Construction Of Suppression Subtractive Hybridization Library And Analysis Of Differential Expressed Genes Related To Gametophytic Self-incompatibility In Citrus Grandis Var. Shatinyu Hort. Style

The Shatinyu(Citrus grandis var. Shatinyu Hort.)is the famous fruit tree of Guangxi,which is the typical gametophytic self-incompatibility(GSI) plant and is the good material for researching the mechanism of GSI. However, its lower yield has greatly limited the development of the Shatinyu production. Studying the mechanism of GSI in Citrus grandis var.Shatinyu Hort. has important value in both Shatinyu production and the scientific research.The self and cross pollinated styles were used in this study, suppression subtractive hybridization (SSH) forward and reverse cDNA libraries were constructed from the tissue of style and expressed sequence tag(EST) was researched and analyzed.The rate of recombination of the forward library was 95% and the average length of inserted fragments was 250bp, which was testified by PCR analysis. The positive clone of the library was randomly picked for sequencing. After abandoning the lower quality ESTs, 67 high quality ESTs were obtained, in which 30 unique ESTs were gained through screening 37 repeat and <100bp ESTs. Searching these ESTs in GenBank non-redundant dates by Blastx and Blastn, 23 ESTs of them had comparatively clear results in function. Analyzing the genes through GO(gene ontology) showed that ten ESTs were relative to catalytic activity, accounting for 43.48%. Three ESTs were relative to enzyme regulating activity, accounting for 13.04%. Two ESTs were relative to transcription regulating activity, accounting for 8.7%. Three ESTs were relative to signal transduction,transporter and physiological process, accounting for 4.35% respectively. Additionally, four ESTs had not clear classification, accounting for 17.39%.Moreover, there were 7 ESTs, which had not comparatively clear results through Blastx, but one EST was highly homologous to Ipomoea trifida self-incompatibility(S-) locus region(Ipomoea trifida DNA,self-incompatibility(S-) locus region,haplotype:S1), its identities were 89%. One EST was homologous to Malus x domestica S9-RNase gene(Malus x domestica MdSFBB9-alpha, S9-RNase, MdSFBB9-beta genes, complete cds, Psi-SFBB9-alpha, Psi-SFBB9-beta pseudo genes), its identities were 89% also. Two of them were similar to Nicotiana benthamiana isomerase (Nicotiana benthamiana isopentenyl /dimeth-ylallyl diphosphate isomerase mRNA, partial cds) and Arabidopsis thaliana lyase(Arabidopsis thaliana LAS1(Lanosterol synthase1); catalytic/lyase (LAS1) mRNA,complete cds), their identities were 85% and 87% respectively.34 clones of the reverse library were picked up randomly and sequenced, of them there were 27 ESTs that sequenced results were good. Removing redundant ESTs, 24 unique ESTs were obtained. The results of Blastx analysis and GO classification showed that they were highly homologous to catalytic activity and enzyme regulating activity, which represented 29.17% respectively, such as glucosyltransferase, chitinase, prolyl oligopeptidase and histidine kinase etc. The rest were some homologous to cellular component(16.67%),binding(8.33%) and physiological process(4.17%) and so on.Above all, we can conclude that GSI of Shatinyu is not only controlled by S-gene, but also are influenced by many factors. The SSH library construction of gametophytic self-incompatibility in shatinyu style will make a foundation for cloning the correlative genes of self-incompatibility(SI) and thoroughly understanding the mechanism of SI of Shatinyu.