| Self-incompatibility is a widespread reproductive isolation mechanism formed by plants during long-term natural evolution.It enables the pistil to refuse self-pollen growth.S-RNase-mediated gametophytic self-incompatibility is the reason why the pollen tube is inhibited and cannot be extended to the ovary to complete the pollination and fertilization process.It is mainly controlled by the style determinant S-RNase and pollen determinant SLF/SFB,and other non-S genes involved in the self-incompatibility of plants.In order to further explore the mechanism of self-incompatibility,Fragaria viridis Ls-S2-49(SaSa)was used as experimental material to construct pollen cDNA library and screen out non-S genes that may be involved in self-incompatibility.Use Sb-RNase to verify whether it has S-RNase haplotype specificity to further study the positive clones;use qRT-PCR technology to analyze the specific expression of interacting genes in different tissues of Fragaria viridis and the expression in the style of different periods after self-pollination;and use bioinformatics to further analyze its sequence.The main results are as follows:1.The cross-pollination of Ls-S2-49 strain can be normal,and self-pollination shows self-incompatibility,and the pollen tube basically stops growing at 24 h after self-pollination.The construction of Fragaria viridis pollen cDNA library is the basis for studying protein interaction using yeast two-hybrid technology.The CTAB method was used to extract the pollen tube RNA,and the pollen cDNA library was constructed by the gateway technique.The results show that the extracted total RNA has good integrity and high purity.The cDNA library constructed by gateway technology has a library capacity of 1.2 × 107 CFU.The library has high coverage,recombination rate and integrity.2.Screen the green strawberry pollen tube yeast cDNA library with the expression vector pGBKT7-Sa as bait plasmid,spread the co-transformed yeast on SD/-Trp/-Leu/-His/-Ade selection medium,and do further verification by SD/-Trp/-Leu/-His/-Ade/X-α-gal mediumt.The results show that the bait expression plasmid pGBKT7-Sa has no toxicity and no automatic activation,and can be used as a bait vector for yeast two-hybrid test.Six interacting proteins were obtained,namely RD21a-like,PRP2-like,UN,AT,CNR8 and ASP.3.Verify the interaction between the six selected proteins and S-RNase whether have haplotype specificity by Sb-RNase;the results showed that these six proteins interacted with Sb-RNase,and there was no S-RNase haplotype specificity.It may play a role in regulating the general metabolic pathway in self-incompatibility.In order to clarify the interaction sites of 6 kinds of interactor proteins,leaf,stem,calyx,petal,pollen and style were selected for tissue-specific expression analysis;the results showed that 6 kinds of interactors all can be expressed in leaves,stem,calyx,petal,pollen and style,but the expression level in pollen is the highest.In order to analyze the possible function of the interactor in the GSI response of green strawberry,the styles at 6 h,12 h,24 h,48 h and 72 h were selected for spatiotemporal expression analysis compare with the style at 0 h after pollination;The expression trend of six kinds of interaction are consistent with S-RNase,showing a trend of increasing first and then decreasing,reaching a peak at 12 h after pollination.And then we make basic bioinformatics analysis to further understand the function of interactors.The results show that RD2 1a-like may be involved in growth and development,seed germination,environmental stress and programmed cell death,and is also related to pollen development,germination and pollen tube growth.PRP2-like and unknown proteins control pollen germination by affecting the normal development of cell walls And pollen tube growth;AT protein is associated with pollen germination;CNR8 may participate in self-incompatibility by inhibiting the growth of pollen tubes;ASP may provide a substrate for basic metabolic pathways. |
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