Study On The Interaction Between Transposase And Non-autonomous Transposon Of Soybean Transposon Tgm9

The synthesis of anthocyanins in soybean is controlled by many genes,of which W₄ gene controls the color of flowers and hypocotyls and encodes dihydroflavone reductase.When the soybean plant contains the wild-type W₄ gene,the soybean plant has purple flowers and the hypocotyl is also purple.When the W₄ gene is homozygous for recessive mutation,the soybean plant has white flowers and the hypocotyl is green.In the unstable soybean line,the color of soybean flower and hypocotyl are found to be mottled（the leaves have white and purple spots）.It was later proved that the active transposon Tgm9 inserted into the DFR2gene.The soybean transposon Tgm9 has a full length of about 20 kb and belongs to the active transposon of the CACTA type.It consists of a non-autonomous transposon element comprising terminal inverted repeat and transposase elements.There are 27 exons in the middle and 2 open reading frames ORF1 and ORF2,encoding the transposase GmTNP2（1060 aa）and GmTNP1（750 aa）,respectively.Its structure is complete and the sequence is clear,but its transposition mechanism remains unclear.Comparing the conserved protein sequences of the transposase GmTNP2,GmTNP2 has 32%similar to TNP2 of Snapdragon Tam1 and 46%similar to TNPD of maize En/Spm.Therefore,it is speculated that the transposition mechanism of soybean transposon Tgm9 is similar to the transposition mechanism of maize En/Spm.In order to testify the hypothesis about the Tgm9 transposition mechanism of soybean transposon,it is mainly explored from two aspects.First,exploring the activity of the transposons in Arabidopsis.im（immutans）and gun5（genome uncoupled）mutants are used as plant materials,and the phenotype is easy to observe.The phenotype of the im mutant exhibits a plaque,which is composed of white tissue and green tissue and is sensitive to light.The white part of the leaf is due to the absence of plastid terminal oxidase（PTOX）leading to white tissue in the leaves.The phenotype of the gun5 mutant showed a yellow-green color.The gun5 gene encodes the H subunit of Mg-chelatase and plays a role in chlorophyll biosynthesis.Insertion of the T-DNA into the promoter region of AtChlH of gun5 reduced the expression of the gun5 gene,resulting in a yellow-green phenotype.There are three non-autonomous transposons with different fragment lengths（dTgm9-1:4.5 kb,dTgm9-2:3.5kb,dTgm9-3:1.6 kb）.Using 35S as a promoter,PTOX or Gun5 as reporter genes respectively,and they were constructed together with non-autonomous transposons of different lengths on the plant binary expression vector to construct a non-autonomous transposon vector.Furthermore,35S and Ubi7pro were used as promoters and the transposases TNP2 and TNP1were constructed together on the plant binary expression vector to construct a transposase vector.Using the transgenic technique,the non-autonomous transposon vector and the transposase vector were transferred into im and gun5 plants,respectively.If the transposon is not active in Arabidopsis,the phenotype of the im and gun5 plants remains the original phenotype.If the transposon jumps during the embryonic stage of the plant,the phenotype of the im and gun5 plants is all green.If the transposon jumps in the somatic phase of the plant,the phenotype of the im plant is varigated,and the phenotype of the gun5 plant has green spots or the leaf is green.The second part of the experiment is to investigate the interaction between transposase and non-autonomous transposon by transient expression of tobacco.The interaction between the transposase and the non-autonomous transposon is explored by plant one-hybrid.The transposase（TNP1/TNP2）and the transcriptional activator（VP16）were constructed in a plant binary expression vector to obtain an activation expression vector.Then,using 2x35S as a promoter,green fluorescent protein（GFP）was used as a reporter gene,and a transposon end was co-constructed into a plant binary expression vector to obtain a reporter gene vector.The interaction between the transposase and the non-autonomous transposon was observed by using a laser confocal microscope.The results are as follows:Activity of transposons in Arabidopsis:The screening and identification results of transgenic plants of transposase are as follows:（1）Through the screening of hygromycin,30 transgenic transposase-positive T₁ lines in gun5background were successfully obtained.Both TNP1 and TNP2 were identified by RT-PCR and expressed in gun5 plants.（2）Through the screening of Basta,48 transgenic transposase-positive T₁ lines were successfully obtained.Both TNP1 and TNP2 were identified by RT-PCR and expressed in im plants.The screening and identification results of transgenic plants of non-autonomous transposon are as follows:（1）Screening of non-autonomous transgenic plants by kanamycin,the non-autonomous transposon plants of different lengths in the background of gun5 were successfully obtained,and the leaf phenotypes of the plants were yellow-green.（2）Screening of transgenic plants by kanamycin,non-autonomous transposon plants of different lengths in im backgrounds were successfully obtained.The leaf phenotypes of T₁ plants of im-Tn1 and im-Tn2 were varigated,and the leaf phenotype of T₂ plants of im-Tn1 and im-Tn2 was varigated or green.The leaf phenotypes of T₁ and T₂ plants of im-Tn3 were all green.（3）5’RACE:The initiation site of the transcription of transposon plant of im-Tn3 is mainly in the UTR region of PTOX,and the 3’end of the transposon.（4）After shortening the UTR of PTOX,the non-autonomous transposon plants of three lengths in im backgrounds were successfully obtained by kanamycin screening.The leaf phenotypes of T₁ plants of im-Tn1（2）,im-Tn2（2）,im-Tn3（2）were varigated or full green.（5）Predicting the transcription initiation site of transposon plants that shorten the 5’UTR of PTOX in the background of im,and found that the transcription initiation site is indeed at the 3’end of the transposon,which gives us a clearer understanding of the structure of the transposon.The screening and identification results of the skipping plants in the background of gun5 were as follows:（1）The hopping plants in the background of gun5 were successfully obtained by DNA identification（both transposase gene and non-autonomous transposon gene）（2）No transgenic plants identical to the expected phenotype were found.（3）It may be due to the fact that the transposon has no transposition in the gun5 plant or the number of transgenic plants screened is not enough.Explore the interaction between non-autonomous transposon and transposase through transient expression of tobacco:（1）Successfully obtained an activating expression vector containing VP16 and transposase.（2）A reporter gene vector containing a non-autonomous transposon end（3’TIR or 5’TIR）and GFP was successfully obtained.（3）TATA of the negative control reporter vector can initiate expression of GFP of the reporter vector.