Development of marker-free transgenic resistance against thrips-borne orthotospoviruses in tobacco plants

栽培煙草(Nicotiana tabacum)植物是捲菸和雪茄的常見煙草。然而，蕃茄班萎病毒屬包括西瓜銀斑病毒（WSMoV）血清群的胡椒斑點病毒（pepper chlorotic spot virus,PCSV），對中國南方雲南省的煙草作物造成嚴重損害。到目前為止，由於缺乏抗性煙草品種，沒有有效的措施控制這種病毒。基於轉錄後基因沉默作用（PTGS）的轉基因抗性是控制病毒性疾病的有效方法;然而，選擇標記的轉基因的存在一直是生態和食品安全的關注議題。因此，我們以前曾開發了一種無標記的T-DNA雙元載體，其中包含一個選擇標記（nptII）和一個單獨構建在兩組T-DNA邊界中的目標基因，以產生無選擇標記的轉基因植物。之前我們實驗室已經產生了以攜帶高度保守的病毒複製酶（L）基因區，NSs編碼序列（NSs）和WSMoV的N編碼序列的反義片段的pBI2T-HpL / NSs / N的髮夾結構轉化的轉基因圓葉煙草(Nicotiana benthamiana)賦予煙草病毒廣譜抗性。在本研究中，我們嘗試使用相同的構建體在栽培煙草品種Yunyan-87的實際作物中開發無標記轉基因抗性。以農桿菌轉殖方法將此轉基因轉殖入菸草葉片組織，並再生成轉基因菸草株系，通過聚合酶鏈反應檢測，我們總共獲得了43個卡那黴素陽性煙草品系，其中28株是為轉基因陽性。在PCSV分別接種後14天（dpi）和25 dpi下進行酶聯免疫吸附測定(ELISA)。總共11個轉基因株系顯示症狀發展延遲7-11天，3個高度抗性轉基因株系顯示25 dpi恢復症狀，3個免疫抗性轉基因株系顯示無症狀，ELISA陰性達25 dpi。我們得到的6 個高度抗性及免疫抗性株系，再以tomato spottd wilt virus（TSWV）接種分析，其中34,43,44株系對PCSV和TSWV均具有免疫抗性，11, 49株系則對PCSV和TSWV均有高度抗性，這5個轉基因煙草株系經過南方墨點法分析後發現當中44號只有一個轉基因插入片斷，而43及49號則只有兩到三個。隨後，我們將通過選擇的轉基因品系的自體受粉，篩選出對不同種類的煙草病毒具有廣譜抗性的無標記F2煙草後代並解決蕃茄班萎病毒屬對雲南煙草業造成的問題。Cultivated Nicotiana tabacum plants are the common tobacco for cigarettes and cigars. However, thrips-borne orthotospoviruses including pepper chlorotic spot virus (PCSV), a member of watermelon silver mottle virus (WSMoV) serogroup, cause severe damages on tobacco crop in Yunnan Province, southern China. So far, there are no effective measures to control these viruses due to lack of resistant tobacco varieties. Transgenic resistance based on post transcriptional gene silencing (PTGS) is an effective approach to control viral diseases; however, the maker genes for regeneration selection are concerns for ecology and food safety. Hence, we previously developed a marker-free two-T-DNA-binary vector, which contains a selection marker (nptII) and a target gene separately constructed in two individual sets of T-DNA borders to generate maker-free transgenic plant. Transgenic Nicotiana benthamiana lines carrying the hairpin construct of pBI2T-HpL/NSs/N, which contains highly conserved tospoviral replicase (L) gene region, NSs coding sequence (NSs) and an antisense fragment of N coding sequence of WSMoV, have been proved conferring broad-spectrum resistance to orthotospoviruses. In this study, we attempted to use the same construct to develop marker-free transgenic resistance in the real crop of cultivated tobacco variety Yunyan-87. By Agrobacterium tumefaciens-mediated transformation, a total of 43 kanamycin positive tobacco lines were obtained and among them 28 lines were transgene positive, as detected by polymerase chain reaction. After challenged with PCSV, enzyme-linked immunosorbent assay (ELISA) was conducted at 14 days post inoculation (dpi) and 25 dpi to check the presence of the virus. A total of 11 lines showed delay in symptom development for 7-11 days, 3 highly resistant lines showed recovered symptom at 25 dpi, and 3 immune lines were symptomless and ELISA negative up to 25 dpi. The selected 6 lines with high levels of resistance to PCSV were challenged with tomato spotted wilt virus (TSWV) to validate the broad-spectrum resistance. Our results indicated that lines 11, 34, 43, 44 and 49 confer high levels of resistance to PCSV and TSWV. Southern hybridization analysis showed that line 44 has a single insert of the transgene and 43 and 49 has 2 and 3 transgene inserts, all can be used for further breeding. As PCSV and TSWV belong to two different serogroups of orthotospoviruses, the borad-spectrum resistance of our transgenic lines may solve the problem of orthotospoviruses infection in Yunan. Following by self-fertilization of selected transgenic lines, marker-free F2 tobacco progenies with broad-spectrum resistance to different species of orthotospoviruses are being screened.Content Acknowledgement i 中文摘要 ii Abstract iii Introduction 1 Materials and Methods 10 Transgene construction 10 Transformation of Tobacco plants 10 Detection of the transgene and the marker gene in plants of transgenic tobacco lines 12 Infectivity tests with WSMoV, TSWV and PCSV for non-transgenic Yunyan-87 tobacco plants 13 Indirect Enzyme-linked Immunosorbent Assay (ELISA) 14 Evaluation of transgenic resistance to PCSV 15 Evaluation of transgenic resistance to TSWV 15 Southern blotting analysis 16 Results 18 Transformation of Tobacco plants 18 Detection of the transgene and the marker gene in plants of putative transgenic tobacco lines 18 Infectivity tests of WSMoV, TSWV and PCSV on non-transgenic Yunyan-87 tobacco plants 19 Evaluation of transgenic resistance to PCSV 20 Evaluation of transgenic resistance to TSWV 21 Southern blotting analysis 23 Discussion 24 References 29 Table 1: All primers used in the study 36 Detection of nptII selection marker 36 Table 2: Evaluation of transgenic Nicotiana tabacum Yunyan-87 tobacco lines carrying transgene HpL/NSs/N resistance of tomato spotted wilt virus (TSWV) and pepper chlorotic spot virus (PCSV) under greenhouse condition 37 Fig.1: Construction 38 Fig.2: Transformation of Nicotiana tabacum tobacco plants (Variety Yunyan 87) with the HpL/NSs/N construct 39 Fig.3: Detection of nptII selection marker by polymerase chain reaction (PCR). 40 Fig.4: Detection of the L fragment of the transgene by polymerase chain reaction (PCR). 41 Fig.5: Non-transgenic tobacco plants of Nicotiana tabacum (Variety Yunyan 87) inoculated with watermelon silver mottle virus (WSMoV), pepper Chlorotic spot virus (PCSV) or tomato spotted wilt virus (TSWV) 42 Fig.6: Indirect Enzyme-linked Immunosorbent Assay (ELISA) to check the presence of watermelon silver mottle virus (WSMoV) after inoculation. 43 Fig.7: Evaluation of transgenic tobacco lines by challenge inoculation with pepper chlorotic spot virus (PCSV) under greenhouse condition. 44 Fig.8: Evaluation of transgenic tobacco lines by challenge inoculation with tomato spotted wilt virus (TSWV) under greenhouse condition 45 Fig.9: Detection of transgene inserts from transgenic Nicotiana tabacum tobacco highly resistant T0 lines for selfing. 4