Efficient Transposition Of The Retrotransposon Tnt1 In Cucumber(Cucumis Sativus L.)

Cucumber（Cucumis sativus L.）is one of the most important vegetables in the world,playing an important role in people’s diet.As the first vegetable completed its sequencing of the whole genome,it has composed the foundation of subsequent molecular breeding,functional genomics and comparative genomics.Tnt1 is a type of retrotransposon from tobacco（Nicotiana tabacum）.Because of its characteristics in the process of tissue culture being induced and transposed,being stably inherited and isolated in sexual reproduction,Tnt1 plays a significant role in the construction of mutant library of many crops.However,study on Tnt1 construction of cucumber mutant library has not been reported till now.In order to explore the applying value in Tnt1 construction of cucumber,transgenic cucumber of T₂generation with Tnt1 was self-crossed to gain T₃generation.Cotyledonary nodes of T₃generation were used for tissue culture,followed by PCR detection to identify if the plants contained Tnt1.Tnt1 copy numbers were analyzed by Southern blot.Genome Walking technique was used for transgenic plants to isolate the flanking sequences of Tnt1,preliminary proved that Tnt1 has a characteristic of"copy"and retransposition.The main results are as follows:NCT10（T₂）with Tnt1 was crossed with the non-transgenic inbred line‘SA0422’,and F₁was back crossed with‘SA0422’to produce BC₁F₁.Among 97 BC₁F₁plants examined with PCR detection,87 contained and10 were absent of Tnt1.Based on this ratio（8.7 presence versus 1 absence）,the estimated copy number was at least 3.T₃generation seeds（NCT10,NCT05）were used for tissue culture.Twenty-three NCT10-2 seeds were used and fifty-six regenerated cucumber plants were gained,each leaf produced 1～2 regenerated buds.Southern blot was used to detect the Tnt1 copy numbers of NCT10,NCT05（T₂）and their regenerated seedlings T10-1～10-x,which reconfirmed that Tnt1 was inserted into the genome of the original material,and it could activate the transposition activity through tissue culture.Genome Walking technique was used for Tnt1 flanking sequence separation of ten plants randomly selected from NCT10,NCT05 and T10-1～10-x.The results showed that Tnt1 insertion was random,inserted into the exons of genes,also the non-gene encoding interval.