Cloning And Function Analysis Of Genes Related To Gibberellin Acid Signaling In Phyllostachys Edulis

Gibberellic acids(GAs)can regulate plant growth and development in many aspects acting as a kind of plant hormones,such as seed germination,stem elongation,the fruit development and so on.Currently,gibberellin signal transduction pathways in plant is a hotspot of research.By mutant research method in plant such as rice and Arabidopsis thaliana,people have found several proteins: the GA receptor protein GID1,the DELLA protein which is the negative regulator of gibberellin signaling pathway,and the protein GID2 which is associated with DELLA protein degradation.DELLA protein inhibits plant response gibberellin signal when the bioactive gibberellins are absent or when the concentration of bioactive gibberellins are low.Bioactive gibberellins could bind to GID1,and then interact with DELLA protein.The interaction between GID1 and DELLA protein promotes DELLA ubiquitination and leading to DELLA degradation through the ubiqitin proteasome pathway.The bioactive gibberellins gibberellins regulate plant growth and development by removal of DELLA repression.Phyllostachys edulis is a kind of bamboo species widely used in the production and life,regenerated mainly by sprouting buds.The bamboo shoots usually grow explosively with high rate,the molecular process underlying this phenomenon remains unclear and has important academic value.Previous research revealed that the content of gibberellic acids had a dynamic change in the rapid growth period of bamboo shoots,which suggested us gibberellic acids may play an important role in the process.So study the gibberellic acids signaling pathways in the phyllostachys edulis is of great significance.Phyllostachys edulis seeds and shoots in the rapid growth period were used and several aspects are involved in this experiment.We researched the effects of gibberellin on seeds germination and seedling growth.We obtained three genes belonging to the gibberellin signal transduction pathway by using homology cloning and analyzed the gene by using bioinformatics.Real-time RT-PCR was performed to analyze the tissue specificity expression of these genes in the rapid growth shoots.Yeast two-hybrid technology was used to study the interactions between the proteins.Agrobacterium-mediated transformation was used to produce the Arabidopsis over-expressing DELLA gene of Phyllostachys edulis to study the function of it.The preliminary results are as following: 1.GA3 can promote the seeds germination and seedlings growth,the function is most obvious whenthe concentration of GA3 was 100 mg.L-1,the seed germination rate and the stem length increasedrespectively by 19.80% and 97.17% comparied with control group.2.Three genes in the Phyllostachys edulis were obtained by homology cloning,PhGID1,PhGID1 andPhSLR1,Full-length ORF of them are 1065bp?648bp and1866 bp,encoding 354,215 and 621 aminoacids respectively.Bioinformatics analysis showed that,PhGID1 contains a hydrolase structuredomain,PhGID1 belongs to the family of F-box proteins,the N-terminal of PhSLR1 contains atypical DELLA structure domain and the C-terminal contains a domain belonging to the GRASsuper family,these structure information are very similar with the homologous proteins in otherplants,.3.Real-time RT-PCR analysis showed that PhGID1,PhGID2 and PhSLR1 were expressed both fromthe root to the top of bamboo shoots,PhSLR1 showed low expression in the top and central whilehigh expression in the root,PhGID1 and PhGID1 had the similar expression,they both had highexpression in the top and central while low expression in the root which is opposite compared withPhSLR1.4.Prokaryotic expression of the proteins PhGID1,PhGID2 and PhSLR1 showed that,when made theE.coli BL21(DE3)as the expression strain and pET-32 a as the expression vector,PhGID2 andPhSLR1 could be expressed and they were soluble while PhGID1 be expressed in the form ofinclusion body.5.The yeast two-hybrid experiments showed that the interaction between PhGID2 and PhSLR1happened on the GRAS domain located in the C-terminal of PhSLR1,the interaction betweenPhGID2 and PhGID1 was also observed.6.The transgenic experiment showed that overexpression of PhSLR1 in Arabidopsis thalianaseedlings could make the plants shorter and make the leaves and capsules smalle and less than thewild-tipe plants.