Rootstock-scion Interaction Induced MicroRNA Affecting The Contents Of Sugar And Acid In Citrus Fruit

Citrus is mainly propagated by grafting.Grafting combines two plants of different genetic backgrounds to generate a new plant.Rootstock-scion interaction has obvious influence on biological and horticultural characters of scion.Rootstock-scion interaction could induce plant dwarf,shorten juvenile stage,improve fruit quality and produce better biotic and abiotic stress resistant.The mechanisms of rootstock-scion interaction were studied in plants at different levels of physiology and biochemistry,nutrient distribution,hormone signaling,and exchange of genetic information(m RNAs,mi RNA and si RNA)between rootstock and scion.Some studies indicated that rootstock-scion interaction could affect fruit quality and the expression of mi RNAs in citrus.However,there was no report on the relationship between fruit quality and differentially expressed mi RNAs caused by rootstock-scion interaction.In this thesis,the contents of sugar,acid,vitamin C and invert sugar in the juice sac(JS)of fruit were investigated in eight different citrus rootstock/scion combinations.Four different citrus scions(Powell,Banfield,Chieslett and Daya)were grafted on both Trifoliate orange(Poncirus trifoliata)rootstock and Hongju(Citrus reticulata)rootstock.Rootstock-scion interaction on fruit quality was investigated.Fruits(130,150,190 DAF(Days after flowering))from the Daya mandarin(C.reticulata)grafted on two genetically divergent rootstocks Trifoliate orange(Z)and Hongju(H)were used for fruit quality,expression of small RNA,and degradome sequencing analysis at different developmental stages.Differentially expressed mi RNAs were identified by comparative analysis of small RNA sequencing data of different fruit developemt stages.Correlation analysis was conducted between the content of sugar or acid and the differential expression level of mi RNA to screen the candidate mi RNAs related to the fruit quality.Combined with the analysis results of degradome sequencing,q RT-PCR,double luciferase reporter gene assay and tobacco transient expression,the potential target genes of mi RNAs were identified.Over-expression vectors were constructed with short tandem target mimic(STTM)and the precursor of mi RNA,respectively.The transgenic plant was obtained by genetic transformation of precocious trifoliata orange.To explore the differential expression of candidate mi RNA between rootstock and scion in citrus,reciprocal grafting was carried out with over-expressed mi RNA plants and wild-type plants.The expression level of mi RNA in the phleom tissues at the above and below of graft union(rootstock and scion)in different scion-rootstock combinations were determined.The main research results of this thesis are as follows:1.The contents of total soluble solid of fruit on Poncirus trifoliata rootstock wassignificantly higher than that of Hongju rootstockThe sugar and acid contents of citrus fruits in eight different combinations of scions and rootstocks(Powell/Z,Powell/H,Banfield/Z,Banfield/H,Chislett/Z,Chislett/H,Daya/Z,and Daya/H)from two citrus orchards were compared.Results showed that the contents of total soluble solid(TSS)in fruits of four scion cultivars grafted on Poncirus trifoliata rootstock were significantly higher than those of grafted on Hongju rootstock.Sucrose contents were also consistently significantly higher than those of grafted on Hongju rootstock.However,only the content of sucrose in the fruit of four scions grafted on Poncirus trifoliata rootstock was consistently higher than Hongju rootstock.The contents of fructose and glucose did not show significant and consistent differences.The contents of total organic acid and titratable acid(TA)in fruits on Poncirus trifoliata rootstock were significantly lower than those on Hongju rootstock.Among the four organic acids detected,only citric acid contents in fruit consistently significantly higher than those of on Hongju rootstock.The contents of other three organic acids(malic acid,tartaric acid and succinic acid)in eight scion-rootstock combinations did not show the consistently difference.There was no consistent difference in the content of vitamin C in the fruits of four scions grafted on two rootstocks.2.Differentially expressed mi RNA induced by interaction of scion-rootstockFruits in different developmental stages from the Daya mandarin grafted on two genetically divergent rootstocks(Trifoliate orange and Hongju)were used for expression of small RNA,and degradome sequencing analysis.The results showed that among 108 known and 1451 novel mi RNAs obtained from mi RNA sequencing,129 of them were differentially expressed(48 known and 81 novel mi RNAs)during the development of fruit.Seventy-two mi RNAs were differentially expressed at 130 DAF,including 35 down-regulated mi RNAs,and 37 up-regulated mi RNAs.Seventy-one DEMs were identified at 150 DAF with 27 DEMs down-regulated and 44 DEMs up-regulated.25 mi RNAs and 37 mi RNAs were up-and down-regulated at 190 DAF.Between D/Z and D/H,25,24 and 20 mi RNAs were up-expressed and 26,15 and 18 were down-expressed at fruit developmental stage 130,150,190 DAF,respectively.Among them,five and two mi RNAs were up-and down-expressed throughout the fruit development.A total 551 target genes were acquired by in silico prediction.Degradome sequencing was applied to identify sliced targets.A total of 107 sliced targets for 53 known and 2 novel mi RNAs were identified based on the analysis of PARE snip.Only 9targets of 6 differentially expressed mi RNAs were found by degradome sequencing.The duplicate target genes obtained by the two methods were removed,555 candidate target genes were found.GO