Cloning,Expression Analysis And Subcellular Localization Of RrSLF Gene From Rosa Rugosa

Rosa rugosa is a famous traditional flower in China.It was found that the self incompatibility mechanism of Rosa rugosa is the same as the self incompatibility mechanism of the Rosaceae plants such as Armeniaca vulgaris Lam.and Pyrus spp,all of which belong to the self incompatibility of gametophyte involved in S-RNase.The plant S gene,including the style S-RNase gene and the pollen SLF gene,controls the self incompatibility.The study of the pollen SLF gene is one of the keys to the exploration of the mechanism of the gametophyte self incompatibility(GSI).In this paper,a self incompatibility gene RrSLF of Rosa rugosa was cloned from Rosa rugosa powder by RT-PCR and RACE technology.The sequence characteristics and bioinformatics characteristics of the gene were analyzed,and the subcellular localization and fluorescence quantitative expression analysis were carried out to lay the foundation for further study of the mechanism of rose self incompatibility.The foundation.The main results are as follows:1.the sequence of RrSLF gene from Rosa rugosa is 1236 bp and open reading frame is 1122 bp,which encodes 373 amino acids.The RrSLF gene from Rosa rugosa encoded protein: relative molecular weight of 43706.15Da;theoretical isoelectric point value PI=6.24;343-741 bits containing F-box conservative domain;no signal peptide cleavage site,non secretory protein;average hydrophobicity index is 0.716,hydrophobic protein;non transmembrane region exists;21 Ser phosphorylation sites,7 Thr acids Phosphating sites,7 Tyr amino acid phosphorylation sites,2 N-glycosylation sites,no O-glycosylation sites,32.17% extended chains,22.25% alpha helix,31.37% irregular curls,and 14.21% beta angle.2.the subcellular localization of the RrSLF gene from Rosa rugosa: the RrSLF gene was constructed through the DNA recombination technique to the PROKII-GFP vector,and the subcellular location vector was successfully constructed and named PROKII-RrSLF-GFP.The RrSLF gene was transferred into the tobacco leaf cells by Agrobacterium tumefaciens injection,and the RrSLF gene was located in the nucleus by laser confocal microscopy.3.RrSLF gene fluorescence quantitative expression analysis: the specific expression of RrSLF gene in pollen;expressed in different rose varieties,Rosa davurica and Rosa chinensis,among which the expression of R.rugosa‘zilongwochi' was significantly higher than that of other rose varieties,Rosa davurica and Rosa chinensis;After pollination 10h(pollination at 10:00 am),the difference of RrSLF gene expression in the style of different flower age was significant.The expression of RrSLF gene in the pre flowering 2d style was the highest,and the RrSLF gene expression in the blooming day(0d)style was the lowest;the expression of RrSLF gene expression in the 3 pollination methods was different in the 4 time periods of self crossing and among the varieties.The hybrid style showed a trend of gradual decline,but it showed a trend of rising and descending in interspecific cross style.The expression of RrSLF gene in interspecific cross style was significantly higher than that of the other two pollination methods in 4 time periods,except for 2h,the RrSLF in the intervariety cross style in his time period.The expression level of F gene was higher than that of RrSLF gene in selfing style.