Molecular Mechanism Of Fruit Length Candidate Gene CsSUN Involved In Regulating Fruit Length In Cucumber

Fruit shape is an important appearance quality trait of cucumber,which directly affects the output and sales.However,the shapes of cucumber fruit are quantitative traits,and its genetic mechanism is complicated,the underlying molecular mechanism needs to be explored further.This study is carried out using a recombinant inbred lines（RILs）population and an F₂segregation population to conducts the gene mapping of cucumber fruit length trait.The CsSUN gene was likely to be the candidate gene controlling fruit length in cucumber.Two residual heterozygous lines sun161 and SUN162 of CsSUN gene were used to phenotype analysis and and RNA-seq analysis.Furthermore,the target protein CsMPKK2was screened by using CsSUN as bait and their interaction was verified.CsMPKK2 target with CsSUN and its molecular mechanism were studied.The main results are as follows:1.Research on the cucumber fruit shape QTL analysis using a RIL6 population.Eight stable QTLs were detected,which were located on chromosomes 1,2,3,4 and 7.Among them,FS1.1 is a major-effect QTL,which has the highest LOD score of 11.65 and contribution rate of 27.05%.A pair of residual heterozygous lines（sun161 and SUN162）of FS1.1 was obtained.The ovary length of sun161 was 15.79 mm,which was significantly decreased 32.85%compared with SUN162.The ovary diameter of sun161 was 8.07 mm,which was significantly increased 32.85%compared with SUN162.2.The FS1.1 candidate gene CsSUN cloning and functional validation.The FS1.1 was fine mapped to a 269.44 kb interval basing on the F₂population.The target interval contained 32 predicted genes,and inferred that CsSUN was the FS1.1 loci related candidate gene.The CRISPR/Cas9-mediated CsSUN mutants cssun-1 and cssun-2 were show a shorter fruit length.The fruit length of.cssun-1 and cssun-2 were significantly decreased 38.42%and 43.64%compared with the 9930.3.RNA-seq analysis showed that calmodulin signaling pathway was involved in the regulation of fruit length by CsSUN.The RNA-seq analysis indicated that the plant hormone signal transduction genes and Ca Ms/CMLs were mainly enriched of DEGs between sun161and SUN162.The content of auxin during ovary fruit development was measured,and the results showed the auxin in sun161 was significantly higher than in SUN162 in the early stage.Further selected CAM11,CAM13,and CML38 proteins for the protein interaction with CsSUN Validation.The verification results showed the interaction between CsSUN and CAM11,CAM13,and CML38 proteins.4.Calmodulin was upstream of auxin signal and promotes cucumber fruit elongation by reduced endogenous auxin content.Ca Cl₂,and IAA applied in sun161 and SUN162.The ovaries’length,diameter,and shape index were significantly increased by Ca Cl₂in SUN162,while it did not change significantly in sun161.After IAA treatment,sun161 and SUN162significantly increased ovaries length and diameter.Ca Cl₂treatment also increased the expression of the three Ca Ms/CMLs both in sun161 and SUN162.The expression of two main auxin efflux genes PIN1,and PIN2 were significantly up-regulated in sun161,and it had no significant changed in SUN162 ovaries after Ca Cl₂treatment.The above tests show that Ca²⁺signaling which acts upstream of auxin.A high Ca²⁺concentration promote the decrease of the auxin in cucumber fruit and the growth of the fruit length.5.The kinase activity of CsMPKK2 plays an important role in regulated fruit length by CsSUN.Using CsSUN protein as the bait,the candidate target protein CsMPKK2 was screened from the CDS library by yeast two-hybrid screening,and they interaction was verified by Y2H,Bi FC and Co IP tests.Subcellular localization showed that the CsSUN was localized in the cell membrane,and the CsMPKK2 was was localized in the cell membrane and nucleus.The CsSUN and CsMPKK2 co-localization was found in the cell membrane.However,the CsMPKK2 phosphorylation states would influenced the CsSUN and CsMPKK2 co-localization.It could be inferred that the CsMPKK2 mediated phosphorylation may regulate the interaction between CsMPKK2 and CsSUN.6.CsMPKK2 negatively regulated cucumber fruit length.The fruit length was significantly decreased and the seed length and width were significantly increased in the CsMPKK2 overexpressed lines CsMPKK2-OE1 and CsMPKK2-OE2,while the internode length weas significantly decreased in the CsMPKK2 gene knockout line mpkk2.CsMPKK2can inhibit CsSUN gene expression,and further to decrease the fruit elongation in cucumber.Transcriptome analysis showed that,the DEGs related to MAPK signaling pathway and plant hormone signal transduction pathways were significantly enriched.The measurement of hormone content in the CsMPKK2 overexpressed line and knockout line implied the auxin may play an important role in cucumber fruit elongation.A model of CsSUN molecular regulatory of cucumber fruit length was proposed based on this study.