蕪菁嵌紋病毒載體表現外源蛋白之開發及具交互保護能力輕症病毒株系之建構

Abstract

蕪菁嵌紋病毒(Turnip mosaic virus，TuMV)為Potyviridae科、Potyvirus屬之病毒，其寄主範圍廣泛，於亞洲被公認為是感染十字花科最重要之病毒。本研究以分離自彩色海芋(Zantedeschia spp. hybrids. Black Magic)的TuMV分離株進行其全長度核酸之選殖與定序，並與其它登錄於GenBank之不同TuMV分離株進行親緣性分析。同時將TuMV-YC5分離株開發成為植物病毒載體系統，於不同寄主作物上表現外源蛋白，探討不同病毒基因體上對應HC-Pro、P3、CIP、NIb及CP N端等五個嵌入位置對表現外源蛋白之效率，並將TuMV-YC5載體建構為輕症型病毒株(mild strain)，探討其對嚴重型病毒株(severe strain)之交互保護效果。 第一章為前人研究，主要是彙整近年來與本研究相關的研究概況。 第二章主要研究分離自彩色海芋之蕪菁嵌紋病毒之分子特性及其親緣性分析，針對TuMV病毒海芋分離株RC4及YC5，以5''-RACE及TuMV之專一性引子對進行反轉錄-聚合酶鏈鎖反應，由相互重疊的cDNA片段選殖解序後，各別解得之基因體RNA均含9832個核苷酸，並登錄於基因庫(GenBank)，其序號分別為AY134473及AF530055。RC4及YC5之基因體架構相同，可對應轉譯出一含3164個胺基酸之大蛋白。5''端之序列包含有四個腺嘌呤核苷酸(adenylated nucteotides)，大蛋白之第一個胺基酸轉譯之起始位(AUG)在5''端起第129-131 nts。與登錄於GenBank上之TuMV分離株比對結果，發現P1、P3 及 6k2等蛋白在TuMV分離株間變異性最大。對應CIP蛋白之第471-490胺基酸出現差異性，在所比對的42個分離株中，僅有Q-Ca (Acc. No. D10927)及C1 (Acc. No. AF394601)分離株呈現GSQPVxMxDxVxMxKIGVTL序列，而RC4、YC5與其他38個分離株則均為WLTASEYARLGANVEDRRDV序列型態。RC4及YC5乃分離自天南星科之海芋分離株，但二者均能感染十字花科且造成植株系統性嵌紋病徵。進一步根據與其他30個具有全長度大蛋白胺基酸序列之TuMV分離株，以及與其他59個TuMV分離株之鞘蛋白胺基酸序列所得之親緣性分析結果顯示，RC4與YC5仍同列於大部分由十字花科白菜屬(Brassica spp.)分離之TuMV所組成的群組，然而RC4與YC5之間仍有一段遺傳距離。此種遺傳距離的差異性是否因RC4及YC5為分離自不同的海芋品系或區域隔離所造成，需藉由更多來源不同的TuMV海芋分離株探討之。 第三章主要研究內容為應用蕪菁嵌紋病毒載體於可鮮食的十字花科蔬菜上表現塵蟎過敏源蛋白Der p 5 (來自Dermatophagoides pteronyssinus Trouessart)。將異源基因構築於病毒載體系統上，隨著病毒載體在植物內之複製而大量表現外源蛋白，為相當有效率之蛋白表現系統。利用反轉錄-聚合酶鏈鎖反應所增幅出之TuMV-YC5全長度cDNA構築於具有Cauliflower mosaic virus (CaMV) 35S啟動子之pCaMVCN載體上，獲得生體內表現(in vivo)之構築體p35SYC5。將p35SYC5質體DNA接種至單斑寄主奎藜(Chenopodium quinoa Willd.)後可於葉片形成單斑，取單斑組織接種於系統性作物後可造成煙草(Nicotiana benthamiana Domin)植株出現萎凋、十字花科蔬菜葉片產生嵌紋、海芋植株葉片形成黃斑及黃化條斑等病徵，均與由TuMV-YC5所引起之病徵相同。在TuMV-YC5基因體對應P1與HC-Pro轉譯架構(open reading frame, ORF)之間突變出限制酶切位，並將水母綠螢光蛋白(GFP) ORF及塵蟎過敏源Der p 5 ORF分別構築於P1/HC-Pro之嵌入位置，得到TuMV-GFP或TuMV- Derp5重組體。經免疫分析證實，重組病毒皆能於受感染植物中成功地表現融合態(fused form)或游離態(free form)的GFP蛋白，以及游離態的Der p 5蛋白，且與ZYMV載體在矮南瓜植株上所表現的Der p 5結果相比，Der p 5 ORF 構築於不含六個histidines的TuMV-YC5載體上，於白菜(Brassica campestris L. var. chinese)和青江白菜(B. campestris L. var. ching-geeng)兩種十字花科蔬菜上所表現的Der p 5蛋白產量，高於ZYMV載體表現者1.7-4.6倍。顯示Der p 5的表現量，TuMV-YC5病毒載體系統顯著地優於ZYMV載體系統。本研究結果顯示TuMV病毒載體系統在可直接鮮食之十字花科寄主上表現Der p 5蛋白，將可提供免疫療法上一重要而簡易的途徑，而TuMV的寄主範圍廣泛，顯示TuMV-YC5病毒載體系統之應用性具有極高之潛力。 第四章主要探討對於TuMV-YC5病毒載體上不同嵌入位置對外源蛋白表現的效率與穩定度。利用以35S啟動子調控之TuMV-YC5病毒載體系統，將病毒基因體上對應HC-Pro、P3、CIP、NIb及CP等蛋白之N端嵌入一組對應NcoI-NheI-CVYHQA組合之序列，其中NcoI-NheI為限制酵素切位，以供構築外源蛋白的ORF；CVYHQA為對應NIb/CP接合處之NIa蛋白酶辨識切位序列，以供游離態外源蛋白的生成。分別將水母綠螢光蛋白(GFP)和塵蟎過敏源蛋白Der p 5 的ORF構築於病毒載體之不同嵌入位，並分別於奎藜(C. quinoa)、煙草(N. benthamiana)、白菜、青江白菜、芥菜(B. juncea L.)和山東白菜(B. campestris L. var. pekinensis)等寄主上，分別探討此二種外源蛋白的表現與穩定性。結果顯示上述五個嵌入位置均能成功於不同寄主中表現GFP；除了CIP的N端外，其他四個嵌入位都適合Der p 5表現。在奎藜、煙草、白菜、青江白菜以及芥菜上，HC-Pro和CP的N端較能使GFP和 Der p 5的表現量相對提高，而P3和CIP的N端則因其表現量均較其他於嵌入位者低，較不適合此二種蛋白之表現，對Der p 5而言，嵌入CIP的N端則無法表現。此外，Der p 5 ORF嵌入NIb的N端時，可於奎藜及煙草上表現Der p 5蛋白，然而無法於各種測試的十字花科寄主中被表現。利用HC-Pro、P3、CIP之N端表現游離態外源蛋白之TuMV-GFP或TuMV-Derp5 (不包括CIP N端無法表現的Der p 5 )病毒重組體於系統性寄主上引起之病徵，普遍比其它病毒重組體或TuMV-YC5所造成的病徵輕微。本研究證明TuMV-YC5病毒載體系統之不同嵌入位置能有效地於不同寄主作物上表現不同的外源蛋白，然而外源蛋白在寄主植物上之表現量或穩定度，則受到嵌入位置、寄主種類以及外源基因種類之影響。 第五章主要研究利用嚴重型(severe strain) TuMV-YC5之協同蛋白(HC-Pro)定點突變所產生之輕症型病毒株(attenuated mutant)，並探討輕症型病毒突變株的交互保護效果。