Cloning And Functional Analysis Of PbABCG11 Gene Of ‘Dangshansuli’ Pericarp Russet Mutant

The ABCG is the largest subfamily of ABC transporter protein,one of the most ancient and largest known protein families,and plays an improtant role in the transportation of suberin in plants.In order to further study whether the PbABCG11 gene is involved in the formation of suberin in the brown peel of ’Dangshanjinsu’,which is the variant of ’Danshansuli’,we analyzed the the ABCG family genes in ’Danshansuli’.In additon,PbABCG11 was cloned and plant expression vectors were constructed,expression characteristics,subcellular localization,and transcriptional activation activity have been studied.And the inhibitor treatment experiment was carried out.What’s more,the metabolomic data of peels from both varieties has been analyzed.The main findings are as follows:1.Through the bioinformatics analysis of ABCG family genes of ‘Dangshansuli’,it was found that the ABCG family genes of pears are very complicated.And the function of these genes has obviously differed from each other in the process of evolution,while some of them have the similar structure and function.The expression of the ABCGs in the peel of‘Dangshansuli’ and ‘Dangshanjinsu’ showed that the formation of suberin substance on the surface of the ‘Dangshanjinsu’ may have a certain relationship with some ABCG genes;2.Transcriptomic profiles of ‘Dangshansuli’ and ‘Dangshanjinsu’ were analyzed by high-throughput sequencing,and the PbABCG11 was screened from the library which expressed differentially in different fruit development stages.We cloned the PbABCG11 from both varieties and found that PbABCG11 is 2061 bp in length and encodes 687 amino acids in ‘Dangshansuli’.Interestingly,there was a 3-bp deletion at the 116 bp position in the variant ’Dangshanjinsu’,and the base G at position 596 is changed to A,leading to early termination of amino acid translation.3.The results of subcellular localization showed that PbABCG11 D was localized to the cell membrane while PbABCG11 X,cloned from ‘Dangshanjinsu’,was unable to localized normally because of the early termination and synthetic disorder of amino acid translation.PbABCG11 Q and PbABCG11 H was localized to the cell membrane and nucleus.We found that in the transgenic lines,the content of the trimethylsilyl octadecanoate and octadecanoyl benzoate,which are the precursor of suberin,decreased obviously,while there was no significant difference in other major components.The results showed that PbABCG11 D gene may reduce the formation of suberin substance by promoting the synthesis of suberin precursors into keratin4.The inhibitor treatment experiment was carride out,and we found that the inhibitors of fatty acid can reduce the accumulation of the suberin layer of the fruit peel of’Dangshanjinsu’ to a certain extent.What’s more,different inhibitors had unequal effects.Among all kinds of inhibitors,pebulate and cerulenin had the best inhibitory effect which significantly reduced the content of the suberin layer in ‘Dangshanjinsu’ by negatively controlling synthesis of fatty acids,especially long-chain fatty;5.Metabolic profiles of peels of ‘Dangshansuli’ and ‘Dangshanjinsu’ were analyzed,the results showde there was a content difference of fatty acids related to the suberin wood composition.What’s more,a large amount of tannins substances,glycerin substances and terpenoids showed great divergence,suggesting that besides fatty acids,all of these substances are also likely to play an important role in the suberin formation in the fruit of‘Dangshanjinsu’.