Primary Functional Ananlysis Of GmMADS28Genes From Soybean

MADS-box genes are the most important genes in plants, which participate in the development of flowering, lateral root, fruiting and embryogenesis. There are more than70MADS-box genes and over100MADS-box genes have been researched respectively in rice and Arabidopsis. However, there is little research about MADS-box genes in soybean. Therefore, the study about MADS-box genes in soybean is meaningful. GmMADS28is one of MADS-box family, which is highest expression in floral organ. Sequence alignment analysis and the phylogenetic tree of GmMAS28show that it belongs to SEP subfamily. Several methods have been used to study the functions of GmMAS28in this thesis.First, we used FISH (florescence in situ hybridization) to investigate the expression of flower development. To design the mRNA probe sequence, which is a specific mRNA fragment of GmMADS28, we did sequencing and analysis to GmMADS28. Then the specific mRNA fragment of GmMADS28was cloned and inserted into pGEM-T Easy vector, which was transformed into Escherichia coli. The mRNA hybridization probe had been built, after sequencing and adding with Digoxin labeling. Then we made mRNA probe to hybridize with paraffin section. By doing analysis on paraffin section which is hybridized by probe, we found that GmMADS28is a flower-enriched gene. The signals, which are detective strongly at the early stage of flowing, were captured at flower bud, anther、carpel、ovule and immature embryo medial during different flowering periods.Second, GmMADS28also was used as preys to screen GmMADS28-interacted protains by yeast two-hybrid system.We used yeast two-hybrid system to build cDNA library and bait vector, which is used to screen GmMADS28-interacted proteins, for probing GmMADS28-involved biological processes during the formation of floral organ. Detecting the reporter gene lacZ show that12interaction proteins have been found, eight of which have strong interaction with GmMADS28. Four of these proteins, the functions of which are unknown, are belong to soybean. Furthermore, SOC1, API and another unknown gene fragment are the member of MADS-box genes family. We speculate that MADS-box transcription factors play their role by interacting with each other.Third, by using Gateway technology, we built RNAi vector and over-expression vector of GmMADS28to investigate its functions. Then we did transformation of them into soybean by Agrobacterium-mediated soybean cotyledonary node method. Meanwhile, we did investigation on the factors which influence the rate of transformation. The result is that transformation rate is depends on soybean species. Nau.88-1has less pollution and stronger vitality than any other species. However, Jack has the highest regeneration rate in the species we used. Forty two over-expression transformed plantlets are obtained by antibiotic selection; a few of which are found to be early flowering and petals can not commence. The result is,Twenty RNAi transformed plantlets are also obtained, and some of them are sterility and with blade-shape leaves.