Cloning And Functional Characerization Of Genes Encoding A Putative DELLA Protein From Flowering Chinese Cabbage

Flowering Chinese cabbage（Brassica Campestris L.ssp.Chinesis var.utilis Tsenet Lee.）is an important vegetable in Cruciferae,and widely grown in south China.Stalk is the major produced organ of flowering Chinese cabbage,and normal bolting is a necessary condition for the stalk formation.In recent years,analysis on bolting and flowering mechanism of flowering Chinese cabbage has attracted much attention.In earlier research have shown that low temperature and gibberellin can promote bolting and flowering of flowering Chinese cabbage,but the molecular mechanisms are not known.DELLA protein is an important negative regulator in the gibberellin signal transduction pathway and plays an important role in the regulatory pathway of GA.Therefore,based on the existing transcriptome database in the laboratory,the molecular characterization of DELLAs,the localization of their proteins,and expression patterns in different organs and upon gibberellin and low temperature treatment were investigated.The main results were as follows:1.Cloning BcRGA1,BcRGA2,BcRGA2-like,BcRGL1,BcRGL2 and BcRGL3 genes from flowering Chinese cabbage and the full-length open reading frames of these six genes were 1722bp,1740bp,1740bp,1524bp,1635bp,and 1578bp,respectively.Phylogenetic tree analysis showed that the DELLAs gene had the closest relationship with Chinese cabbage,and had the furthest with grape.Predicted the protein domain,motifs and secondary structure of these six genes by the amino acid sequences,and the result showed that these six genes function were conserved,and had the DELLA and GRAS domains,which were the typical domain of DELLA family.2.The BcRGA1,BcRGA2,BcRGA2-like,BcRGL1,BcRGL2 and BcRGL3 genes were constructed the recombinant plasmids fued with EGFP and transformed into onion epidemics cells,the results showed that BcRGA1,BcRGA2,BcRGA2-like,BcRGL1,BcRGL2 and BcRGL3 genes were localized in cell membrane,cytoplasm and nucleus,but the signal strength is not the same.BcRGA2 were localized in cell membrane and nucleus.3.BcRGA1,BcRGA2,BcRGA2-like,BcRGL1,BcRGL2 and BcRGL3 genes were expressed in different tissues of flowering Chinese cabbage,include root,shoot tips,leaf and flower.The results showed that BcRGA1,BcRGA2,BcRGA2-like,BcRGL1,BcRGL2and BcRGL3 genes were all expressed in the roots,stem tips,leaves and flowers of flowering Chinese cabbage.And the expression patterns of these genes in the cotyledons,three-leaf,six-leaf and flowering period were similar,with the highest expression levels in stems and leaves.Among them,the expression levels of BcRGA1,BcRGA2-like,and BcRGL1 were higher than those of the other three genes.4.By paraffin section we observed the shoot tip structures of two different maturing（early-maturing variety‘youlv501’and lately-maturing variety‘youqing 80 day’）of flowering Chinese cabbage.From the longitudinal section of the stem tips,it can be seen that the‘youlv501’has developed faster than the‘youqing 80 day’at all times,especially during the four-leaf period.In the period of two leaves and one heart,both of them had not completed the flower bud differentiation,but the flower bud differentiation of‘youlv501’was completed in the four-leaf and one-heart period.Through quantitative PCR that we found that only the expression level of BcRGL1 was significantly different between the two cultivars at both leaves and shoot tips.There were no significant differences in other genes.During the four-leaf period,the expression levels of all genes were different.From this we conclude that BcRGL1 may play an important role in flower bud differentiation.5.In order to study the effect of gibberellin on flowering Chinese cabbage,treated flowering Chinese cabbage with 200 mg/L gibberellins in two-leaf and one-heart and three-leaf and one-heart,the results showed that two-leaf and one-heart spraying GA₃treatment did not significantly lead to flowering and blooming of the flowering Chinese cabbage,and the three-leaf and one-heart spraying GA₃ treatment significantly advanced the flowering and blooming of the flowering Chinese cabbage.Whether it is two-leaf and one-heart or three-leaf and one-heart spraying uniconazole treatment,significantly inhibited flowering and blooming of the flowering Chinese cabbage.From relatively expression,the two-leaf and one-heart spraying gibberellin treatment increased the expression of DELLAs gene,and the treatment of three-leaf and one-heart spraying gibberellin reduced the expression of DELLAs gene.6.In order to study whether temperature affected the DELLAs,we used a low temperature of 15°C to treat flowering Chinese cabbage.Quantitative results showed that the DELLAs gene was significantly down-regulated in stem tips,leaves of flowering Chinese cabbage at 15°C for 6 h.But the BcRGL2 gene was not down-regulated after 6hours of treatment.7.In order to verify the interaction between DELLA protein and gibberellin receptor GID1 in flowering Chinese cabbage,we used the yeast two-hybrid technique.The yeast double-hybrid result showed that when GA existed,6 DELLA proteins were only associated with BcGID1b.It is also found that none of the 6 DELLA proteins interact with GA receptors BcGID1a and BcGID1c.In summary,this study showed that three-leaf and one-heart spraying of gibberellin increased the plant height,stem diameter,budding and flowering rate,and quantitatively detected the expression level of DELLAs gene by fluorescence,except for BcRGL2.The expression levels of the five genes were significantly down-regulated.Yeast double-hybrid assay was used to detect the relationship between DELLA protein and gibberellin receptor BcGID1.It was found that DELLA protein interacts with BcGID1b.Therefore,it can be speculated that DELLA protein exists when gibberellin is present.When combined with the gibberellin receptor BcGID1b to form a hydrophobic surface,it is easy to bind with the E3ubiquitin ligase,so that the DELLA protein is ubiquitinated and thus is degraded by the 26S proteasome,so that the flowering hearts are twitched and flowered in advance.