[讨论交流] 病毒侵染性克隆构建经验交流与学习
最近和几位网友在讨论关于侵染性克隆构建的问题，在这先开个头，希望能有经验的来说说自己的经历。毕竟侵染性克隆能够解决越来越多的问题。
载体选择
启动子选择
构建策略
接种方法
troubleshooting？？问题与解决方案
比如，序列在大肠杆菌里面有毒性，一个解决方案是在相应序列里面加入内含子。
一些关于构建的文献：
1. Barrett, K. J., S. E. Gold, and J. W. Kronstad. 1993. IDENTIFICATION AND COMPLEMENTATION OF A MUTATION TO CONSTITUTIVE FILAMENTOUS GROWTH IN USTILAGO-MAYDIS. Molecular Plant-Microbe Interactions 6:274-283.
2. Candresse, T., A. Gora-Sochacka, and W. Zagorski. 2001. Restoration of secondary hairpin II is associated with restoration of infectivity of a non-viable recombinant viroid. Virus Res. 75:29-34.
3. Edwards, M. C., and J. J. Weiland. 2010. First infectious clone of the propagatively transmitted Oat blue dwarf virus. Archives of Virology 155:463-470.
4. Fenaux, M., P. G. Halbur, G. Haqshenas, R. Royer, P. Thomas, P. Nawagitgul, M. Gill, T. E. Toth, and X. J. Meng. 2002. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: Characterization of clinical disease, virus distribution, and pathologic lesions. Journal of Virology 76:541-551.
5. Fernandez-Delmond, I., O. Pierrugues, M. de Wispelaere, L. Guilbaud, S. Gaubert, Z. Diveki, C. Godon, M. Tepfer, and M. Jacquemond. 2004. A novel strategy for creating recombinant infectious RNA virus genomes. Journal of Virological Methods 121:247-257.
6. Ferreira, P. d. T. d. O., T. O. Lemos, T. Nagata, and A. K. Inoue-Nagata. 2008. One-step cloning approach for construction of agroinfectious begomovirus clones. Journal of Virological Methods 147:351-354.
7. Flatken, S., V. Ungewickell, W. Menzel, and E. Maiss. 2008. Construction of an infectious full-length cDNA clone of potato virus M. Archives of Virology 153:1385-1389.
8. Gharsallah Chouchane, S., F. Gorsane, M. K. Nakhla, D. P. Maxwell, M. Marrakchi, and H. Fakhfakh. 2006. Complete nucleotide sequence and construction of an infectious clone of a Tunisian isolate of Tomato yellow leaf curl Sardinia virus. Journal of Phytopathology 154:626-631.
9. Istomina, E. A., P. B. Snegireva, and A. N. Shiyan. 2004. Construction of a full-length cDNA of tobacco mosaic virus strain V-69 genome. Russian Journal of Genetics 40:1356-1363.
10. Jenner, C. E., F. Sanchez, S. B. Nettleship, G. D. Foster, F. Ponz, and J. A. Walsh. 2000. The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene TuRB01. Molecular Plant-Microbe Interactions 13:1102-1108.
11. Lan, X., Z. Yao, Y. Zhou, J. Shang, H. Lin, D. L. Nuss, and B. Chen. 2008. Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency. Current Genetics 53:59-66.
12. Lee, M. Y., Y. S. Song, and K. H. Ryu. 2011. Development of infectious transcripts from full-length and GFP-tagged cDNA clones of Pepper mottle virus and stable systemic expression of GFP in tobacco and pepper. Virus Res. 155:487-494.
13. Liang, D., S. M. Gray, I. Kaplan, and P. Palukaitis. 2004. Site-directed mutagenesis and generation of chimeric viruses by in yeast to facilitate an recombination it-virus interactions. Molecular Plant-Microbe Interactions 17:571-576.
14. Lin, S. S., R. F. Hou, and S. D. Yeh. 2002. Construction of in vitro and in vivo infectious transcripts of a Taiwan strain of Zucchini yellow mosaic virus. Botanical Bulletin of Academia Sinica 43:261-269.
15. Lopez-Moya, J. J., and J. A. Garcia. 2000. Construction of a stable and highly infectious intron-containing cDNA clone of plum pox potyvirus and its use to infect plants by particle bombardment. Virus Res. 68:99-107.
16. Marquet, S., P. Lepage, T. J. Hudson, J. M. Musser, and E. Schurr. 2000. Complete nucleotide sequence and genomic structure of the human NRAMP1 gene region on Chromosome region 2q35. Mammalian Genome 11:755-762.
17. Miyanishi, M., S. H. Roh, A. Yamamiya, S. Ohsato, and Y. Shirako. 2002. Reassortment between genetically distinct Japanese and US strains of Soil-borne wheat mosaic virus: RNA1 from a Japanese strain and RNA2 from a US strain make a pseudorecombinant virus. Archives of Virology 147:1141-1153.
18. Nagyova, A., and Z. Subr. 2007. Infectious full-length clones of plant viruses and their use for construction of viral vectors. Acta Virologica 51:223-237.
19. Nicolas, O., T. P. Pirone, and G. M. Hellmann. 1996. Construction and analysis of infectious transcripts from a resistance-breaking strain of tobacco vein mottling potyvirus. Archives of Virology 141:1535-1552.
