| In this experiment,based on the previous studies,the formula of callus medium of’Dangshansu pear’ was further optimized.The pulp of ’Dangshansu pear’ was induced by callus,and the callus of ’Dangshansu pear’ was used as the main material to carry out calcium treatment of different concentrations,combined with the physiological indexes such as lignin content,calcium content,phenotype,etc.,to screen the inhibition wood in order to provide theoretical guidance for the field application of calcium fertilizer to inhibit stone cells.In order to obtain the genes related to calcium binding protein that may be involved in the pathway of calcium regulated lignin synthesis and further explore the regulatory network of calcium affecting lignin formation,we screened out the callus treatment groups with significant physiological differences after calcium treatment and sequenced the transcriptome.Based on the transcriptome data,the candidate gene family PbrCMLs were selected and analyzed,which provided resources for further study on the function of PbrCML gene in Pear development and whether it played a regulatory role in lignin synthesis.Combined with real-time fluorescence q-PCR technology,the key genes obtained from transcriptome data were cloned.Through the technology of instantaneous infection and subcellular localization of pear young fruit,the function of the key genes of pbrcml was verified,providing the molecular basis for revealing the pathway of calcium regulated lignin synthesis.The main results are as follows:1、The medium formulation of Dangshansu pear callus was further optimized,that is,MS 4.74 g/L+1.5 mg/L 2,4-D+0.5 mg/L NAA+0.2 mg/L TDZ+7 g/L agar+30 g/L sucrose+10 g/L sorbitol+0.1 mg/L VC.The callus of ’Dangshansu pear’ was successfully induced and a relatively perfect callus culture technology system was established.Based on the previous field experiments of calcium chloride spraying,the calli of Dangshan pear were treated with different concentrations of calcium nitrate to verify the effect of calcium on the lignin content of calli in vitro,and further explore the appropriate concentration of inhibiting the formation of stone cells.The results showed that calcium nitrate treatment had a certain effect on the calli of pear fruit.The calli treated with 0.1%calcium nitrate had a fast growth rate,a good growth state and a wet surface.With the increase of treatment time,the lignin content in the calli gradually decreased,while the calli treated with 0.5%calcium nitrate had a slow growth,a poor growth state and browning.The growth of callus almost stagnated after 15 days treatment.Comprehensive comparative analysis shows that 0.1%calcium nitrate concentration has a significant effect on reducing lignin content.2、Based on the significant difference in lignin content of callus at 10 days,we sequenced the transcriptome of callus treated with 0.1%calcium nitrate,0.5%calcium nitrate and CK.The results showed that 2889 differentially expressed genes were identified in callus treated with different concentrations of calcium nitrate for 10 days,which were anchored in 47 go groups and 16 KEGG pathways at the same time.In addition,we found a large number of structural genes and transcription factors related to phenylalanine and lignin synthesis in this process,most of which were significantly up-regulated under 0.5%calcium nitrate treatment,indicating that 0.5%calcium nitrate treatment activated the transcription related to lignin synthesis.Calcium binding proteins,CAM/CML,CDPK and CBL,which are Ca2+ receptors,are involved in Pear callus treated with different concentrations of calcium nitrate,among which a large number of CML genes are expressed differently.Finally,we selected 15 differentially expressed genes in phenylalanine pathway and 17 CMLs differentially expressed genes in pears,and verified the results of transcriptome sequencing by real-time fluorescent quantitative PCR.The results showed that the expression difference in tissue was basically the same as that in transcriptome.3、The whole genome of pear PbrCML gene was analyzed.The results showed that 56 PbrCML genes were identified from pear,and they were divided into 8 subfamilies according to their structural characteristics and phylogeny.The members of PbrCML gene family were distributed on 15 chromosomes,46 PbrCML genes were located on chromosomes,and the rest 10 genes were distributed on scaffolds.The results of collinearity analysis showed that there were 21 pairs of collinearity genes in PbrCML gene,and Ka/Ks value indicated that all the 21 pairs had undergone purification selection.Through the bioinformatics analysis of PbrCML gene,it provides resources for further study on the function of PbrCML gene in Pear development and whether it plays a regulatory role in lignin synthesis.4、According to transcriptome and bioinformatics analysis,PbrCML41 may play an important role in lignin synthesis.The results of fluorescence q-PCR showed that the relative expression of calmodulin gene PbrCML41 was the highest in Dangshansu pear fruit 45 days after flowering,which was consistent with the expression of stone cells and lignin in pear fruit.The results of subcellular localization showed that PbrCML41 was a membrane protein.The results showed that the instantaneous overexpression of PbrCML41 in the young fruit of Dangshansu pear 35 days after flowering could increase the content of lignin,and the difference was significant.The expression of PbrCML41 in the area of instantaneous injection of PbrCML41 in the fruit was significantly higher than that in the area without injection. |
