Construction of RNAi Expression Vector Targeting Vernalizational Gene FaCONSTANS in Tall Fescue

• 作 者: Xiaoxia LIU Jiahai WU Jianhong SHU Xiaoli WANG Qiong MOU
• 机 构: Guizhou Institute of Prataculture,
• 出 处: Agricultural Biotechnology
• 发表日期: 2014年01期
• 关键词: digestion inserted BamHI intron Arabidopsis breeding hairpin flowering targeting

[Objective] This study aimed to construct RNAi expression vector targeting vernalizational gene FaCONSTANS( GenBank accession number:GU214996) in tall fescue. [Method] A 145 bp long Arabidopsis actin gene intron was inserted into the expression vector to construct an intermediate vector pBI121-M-INT. Two pairs of specific primers with restriction sites were designed to amplify a 351 bp long cDNA conserved sequence fragment of vernalizational gene FaCONSTANS for RT-PCR. After restriction enzyme digestion,the amplified fragment was inserted forwardly and reversely at two sides of the intron of intermediate vector to construct an RNAi expression vector with hairpin structure. [Result]Double digestion( HindIII + BamHI) showed that the intron was successfully inserted into the vector pBI121. PCR amplification and double digestion indicated that target fragment FaCONSTANS was successfully inserted forwardly and reversely into the intermediate vector. [Conclusion] This study laid foundation for breeding novel flowering-inhibited tall fescue cultivars.

[Objective] This study aimed to construct an RNAi expression vector targeting vernalizational gene FaCONSTANS (GenBank accession number: GU214996) in tall fescue. [Method] A 145 bp long Arabidopsis actin gene intron was inserted into the expression vector to construct an intermediate vector pBI121- M-INT. Two pairs of specific primers with restriction sites were designed to amplify a 351 bp long cDNA conserved sequence fragment of vernalizational gene FaCONSTANS for RT-PCR. After restriction enzyme digestion, the amplified fragment was inserted forwardly and reversely at two sides of The intron of intermediate vector to construct an RNAi expression vector with hairpin structure. [Result] Double digestion (HindIII + BamHI) showed that the intron was successfully inserted into the vector pBI121. PCR amplification and double digestion indicated that target fragment FaCONSTANS was successfully inserted forwardly and reversely into the intermediate vector. [Conclusion] This study laid foundation f or breeding novel flowering-inhibited tall fescue cultivars.