The Cytological Observation And Analysis The Sterility Related Genes Via Comparative Transcriptome Sequencing Of The Male Sterility Moringa Oleifera MMS1

Moringa oleifera Lam.is a deciduous,multi-purpose woody plant of the Moringa genus in the Moringaceae family.M.oleifera has high economic value and is rich in various nutrients.Its leaves and seed oil are edible,and its seeds can also be used as water purifiers.The research team conducted preliminary research and obtained male sterile MMS1 from thousands of materials in M.oleifera.Its anthers degenerated while the pistils were normally fertile,making it a very rare male sterile material for woody plants.In this study,the normal fertile M.oleifera PKM1 was used as the control,and paraffin sections were taken at each stage of the development of the MMS1 flower buds.Microsporogenesis and the development of male and female gametophyte were observed and analyzed,and the cytological basis of the male sterility of MMS1 was clarified.Artificial pollination and fluorescence staining were used to observe the receptivity of MMS1 pistil stigma.Through the comparative transcriptome sequencing and analysis of flower development stages of MMS1 and PKM1 of M.oleifera,differential expression genes were obtained,and male sterility related genes were searched to reveal the molecular basis of male sterility.The main research findings are as follows:1.PKM1 buds are white or milky white,with a mature bud length of 13 mm ±1mm;The flower buds of MMS1 are mostly light green and a few are white,smaller than PKM1 and within 10 mm in length.The microscopic observation results of paraffin sections during the flower development stage indicate that during the pollen development process of MMS1,abnormalities occur when the tetrad develops to the microspores,and the microspores gradually disappear,ultimately exhibiting no anthers.The microstructure of the pistil is normal.The developmental order of PKM1 flowers is to first develop stamens,then develop pistils.The stamens mature first,and the pistils mature later.The pistil of MMS1 develops earlier than the stamen,and the pistil development time is advanced.2.The pollen vitality of PKM1 reached its highest at 95.23% during flowering,and gradually decreased to 0 within the following three days;There was no significant difference in stigma receptivity between PKM1 and MMS1,and both showed a trend of increasing and then decreasing vitality within 3 days after flowering;The stigma has the highest receptivity on the 2nd day after flowering.The fluorescence staining observation results of PKM1 and MMS1 after pollination showed that the pollen was recognized and germinated on the stigma 1 hour after pollination,and the pollen tube extended to the style within 2-6 hours,and finally reached the ovule after 6 hours;MMS1 pistil column hair is normal and can be fertilized.3.The comparative transcriptome sequencing of nine samples yielded 70.47 Gb of raw data.After filtering out unqualified reads,58.16 Gb of effective reads were obtained,and Q30 was above 93%.By removing redundancy from effective reads clustering,a total of 21597 Unigenes were obtained,and further 3480 differentially expressed genes were obtained,of which 1550 were upregulated and 1930 were downregulated.The GO enrichment analysis results showed that 148 Unigenes were found to be enriched in biological processes.Moreover,the processes of biological regulation and sucrose metabolism are more enriched.There are 16 Unigenes enriched in the cell components,and the most enriched ones are the membrane components and the cell intima.There are 67 Unigenes enriched in molecular functions,and the most enriched ones are catalytic activity and hydrolase binding.4.KEGG analysis mapped differentially expressed genes to 88 biological pathways,mainly glycolysis,pentose phosphate pathway,and non oxidative stage.Map the screened Unigenes into metabolic pathways,focusing on starch and sucrose metabolism as well as amino acid biosynthesis pathways.5.The results of m RNA variable splicing analysis showed that: GO enrichment analysis and KEGG analysis of differentially variable splicing genes showed a total of15705 variable splicing genes in SE type,with a ratio of 112:362 detected differentially regulated variable splicing genes in terms of expression levels;There are a total of 2017 variable splicing genes in the MXE type,and the proportion of up-regulation and down-regulation of detected variable splicing genes with differences is 120:411.Differentially expressed genes are enriched to 100 different biological pathways,mainly including small molecule metabolism,organic acid metabolism,membrane binding organelle,membrane binding organelle,catalytic activity,etc.Differentially expressed genes are mapped into 41 different metabolic pathways,mainly including amino acid biosynthesis,carbon metabolism,ubiquitin mediated proteolysis,RNA transport,and spliceosome.This study reveals that the stamens of PKM1 mature first,and the pistils mature later.The development of MMS1 stamens is delayed,and abnormalities occur during the tetrad development to the microspore stage.Subsequently,the microspores gradually disappear,ultimately exhibiting no anthers;MMS1 pistils mature earlier than stamens and are normally fertile.Compared with transcriptome sequencing,3480 differentially expressed genes were obtained,of which 1550 were up-regulated and1930 were down regulated,focusing on starch and sucrose metabolism and amino acid biosynthesis pathways;MRNA alternative splicing analysis mapped differentially expressed genes into 41 different metabolic pathways,mainly including amino acid biosynthesis,carbon metabolism,ubiquitin mediated proteolysis,RNA transport and spliceosome.