Molecular and morphological analyzes of floral organs in an asparagus homeotic mutant

1966年，两个无性系“金沙茨”芦笋从东德引进日本，并通过无性繁殖在北海道大学维持。其中一个系的花被片很小，呈绿色。未发现雄蕊，但在第3轮（雄蕊轮）分化出类似心皮的花器官。这些不定心皮分化出胚珠，包括胚囊和卵细胞。这些形态特征表明，“金沙茨”系可能是由于b类MADS-box基因功能缺失而导致的花器官发生同源突变体。Southern blot分析表明，用b类和c类MADS-box基因的DNA片段进行探针时，野生型和突变型植物的DNA片段带型没有差异。然后，通过Northern blot分析MADS-box基因（c类为AOAG1和AOAG2， b类为AODEF， AOGLOA和AOGLOB）在野生型和突变型花中的表达情况。结果表明，b功能基因仅在野生型中表达，在突变花中几乎没有表达。在两系花中均检测到c功能基因的表达。原位杂交实验表明，AODEF和AOGLOB在野生型花的内花被片和雄蕊原基中只表达。在发育早期，AODEF的表达似乎保持不变，而在突变花的发育早期，AODEF的表达迅速消失。在整个发育过程中，突变花中未检测到AOGLOA转录本。这些结果表明，AOGLOA的表达降低导致了AODEF和AOGLOB基因的表达下降，B功能基因的失能导致了芦笋“金沙茨”同源突变体的表型。

Two vegetative lines of 'Gold Schatz' asparagus were introduced to Japan from the East Germany in 1966 and maintained in Hokkaido University through a vegetative propagation. One of these lines developed flowers with small tepals showing green color. Stamens were not found, but floral organs resembling the carpel differentiated at whorl 3 (stamen whorl). These adventitious carpels differentiated ovules including an embryo sac and egg cell. These morphological features suggested that the 'Gold Schatz' line might be a homeotic mutant of floral organogenesis, caused by a functional loss of class-B MADS-box genes.Southern blot analysis indicated that there was no difference in banding patterns of DNA fragments between wild type and mutant plants, when probed with DNA fragments specific for each of class-B and class-C MADS-box genes. Then, the expression of the MADS-box genes (AOAG1 and AOAG2 for class-C and AODEF, AOGLOA and AOGLOB for class-B genes) in wild type and mutant flowers was examined by Northern blot analysis. As a result, the expression of the B-functional genes were detected only in the wild type or scarcely detected in the mutant flowers. The expression of C-functional gene was detected in flowers from both lines. The hybridization experiments in situ displayd the exclusive expression of AODEF and AOGLOB at the primordia for inner tepals and stamens in wild type flowers. It appeared to remain during the early developmental stage, while the expression of AODEF in the mutant flowers rapidly disappeared in the early developmental stage. The transcripts for AOGLOA were not detected in the mutant flowers throughout the development.These results suggest that the lowered expression of AOGLOA causes a decline of the expression of AODEF and AOGLOB genes, and that the inability of B function genes leads to the phenotype found in an asparagus 'Gold Schatz' homeotic mutant.