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Bulletin of Botanical Research ›› 2011, Vol. 31 ›› Issue (5): 537-542.doi: 10.7525/j.issn.1673-5102.2011.05.005

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Construction of ihpRNA Expression Vector of Sugarcane Yellow Leaf Virus CP Protein Gene and Genetic Transformation in Tabacco

ZHANG Yu-Liang;XIONG Guo-Ru;WANG Jian-Hua;ZHANG Shu-Zhen;GUAN Mei-Hua;YANG Wen-Jun;LIU Zhi-Xin*   

  1. Ministry of Agriculture Key Laboratory for Tropical Crop Biotechnology,Institute of Tropical Bioscience and Biotechnology,CATAS,Haikou 571101
  • Received:1900-01-01 Revised:1900-01-01 Online:2011-09-20 Published:2011-09-20
  • Contact: LIU Zhi-Xin
  • Supported by:

Abstract: To improve the sugarcane resistance against disease, a suagrcane yellow leaf virus CP protein mediated RNA interference technology was set up. In the paper, a RNAi expression vector harboring CP protein gene fragment of sugarcane yellow leaf virus was constructed. On the basis of the complete genome sequence of sugarcane yellow leaf virus Hainan isolate (GenBank accession no. HQ342888), two pairs of specific primers, CPsf1/CPsr1 and CPasf1/CPasr1, containing different enzyme sites were designed. With the template of pMD19-T/CP plasmid constructed and middle vector of pRNAi1017, positive sense strand and antisense strand were obtained, which were separately inserted into the plant expressional vector pCAMBIA2300. The RNAi vector p2300-CP-F-R containing a hairpin structure was confirmed by the digestion of restriction enzyme PstⅠ. The p2300-CP-F-R was transformed into tobacco by Agrobacterium-mediated transformation system. The results revealed that 12 transgenic tobacco plants were obtained by PCR, Southern blot and semiquantitative RT-PCR. The CP gene was succeeded in being integrated into genome of tobacco and transcripted. This work laid the foundation for breeding of plant mediated RNAi technology in sugarcane against disease.

Key words: sugarcane yellow leaf virus, CP protein, RNA interference, ihpRNA expression vector, tobacoo transformation

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