Expression Pattern Analysis Of The Regulatory Genes Associated With Fruit Development In Isatis Indigotica And Functional Identification Of The Key Gene IiSHP2 Related With Fruit Dehiscence

The expression patterns of six genes involved in controlling of fruit development in Isatis indigotica were analyzed systemically in the present work,and the functional identification of the key gene IiSHP2 related to fruit dehiscence was carried out simultaneously.Multiple sequence alignment and protein modeling analysis showed that Ii SHP1,IiSHP2,Ii AG and Ii FUL are typical MIKC-type MADS-box transcriptional factors.Both Ii ALC and Ii IND are typical b HLH family transcriptional factors.The construction of the evolutionary tree further elaborated the evolutionary relationship between these Isatis indigotica fruit development proteins and their homologous proteins.In analysis of the expression pattern by q RT-PCR,it was found that the transcripts of Ii SHP1,IiSHP2 and Ii IND were only detectable in valve,whereas Ii AG,Ii FUL and Ii ALC could express in valve and fruit wing.The genomic DNA of IiSHP2 is constituted by seven exons,six introns and a 2100 bp regulatory sequence before transcription start site.It forms the 2502 bp promoter sequence together with the 5’untranslated region.In the promoter sequence,there are only three CAr G cis-acting elements.The result of subcellular localization experiment showed that IiSHP2 existed in nucleus,further confirming that this protein is a MADS-box transcriptional factor.In order to determine the function of IiSHP2,35S::IiSHP2 overexpressing Arabidopsis lines and At2056pro::IiSHP2 functional complementing Arabidopsis lines were prepared by genetic transformation mediated by Agrobacterium in the present work.Constitutive expression of IiSHP2 in Arabidopsis resulted in early flowering,accompanied by upward curling of the rosette leaves and the cauline leaves,reduction of leaf area,conversion of sepals to carpels,transformation of petals to stamens,shortening of silique length and sterility phenotype of seeds.Quantitative PCR analysis showed that constitutive expression of IiSHP2 could upregulate the activity of FT,SOC1,SHP2,IND,,STK and AGL15,and downregulate the activity of FLC,FUL,SERK1,CLF and ICU1,in consistent with the results of phenotypic observation.The functional complementing Arabidopsis lines in the genetic background of shp1shp2 double mutant were further analyzed by phenotype and paraffin section technology.The results indicated that the indehiscent phenotype of the mutant silique was restored to the dehiscent phenotype,and the lignified layer in the dehiscent zone also was restored.Further,ALC and IND was found that their expression levels were significantly up-regulated compared to shp1shp2 double mutant.Data of yeast two-hybrid experiments also illustrated that IiSHP2 possessed strong interaction with E-class MADS-box proteins Ii SEP1,weak interaction with Ii SEP4,and could not interact with other woad MADS-box proteins.Compared with the Arabidopsis At SHP2,IiSHP2 has conserved functions.In conclusion,the indehiscent phenotype of the silicles in woad was associated with the change in the number of CAr G elements of the IiSHP2 upstream regulatory sequence and the resulting changes in spatio-temporal pattern,but not associated with the conserved structure and function of the IiSHP2 protein.