Cloning And Analysis Of Aocrt4, A Ty1-copia-like Retrotransposon From Asparagus Officinalis L.

Asparagus officinalis L. is a dioecious plant with2n=20chromosomes, and its homotypic sexchromosomes XY are in the early stages of chromosomes evolution, but there is still no exact karyotypeand direct cytological evidence of the homotypical sex chromosomes. The genome of Asparagus officinalisis not stable, mainly in the presence of a pair of heterogenic autosome, the number of B chromosome is alsonot fixed, karyotypes of differrent breeds are differ in the positions of centromere and satellite.Therefore,Asparagus officinalis not only can be used as model plant to study sex chromosome evolution, origin ofdioecious plant and sex determination of dioecious plant, but also a good material for researching thestability of plant genome and chromosomes.Retrotransposons are DNA fragments that can move between different chromosomes, and theiractivities are closely related to gene expression, chromatin remodeling and the stability of genomics andgenome.Therefore, in this paper, a Ty1-copia group retrotransposon was separated from Asparagus officinalis.Its sequence features, copy number; transcriptional activity and chromosomal localization had beenresearched by the technique of PCR, RT-PCR, fluorescence in situ hybridization of chromosome andrelated bioinformatics. And its role in the evolution of Asparagus officinalis chromosomes was discussed.The Ty1-copia group retrotransposon was analysed to provid basic information for the genome evolution ofAsparagus officinalis. The results were as follows:1. Four Ty1-copia retrotransposons were separated and cloned from Asparagus officinalis and twofrom A. setaceus by degenerate primers, and sequences are about260bp. Sequencing and sequence analysisshow that they are conserved regions of Ty1-copia reverse transcripatase.2. By fluorescence in situ hybridization and RT-PCR technique, ranscriptional activity and copynumber of Aocrt4and Ascrt1were analyzed. Both Aocrt4and Ascrt1had been obtained positive bands fromtranscriptome of Asparagus officinalis, indicating that Aocrt4is with transcription activity, and with nodifferention between sex; the total copy number of Aocrt4in female and male Asparagus officinalis are2996and4572, respectively. Copy numbers of1C are558and589, respectively. Indicating that thedistribution of Aocrt4has no significant correlation with the sex of Asparagus officinalis. 3. In this paper, the intraspecific heterogeneity of Aocrt4were analysed. One A.setaceus, male andfemale Asparagus officinalis each for three were amplicated by specific primers. For each PCR products,eight clones were randomly selected and sequenced. Sequences were244-245bp. Their average similarity is97.72%, showing a lower heterogeneity. There is no significant difference between male and female plants,and among plants with the same gender.266mutation sites were found in the48sequences. In addition totwo sites are single T deletion, the rest are base substitutions, moreover, mostly were G/C A/T.Sequence alignment revealed that the interspecific heterogeneity of Aocrt4is higher thanintraspecific.heterogeneity. By amino acid sequence alignment of Aocrt4, which encode83amino acid,12sequences were found to have occured stop codon mutation, and mutation sites concentrated at47or48amino acid positions;2amino acid sequences occured frameshift mutation; there are two kind amino acidat43,49and73amino acids position, namely in turn histidine (H)/tyrosine (Y),85.2%of histidine; valine(V)/isoleucine (I), valine accounting for72.8%; isoleucine/threonine (T), isoleucine93.8%. In addition,all of the amino acid sequence showed a high degree of conservation.4. Chromosomal localization of Aocrt4were finished by fluorescence in situ hybridization. Aocrt4islocated on chromosome7of Asparagus officinalis, which exhibit heteromorphic features. Weakerfluorescence signal had also been found in all the other chromosomes, which shows that it may be withtranscriptionally activity. In addition, when45S rDNA were used as probes, hybridization signalspositioned to the1st, the2nd and the5th chromosome. If both Aocrt4and45S rDNA as dual hybridizationprobes, hybridization signals would locate in the1st, the2nd, the5th and the7th chromosome. Combinedwith the results of our laboratory study of5S rDNA used as hybridization probes, accurate chromosomepairing of Asparagus officinalis can be obtained.