Two pairs of primers containing restriction enzyme site were designed and used to amplify sequenced plasmid.Two PCR products were digested by the corresponding restricted enzymes respectively,and connect to be a long reverse complementary fragment.The fragment was double digested by corresponding enzymes and inserted between CaMv 35S promoter and NOS terminator of expression vector pSMAK311.Confirmed by restriction endonucleases,the RNAi expression vector was transformed into Agrobacterium tumefaciens strain EHA101 by freezing-thaw method.Using Uenishiwase′ persimmon in vitro plantlet leaves as explant,Agrobacterium-mediated transformation system was optimized.The results showed that adding 200 μmol/L of AS in both agrobacterium suspension liquid and culture medium and Spc concentration gradient screening could increase the frequency of transformation.7 strains of transformed Uenishiwase persimmon plantlets were verified by PCR.
YU Cong-cong1
,
MA Jun-lian1
,
SONG Chun-li2
,
CHEN Jia1
. Uenishiwase Persimmon ETR5 Gene RNA Interference Expression Vector Construction and Genetic Transformation[J]. Acta Agriculturae Boreali-Sinica, 2010
, 25(1)
: 84
-87
.
DOI: 10.7668/hbnxb.2010.01.016
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