Uenishiwase Persimmon ETR5 Gene Cloning And Its Plant Expression Vector Construction

Persimmon fruit,a climacteric fruit,is very defficult to be storaged and transported because it is easy ti be soften after harvested.A large body of evidence has demonstrated that ethylene is the hormone of catalyzing fruit ripening.After the start of rthylene biosymthsis in fruit,enthylene production incresae fast.Make use of transgenic technique to inhibit or decrease the ethylene biosynthsis of climacteric fruit,can descrease the content of ethylene in fruit.And the ethylene must combined with the ethlene responses to work.In the present study,we have cloned the ETR5 gene frament using total RNA extracted from the persimmon fruit,identified by sequencing and further constructed in the plant expression vector.The results were as fellow:1.The quality RNA was extracted by Rneasy^?Plant Mini Kit.The value of OD₂₆₀/OD₂₈₀ was 2.0896.2.Reverse translation of mRNA was conducted with Omniscript^TMRT Kit.High quality RNA was obtainded by the kit,which was base for successful PCR.3.According to the amino acid sequences of ETR of other plants,a pair of specific primers were designed.One piece of ETR5 DNA sequence from persimmon fruit was cloned,which was amplified by RT-PCR from ripening persimmon fruit.4.According to the ETR5 aequences analysis,using PCR to remove the intron.The gene segment was transformated in Ecoli DH5αand indentified.The sequencing result showed that the intron were removed from the gene segment.5.Two pairs of specific primers were designed,and the one had the restrictions enzyme Xbaâ… and Sacâ… ,the other had the restrictions enzyme Smaâ… and Sacâ… .Cloned these restrictions enzyme to ETR5 gene with PCR.6.The two PCR products digested by Sacâ… and then ligased as one gene segment by T4 DNA ligase.,.The gene segment was transformate in DH5αand identified.7.The plasmids contained ETR5 gene and pSMAK311 plasmids were disgested by Xba â… and Smaâ… .The ETR5 gene were3 inserted into pSMAK311 plasmids of 35S promoter contained.The RNAi espression vector was constructed which was named as pSMAK-ETR5-RNAi.8.The plant expression vector 133 was transformed into Agrobacterium EHA101 by freezing and melting method.Using the specific primers and transformed Agrobacterium EHA101 as template to PCR respectively,and the result proved recombined plasmids having been transformed to EHA101.