functional analysis was used to annotate the functions of mi RNAs.The targets were enriched in 17 biological processes,7 molecular functions,and 10 cellular components.Meanwhile,KEGG pathway analysis revealed that 231 genes were involved in 70 pathways,including metabolic pathways,pantothenate and Co A biosynthesis,plant-pathogen interaction,and so on.3.mi RNAs were related to interior quality of fruitCorrelation analysis was conducted between the contents of fruit quality(TSS or TA)and the differentially expressed levels of seven mi RNAs during different fruit development stages.Four mi RNAs(Cre-mi R159-3p,Cre-mi R160 a,Cre-mi R171 b,and Cre-mi R399-3p)were significantly correlated with fruit quality.The target genes of these mi RNAs may be involved in the formation of fruit quality,such as transcription factors SCL,GAMYB,ARF.The expression levels of the four candidate mi RNAs had a significant correlation with fruit quality in the combinations of three navel orange varieties grafted on two different rootstocks(Trifoliate oragne and Hongju).When the three navel orange varieties graft on Hongju rootstock,the content of TSS in the fruit was significantly positively correlated with Cre-mi R399-3p.The expression level of Cre-mi R399-3p was significantly positively correlated with TA in Banfield/H and Chislett/H.The results showed that rootstock may affect fruit quality by regulating the expression of mi RNA in scions.The expression of four candidate mi RNAs and their predicted target genes were detected by q RT-PCR in 8 different scion-rootstock combinations.The results showed that when three scions(Banfield,Chislett and Daya)were grafted onto Poncirus trifoliata rootstock.The expression level of Cre-mi R159-3p was significantly increased,but the target gene GAMYB showed an opposite expression pattern only in Chislett/Z.The trends of Cre-mi R160 a expression were not consistent in four scion varieties grafted on the two rootstocks,indicating that Cre-mi R160 a was not associated with fruit quality on interaction of scion-rootstock.The expression of Cre-mi R171 b was significantly higher in two scions(Powell and Banfield)grafted on Poncirus trifoliata rootstock,but the expression of its target gene SCL only showed opposite pattern in the Banfield/Z.Cre-mi R399-3p were significantly lower in three of the four scion varieties(Banfield,Chislett and Daya)grafted on Poncirus trifoliata rootstock,and expression of its target gene INVE showed opposite patterns in two scion varieties of Banfield and Chislett.Among the four candidate mi RNAs and their targets,only the expressions of Cre-mi R399-3p and its target gene INVE demonstrated consistently negative correlation in the scions grafted on Poncirus trifoliata rootstock.Therefore,it could be presumed that Poncirus trifoliata rootstock might promote the expression of INVE by suppressing the expression of Cre-mi R399-3p in scion varieties to affect the metabolism of sugar.So,Cre-mi R399-3p and its target gene INVE may be one of the important factors to affect the fruit quality by Poncirus trifoliata rootstock.4.Verification the target gene of Cre-mi R399Three target genes(Ccl LAC,Ccl INVE and Ccl DSR)of Cre-mi R399 were predicted by bioinformatics prediction,q RT-PCR and degradome sequencing.To more accurately screen the target gene of Cre-mi R399,explore whether or not Cre-mi R399 possiblly supperssing the expression of its target gene in vivo,double luciferase reporter gene detection was conducted.The results showed that Cre-mi R399 had a certain degree of inhibition effect on the expressions of three potential target genes(Ccl LAC,Ccl INVE and Ccl DSR).The inhibition effect of Cre-mi R399 on the target gene INVE was significantly higher than on other target genes.In the results of transient expression verification system of tobacco indicated when co-injection of Cre-mi R399 and target gene INVE into tobacco,the fluorescence signal in the leaves was significantly weakened.Therefore,it was speculated that INVE should be one of the important target genes of Cre-mi R399.5.Cre-mi R399 was involved in rootstock-scion interaction of transgenic citrusTwo recombintant vectors(precursor of Cre-mi R399 and STTM-mi R399)were constructed in this study.Transgenic plants were obtained by genetic transformation.Compared to the wild-type plants,the expression of Cre-mi R399 was down-regulated and up-regulated in silenced and over-expressed transgenic plants,respectively.Reciprocal grafting was performed with over-expressed transgenic(TP)plants and wild-type(WT)plants to produce combinations of TP-WT and WT-TP plants.The scion and rootstock phloem tissues at upper and lower part of the graft union,respectively,were collected at different time after grating to detect the relative expression level of Cre-mi R399.In the combination of wild-type on transgenic plant,the level of Cre-mi R399 ecpression in the scion was significantly increased at the 14 th day after grafting,and continuously increasing until 40 th day after grafting.However,the expression of Cre-mi R399 did not significantly change when the over-expressed plants as scion grafted onto wild-type plants.There were also no significantly changes on Cre-mi R399 expressions in both self grafted wild-type and transgenic plants.This suggested that Cre-mi R399 was involved in the interaction of rootstock to scion.The rsults also implied that between the Cre-mi R399 differentially expressed scion and roostock,the Cre-mi R399 highly expressed rootstock could induce the higher expression of Cre-mi R399 in scion,or Cre-mi R399 could move from higher expreesed rootstock to lower expressed scion.However,the mechanism underlying this event still needs to be proved in the future work.