Tissue-specific Expression Activity Of Mdsolo-LTR1 And Its Deletion Mutagenesis From Malus×Domestica

The apple(Malus × domestica Borkh.)belongs to the Malus genus of Rosaceae and is rich in transposons and other transposable elements that promote the evolution of the genome.Previous research showed that there was a specific length on the chromosome 4 chromosomes of “ Delicious” spur sports ‘Chinese Marshal',‘Oregon Spur ‘Delicious' ',‘Meiguihong' and ‘Show Red'.The 2190 bp solo-LTR(named Mdsolo-LTR1)was further analyzed and found to be inserted in another solo-LTR(named Mdsolo-LTR2,length 1536 bp);Mdsolo-LTR1 was specifically expressed in the stem.,With two-way start function;Mdsolo-LTR2 sense and antisense strands have no obvious promoter activity.On this basis,this study used the PlantCARE prediction results to segment the sense and antisense strands of Mdsolo-LTR1 into 20 fragments based on the full-length sequences of Mdsolo-LTR1 sense and antisense strands.The activity of the sense strands tests were D2(699 bp ~ 2190 bp),D3(1179 bp ~ 2190 bp),D4(1643 bp ~ 2190 bp),D5(2041 bp ~ 2190 bp),and D6(2102 bp ~ 2190 bp).The activity of the antisense strands was designated as-D2(2190 bp ~ 699 bp),-D3(2190 bp ~ 1179 bp),-D4(2190 bp ~ 1643 bp),and-D5(2190 bp ~ 2041 bp).-D6(2190 bp ~ 2196 bp);double-stranded activity assays were named dF1(175 bp ~ 2190 bp),dF1(336 bp ~ 2190 bp),dF3(510 bp ~ 2190 bp),dF4(852 bp)~ 2190 bp),dF5(1040 bp ~ 2190 bp),dF6(1313 bp ~ 2190 bp),dF7(1486 bp ~ 2190 bp),dF8(1750 bp ~ 2190 bp),dF9(1910 bp ~ 2190 bp),dF10(2156 bp ~ 2190 bp).The results of tobacco transient expression assay showed that the positive and antisense strands of Mdsolo-LTR1 and its deletion fragments of different lengths,Mdsolo-LTR2 positive and antisense strands,PEAR-1-sense strand and PEAR-1-antisense strand have promoter expression activity.Further analysis revealed that the deletion fragments of different lengths of Mdsolo-LTR1 at the 5' end was: dF4(1339 bp)(852 bp ~ 2190 bp)(a)> dF2(1855 bp)(336 bp ~ 2190 bp)(b)> dF6(878 bp)(1313 bp ~ 2190 bp)(c),dF5(1151 bp)(1040 bp ~ 2190 bp)(c)> dF1(2016 bp)(175 bp ~ 2190 bp)(d),dF7(705 bp)(1486 bp ~ 2190 bp)(d),dF9(281bp)(1910 bp ~ 2190 bp)(d),dF3(1681 bp)(510 bp ~ 2190 bp)(d),dF8(441 bp)(1750 Bp ~ 2190 bp)(d),dF10(35 bp)(2156 bp ~ 2190 bp)(d);Antisense strand 3'-end deletion fragment of different length Expression activity: dF5(1151 bp)(1040 bp ~ 2190 bp)(a)> dF10(35 bp)(2156 bp ~ 2190 bp)(ab),dF3(1681 bp)(510 bp ~ 2190 bp)(ab)> dF4(1339 bp)(852 bp ~ 2190 bp)(b)> dF2(1855 bp)(336 bp ~ 2190 bp)(bc)> dF9(281bp)(1910 bp ~ 2190 bp)(cd)> dF6(878 bp)(1313 bp ~ 2190 bp)(d),dF1(2016 bp)(d)175 bp ~ 2190 bp(d),dF8(441 bp)(1750 bp ~ 2190 bp)(d),dF7(705 bp)(1486 bp ~ 2190 bp)(d),indicating that the Mdsolo-LTR1 sense chain 189 bp at the 3` end,35 bp at the 5' end of the antisense strand and 274 bp at the 3' end have promoter expression activity.There was no transient expression activity of the Mdsolo-LTR2 positive and antisense strands,but there was a little blue spots on the tobacco petiole wounds,possibly due to the injury stress caused by the wound to stimulate its expression activity.Arabidopsis thaliana was transformed by Floral-dip method.T3 transgenic lines of each fragment were obtained after resistance screening and PCR identification.Histochemical staining and quantitative analysis of GUS activity were used to detect the expression activity of each fragment in different organs at different developmental stages of transgenic lines.The results showed that the 88 bp 3'-end of the sense strand of Mdsolo-LTR1 expressed active leaf>root>stem in 30 d seedling transgenic lines;The 547 bp 5'-end of the antisense strand of Mdsolo-LTR1 expressed in roots,stems and leaves,among them,the expression of active root> leaf> stem;the activity of Mdsolo-LTR2 positive and antisense was detected only in the above-ground part of the 15 d seedling transgenic lines,and the activity was comparable,In the 30-day-old transgenic lines,the activity of Mdsolo-LTR2 sense strands was higher in stems and leaves than in roots,and the Mdsolo-LTR2 antisense strands were only weakly expressed in stems and leaves;Both the PEAR-1 positive and antisense strands have expression activity in the root part of the roots of the 30 days old transgenic lines.