| The normal growth of the pollen tube is the prerequisite for plants to complete pollination and fertilization.Pollen tube growth is controlled by a variety of factors,in addition to being controlled by external factors,it is also affected by hormones,proteins,peptides and other signal substances secreted inside the pollen tube.Plant sulforaphane PSK is a sulfonated short peptide composed of 5 amino acids.It was originally discovered during cell suspension culture and it can promote cell division.Later research revealed that PSK also played an important role in promoting cell differentiation and somatic embryo formation,regulating the pollen group effect,improving stress resistance,and stimulating the production of secondary products.At the same time,it was found that proteins containing the C2 conserved domain play an important role in many processes of plant growth and development.This study mainly focused on the mechanism of PSK peptides and proteins containing C2 conserved domains in regulating the growth of pear pollen tubes.The main results are as follows:1.36 PSK genes were identified in five Rosaceae species(pear,apple,strawberry,peach,plum)and divided into 3 subfamilies based on phylogenetic analysis.The results of gene structure analysis and motif analysis found that the gene similarity between the same subfamily is higher,and the structural similarity of conservative motif is also higher.Analysis of duplication pattern found that whole genome duplication was the main driving force for PSK family gene expansion in pears,apples,peaches and plums,while among strawberry PSK family members,50%are dispersed duplication and 33.3%are proximal duplication.Ka/Ks value calculation analysis found that purifing selection played a key role in the evolution of the Rosaceae PSK genes,so it is concluded that the Rosaceae PSK family is very conservative.Through tissue location analysis,it was found that there were six PSK genes expressed in pear pollen,of which PbrPSK2 was the highest expressed in pollen;combined with the results of the qRT-PCR of the dynamic development process of pollen,it was found that the expression level of PbrPSK2 during the entire pollen development process very high.Therefore,it is speculated that PbrPSK2 may play an important role in the process of pollen tube growth and development.2.In order to further study the effect of PbrPSK2 on the growth of pear pollen tube,this study used prokaryotic recombinant PbrPSK2 protein to treat pollen tube cultured in vitro.The results showed that PbrPSK2 could promote the growth of pear pollen tube,and the length of pollen tube increased significantly.And PbrPSK2 protein was treated with different concentrations of pear pollen and found to be concentration-dependent.Meantime the antisense oligonucleotide technology(ODN)was used to knock down the expression of PbrPSK2 in pear pollen.Compared with the control,the growth of pollen tube treated with ODN was significantly inhibited.It was reversely verified that PbrPSK2 promoted the growth of pear pollen tube.In addition,exogenous application of PbrPSK2 protein can increase the ROS content at the tip of pear pollen tube,and it is inferred that ROS may play an important role in the process of PbrPSK2 promoting pollen tube growth.3.The protein containing the C2 conserved domain play important roles in many processes of plant growth and development.A total of 166 genes containing C2 domains were identified in pears,which were divided into 5 families,namely PKC-C2(PF00168,135),PTEN-C2(PF10409,8),NT-C2(PF10358,17),YEATS(PF03366,5)and DOCK-C2(PF14429,1).Among them,38 genes with multiple C2 domains were found,and they were divided into two types:proteins with transmembrane structure(27)and soluble proteins(38).Analysis of duplication pattern found that WGD and tandem duplication events were the main driving forces driving the expansion of the pear C2 gene family.By calculating the Ka/Ks value,it is found that the Ka and Ks values of genes containing only the C2 conserved domain are significantly higher than the Ka and Ks values of genes containing all types of C2 genotypes in the pear genome.Moreover,the selection pressure of C2 subfamily is significantly higher than that of C2 superfamily in the process of evolution.The C2 family functions are more conservative.In order to adapt to its functional requirements,it has a high evolution rate.Analysis of transcriptome data during the dynamic development of pollen revealed that 104 C2 genes were expressed during the development of pollen,of which 34 were relatively high(RPKM>10).RT-PCR analysis of 15 genes with high expression levels revealed that 7 genes were found to have high expression levels in pollen tube and other tissues,of which Pbr020577.1 and Pbr008359.1 were specifically expressed in pollen.Combining with the results of qRT-PCR analysis,it was found that the expression trend of six genes during the dynamic development of pollen was consistent with RPKM,indicating that the C2 gene plays an important role in the growth and development of pear pollen.The effects of PbrPCCP3 on the growth of pollen tube were further studied by experiments such as subcellular localization and ODN.Subcellular localization results showed that PbrPCCP3 is a membrane-localized protein.ODN results showed that when the expression of PbrPCCP3 in pollen tube was knocked down,the growth rate of pollen tubes slowed down,and the length of pollen tubes became significantly shorter,which in turn indicates that PbrPCCP3 can promote the growth of pollen tube,which is consistent with the results of previous studies in the laboratory.In summary,this study clarified that WGD is the main driving force for gene expansion of the PSK family of Rosaceae,and clarified that PbrPSK2 promotes the growth of pear pollen tubes by regulating active oxygen concentration.At the same time,a systematic analysis of the pear C2 family genes revealed that the plasma membrane PbrPCCP3 protein is a pear pollen tube growth-promoting factor.The related research results provide a reference for exploring the mechanism of pear pollen tube growth and development. |
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