由PCR突變選殖過程中所獲得之二個輕症型病毒選殖株pHCNIE16及pHCIG5為基礎，進行一系列定點突變所獲得之不同輕症型病毒株，此等病毒株感染葵藜形成淡綠色單斑(pale-green lesions)，感染煙草僅引起輕微嵌紋且不會造成植株萎凋，感染十字花科作物也僅引起輕微之斑駁甚或無病徵，有別於原始(wild-type)嚴重型病毒株所引起之明顯病徵。由對應HC-Pro區域內之部分片段進行嚴重型及輕症型病毒之同區塊之基因片段相互交換後，使得彼此之病原性也因此而互換。HC-Pro蛋白之胺基酸突變結果使得本研究之TuMV嚴重型病毒株變成為輕症型病毒株。在對應HC-Pro蛋白之第7 (F7/I7)與第171個(E171/G171)胺基酸，以及是否構築具備NIa蛋白酶辨識切位(CVYHQA)等組合下所構築之各種病毒株，顯示第7個胺基酸單獨由(F7) 改變為 (I7)，需配合CVYHQA切位之構築才足以形成輕症型病毒株，而第第171個胺基酸單獨由(E171)改變為(G171)便足以形成輕症型病毒株，但若配合第7個胺基酸突變為 (I7)或有CVYHQA切位之構築，則可以獲得更為弱毒性之病毒株。利用輕症型病毒突變株pHCIG5 (I7/ G171)於煙草(N. benthamiana)及白菜進行對嚴重型病毒株之交互保護試驗結果顯示，此輕症型病毒株均具有保護作物不受嚴重型病毒株之感染，同時也印證pHCIG5等不同的輕症型病毒突變株，在煙草上均具有保護植株不被嚴重型TuMV-YC5感染的效果。 Turnip mosaic virus (TuMV), a member of the genus Potyvirus, is the most important virus infecting field-grown crucifers world-wide, and with a wide host range including 318 plants species in 156 genera of 43 families. In this study, the molecular characterizations of two TuMV isolates, YC5 and RC4, isolated from calla lily (Zan- tedeschia spp., Araceae), and compared their genetic variablitiy to those TuMV strains isolates available in GenBank based on the full-length RNA genomic sequences and coat protein coding regions were analyzed. The infectious cDNA clone of TuMV-YC5 driven by the 35S promoter was constructed, and modified as a viral vector to express the open reading frame (ORF) of GFP or a dust mites allergen Der p 5 (from Dermatophagoides pteronyssinus Trouessart). Five different insertion sites in the TuMV- YC5 vector were created. The effectiveness and stability of the expressed heterologous proteins in plants by TuMV-YC5 vector at these different insertion sites were compared. The ORFs of GFP and Der p 5 were successfully expressed by TuMV-YC5 vector in various host plants. Finally, the attenuated mild strains with the ability to protect plants of Nicotiana benthamiana Domin and Brassica campestris L. var. chinese from the infection of severe-strain TuMV-YC5 were constructed. The Chapter 1 reviews all relevant references for this study. This chapter is written in Chinese. The other chapters are written in English for publication in journal. In the Chapter 2, the complete nucleotide sequences of TuMV-RC4 and TuMV-YC5 isolates were determined from overlapped viral cDNA clones derived from reverse-transcription polymerase chain reaction (RT-PCR) using the TuMV specific primers. Both TuMV-RC4 and TuMV-YC5 showed similar genomic features. When compared with TuMV isolates available in GenBank,sequences coding for P1, P3 and 6k2 proteins were identified as the most varible regions. Based on the variablilty of amino acid sequences at the amino acids positions of 471-490 in CIP region, TuMV isolates were divided into two groups. Among 42 TuMV isolates compared, only Q-Ca (Acc. No. D10927) and C1-isolate (Acc. No. AF394601) have a sequence of GSQPVxM- xDxVxMxKIGVTL, while RC4, YC5, and other 38 isolates with a sequence of WLTASEYARLGANVEDRRDV. The protease recognition tetrapeptides for P1 protease and nuclear inclusion a protease (NIa-Pro) are variable among TuMV isolates, but both of RC4 and YC5 are identical. The phylogenetic analyses based on the comparison of the deduced amino acid sequences of the complete polyproteins with those of other 30 