The Cloning And Expression Analysis Of Pollen AcSFB Genes From Almond (Amygdalus Communis L.)

Almond (Amygdalus communis L) is the diploid member of Rosaceae and is cross-pollinatd and self-incompatible plant. Style S-determinants have been determined to be S-RNase and the SFB (S-haplotype-specific F-box protein gene) alleles are considered to be the first candidates of the pollen S-determinants. Now there is not any research of almond pollen GSI in Xinjiang. In this study, the initial aim was to isolate and characterise AcSFB allele genes from almond cultivar in Xinjiang, China, to gain further understanding of the evolutionary relationships and phylogeny of SFB alleles within Prunus. Expression analysis and semi-quantitative RT-PCR were used to find which tissue could express SFB protein and which flowering period expressed more. 5'UTR and 3'UTR analysis and bioinformatics analysis still had studied in this experiment. The study of almond self-incompatible mechanism using molecular biological technology would fast promote the industry of almond. All results were summarized as follows:1. Total RNA from almond anther was extracted with the improved CTAB method. The almond anther RNA obtained was good integrity and without stain of polysaccharide and protein, whose yield was very high.2. In this study, fifteen novel AcSFB genes (GenBank accession numbers:EU909685, FJ362524~ FJ362529, FJ231204, FJ415321 and FJ514935~FJ514940 ) were identificated using RT-PCR and RACE technolgy in A. communis mainly planted in China for the first time. The identified AcSFB genes showed S-haplotypespecific polymorphism. These alleles encoded proteins with the F-box motif in the N-amino terminus, and with four (hyper) variable regions, i e., V1, V2, Hva and Hvb, considered to be recognition site between SFB and S-RNase and determined GSI. With the bioinformatics analysis, the AcSFB proteins were instable, hydrophilicity and non-secretory protein and had ligase or lyase enzyme activity.3. Using the DNAMAN soft, phyletic evolution tree was constructed. The result showed the interspecies amino acid identities obtained by comparison with other Prunus SFBs were often higher than intraspecies identities. The evolution relationship of pollen S genes between Chinese and American almonds was reciprocal chiasmata and there were no places differences.4. AcSFB gene was expressed only in pollen and not in other organ, which showed AcSFB expression had organspecificity. The AcSFB was expressed more in ball stage than in other stage, which would give a reference for arraying pollenizer. Study on express pattern of AcSFB may contribute to confirmation that the AcSFB gene is the pollen S-determinant. Still we proved there was no intron in the AcSFB genes and a single intron had been found in the 5'untranslated region of the AcSFB genes of almond.5. The vector of pET-AcSFB was constructed, after transformed to Escherichia coli BL21 and induced by isopropyl-β-D-thiogalactopyranoside (IPTG), recombinant protein about 60 kD was expressed in pET-AcSFB system and separated by SDS-PAGE electrophoresis. These results would provide a foundation for further purifying and identifying SFB protein.