Clone And Expression Of SRK And SCR In Ornamental Kales And Some Study About Overcoming Its Self-incompatibility

Ornamental kale (Brassica oleracea var. acephala) is a kind of typical sporophytic self incompatible plant. The sporophytic self-incompatibility (SSI) is genetically controlled by multi-allelic S loci, and the male SI and female SI are controlled by S-locus receptor kinase (SRK) and S-locus cysteine-rich protein (SCR)/S-locus protein 11 (SP11), respectively. In this study we obtained four clones, i.e., those of SRK kinase domain, the full sequence of SRK expression fragment, SCR cDNA sequence, and SCR DNA sequence. It was confirmed that the genotype of the self-incompatible materials we used in this study was S_13b haplotype. In addition, some researches about overcoming the pollen self-incompatibility were undertaken. Results are as follows:(1) The expression of SRK gene in stigmas at five developmental stages as defined in bud length was examined by Northern blotting. SRK was expressed in stigmas of buds, the length of which were longer than 10mm and between 8-10mm, but no expression in smaller buds. The expression quantities in stigmas of buds with＞10mm, which produced mature stigmas, was greater than that of 8- 10mm buds, which was estimated to open next day.(2) A new gene SCR_13b was obtained, which had a high sequence identity with SCR₁₃. The comparison between the SCR genomic sequences and cDNA sequences revealed that SCR_13b gene contained two exons interspaced with one 758 bp intron, and that this gene encoded a polypeptide of 78 amino acids containing a conserved signal peptide of 54 amino acids at the N-terminal. We produced a glutathione-S-transferase (GST) fusion protein in Eschichia coli using the pGEX-KG vector to express SCR_13b without the signal peptide, and purified the GST SCR_13b fusion protein of about 32 kDa by affinity chromatography, which provided foundation for the study of function of SCR_13b in the self-incompatibility response.(3) It was confirmed that CO₂ laser (a single irradiation for 5 min and high-voltage electrostatic field (HEF) (75kV/m) were helpful for overcoming the self-incompatibility of pollens.(4) According to several measurements of the pollen germination rate and the length of pollen tubes it was shown that pollens in the germination medium stopped growing by adding incompatible stigmatic proteins. When incompatible stigmatic proteins were put into the medium before pollen germination, the germination rate of pollens pre-treated with HEF was higher than that of the control. When incompatible stigmatic proteins were added after germination, the pollen tubes pre-treated with HEF continued to grow, but the pollen tubes without HEF pre-treatment stopped growing.(5) Ca²⁺ gradient in pollen tubes, which was labeled by fluo-3 probe, was observed with a confocal laser scanning microscope. Ca²⁺ gradient in the pollen tube was maintained, when pollen grains were pre-treated with HEF, but the gradient was lost without HEF treatment, i.e., Ca²⁺ dispersed in the pollen tube.(6) The protein expression analysis by 2D electrophoresis was performed using proteins extracted from self-pollinated stigmas, non-pollinated stigmas, cross-pollinated stigmas, and stigmas pollinated with HEF-treated pollen. It was found that spot distribution of proteins prepared from self-pollinated stigmas was similar with that from non-pollinated stigmas, and that spot distribution of proteins prepared from cross-pollinated stigmas was similar with that from stigmas pollinated with HEF-treated pollen. And the latter two samples had some spots more than the former two samples. It was suggested that the different pattern of spots were caused by some specific proteins that were involved in the self-incompatible response. It may be possible that lacking of these proteins brings about the self-incompatible response, but that HEF-treated pollens with these proteins expressed can overcome the sell-incompatible response.