Transgenic lines of melon (Cucumis melo L. var. makuwa cv. “Silver Light”) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens

東方甜瓜 (Cucumis melo L. var. makuwa cv. “Silver Light”）為一重要之水果作物，其主要分佈於熱帶及亞熱帶地區。然而，真菌引起的疾病會造成其產量下降。 本研究將抗真菌蛋白（AFP）與幾丁質酶（CHI）之融合基因轉殖入東方甜瓜中，探討該基因轉殖作物對於真菌枯絲核菌（Rhizoctonia solani）及尖孢鐮刀菌（Fusarium oxysporum）所引起疾病之抗性。轉殖方式乃利用農桿菌LBA4404（Agrobacterium tumefaciens strain LBA4404）行之，該農桿菌含有抗真菌蛋白（AFP）與幾丁質酶（CHI）基因，其啟動子為椰菜嵌紋病毒35S啟動子（Cauliflower mosaic virus, CaMV），篩選標記為新黴素磷酸轉移酶（neomycin phosphotransferase, nptII）基因。 利用農桿菌將pBI121- AFP- CHI基因轉殖入懸浮培養中之東方甜瓜子葉體後，再建立植物培養系統。以聚合酶鏈反應（polymerase chain reaction, PCR）確認基因是否轉殖入。南方墨點法（Southern blot）之結果確認PCR陽性轉殖株之AFP-CHI融合基因確實被轉殖入。RT-PCR分析結果則確認於個別基因轉殖株之AFP-CHI融合基因確實有表現。 西方墨點法（Western blot）分析結果顯示CHI蛋白累積於葉片上。分離分析（segregation analysis）顯示該基因可被遺傳至第一子代（T1）。 研究結果指出，本系統可有效建立具抗枯絲核菌和尖孢鐮刀菌之AFP-CHI基因轉殖甜瓜。The oriental melon (Cucumis melo L. var. makuwa cv. “Silver Light”) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases. In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. “Silver Light”) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR–positive lines. RT–PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T1 generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.TABLE OF CONTENTS Page Acknowledgments I Absract (Chinese version) II Absract III Table of contents IV LIST OF FIGURES VII LISTOF TABLES VIII Chapter 1: Literatures review: 1 1.1 Botany of Cucumis melo 1 1.2 Biotechnology and in genetic engineering in plant 3 1.3 Tissue culture of Cucumis melo 4 1.4 An Overview of Agrobacterium-mediated transformation 6 1.4.1 Biology of Agrobacterium-tumefaciens 6 1.4.2 Tumor inducung (Ti) plasmid 8 1.4.3 Transfer DNA (T-DNA) of Ti plasmid 9 1.4.4 Virulence genes 10 1.4.5 Plant transformation via Agrobacterium tumefaciens 11 1.4.6 Binary vectors of Ti plasmid 12 1.5. Agrobacterium-mediated transformation of Cucumis melo 15 1.6 Selectable marker genes 16 1.7 Antifungal proteins 17 1.8 Chitinases and their use in genetic engineering 18 1.8.1 Chitinases 18 1.8.2.Chitinases in nature 19 1.8.3 Plant chitinases and their classification 20 1.8.4 Role of chitinases in plant defense against pathogens 25 1.8. 5 Expression of AFPCHI genes in transgenic plants 27 Chapter 2. Objectives 29 Referencess 30 Chapter 3. Enhanced tolerance of transgenic melon expressing chitinase and antifungal protein to fungal pathogen 3.1.Introduction 49 3.2. Material and Methods 51 3.21 Plant material 51 3.2.2 Tissue culture media and culture conditions 51 3.2.3 Agrobacterium strain and vector construction 52 3.2.4 Infection of explants and Co-cultivation 53 3.2.5 Selection of explants 53 3.2.6 Elongation of explants 54 3.3 Molecular analysis 55 3.3.1 Genomic DNA isolation 55 3.3.2 DNA quality measurement 56 3.3.3 Polymerase Chain Reaction (PCR) 57 3.3.4 Southern blot analysis 58 3.3.5 Total RNA isolation 59 3.3.6 Measuring RNA concentration 60 3.3.7. RT-PCR 61 3.3.8 Formaldehyde Agarose Gel Electrophoresis 62 3.3.9 Western blot analysis 63 3.4 Disease resistance assay with R. solani and F. oxysporum 64 3.5 Segregation analysis 65 4. Result 66 4.1 Production of transgenic melon lines 66 4.1.1 Callus induction 66 4.1.2 Shoot development 66 4.1.3 Acclimization 67 4.2 Molecular analysis 68 4.2.1 Integration of transgene host genome 68 4.2.2 Expression of transgene 68 4.3 Antifungal assay 69 4.3.1 Disease resistance assay with R. solani 69 4.3.2 Disease resistance assay with F. oxysporum 69 4.4 Segregation analysis 70 5. Discussion 71 Chapter 4 Conclusion 75 Referencess 98 LIST OF FIGURES Chapter 1 Figure 1 Agrobacterium-mediated plant transformation process 11 Figure 2 The classification of plant chitinase 24 Chapter 3 Figure 3.1 Partial map of the plasmid vector used for transformation of Cucumis melo L. var. makuwa cv. “Silver Light” AFP-CHI gene isolated from papaya.. 76 Figure 3.2 Inoculation procedure of Rhizoctonia solani 77 Figure 3.3 Inoculation of procedure of Fusarium oxysporum 78 Figure 4.1 Infection procedure of Agrobacterium tumefaciens 79 Figure 4.2 Shoot and root development of transgenic melon lines 80 Figure 4.3 Acclimization of transgenic melon lines 81 Figure 4.4 PCR analysis of transgenic melon lines 82 Figure 4.5 Southern blot analysis of individual transgenic lines 83 Figure 4.6 Fungal resistance test of transgenic melon expressing AFP-CHI gene against R. solani 84 Figure 4.7 Plant survival of the wild-type, P1 and individual transgenic lines inoculated with R. solani, measured 7 days after inoculation 85 Figure 4.8 Representative photographs of the wild type, P1, B4, D1, and M1 transgenic plants grown in greenhouse at 15 days after inoculation F. oxysporum 86 Figure 4.9 RT-PCR analysis of individual transgenic lines 87 Figure 4.10 Western blot analysis individual transgenic lines challenged with R. solani 88 LIST OF TABLES Chapter 1 Table 1. Plant chitinases that have been isolated and cloned 21 Chapter 3 Table 3.1 The compositon of tissue culture medium 89 Table 3.2 Buffer and Solutions of Genomic DNAisolation 90 Table 3.3 Buffer and Solutions of Southern blot analysis 91 Table 3.4 Wash solutions of Southern blot analysis 92 Table 3.5 Compositon of formaldyehde agarose gel electrophoresis 93 Table 3.6 1X FA Gel running buffers 94 Table 3.7 5X FA Gel running buffers 95 Table 4.1 Resistance of individual transgenis lines after inoculation with F. oxysporum 96 Table 4.2 Segregation analysis of individual transgenic lines selection on MS medium with supplemented on kanamycin 9

