Characterization And Studies On Associated Genes Of LINE Retrotransposons In Mulberry(Morus Notabilis)

Transposable elements are DNA fragments which are able to move from one location to another in a chromosome or move from one chromosome to another. They are universal components in the prokaryotic and eukaryotic genomes and play important roles in the structural organization and evolution of genomes. Long interspersed nuclear element retrotransposons（LINEs） belong to Class I transposon which move to another chromosomal location via an RNA intermediate. LINEs are not flanked by long terminal repeats. They play important roles in the evolution of genes and genomes including size, structure and function.Mulberry is a deciduous and perennial woody plant and widely planted for breeding silkworm. It has important economic, medicinal and ecological value. With completion of mulberry genome sequencing and the establishment of mulberry transposons database, a platform was provided for the studies on LINEs. The studies on mulberry LINEs still stayed in the identification and classification without report for the further studies. In the present study, LINE reverse transcriptase（RT） fragments were amplified using degenerate primer pairs and their characteristics were analyzed. In addition, we analyzed the structure, preferences of insertion, and functions of associated genes of mulberry LINEs in genome-wide. Alternative splicing genes caused by the LINE insertions were screened out and verified by experiments. The purpose of this work is to provide candidate genes to further studies on how LINEs influence the gene regulation. The main results of this study are as follows:Isolation and characterization of mulberry LINE RTsA total of 43 clones of LINE RTs were isolated and identified by using degenerate primers from the mulberry genome. Sequences analyses showed that the length of RT nucleotide sequences ranged from 557 to 595 bp. All RT fragments were rich in A and T base and the ratio of AT / GC ranged from 1.26 to 1.98. Pairwise comparisons of the RT sequences revealed the identities ranged from 31.8% to 99.4%. The results indicated LINE RTs were highly heterogeneous. Furthermore, amino acid sequences analyses indicated that except Mno L₁0, stop codons exist in all sequences. In addition, frameshift mutation had occurred in 93%（40/43） sequences. The results showed that the stop codons and frameshift mutations were the main reasons resulted to the heterogeneity of LINE RTs.Sequence clustering analysis showed the 43 of RT fragments were divided into eight groups. The result of classification was consistent with classification based on sequences similarities. Group VIII and III accounted for 72%（31 / 43） of the total number of clones, which likely means Groups VIII and III would be the primary mulberry LINE families. The distribution of the stop codons and multiple sequence alignment analyses showed that the stop codons were distributed consistently among sequences of the same group. The results indicated that family members had similarly evolved, and had undergone a relatively stable amplification process at the same time.Furthermore, the phylogenetic analyses of the isolated RT fragments together with LINE RTs from 13 plant species showed that some mulberry LINE RTs were clustered with exogenous plants’, implied LINE retrotransposons not only transfer vertical, but also transfer horizontal between different species.Fluorescence in situ hybridization showed that LINEs were scattered, not randomly distributed. Hybridization signals were mainly visualized in subtelomeric regions with a little signal detected in the pericentromeric regions.Characteristics of mulberry LINEThe mulberry 36 intact L1 and RTE superfamilies sequences of LINE’s were downloaded from transposable element database. Sequences analyses indicated that their structure contained EN, RT, RNase H elements, which are consistent with thegeneral structure of LINEs. In addition, some families also contained CCHC zinc finger and DUF4238. LTR-type nested inside GAG, RT, RNase H and so on.The multiple sequence alignment analyses of the mulberry RT and EN fragments together with other plant species showed that the RT and EN fragments were highly homologous with corresponding conserved domains. The phylogenetic tree constructed based on RT and EN sequences showed that L1 and RTE were clustered in each clade, suggesting that after the differentiation, plant L1 and RTE superfamilies evolved in different directions.Though the analyses of insertion propensity of LINEs, we found that LINEs trended to insert into the internal genes. Functional annotation and pathway analyses for the genes inserted by LINE showed that these genes were mainly involved in the cell（37.41 %）, organelle（25.37%） and membrane（20.37%） of the cellular components; the catalytic activity（48.35%） and binding（38.07%） of the molecular function; the metabolic process（23.36%）, cellular process（19.97%） and single-organism process（17.07%） of the biological function. Furthermore, Pathway analyses showed that these genes were involved in 81 pathways, suggesting that LINEs may have potential regulating functions for these genes.Analysis of mulberry LINE retrotransposons associated genesThe 560 genes with LINE insertions（called LINE associated genes） were screened out based on the location information of mulberry genes and LINEs. Furthermore, 57 alternative splicing genes with LINE insertions were selected combined the alternative splicing location information. Some intron-exon, exon-intron, intron retention or skipping exon splice junction were contributed by LINE. These alternative splicing genes called LINE alternative splicing associated genes. Finally, 10 alternative splicing genes which alternative splicing forms caused by LINE insertions were obtained.The protein sequences of 10 LINE alternative splicing associated genes were analyzed. Among them, Morus009604, Morus023788 and Morus026596 were defined as hypothetical proteins without prediction results in the mulberry whole genome sequencing database. The annotation results showed that they had high similarities withtransporter protein（MATE） family ALF5（pear similarity 74%） from multidrug and toxic compounds row, the circadian clock output gene（GIGANTEA）（almond similarity 84%）, and acyltransferase（partial plum sequence similarity 75%）, respectively. Other seven predicted proteins were consistent with the results of the comparison in other species with high sequence similarities.Primers were designed according to the sequences of 10 LINE alternative splicing associated genes. The corresponding fragments were amplified which containing LINE retrotransposons and splicing regions. The 10 LINE associated genes were verified through experiment and the results showed that the alternative splicing of the five genes Morus008188, Morus012782, Morus013014, Morus006083 and Morus014786 were caused by LINE insertions. They can be used as candidate genes in studies on effects of LINEs to gene function.