was decreased. The income of the guesthouse manager and firefly watching tour operators in the Mt. Ali area was collected by questionnaire from Jun, 2007 to Feb, 2008. The data from 31 operators were collected. The average operators were 7.5 years. Tours were mainly conducted mainly during the spring and summer months. Firefly watching tours were conducted between 6-9:00 p.m., lasted about 1 hour, and the guesthouse guests were given one free tour per evening. The total travel business income in the Mt. Ali area amounted to NTD 931,000,000 annually. The annual income of the guesthouse manager during the firefly watching season (from Apr. to Jun.) and the rest of the year were NTD 52,654,560 dollars (28.67%) and NTD 131,011,920 dollars (71.33%), respectively. The monthly incomes during the firefly watching period and the non-firefly watching months were NTD 17,551,520 and NTD 14,556,880 respectively, which was a significant difference (p = 0.023, δ < 0.05). The results show that firefly watching in the Mt. Ali area is beneficial to the guesthouse manager. This study suggests that a limited firefly-watching activity, well-planning firefly-watching season project and the continuing firefly ecological education are relative suitable methods for the conservation of L. cerata.