Characterization and expression analysis of a MADS box gene, HoMADS2, in Hyacinthus orientalis L.

A MADS box gene HoMADS2 was cloned from Hyacinthus orientalis L. in this study. Sequence comparison revealed that HoMADS2 was highly homologous to the class B MADS box genes. Furthermore, phylogenetic analysis showed that HoMADS2 was closely related to PI gene family and this was also supported by the presence of specific diagnostic sites of PI homologs in K box domain and C terminal region,suggesting that HoMADS2 might be a PI-like gene. HoMADS2 mRNA was accumulated in all floral organs,different from the expression patterns of PI homologs in dicots. During in vitro flower development, HoMADS2 expression was constitutively expressed and not affected by the presence of cytokinin and auxin in the regenerated flowers. Our results indicated that the expression of HoMADS2 is different from those of both HAG1 and HoMADS1 in responding to plant hormones during in vitro flower development.

RNA silencing of Waxy gene results in low levels of amylose in the seeds of transgenic wheat (Triticum aestivum L.).

In this study, the level of amylose was reduced in wheat seeds by RNAi strategy. Because the synthesis of amylose is catalyzed by the granule-bound starch synthase I (GBSSI or WAXY protein), the Waxy gene of wheat was isolated from wheat seeds by using RT-PCR. Southern analysis confirmed that there were three Waxy genes in wheat genome. Northern hybridization showed that Waxy mRNA accumulated in seeds following pollination. By RNAi strategy,the 683 bp sense and antisense fragments in reverse orientation separated by a 150 bp intron were cloned into pCAMBIA 3300 just downstream of the maize ub/1 promoter. By Agrobacteriurn-mediated wheat transformation method, four transgenic plants (Cultivar Yangmai 10) were identified by PCR, RT-PCR and leaf painting assay. The level of amylose in the endosperm were significantly reduced in transgenic seeds as checked by iodine staining and analysis of amylose content. The results indicated that RNA silencing of Waxy gene resulted in low level of amylose in the seeds of transgenic wheat.

Characterization of HoMADS 1 and its induction by plant hormones during in vitro ovule development in Hyacinthus orientalis L.

To understand the molecular mechanism of ovule development, a MADS box gene, HoMADS 1 , has been isolated from the ovule tissues of Hyacinthus . Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.

Isolation of HAG1 and its regulation by plant hormones during in vitro floral organogenesis in Hyacinthus orientalis L.

Floral organs have been successfully induced from the regenerated floral buds of Hyacinthus orientalis L. by precisely controlling exogenous hormones in the medium. Under high concentrations of cytokinin and auxin, the regenerated floral bud produces only tepals. However, at reduced levels of the hormones, the regenerated floral bud can produce stamens and/or carpels with ovules. To understand the molecular mechanism of hormone-regulated flower development, a MADS-box gene, HAG1, which is homologous to AGAMOUS (AG) in Arabidopsis, was isolated from the floral tissues of Hyacinthus. Overexpression of HAG1 in Arabidopsis created flower phenotypes resembling those of the apetala2 mutant and AG transgenic Arabidopsis plants. Furthermore, the HAG1 expression pattern was similar to that of AG, confirming that HAG1 is the ortholog of AG in Hyacinthus. HAG1 mRNA was first detected in cultured explants at day 5 in the medium containing high levels of cytokinin and auxin, which could induce floral regeneration in vitro. However, no HAG1 mRNA was detected in the cultured explants until day 10 in media with low or no hormones. Further, HAG1 mRNA was detected in the stamens and carpels of regenerated floral buds, but not in the tepals. Our data support the hypothesis that hormone-regulated HAG1 activity is required for the induction of floral buds and the determination of floral organ types during the regeneration of floral buds.