Isolation And Functional Analysis Of Auxin Responsive Factors Related To Floral Development In Prunus Mume

Prunus mume Sieb.et Zucc is a traditional fruit tree in China and enjoys the reputation of a national flower.Researches show that its fruit contains B1(C19H22O6)substance,which can inhibit cancer.And it often has a good effect on the prevention of gastrointestinal cancer.However,the pistil abortion phenomenon is serious.In the process of blossom growth and development of Prunus mume,the abortive phenomenon often occurs due to extreme climate,adverse conditions and other environmental factors.Studies have shown that the pistil abortion phenomenon has an important relationship with its own regulatory genes.Auxin response factors can act as auxin receptors in plants and directly or indirectly regulate plant growth and development.Many researches have shown that there is a certain relationship between abortion of plants and changes in endogenous hormone content.However,there are few study reporting about the relationship between sterility of Prunus mume and hormone in China and abroad.Based on previous studies,this study used RT-PCR to isolate the PmARF13 and PmARF17 genes from the early floral buds of the 'Daqiandi',a cultivar of Prunus mume,and analyzed their gene structure and functional characteristics through bioinformatics methods.Real-time quantitative PCR had detected the expression of PmARF13 and PmARF17 genes in flower buds and leaf buds of the'Daqiandi' and 'Longyan',flower buds of different pistils and different mature floral organs of f'Daqiandi'.Subcellular location technology determined the location of PmARF17 protein in tobacco leaf epidermis cells.The determination of hormone content in the early flower bud and leaf bud,the different flower organs at the flowering stage,and the correlation analysis with the expression of PmARF13 and PmARF17 genes,provided important reference for the regulation mechanism between the auxin response factors and the plant hormone,and offered an important theoretical basis for the mechanism of pistil abortion.Promoter contains a variety of important cis-acting elements,which regulated the expression of corresponding genes at the transcriptional level,so that plants can resist external environmental stress.Therefore:functional analysis of PmARF17 promoter is helpful to understand the transcriptional regulation mechanism of auxin response factors in pistil development of Prunus mume.The main results are as follows:1.2600 bp of PmARF13 gene was cloned from 'Daqiandi'.Results showed that the encoded protein was unstable,water-soluble,non secreted transmembrane protein and nuclear protein.Phylogenetic tree and homologous comparison showed that the PmAR13 protein was highly similar to the protein sequence of the auxin response factor in other plants.The N terminal of PmARF13 had a specific B3-DNA binding domain of higher plant transcription factors,the C terminal had a AUX/IAA dimerization domain,and the auxin response factor component.The conservative area is very similar to the other species.2.2342 bp of PmARF17 gene was cloned from 'Daqiandi'.Results showed that the protein encoded by the gene was unstable,water-soluble and non secreted transmembrane protein.The phylogenetic tree and homologous comparison showed that the PmARF17 protein was very similar to the protein sequence of the auxin response factor in other plants.The PmARF17 N and the C terminal protein sequence were more conservative,and the similarity was high with other plant ARF protein.3.PmARF13 gene plays an important role in the regulation of flower buds in October and January.It may play a role in promoting the stamen development and may affect pistil development by regulating the growth of stamen and sepals.PmARF17 gene plays an important regulatory role in the important stages of pistil development in November,December and January.PmARF17 gene may promote or maintain the normal pistil development,which can reduce the pistil abortion to a certain extent,and it is a positive regulation gene for pistil development.PmARF17 gene also plays an important role in regulating the stamens development of Prunus mume.It may affect the floral development and the process of abortion by controlling the growth of stamens.Laser scanning confocal method showed that the PmARF17 protein were located on the nucleus and cell membrane.4.The determination of endogenous hormones in different flower buds9 leaf buds and flower organs and their correlation analysis with the expression of PmARF13 and PmARFll genes showed as follows:In the process of blossom growth and development,PmARF13 gene may play a synergistie role with IAA,ABA and ZT,and it may affect the pistil development by regulating the IAA level of stamens,affecting the petal growth and development by negatively regulating GA3 contelt,and thus affecting the floral development process.PmARF13 gene may affect the floral development process by negatively regulating the content of ZT in pistils,stamens,petals and sepals.The change of ABA content and the expression of PmARF17 gene in 'Daqiandi' flower buds and leaf buds generally showed the opposite trend,and may be antagonistic to each other in pistil development.PmARF17 gene and ZT may be antagonistic to each other during the blossom growth and development.The expression of PmARF17 gene and GA3 may play a synergistic role in pistil development by regulating GA3 content in pistil and stamen,it influences the floral development and the abortion process.PmARF17 gene may affect the floral development and the pistil abortion process by negatively regulating the ABA content in pistil,stamen and petals.PmARF17 gene may affect the floral development through positive regulation of ZT content in pistil,stamen and sepals.5.2037 bp sequence of PmARF17 promoter was cloned from 'Daqiandi'.The informatic analysis showed that the promoter contains the GARE-motif,the salicylic acid response element,the salicylic acid response element TCA-element,the core promoter element TATA-Box,light response element Box I,GAG-motif and so on.Agrobacterium-mediated transient infestation of PmARF17 promoter in Arabidopsis experiments showed that the PnARF17 promoter has activity and can initiate the expression of its downstream GUS gene,rendering the stamen,petals and roots of Arabidopsis appear blue.It can be inferred that the PmARF17 promoter plays an important regulatory role for downstream genes in floral development,providing an important reference for the study of signal transduction pathway of ARF in pistil abortion of Prunus mume.