Study Of Mechanism On Regulation Of Curd Development

Cauliflower curd is a white smooth semi-spherical structure, with shortened branches inside and a mass of proliferated meristems outside as surface. Most of the meristematic tissues outside have been identified as indeterminate inflorescence meristems. In contrast, the surface of broccoli curd is composed of much proliferated developing flowers, which would not bloom until the curd begins to bolt. After developing for a period of time, both the curds of cauliflower and broccoli will bolt and produce normal flowers. Previous studies have shown that the formation of cauliflower curd is related to the stop mutant in BobCAL gene. But how the morphogenesis of the curd is regulated by BobCAL is still under dispute because of no direct evidences. The study on the molecular mechanisms underlying curd formation would be useful to catch on the regulatory mechanism of floral meristem determination, and could provide theoretical basis for genetic engineering of this important agricultural trait.In a lot of homozygous cauliflower lines, we have identified four genotypes of cauliflower: smooth curd (sc), thorny curd (tc), crystal curd (cc) and hairy curd (hc). Microscopy analyses indicate that there are numerous inflorescence meristems inserted on the surface of the smooth curd of sc, and some little cauline leaves except for a lot of inflorescence merstems found on that of thorny curd of sc. However, naked floral mersitems have been found besprinkled on the surface of the crystal curd of cc. As to hc, sepal primordia are found on a portion of the floral mersitems composed of the hairy curd. To prove it is the existence of the stop mutant in the fifth exon of BobCAL gene that leads to curd formation, we have introduced the full length BoCAL gene into cauliflower to complement the lost function of BobCAL. The result shows that the transgenic cauliflower lines give rise to inflorescence composed of green flowers instead of white curd. It indicates that the curd phenotype caused by the loss of function of BobCAL has been complemented, suggesting it is the premature stop mutant in BobCAL that leads to the formation of cauliflower curd. However, sequencing analyses have revealed the existence of two copies of BobCAL genes: one BobCAL-T with premature stop codon and one BobCAL-G without in hc and cc cauliflower, while one BobCAL-T in sc and tc cauliflowers. In broccoli, there are two copies of BoiCAL genes: BoiCAL-G and BoiCAL-T, too, which may contribute to the similarity between the curd phenotype of hc cauliflower and broccoli. The formation of curd in hc cauliflower and broccoli is possibly because the function of Bob/BoiCAL-G has been interrupted by the existence of Bob/BoiCAL-G, although other genetic factors still could not be excluded as determinants of curd formation.Expression analyses of flowering-related genes have shown that, BobTFL1 gene expresses at a high level, while BobLFY at a low level, at the curd enlargement stage in sc and cc cauliflower. In contrast, expression of BobTFL1 shows a tendency of decrease, while that of BobLFY shows increase, in tc and hc cauliflowers. Higher expression level of BobLFY and the higher ratio of BobLFY/BobTFL may result in the morphogenesis of crystal curd and hairy curd. In the curd of hc and cc, the ratio of BobLFY/BobTFL may be upregulated by BobCAL-G.The alternative splicing of BobCAL gene is existed in cauliflower. In both sc and tc cauliflowers, we have found 4 alternative splicing variants and, according to their length, named them as BobCAL-Ta,BobCAL-Tb,BobCAL-Tc, and BobCAL-Td respectively. While in hc and cc cauliflowers, 8 splicing variants have been found to be resulted from both BobCAL-T and BobCAL-G. Sequence analyses have shown, besides BobCAL-Ga/Ta, all the other splicing variants have intronic sequence inserted. Expression analysis have shown the relative abundance of BobCAL splicing variants are different developmental stage of cauliflower curd, suggesting BobCAL regulate the development of curd through producing different proteins.In broccoli, BoiCAL also produces 8 splicing variants. RT-PCR analysis indicates that the expression pattern of the splicing variants in floral organs of curd flowers is different from that of flowers at bolting stage. The different expression pattern may act to inhibit flowers bloom in curd enlargement stage but promote at curd bolting stage.In order to study the function of proteins encoded by the splicing variants of BobCAL gene, we have tried to introduce the coding sequence of the four BobCAL proteins into Arabidopsis Ler and Ws ecotype to study the effects when they are over expressed; and into ap1 mutant and ap1 cal double mutant lines to study to what extent they can recover the mutant phenotype. Through the floral dipping method, we have obtained the transgenic lines with coding sequence for each BobCAL protein in different background. Further phenotypic analyses are ongoing. SEPALLATA 3(SEP3) gene acts to determinate the identity of petal, stamen and carpel. While in hc and cc cauliflower curds, the three inner floral whorls are all inhibited from differentiating. In order to study whether the BobCAL proteins of different length still can interact with BobSEP3, we have cloned the BobSEP3 genes from cauliflower, and carried out yeast two hybrids analysis aiming at the interaction between BobCAL and BobSEP3. Preliminary results have indicated the three truncated BobCAL proteins can interact with BobSEP3. In contrast, the binding activity to BobSEP3 is weaker for the whole BobCAL protein. The complex of the truncated BobCAL and BobSEP3 is nonfunctional and therefore could impact on the action of BobSEP3 in the determination of the three inner whorls of petal, stamen and carpel.In addition, we have cloned the homologous genes of CAL from subspecies of B. olearcea and B. campestris species, and carried out polymorphic and phylogenic analysis. The results indicate that the varieties of B.oleracea can be attributed into two groups that have evolved independently. We have also found that flowering Chinese cabbage are much more related to B.oleracea than to B.campestris.