Study On Morphological And Anatomic Characteristics And Its Regulatory Mechanism Of Pistil Development In Radish(Raphanus Sativus L.)

Flower and fruit are not only the reproductive organ,but also the important product organ for some plants.The development of flower and fruit is one of the most complex courses in plant.There are many fruit shapes in Cruciferae,such as cabbage and Chinese cabbage of which the siliques dehiscent after matured,which would reduce the yield destructively if not harvesting in time;however,the shape of radish silique is significantly different and it is difficult to threshing for indehiscence of radish silique.In the present research,RNA sequencing and silique morphological at different development stage in radish,Chinese cabbage and their distant hybrids was analysised which will contribute to the better understanding of radish silique development and to promote comprehensive utilization of silique genetic improvement.The selection of stable reference genes in qPCR analysis of radish,Chinese cabbage and their distant hybrids will contribute to the accurately analyze of target gene.The main results were as follow:1.To analyze the silique development morphological differentiation among radish,Chinese cabbage,and the distant hybrids,the morphology,anatomy and cytology analysis were carried out in the present research,we found that,the silique development of radish,Chinese cabbage,and the distant hybrids was basically consistent,and there is no significant ventral suture and dorsal suture in radish silique,which had two thick valves and no carpel margin,moreover,there is two tubers at the basic of radish silique.Chinese cabbage had thin valves and carpel margin when rape,the silique dehiscent when ripe.The morphology of distant hybrids was between radish and Chinese cabbage,the top part was the same as radish,and the bottom part was the same as Chinese cabbage.2.To analyze the mechanism of silique morphological construct of radish,RNA-seq was carried out in the present research with pistils at different development stage in radish,Chinese cabbage,and the distant hybrids.The results show that,differentially expressed gene were enriched in the following terms:"cell process","metabolic process","single-multicellular organism process","developmental process","cell","cell component","organelle","binding","catalytic activity","nucleic acid binding transcription factor activity".Moreover,indole alkaloid biosynthesis,zeatin biosynthesis,brassinolide biosynthesis,cell wall hydrolase and some transcription factors related to floral development were elevated as playing crucial roles in pistil development and silique dehiscence.3.Accurately results of qPCR can be obtained by selecting stable reference genes,but there is few researches about reference genes selecting of radish,therefore,in the present research,9 traditional reference genes(GAPDJH,TEF2,ACTIN2/7,TUA,TUB,RPII,18SrRNA,UBC9andUP2)and 3 new reference genes(GSNOR,OUR? DSSI)were selected,their expression in 28 samples,including 20 radish samples from different organs and conditions,four Chinese cabbage organs and four organs of their distant hybrid,were assessed by qPCR and then five software tools(?Ct,geNorm,NormFinder,BestKeeper and RefFinder)were used to compare their expression stability.The most suitable reference genes were changeable in different organs and conditions.GAPDH,DSS1 and UP2 were optimal reference genes for expression analysis in all organs and conditions in radish.UPR,GSNOR1 and ACTIN2/7 were the most stable reference genes in different radish organs.UP2 and GAPDH were suitable reference genes for radish pistil development studies.RPII,UBC9 and GAPDH had the most stable expression in radish under various stresses.DSS1,UP2 and TEF2 were the optimal reference genes for Chinese cabbage organs,whereas TUA was optimal for the distant hybrid.UP2,GAPDH and TEF2 were appropriate reference genes for all of the samples together.The optimal reference genes we identified,UP2,GAPDH,UPR and GSNOR1 were verified by normalizing the expression patterns of YAB3,RPL and FUL,the profile of target genes were distorted while selecting the unstable gene-GSNORlas reference gene,which show the necessity for the application of suitable reference genes.