Analysis Of Plant PME Gene Families And Functional Identification Of PME37c In Brassica Campestris

Pollen development is an essential part of sexual reproduction,and the development of pollen wall is significantly crucial to the maintaining of pollen morphology and function.The intine is mainly composed of pectin,and the abnormal synthesis and modification processes of pectin will directly influence the development of intine and lead to abnormal pollen germination and pollen tube growth.Pectin methylesterase(PME)is an important modification enzyme of the side chains of pectin,and it has been reported that there are some genes involved in intine development in Arabidopsis thaliana and Chinese cabbage(Brassica campestris L.subsp.chinensis Makino,syn.B.rapa subsp.chinensis).At least 15 PMEs participate in pollen development in Arabidopsis thaliana,and dozens of PMEs in Chinese cabbage are related to pollen development.However,the specific functions of the major pollen development related PME genes remain unclear.The traditional approaches to study the gene function in Chinese cabbage are mutagenesis,antisense interference,RNA interference(RNAi),overexpression,etc.As Chinese cabbage has undergone a genome-wide tripled event during evolution,resulting in two or more homologous genes in its genome.So in actual use,the above mentioned methods have many limitations.The emerging CRISPR/Cas9 gene editing technology can solve these problems well,but the CRISPR/Cas9 system has not yet been established in Chinese cabbage.In this paper,we first identified PME family genes using bioinformatics tools in nine plants,such as Chinese cabbage,and then analyzed the expression of these PME genes by processing the transcriptome data from the Internet.Then we found out the PMEs with specific or superior expression in flower development and then performed sequence alignment and promoter cis-acting element prediction;furthermore,we confirmed the expression characteristics of three genes,BcPME37a(Bra007665),BcPME37b(Bra014410)and BcPME37c(Bra003491)in ’Bcajh97-01B’flower by qRT-PCR in Chinese cabbage ’Bcajh97-01A/B’(’Aijiaohuang’genic male sterility,ajhGMS);afterwards,three PME genes were edited by CRISPR/Cas9 in B.campestris subsp.chinensis var.parachinensis cv.Youqing 49.Then,using morphological,molecular biology,cytology and other methods to analyze the function of BcPME37c in pollen development in Chinese cabbage.The main results and conclusions are as follows:(1)A total of 709 PME genes were identified in nine plants including Chinese cabbage,and all the PME families in nine plants were very large.Expression analysis of the PME family genes revealed that a significant portion of the 709 PME genes was involved in pollen development.The MEME analysis showed that these flower development-associated PMEs shared many conserved motifs in protein sequence.Further analysis of promoters in the PlantCARE revealed that these PMEs also had many identical cis-acting elements,indicating that pollen development related PME genes were conserved in different plants.(2)The primers were designed according to the reference sequence in Brassica database,and the DNA sequences and CDS of BcPME37a,BcPME37b and BcPME37c were cloned in ’Youqing 49’.The alignment results showed that the three genes were highly similar and they had high similarity to PME37(At3g62170)in Arabidopsis.Further results revealed that BcPME37a,BcPME37b and BcPME37c all showed high expression levels during mature pollen stage by qRT-PCR.BcPME37a and BcPME37b also had high expression in gynoecium a few hours after pollination,but BcPME37c showed low expression in this process.The promoter-GUS fusion transformation of Arabidopsis further confirmed that BcPME37c was highly expressed during mature pollen stage.BcPME37a,BcPME37b and BcPME37c were homologous genes of Arabidopsis PME37 in Chinese cabbage,but their expression characteristics were significantly different,suggesting that they might have differentiation functions.(3)The Arabidopsis CRISPR/Cas9’vector was adjusted for genetic transformation of Chinese cabbage.Three sgRNAs targeting BcPME37a,BcPME37b and BcPME37c were designed,and the corresponding CRISPR/Cas9 vectors of pBI121 backbone were successfully constructed.’Youqing 49’was used for genetic transformation.A number of genetically edited bud lines were obtained,and the editing efficiency could reach 50%.The genome editing of a single target(BcPME37c)was observed,while the simultaneous editing of two targets(BcPME37b and BcPME37c)was also achieved.Sequencing of the predicted potential off-target sites of the designed sgRNAs revealed that there was no off-targeting in the genetically edited bud lines.Knockout mutants of BcPME37c were then self-crossed,and mutants containing no T-DNA fragments were detected in the progeny.(4)The knockout mutant of BcPME37c showed no significant difference in vegetative development stage compared to the control plants.The flower organs were also normal,and the seeding rate did not change.Furthermore,the pollens was stained with Alexander staining,and about 5%of the pollens of BcPME37c knockout mutants showed significantly increase in the diameter.Scanning electron microscopy showed that the mutants had about 5%malformed pollens,and the proportion of malformation pollens was identical to the proportion of pollens with increased diameter after swelling.Transmission electron microscopy showed that the intine of the mutants were abnormally thickened during the binuclear pollen and trinuclear pollen stages.After knocking out BcPME37c,the degree of methylation of homopolygalacturonic acid(HG)in pollen inner wall was likely to increase abnormally,and other PMEs with specific expression in pollen might be up-regulated,eventually leading to abnormal thickening of intine,and thus affecting the swelling and the morphology of pollens.