| The tea plant is a widely planted commercial plant in the world.However,selfincompatibility and low seeding rate hindered the breeding research and genetic study in this plant.In order to uncover the molecular mechanism of fertility in tea,RNA-seq was employed to explore the molecular mechanisms of SI and to identify SI-related genes in C.sinensis.Several genes were cloned and characterized.The results are as follows:1.The growth of pollen tubes following selfing and crossing was observed using fluorescence microscopy.At 24 h after pollination,a small number of tubes from the cross-pollination reached the style base.However,pollen tubes from self-pollination grew more slowly,reaching just the middle of the style.At 48 h after pollination,most tubes from cross-pollination reached the style base,but the pollen tubes from the selfpollination took 72 h to reach the same place.The delay in pollen tube growth may be critical for the SI reaction.2.RNA-seq was employed to explore the molecular mechanisms of SI and to identify SI-related genes in C.sinensis.Self and cross-pollinated styles were collected at 24 h,48 h and 72 h after pollination.Six RNA-seq libraries(SP24,SP48,SP72,CP24,CP48 and CP72;SP=self-pollinated,CP=cross-pollinated)were constructed and separately sequenced.In total,299.327 million raw reads were generated.Following assembly,63,762 Unigenes were identified.To identify SI-related genes,the fragments per kb per million mapped reads(FPKM)values of each Unigene were evaluated.Comparisons of CP24 vs.SP24,CP48 vs.SP48 and CP72 vs.SP72 revealed differential expression of 3,182,3,575 and 3,709 genes,respectively.Consequently,several ubiquitin-mediated proteolysis,Ca2+ signaling,apoptosis and defense-associated genes were obtained.The temporal expression pattern of genes following CP and SP was analyzed.The results show that cross-and self-pollination result in different gene expression responses in the style.Expression of “oxidation-reduction process”-related Unigenes steadily increased from 24 h to 72 h in cross-pollinated styles.The higher expression levels of these genes might promote pollen tube elongation during compatible styles.3.Pollen tubes are captured in ovaries in Camellia sinensis.Pollen-pistil communication and plant hormone signaling in ovaries are crucial for pollen tube inhibition.Therefore,to obtain candidate genes associated with these processes,transcriptomes of self-(Sp O)and cross-pollinated(Cp O)ovaries have been reported.In total,51,200 Unigenes were assembled.99 genes exhibited increased transcription in ovaries compared to other tissues.These genes might be crucial for pollen-pistil communication and double fertilization in tea.After data collection,the expression patterns of 51 signaling-related genes in self-and cross-pollinated ovaries were identified using quantitative real-time PCR.The results showed that several putative genes,GID1,SAUR,GH3.6,GH3.1,IAA29 and ARF1,exhibited dramatically different expression profiles between the two treatments,which suggested that these genes might play important roles in the SI process in C.sinensis.4.S-RNase is the female determinant in GSI(gametophytic self-incompatibility)plants.A putative Cs S-RNase gene was selected and cloned basing on the transcriptome data.The full-length c DNA of Cs S-RNase(KU852488)is 1121 bp,which encodes 283 amino acids.Cs S-RNase was primarily expressed in the styles,and the expression level in self-pollinated styles was higher than in the cross-pollinated treatment group at 24 hours after pollination.The genotypes of Cs S-RNase in 10 cultivars and one breeding line of C.sinensis were analyzed.Two polymorphic amino acid residues were identified in the strains ‘Fuding Dabaicha' and ‘Fuyun6',which might affect the activity of RNase.Two and four cultivars have the same amino acid sequences.Afterwards,a single nucleotide polymorphism(SNP)marker of Cs S-RNase was developed and genotyped in an F1 segregating population(‘Longjing43' × ‘Baihao Zao').Eighty one homozygotes and 85 heterozygotes were distributed in F1 individuals,which corresponded to a Mendelian ratio of 1:1.Then,the Cs S-RNase was mapped onto linkage group 14(LG14)of the reference genetic map of tea plant.Finally,the Cs S-RNase protein was prokaryotic expressed and purified.The protein can dramatically inhibite the elongation of self-pollen tubes.Therefore,we believe that the molecular mechanism of self-pollen tube inhibition is controlled by GSI system.5.Calcium is a crucial regulator of a large number of biological events,particularly in the polar growth modulation of pollen tubes.Calmodulin-dependent protein kinases(CDPKs)are one of the most important Ca2+ sensors in plants that sense and decode the Ca2+ signal during tip-growth regulation.Pollen germination and pollen tube growth are crucial steps for successful fertilization.In particular,in the tea plant,it is important that the flower stage go through improper weather conditions.In this study,5 pollen-specific expressed Cs CDPKs,namely,Cs CPK1,Cs CPK2,Cs CPK3,Cs CPK4 and Cs CPK5,were isolated.The homologous genes in Arabidopsis and Oryza sativa are associated with pollen tube development.Time-series expression patterns of these genes in pollen tubes were detected.All the genes were up-regulated over culture time,Cs CPK3,Cs CPK4 and Cs CPK5 exhibited a rapid response at 15 min after culture initiation.In addition,the expression profiles of the 5 genes were also detected in different media with a series of Ca2+ concentrations.The results indicate that the expression profiles were consistent with the variation of germination rate and pollen tube length.In summary,the 5 pollen-specific Cs CDPKs might be directly associated with pollen germination and tube growth in C.sinensis.Finally,an specific antisense oligonucleotides of Cs CPK5 was used to reduce the expression levels of this gene.The results show that,the average length of antisense oligonucleotides treated pollen tubes was significantly lower than sense oligodeoxynueleotides treated sample and the blank control sample. |
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