Isolation Of Vacuoles From Pollen Tubes Of Pear And Functional Analysis Of CPA And TPC1 Genes In Regulating Pollen Tube Growth

Pollen tube development is important for sexual reproduction in flowering plants.Pollen tube extension is very rapid and death of the pollen tube occurs within 24 h in vitro,progress during the late stage of pollen tube development was an ageing process,pollen tube growth cessation and death during this stage.Pollen tube could be used for studying the mechanism of plant growth,senescence and death.In this study,the pollen grains and pollen tubes of 'Dangshansuli' are selected as material,'Dangshansuli' is the most widely grown cultivar of pear in china and had been used for whole-genome sequencing.We investigated the changes of growth-related molecules and organelle during pollen tube development.RNA sequencing(RNA-Seq)technology was used to characterize the expression of genes during pollen tube growth at the transcriptome level.Based on the genome data and gene expression data,Two pore channel 1(TPC1)and cation proton antiporter(CPA)families were analyzed by bioinformatics method.The functions of vacuolar ion transports were primary studied during pollen tube development.The main results were as follows:1.We investigated the changes of signaling molecules and organelle in the pollen tube during the late satge of pollen tube development.Cytoplasmic calcium ion concentration and ROS level were increased and vesicular transport was disturbed in SPT.The volume of vacuoles increased and the number of vacuoles decreased during pollen tube development.The pH of vacuoles in partial ageing pollen tubes increased and the vacuoles were collapse in SPT.Finally,nuclear DNA was degraded and PCD occurred in SPT.2.RNA sequencing(RNA-Seq)technology was used to characterize the expression of genes during four development stages of pear pollen:mature pollen grains(MP),hydrated pollen grains(HP),growing pollen tubes(PT)and stopped-growth pollen tubes(SPT).Data of the four libraries were assembled into 21,394 genes.Transcripts from the four stages were classified into 38 functional subcategories.Between MP and HP,305 genes were differentially expressed,and 502 genes were differentially expressed between HP and PT,and 2208 genes were differentially expressed between PT and SPT.Quantitative real-time PCR(qRT-PCR)was used to confirm the RNA-Seq results.We also found a number of genes associated with pollen tube development,these genes discovery will help us to explain the signaling molecules changes at the late stage od pollen tube development.This research also provides a wealth of data to study the deveoplent of pollen tube and will help us to understand the mechanism of pollen tube growth cessation and death at the late stage od pollen tube development.3.220 CPA genes were identified from five Rosaceae species(Pyrus bretschneideri,Malus domestica,Prunus persica,Fragaria vesca and Prunus mume),and 53 of which came from P.bretschneideri.Phylogenetic,structure,collinearity and gene expression analyses were conducted on the entire CPA genes of pear.Gene expression data showed that 35 and 37 CPA genes were expressed in pear fruit and pollen tubes respectively.Then we preliminarily studied the function of PbrCHX16 in pollen tube.PbrCHX16 contributed to pollen tube growth and pH homeostasis.4.Two pore channel 1(TPC1)are major Ca2+ channel in vacuole,one TPC1 gene was identified in pear genome,PbrTPC1 protein was localized in large vacuole.Specific gene knockdown mediated by the antisense oligodeoxynucleotides(as-ODN)strategy were used to study the function of PbrTPCl in pollen tube growth.The pollen tubes were more sensitive to NaCl when treated with as-ODN,suggested that TPC1 function in salt stress response.