Function Analysis Of YABBY5 And CRC Gege In Fruit Shaping Of Fingered Citron

Fingered citron(Citrus medica L.var.sarcodactylis Swingle),is one variaty of citron(C.medica L.).Both of them areclose in genetic relationship but are noticeably different in fruit shape.CRABS CLAW(CRC)and YABBY5 all belong to the YABBY gene family.YABBY is an important transcription factor in plants,and plays an important biological function for lateral organdevlopment and fruit morphogenesis in higher plants.In this study,the fruit morphology of Fingered citron was studied from the stage of flower primodium to mature fruit through morphological observation.The sequences structure of CRC and YABBY5 gene were identified and analyzed in Jinhua's Fingered citron and citron.The expression and functional characteristics of the two YABBY gene family members were analyzed by quantitative real-time PCR and virus induced gene silencing(VIGS).1.Collecting the fingered citron's flower buds at lmm?1.5 mm?2 mm?2.5mm?3 mm?3.5 mm?4 mm?5 mm?6 mm?7mm?8 mm?9 mm?10 mm from the day of blooming flowers to the day of bearing young fruits.The fruit morphology of Fingered citron was tracked from the stage of flower primodium to mature fruit by super depth of field microscope and scanning electron microscope.The results showed that most 1 mm buds were on the calyx differentiation stage,and 2 mm buds were on the stamen differentiation stage(SD).As for the gynoecium differentiation stage(GD),the buds were at about 3?4 mm.The carpel primordia of early buds have appeared the finger-like protrusions,which contain stigma and style.With the opening of flowers,the stigma dropped and the ovary also appeared finger-like bulge.The results indicate that the finger part of Fingered citron's came from the ovary,which means fruits built their shape before flowering.2.The sequences of CRC gene promoter and YABBY5 gene were isolated from fingered citron and citronl.Compared the sequence of fingered citron and citron through NCBI,fingered citron's CRC promoter gene sequence,showed the 98%identity with citron's.Analysis of the CRC promoters by PlantCARE demonstrated that both two sequences contained similar conserved functional components of the higher plant,conjecturing that the sequence had promoter activity.However,two TATA-box element were absent in CRC promoters of fingered citron compared to citron,suggesting that the deletion may change the expression lever of the CRC gene.The cDNA sequence and protein sequence of the fingered citron YABBY5 were compared with the homologous sequence of citron.21 bp fragment were absent in YABBY5 cDNA sequence of fingered citron,and also less 7 amino acids in protein sequence of fingered citron.This deletion appeared in its conserved region(CDs).In summary,The expression difference of CRC and YABBY5 gene between fingered citron and citron may be related to the change of fruit shape.3.Collecting the floral organs at Stamen differentiation stage(SD),gynoecium differentiation stage(GD),6 days before flowering,flowering day,3 days after anthesis,6 days after anthesis and 9 days after anthesis.The expression of the two genes was assessed by qRT-PCR.The results showed that the expression of CRC gene was lower since the day of blooming in fingered citron,and all the expression level of floral organs was significantly lower than that in citron.The expression of YABBY5 gene was up-regulated since flowering in fingered citron,but it was also significantly lower than that in citron.Previous research has shown that finger citron cultured under high temperature can produce a wrist-like fruit shape,After culturing the fingered citrons with new shoots o in 20? and 5? incubator for 60 days,the plants were then transfered to normal incubator of 25? day/18? night temperatures.Then the expression of CRC and YABBY5 in floral organs in different time was evaluated by qRT-PCR.The results showed the expression of CRC in wrist-like fingered citron is significantly higher than that in finger-like fingered citron at the 3th days before flowering,flowering and flowering day after 3 days,respectively.However,the expression level in wrist-like fingered citron became lower after flowering.The expression levels of YABBY5 gene under higher temperature were higher than that under low temperature,particularly significantly at the 3th days of flowering,similar with citron fruit.Based on the expression analysis,high temperature induces made the expression of CRC and YABBY5 in fingered citron a little closer to citron.Those results revealed that the two YABBY genes are related to the fruit shaping of fingered citron.4.Virus-induced Gene Silence(VIGS)was emplyed to silence SlYABBY5b gene in Micro-Tom tomato after the cotyledons have emerged for 30 days.The results presented the plantlet still set fruits 30d after Agrobacterium infiltration,and the fruit tip elongated to become a sharp angle like pear or bulged.The phenotype continued until 60 d after treatment,but the color transition of the fruit was not influenced.Twenty-five tomato fruits were selected from the negative control group and the experimental group.After analyzing by Tomato Analyzer 3.0 software,the silence tomato fruit about 30 to 60 d were elongated,and the angle of the fruit tip in silencing group was significantly smaller than that of the control group(P<0.01).To the height of 46-63 mm,the fruit number of silence group was greater than the control group.Betweenthe angle of 80°-119°,the fruit number of silence group was two times than the control group.Based on the precidtion of STRING website,KAN,KAN2,ETT,WOX1,AT4G38840.1,SUN and OVATE may interact with YABBY5 in Arabidopsis thaliana.The expression levels of YABBY5 and their interaction genes in the control and silencing groups were quantified by qRT-PCR.The results showed that the expression level of SIYABBY5 gene in silencing fruit decreased a lot,and the expression level of other genes also changed in silencing lines.It is obvious that the down-regulation of YABBY5 gene affects fruit shape,as well as the expression of downstream gene.To sum up,based on the analysis of gene structure and the expression level and VIGS,we suggest the YABBY5 is sufficient to be involved in fruit shape development in fingered citron,probably crucial for fruit pattern formation.Whether the deletion of CRC promoter is related to the fruit shape needs further study.