Rapidly Cloning And Bioinformatics Analysising Of Specific ESTs On Spinach Y Chrommosome

Spinach (Spinacia loeracea L.), also named as Persian cuisine, field vegetables, usually are dioeciousplants. There have significant morphological differences between male and female, which is that the flowerof male plants are always as spica on the top of the stem or leaf axils, while the flower of female plants arealways born into a group at the leaf axils. The sex-determining mechanism of spinach is XY type. Spinachis in the second stage of the sex chromosomes evolution level. The genome of spinach is small (2n=12),and there are about1000Mbp genes in a single chromosome complete. So the spinach is the bestexperimental material in plant sex differentiation research of dioecian plant. At present, cytologicalresearch have shown that the largest chromosome of spinach (chromosome1) has three gender-relatedmultiple alleles which are the X, Xm, Y and is the sex chromosome of spinach. The gene type of malespinach is the XY, and the female is the XX. At present, the X, Xm, Y not yet have been successfully cloned,and there not yet have been reported clons of the sex chromosome linked function gene in spinach, whilethe sex-determining gene of spinach has not been found, the molecular mechanism of sex determination isunclear.Based on the problems above, this study used a relatively new technique that combine thechromosome microdissection method and the hybrid sequence amplification (HSA) technique to hybrid theDOP-PCR products of spinach Y chromosome and the cDNA in male spinach flower on the floweringperiod, when used the genome in female spinach as the blocking DNA, and to construct a cDNA library ofthe Y chromosome-specific expressed, then cloned, sequenced and bioinformatics analyzed the ESTs in thecDNA library. In this paper, a number of ESTs that spinach Y chromosome-specific expressed wereobtained, and can provide a theoretical basis to the research of the sex determination and differentiationmechanism, while can provide a foundation for the study of plant sex chromosome evolution. The mainresults are as follows:1. The Y chromosome of spinach was successfully microdissected and DOP-PCR products wereamplified, and the source of DOP-PCR products of Y chromosome in spinach was verified and analyzed;2. A new technical methods for rapidly cloning ESTs that Y chromosome-specific expressed in male spinach were successfully established;3. A cDNA library that Y chromosome-specific expressed in male spinach was construced, and90ESTs were obtained, the length of which are between53~486bp. Blastn analysis against the GenBankdatabase revealed that12ESTs showed significant homology to known genes which distributed in spinach,oil camphor, Arabidopsis, weeping birch, castor or other plants. Blastx analysis against the GenBankdatabase revealed that25ESTs share homology with putative proteins which distributed in grapes, rice,Oceania saltbush or other species. Gene Ontology (GO) annotation of the EST sequences was performedusing Blast2Go with a cutoff value of1E-10.18ESTs have523GO functional annotations, whichcontained367biological pathways results, accounting for70.17%;44cell component annotation results,accounting for8.41%;112molecular function results, accounting for21.42%.4. Consolidating the Blastn and Blastx homology analyzing results, we found that SP1-60has98.60%similarity with the mRNA encoding the peroxidase in spinach; SP2-52has98.96%similarity with the mRNAencoding the mitochondrial half-ABC transporter-related protein in Arabidopsis, ABC transporter proteinsare required for plant growth, plant development, resisting adversity while living and other physiologicalactivities; SP3-12has87.64%similarity with the mRNA encoding the histidine kinase3in Betula verrucosa;SP4-42has96.58%similarity with the eDMC1mRNA encoding RecA homolog DMC1in Anguilla japonica,DMC1are required for male sexual development, and lack of DMC1gene may cause male sterilityphenomenon; SP5-13has99.80%similarity with the mRNA for iron transport protein in Ricinus communis,iron transport protein are required for plant growth and embryonic development; SP5-62has81.88%similarity with the AnSAMS4mRNA for S-adenosyl-L-methionine synthase4in Atriplex nummularia.The previous reports showed: we infered that SP2-52, SP4-42and SP5-13have played a role in the growthand sex differentiation processes of male spinach plants, and may play role in the male sex determinationprocess of spinach, which remains follow-up experimental study to analyze and validate. And the expressedsequences without homology analyzing results obtained might be newly discovered in spinach, and needfuther exploration for their functions.