| The frequency of apple bud mutation which plays an important role in apple’s breeding work is high. Many excellent cultivars planted widely have been obtained by bud mutation breeding,and spur mutation is a type of bud mutation. The results of the previous research showed that retrotransposon atrl was involved in apple’s spur mutation and partial sequence of 3’-LTR and RT in atrl were isolated by using IPCR, a specific 1722 bp fragment in "Delicious" spur sports "Meiguihong", "Aihong", "Oregon Spur’Delicious’ "and "Huashuai 1" was isolated by using IRAP based on primers designed from the long terminal repeat sequence of atrl (atrl-LTR) and genome walking. Base on the previous research, we obtained full sequence of atrl by using genome walking and designing primers based on sequence characteristics in retrotransposon atrl. The alignment result indicated that atrl is a typical Tyl-copia-like retrotransposon. we used genome walking to obtain the flanking sequence of specific 1722 bp fragment from "Delicious" spur sports "Meiguihong", "Aihong", "Oregon Spur’Delicious’" and "Huashuai 1", and a specific 4345 bp fragment was isolated and cloned in the four spur sports. The alignment result showed that they are the same for the specific fragments in the four spur sports. We submitted the new sequence to the apple genome database and the result suggests that it may be caused by retrotransposon carrying its flanking sequence inserted into the new loci.1. The isolation of full sequence in atrl1.1 The amplification of atrl based on its sequence characteristicsOn the basis of the previous research, we have obtained atrl-3’-LTR, RT and 3’partial flaking fragment of 2124bp. Using the sequence characteristics of Tyl-copia-like retrotransposon, the forward primer was designed according to the sequence of 3’-LTR, while the reverse primer was designed according to the sequence of 5’-RT and finally most of the sequence of atrl was obtained by using nested PCR, only missing a few base pairs at the 5’ end in 5’-LTR.1.2 The amplification of 5’-LTR and its flanking sequence in atrl by using IPCRGenomic DNA extracted from " Meiguihong" was digested adequatedly by restriction enzymes Vsp I and self-looped and ligated by T4 DNA ligase and the reaction products were used as templates for PCR. A fragment including 5’-LTR and its flanking sequence in atrl was amplified after two circles of nested PCR.1.3 The identification of retrotransposon atrl and its distribution in the apple genomeRetrotransposon atrl which is a full-length of 4996 bp was obtained and cloned from "Meiguihong" genomic DNA using the forward primer designed based on the 5’-flanking sequence and the reverse primer designed by adding adaptor in 3’-LTR. The sequence analysis indicated that it contains "5’-LTR, GAG region, integrase (INT), reverse transcriptase(RT), RNsesH and 3’-LTR"The sequence was submitted to the apple genome and analyzed and the result suggested that there are more homologous sequences in the apple genome, which it indicated that the retrotransposon atrl with transposition activity maybe involved in the evolution of apple genome. Ten sequences with high similarity to atrl were selected and analyzed, and the result indicated "MDC018640.562" included a typical Tyl-copia-like retrotransposon, the homogeneity in it’s enzyme region with atrl ribbon was up to 96.4%, containing 5 bp insert site(AGACA) in the flanking sequence of the retrotransposon, but its homogeneity in LTR with atrl-LTR was only 65.24%. However, flanking sequence of other retrotransposon which have a high homogeneity in both enzyme region and LTR with atrl has no insert site.2 The isolation of the specific amplification fragments from "Delicious "spur sports "Meiguihong", "Aihong", "Oregon Spur’Delicious’ "and "Huashuai 1"2.1 The isolation of the 5’-flanking sequence based on the specific amplification 1722 bp fragments from "Delicious" spur sports by TAIL-PCR5’-flanking sequence in "Delicious " spur sports was obtained by TAIL-PCR based on the specific amplification fragment with 1722 bp in length, and a 1964 bp fragment was obtained after splicing. The alignment result showed that they are the same for the specific fragments in the four spur sports. The PCR result indicated that 1964 bp is the specific amplification fragment in four "Delicious " spur sports.2.2 The isolation of the 3’-flanking sequence based on the specific amplification fragment with 1722 bp in length from "Delicious" spur sports using apple genomeWe submitted the specific amplification fragment (1722 bp in length) to the GeneBank and aligned with apple genome, and found that 1148 bp in the 3’ end of the fragment can match many sequences with homogeneity of more than 90%. We selected one common sequence from these homologous fragments and spliced with 1722 bp at the 3’end, a 2271 bp was isolated using the primer based on 270 bp at the 5’end of the 1722 bp, and the length of new obtained sequence is 859 bp. The alignment result showed that they are the same for the specific fragment in the four spur sports. The PCR result indicated that 2271 bp is the specific amplification fragments in four "Delicious" spur sports.2.3 The isolation of the 3’-flanking sequence based on the specific amplification fragment with 1964 bp in length from " Delicious" spur sports by hiTAIL-PCRThe 3’-flanking sequence of atrl in "Delicious" spur sports was obtained by hiTAIL-PCR based on the specific amplification fragments with 1964 bp in length. After splicing with the specific fragment, a 3032 bp fragment was obtained, and the length of new obtained sequence is 1010 bp.The alignment result showed that they are the same for the specific fragment in the four spur sports. The PCR result indicated that the length of the specific amplification fragments in four "Delicious " spur sports are 3032 bp.2.4 The isolation of the 5’-flanking sequence based on the specific amplification fragment with 3032 bp in length from "Delicious " spur sports by IPCRAfter genomic DNA extracted from "Meiguihong" was digested adequatedly by restriction enzymes Sph I, the flanking sequence at 5’end of the specific amplification fragment with 3032 bp in length was amplified by using IPCR. After splicing, a sequence with 4345 bp in length was obtained. The alignment result showed that they are the same for the specific fragments in the four spur sports. The PCR result indicated that the specific amplification fragments in four " Delicious " spur sports are 4345 bp in length. |
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