Identification And Functional Study Of Differential MiRNA In Cytoplasmic-nuclear Male Sterile Line NJCMS1A And Its Maintainer,Restorer Of Soybean(Glycine Max(L.)Merr.)

Plant cytoplasmic-nuclear male sterility(CMS)is mainly caused by cytoplasmic gene(s)and nuclear gene(s)interactions,which lead to pollen abortion.CMS is a simple and effective pollination control system,which provides crucial breeding tools to harness heterosis for crop.Although extensive researches have been conducted on soybean CMS included genetics,cytology,transcriptomics and proteomics etc.in China and abroad,but compared with other crops,the molecular genetic mechanism of soybean CMS remained largely unknown.MicroRNA(miRNA)is an important transcription factor in plants,which can play a key role in the process of plant growth and development,either via degradation or translational inhibition of target mRNAs.In this study,differential miRNA identification and functional study were conducted between CMS line NJCMS1A and its maintainer line NJCMS1B,restorer line NJCMS1C of soybean,to explore the potential function of miRNA and its targets in fertility regulation of soybean CMS.The main results were as follows.1.A total of 571 known miRNAs,107 new miRNA-5p/miRNA-3p on the other arm of known pre-miRNA,24 new members of known miRNA family and 207 novel miRNAs were identified in the flower buds of CMS line NJCMSIA and its maintainer line NJCMS1B,restorer line NJCMS1C of soybean using small RNA sequencing.Differential expression analysis showed that there were 102 differential miRNAs between NJCMS1A and NJCMS1B,140 differential miRNAs between NJCMS1A and NJCMS1C.A degradome analysis showed that 241 target genes were predicted to be targeted by 250 miRNAs,including squamosa promoter-binding protein-like(SBP domain)transcription factor family protein,GAMYB protein,auxin response factor,homeobox-leucine zipper protein,transcription factor APETALA2 and pentatricopeptide repeat-containing protein etc.According to GO analysis and previous reports,20 miRNA families included gma-miR156,gma-miR2119 etc.that potentially participated in fertility regulation of soybean CMS.2.In view of the fact that miR156 and SPL family are the regulatory centers for plant growth and development,therefore,bioinformatics and functional study of soybean miR156 and SPL family were carried out in this study.Small RNA sequencing identified 25 soybean miR156 family members and 2 new soybean miR156 family members.Degradome analysis showed that most targets of gma-miR156 were SPL family members.Functional study of gma-miR156b and its target genes GmSPL9 and GmSPL13A by genetic transformation in Arabidopsis thaliana was conducted and found that,compared with wild-type Arabidopsis thaliana,gma-MIR156b transgenic Arabidopsis thaliana increased number of leaves and branches,delayed flowering,and decreased plant height and seed number,shorted silique length,GmSPL9 transgenic Arabidopsis thaliana increased plant height and silique length,and GmSPL13A transgenic Arabidopsis thaliana decreased leaves number,changed leaf shape,increased plant height and earlied silique maturity.All these results indicated that miR156 and SPL gene may play an important role in plant growth and development.3.The results of small RNA sequencing showed that gma-miR2119 was up-regulated in CMS line NJCMS1A,degradome results showed that alcohol dehydrogenase 1(ADH1)was the only target gene of gma-miR2119,and the results of qRT-PCR showed that GmADHl was down regulated in NJCMS1A,suggested that there might be a negative relationship between gma-miR2119 and GmADH1.Over-expression of gma-MIR2119 in Arabidopsis thaliana showed male sterility,included pollen sterility,shorted silique length,narrowed silique width,decreased seed number etc.qRT-PCR results showed that gma-miR2119 was up-regulated and AtADH1 was down-regulated in 35S::gma-MIR2119 transgenic Arabidopsis thaliana,and alcohol dehydrogenase activity was decreased in 35S::gma-MIR2119 transgenic Arabidopsis thaliana,indicated the pollen sterility may be related to the down-regulated of AtADH1 and decreased of alcohol dehydrogenase activity.According to the above results speculated that gma-miR2119 and GmADHl might participate in the fertility regulation of soybean CMS.