TuMV isolates, and the coat protein regions with those of other 59 TuMV isolates revealed that RC4 and YC5 are both classified in the group that contains isolates belonging to the Brassica pathotype. In Chapter 3, a dust mite allergen Der p 5 expressed in edible crucifers by TuMV- YC5 vector was investigated in this study. Our previous study indicated that oral feeding of mice with dust mite allergen Der p 5 produced by Zucchini yellow mosaic virus (ZYMV) vector in squash provides a novel approach for immunotherapy of allergic asthma. However, the non-edibility of squash leaves sparked a biosafety concern. In this investigation, we expressed the dust mite allergen by TuMV vector in crucifers that are consumed as fresh edible vegetables to minimize the biosafety concerns for humans. An infectious cDNA clone driven by a 35S promoter was successfully constructed by ligating overlapping cDNA clones generated from RT-PCR amplified fragments of TuMV-YC5. The ORFs of GFP or Der p 5 allergen were in-frame inserted at the N-terminal region of the HC-Pro of TuMV-YC5, respectively, to generate a free-form or fused-form proteins, and expressed in Chenopodium quinoa Willd., N. benthamiana, or Brassica spp. Expression of free-form Der p 5 by TuMV-YC5 vector in crucifers (such as B. campestris L. var. chinese and B. campestris L. var. ching-geeng) is 1.7- to 4.6-folds higher than those by ZYMV vector in squash. Since oral feeding of high dose allergen is essential for effective immunotherapy, we consider that the expression of the dust mite allergen by TuMV vector in cruciferous crops that are consumed as fresh edible vegetables is a more feasible approach than the ZYMV-squash system. In Chapter 4, the effectiveness and stability of heterologous proteins expressed in plants by TuMV-YC5 vector at five different insertion sites was analyzed. The N-terminal regions (NT) of HC-Pro and CP reading frames are generally used for the in-frame insertion of large heterologous ORFs in potyviral vectors for protein expression in plants. The infectious cDNA clone of TuMV-YC5, driven by a 35S promoter, was modified to carry foreign ORFs at the possible sites to investigate the expression efficiency. A sequence cassette including directional cloning sites and the coding sequence for the NIa protease recognition site was separately constructed in the NT regions of HC-Pro, P3, CIP, NIb and CP proteins in TuMV-YC5, by a PCR-based site-directed mutagenesis or PCR-modification. The ORFs of GFP and Der p 5 allergen were individually in-frame inserted at these sites in TuMV-YC5 vector to generate free-form proteins. The results revealed that the five insertion sites are effective for the expression of heterologous ORFs of GFP and Der p 5, but the CIP-NT site is not feasible for the latter. The significant higher yields of GFP expressed by CP-NT are 1.2-3.6 folds higher than that expressed by HC-Pro-NT in plants of C. quinoa, N. benthamiana, and several species of Brassica crops. Lower yields of GFP expressed by P3-NT and CIP-NT, 0.2-0.9 and 0.3-0.8 folds, respectively, were compared to that expressed by HC-Pro-NT in most of the tested hosts. Yields of Der p 5 expressed