Cloning And Functional Analysis Of IiFUL And IiSHP2 In Isatis Indigotica Fortune

The present work mainly focuses on the cloning and functional analysis of IiFUL and liSHP2,two MADS family genes in Isatis indigotiaca Fortune.Firstly,the full-length cDNA sequences of IiFUL and IiSHP2 were obtained through 5-and 3Y-RACE.After that,the coding sequences of these two genes were cloned by using the designed gene-specific primers.Subsequently,over-expression and GFP subcellular localization vectors of IiFUL and IRSHP2 were constructed and were used in transformation of Col-O wild-type Arabidopsis plants and leaf explants of Qinyan 95 through Agrobacterium-mediated method.Finally,transgenic plants were obtained by screening with antibiotics.By observation to the phenotypic characteristics in different parts of the transgenic plants overexpressing IiFUL in this work,the possible functions of liFUL in reproductive stage were determined.In the meantime,quantitative real-time PCR and in situ hybridization were applied to detect the expression levels of IiFUL and IiSHP2 in different tissues.Moreover,the relation of IiFUL and IiSHP2 with the indehiscent phenotype of fruits was analyzed depending on the expression patterns of these two genes in different parts and in different developmental stages of woad silicle.In online sequence alignment in NCBI database,IiFUL and IiSHP2 showed high homology with AtFUL and AtSHP2 in Arabidopsis thaliana,and the identities between IiFUL and AtFUL,and between IiSHP2 and AISHP2,are 93%and 89%,respectively.IiFUL and IiSHP2 have typical structure of MIKC-type MADS transcription factors,and their identities with AtFUL and AtSHP2 reach 100%in MADS-box domain.In the whole aminoacid sequences,the identities between IiFUL and AtFUL,and between IiSHP2 and AtSHP2,are 95.06%and 93.17%,respectively.The transgenic Arabidopsis plants were observed and abnormal phenotypic changes,especially in reproductive stage,were detected in this work.In comparsion with the wild-type plants,transgenic Arabidopsis plants overexpressing IiFUL exhibited early flowering phenotype,but the activeness of the inflorescence meristem in the apex of the main stem was reduced significantly,which leading to the decrease of flower number on the top of the main stem.In development process,the flowers located on the top of the main stem generate a lot of variations in phenotype,including abnormal swelling of pistil,withering and number changing of stamens and petals,appearance of stigmatoid tissues and naked ovules at the end or inside of sepals.Besides,secondary flower could be formed within the flowers on the top of the main stem.These observations illustrated that IiFUL could affect the development of inflorescence meristem and pistils,but its ectopic expression could disturb the normal growth of sepals,petals and stamens.Moreover,the fertile siliques produced by the lateral inflorescences showed indehiscent phenotype in IiFUL overexpressing Arabidopsis plants,owing to the differentiation of the dehiscence zone between valve and replum could not be carried out normally.IiFUL was also associated with leaf morphogenesis,and its constitutive expression could alter the shape of Arabidopsis cauline leaves and the length-width ratio of tobacco leaves.The results of quantitative real-time PCR revealed that higher transcriptional levels of IiFUL and IiSHP2 could be detected in flowers and silicles ofL indigotica,indicating these two genes chiefly affect flowering and fruit development processes.In silicles,IiFUL mainly expressed in valves and fruit wings.It means that IiFUL possibly plays an important role in regulation of the morphogenesis of the external structures in fruits.In contrast,IiSHP2 mainly expressed in seeds,which reflected that IiSHP2 was probably involved in regulation of ovule and seed development.The results of in situ hybridization experiments showed that IiFUL expressed in apical inflorescence of I.indigotica at the early stages and its activity also could be detected in lateral inflorescences.Nevertheless,the transcript of IiFUL almost could not be found in floral meristems and in early flower buds,which denotes that IiFUL mainly plays a role in the early stages of reproductive growth.The results of in situ hybridization experiments also showed that IiFUL was expressed in the outer layer cells of valves and fruit wings,whereas the expression of IiSHP2 was repressed in these positions.In comprehensive consideration of the previous reports about the dehiscence phenotype of Arabidopsis siliques and the fact that the siliques of IiFUL overexpressing Arabidopsis plants are indehiscent in the present work,it can be speculated that high expression of IiFUL in outer layer cells of silicle valve is likely to depress the expression of IiSHP2 as well,this type of regulation probably is the reason why the silicles of I.indigotica possess an indehiscent phenotype.