20. Ohkawa, A., N. Ishikawa-Suehiro, S. Okuda, and T. Natsuaki. 2008. Construction of an infectious full-length cDNA clone of Chrysanthemum virus B. Journal of General Plant Pathology 74:434-437.
21. Osman, T. A. M., P. J. Ingles, S. J. Miller, and K. W. Buck. 1991. A SPONTANEOUS RED-CLOVER NECROTIC MOSAIC-VIRUS MUTANT WITH A TRUNCATED MOVEMENT PROTEIN. Journal of General Virology 72:1793-1800.
22. Predajna, L., A. Nagyova, and Z. Subr. 2010. Simple and efficient biolistic procedure for the plant transfection with cDNA clones of RNA viruses. Acta Virologica 54:303-306.
23. Purkayastha, A., S. Mathur, V. Verma, S. Sharma, and I. Dasgupta. 2010. Virus-induced gene silencing in rice using a vector derived from a DNA virus. Planta 232:1531-1540.
24. Sano, T., T. Candresse, R. W. Hammond, T. O. Diener, and R. A. Owens. 1992. IDENTIFICATION OF MULTIPLE STRUCTURAL DOMAINS REGULATING VIROID PATHOGENICITY. Proceedings of the National Academy of Sciences of the United States of America 89:10104-10108.
25. Shi, B. J., S. W. Ding, and R. H. Symons. 1997. Plasmid vector for cloning infectious cDNAs from plant RNA viruses: High infectivity of cDNA clones of tomato aspermy cucumovirus. Journal of General Virology 78:1181-1185.
26. Stephan, D., and E. Maiss. 2006. Biological properties of Beet mild yellowing virus derived from a full-length cDNA clone. Journal of General Virology 87:445-449.
27. Su, P. Y., and T. A. Brown. 1997. Ty3/gypsy-like retrotransposon sequences in tomato. Plasmid 38:148-157.
28. Truniger, V., C. Nieto, J. A. Diaz-Pendon, M. A. Aranda, and medimond. 2004. A melon necrotic spot virus untranslated RNA sequence is the genetic avirulence determinant for both cultivar and non-host resistances.
29. Uelper, L., I. Sarand, K. Rausalu, and A. Merits. 2008. Construction, properties, and potential application of infectious plasmids containing Semliki Forest virus full-length cDNA with an inserted intron. Journal of Virological Methods 148:265-270.
30. Wu, C.-Y., Y.-C. Lai, N.-S. Lin, Y.-H. Hsu, H.-T. Tsai, J.-Y. Liao, and C.-C. Hu. 2008. A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification. Journal of Virological Methods 147:355-359.
31. Yamamiya, A., and Y. Shirako. 2000. Construction of full-length cDNA clones to Soil-borne wheat mosaic virus RNA1 and RNA2, from which infectious RNAs are transcribed in vitro: Virion formation and systemic infection without expression of the N-terminal and C-terminal extensions to the capsid protein. Virology 277:66-75.
32. Yang, G., X. G. Liu, and B. S. Qiu. 2000. Recombinant constructions and infectivity analysis of tobacco mosaic virus and attenuated tomato mosaic virus N14 genomes. Chinese Science Bulletin 45:1399-+.
33. Yang, S. J., F. Revers, S. Souche, H. Lot, O. Le Gall, T. Candresse, and J. Dunez. 1998. Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants. Archives of Virology 143:2443-2451.
34. Zhu, H. Q., J. P. Chen, and S. Q. Yu. 2006. Construction of an infectious cDNA clone of Ribgrass mosaic virus Shanghai isolate and its modification to express an epitope of Mycobacterium tuberculosis. Archives of Virology 151:1853-1861.
plant virus protocol 2008 书中介绍过几种病毒侵染性克隆的构建，总体思路可以参考里面的，但是有些具体的细节我们可以在这里讨论。
一只蜗牛回复：
重要的是提供一些病毒的元件。其他都是配角。 感觉病毒的元件好准备，就是配角不好办。比如说potyvirus的有的需要在侵染性克隆的序列前面加G，有的对polyA也有要求。有的对UTR的完整性要求很高，有的则不那么重要。 一只蜗牛回复：正常啊，病毒可没有通则，具体病毒都是具体设计的。
是啊，一般就是遇到问题解决问题，解决问题的办法参考别人已经尝试过，并且成功的。
对植物病毒侵染性克隆构建，我的一点点意见是，以potyvirus为例，
首先如果能够提纯病毒
第二步从提纯病毒里面提取病毒RNA，反转录，PCR， 在这一步会体会到提纯病毒的必要性。
第三步，做5‘RACE，确保能够拿到全长。由于potyvirus的都含有polyA，所以3’末端很容易解决。
第四部，得到全长序列。设计引物吧基因组分几步放到载体里面。如果个别片段产生对大肠杆菌的毒性基因 ，尝试插入内含子，可以参考以前的文献选择位置。这里又涉及到载体和启动子的选择。
第五步，得到含有全长的质粒，接种寄主看是否有侵染性，如果有，皆大欢喜，如果没有，进一步验证序列，比如测序或酶切。如果序列没有问题，则尝试其他方法，比如，接种方法，换启动子，加G等等。 关于启动子，我个人倾向于使用35s的启动子。这样等拿到质粒以后就可以直接接种不用再做体外转录。另外我们实验室改造的handygun简直是又便宜，又好用。爱死它了。请个电工，买几样东西就能搞定。强烈推荐！！这个handygun优点多多，便宜，方便，快速，高效。
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