by CP-NT and HC-Pro-NT were similar and significantly higher than those expressed by other sites, however, lower yields of Der p 5 were expressed when its ORF was inserted at P3-NT (0.2-0.6 folds to HC-Pro-NT) or NIb-NT (0.6-0.9 folds to HC-Pro-NT). Moreover, Der p 5 ORF inserted at NIb-NT did not produce detectable protein in plants of Brassica spp. Symptoms on most of the tested hosts infected by the TuMV-GFP or TuMV-Derp5 recombinant, expressing free-form GFP or Der p 5 by the HC-Pro-NT, P3-NT, and CIP-NT, were delayed or milder than those caused by other recombinants. Our results revealed that the engineered TuMV-YC5 is an efficient vector for the expression of foreign proteins by inserting their ORFs at the five different NT sites. However, the yield and the stability of the foreign ORFs, and the symptomology of TuMV recombinant-infected hosts are affected by the insertion sites, host plants, and the foreign ORFs themselves. In Chapter 5, HC-Pro protein has pronounced effects on replication and symptom expression of potyviruses. In this study, a PCR-modified mutant derived from p35SYC5 was recognized as a mild strain, for it only induced mild symptoms on the hosts of Chenopodium quinoa Willd., Nicotiana benthamiana Domin, and Brassica spp. The recombination analyses between the mild mutant and the parental strain indicated that the changes in HC-Pro region were critical for the attenuation. Further analyses by different constructs with amino acid changes in the N-terminal region of HC-Pro indicated that the amino acids (I7) and (G171), and the additional NIa protease recognition site (CVYHQA) added between P1 and HC-Pro regions of TuMV-YC5 were critical for the attenuation. When only (F7) was changed to (I7), the recombinant pHCIE9 still induced severe symptoms on tested host plants. However, when this change was combined with the added NIa cleavage site to generate the infectious construct pHCNIE16, the recombinant became a mild strain. Another construct pHCFG47 with the only change of E171 to G171 also induced mild symptoms. If this construct contained the NIa protease site in addition, the recombinant pHCNFG8 showed even milder symptoms on plants of N. benthamiana. Based on this, the construct pHCIG5 containing double point mutations of F7 to I7 and E171 to G171 in the HC-Pro region, without the NIa cleavage site, was generated and it induced symptoms on plants of N. benthamiana milder than those induced by the construct with only mutation of E171 to G171. In cross-protection assay, the infectious TuMV-YC5 clone carrying a reporter GFP gene was used as severe-strain challenger. By western blotting and RT-PCR at 14 days after the challenge inoculation, GFP was not detected from the plants of N. benthamiana protected with the mild strain TuMV-HCIG5 derived from pHCIG5 and subsequently challenged by severe strain at 10 days after the protective inoculation. Similar results were shown in the plants of Brassica sp. protected with the mild strain TuMV-HCIG5. Moreover, our results demonstrate that various mild mutants provide effective cross-protection on plants of N. benthamiana against the infection of the severe-strain TuMV-YC5.