2nd International Conference on Agricultural and Biological Sciences (ABS 2016), Shanghai, China, July 23-26, 2016 Abstracts

Table of contents 01 The influence of soil salt content on the photosynthetic characteristics of spring wheat with trickle irrigation Lei Pei, Zhenhua Wang, Jinzhu Zhang, Wenhao Li 02 Comparing growth of fast-growing and slow-growing endophytes in plants via ergosterol quantification Adeline SY Ting, Yiing Y Chow, Sadequr Rahman 03 Transcriptome and digital gene expression analysis identifies putative triterpene saponin-biosynthetic genes of Panax notoginseng Rongchang Wei 04 Chitosan-assisted isolation and antioxidant evaluation of flavonoids from Sophora japonica L. Zhang Hu, Sidong Li, Chuyan Li, Zijuan Li 05 Two kinds of new characteristics of the ektexine ornamentation of ginkgo pollen Guoxia Wang, Yuzhen Yang, Gang Chen, Qing Luo 06 Analysis of nutrient and medicinal ingredients of Ginkgo pollen in different regions Guoxia Wang, Ruixia Liu, Yuzhen Yang, Lipei Chen 07 Photosynthetic performance of greening seedlings of seven species to drought stress Zhiyang Lie, Tongtong Zhou, Weilong Huang, Li Xue 08 Changes of fluorescence parameters of greening seedlings of seven species under drought stress Jie Li, Zhuomin Wang, Li Xue 09 Mammalian sex hormone affects regeneration capacity and enzymes activity of Triticale L in vitro culture Ismail Bezirganoglu, Pınar Uysal 10 Fractional Fourier entropy increases the recognition rate of fruit type detection Shuihua Wang, Zhihai Lu, Jiquan Yang, Yudong Zhang, John Liu, Ling Wei, Shufang Chen, Preetha Phillips, Zhengchao Dong 11 Banana-peanut intercropping reduces Fusarium wilt disease in banana from enhancing soil bacterial microorganisms and leaf nutrition Hong Li, Xutong Wang, Fengliang Zhao, Guisheng Yang 12 Manganese stress impairs stem ureide nitrogen fixation in yardlong-bean plants in the acidic environment Hong Li 13 A new pest control method for Rhytidodera bowringii Larvae Haijie Huang, Li Zhao, Weijian Huang, Jinhui Wang, Zhongrun Zhang 14 Research on the seed-like Fruits of Subg. Sclarea of Salvia of Labiatae in China Xiaojuan Li, Ning Xu, Guofu Zhou, Ming Wan, Qi Lin, Fanyun Meng, Jianxiu Li 15 Three pulling resistance models of pioneer plant in landslide area Yichang Chen, Koayung Yu, Chunpin Chang 16 The comparison of physiological and biochemical mechanisms of Reaumuria soongorica and Salsola passerine in different growth pattern Zijuan Zhou, Peixi Su, Rui Shi, Tingting Xie 17 Resources use efficiency of the cosmopolitan plant Potentilla anserina L. in different alpine habitats in China Rui Shi, Peixi Su, Zijuan Zhou 18 Cloning of PPDK gene from Red Amaranand transformation of Alfalfa Xuelan Liu, Yan Zhang, Xiangfa Wei 19 Variation and cluster analysis of morphological characters and nutrient content of Chucrasia tabularis seed Chong Wu, Yanlei Yin, Lijuan Feng, Xuemei Yang, Fei Wang 20 Effect of the planting density of the areca nut on the growth of intercropped Vanilla Hua Wang, Huifa Zhuang, Zihui Zhu, Hui Wan