Mechanism Of Eggplant SmMYB108 Gene Regulating Anther Dehiscence

Eggplant(Solanum melongena L.)is a vegetable crop of the Solanaceae family,and it is grown all over the country.According to FAO statistics,the cultivation area of eggplant in our country in 2019 was 783,000 hm~2,accounting for 42.37%of the world’s total cultivation area.The heterosis of eggplant is obvious.The production of seed hybridization is mainly based on artificial emasculation,which is time-consuming,laborious and costly.At the same time,inappropriate selection of flower buds or incomplete emasculation during emasculation can easily form false hybrids,reducing the purity and quality of seeds and bring certain losses.Using the male sterile line as the female parent to produce hybrid seeds and bring can not only simplify the procedure of seed production,but also reduce the cost and improve the purity of the seeds.Male sterility refers to the phenomenon that stamens cannot grow normally and produce viable pollen during sexual reproduction,while pistils can be pollinated and fertilized normally.As a kind of male sterility,functional male sterility can produce normal pollen,but due to various obstacles,such as anther not dehiscent,pollen outer wall is not developed or has no outer wall,resulting in pollen can not germinate on stigma,pollen tube growth is not normal,the pollination and fertilization process can’t be completed.In recent years,some achievements have been made in the breeding of eggplant male sterile line materials,but so far it has not been put into production in a large area.The functional male sterile line has its own fertile pollen,so there is no need to breed the maintenance line,which is convenient for breeding work.In addition,the research on the mechanism of male sterility in eggplant mostly focuses on the genetic,cellular,physiological and biochemical aspects,and there are few reports on the molecular mechanism.Therefore,it is of great significance to study the molecular mechanism of eggplant anther dehiscence.In this study,the eggplant fertile line F142 was used as the test material,and based on the previous transcriptome sequencing data of the research group,through differential expressed gene analysis,it was found that the Sm MYB108 gene was in different stages of eggplant flower bud development(8 days before flowering,5 days before flowering and flowering.The research focuses on the transcription factor Sm MYB108,and adopts a variety of research methods and technical means to conduct research to analyze its role and molecular regulation mechanism in eggplant anther development,laying a solid foundation for analyzing the molecular regulation mechanism of eggplant anther dehiscence.The main results of the test are as follows:1.A total of 5549 differential expressed genes(DEGs)were obtained by comparing and analyzing the transcripts of the fertile line F142 flower bud development in three stages(8 days before flowering,5 days before flowering and the day of flowering).Among them,most of the DEGs were concentrated in the combination of 8days before flowering vs the day of flowering(8DBF vs FD)and 5 days before flowering vs the day of flowering(5DBF vs FD),and 2562 genes were DEGs common to 8DBF vs FD and 5DBF vs FD,1745 differential up-regulated genes and 817differential down-regulated genes,respectively.89 DEGs encoding transcription factors were screened,which including 11 MYB transcription factor DEGs.Among them,the Sm MYB108 transcription factor was significantly up-regulated on the day of flowering.2.Sm MYB108 gene was cloned from F142 flower bud.Sm MYB108 contained a highly conserved R2-R3 MYB domain and belonged to a typical R2-R3 MYB transcription factor.Using real-time fluorescence quantitative PCR,the expression level of Sm MYB108 gene was higher in eggplant roots and flowers,lower in the early stage of anther development,and significantly up-regulated in mature anthers.Subcellular localization found that Sm MYB108 protein was located in the nucleus.3.The Sm MYB108 gene overexpression vector p CAMBIA-2301G-Sm MYB108was genetically transformed into Nicotiana benthamiana,and 13 transgenic tobacco plants were obtained.Compared with wild-type tobacco,there was no difference in the flower organs of transgenic tobacco,and the anthers dehiscent normally and released vigorous pollen.By comparing the degree of anther dehiscence in the same period,the anther dehiscence degree of transgenic tobacco was greater,and it was speculated that the anther dehiscence of transgenic tobacco was earlier.4.The Arabidopsis mutant myb108 had shorter filaments,delayed anther dehiscence,and decreased pollen viability.The overexpression vector p Bin35SRed3-Sm MYB108 of the Sm MYB108 gene was transformed into the Arabidopsis mutant myb108,and the red light selection marker carried by the vector was used to carry out primary screening of transgenic plants,and then the positive transgenic plants were identified by PCR and RT-PCR identification.The results showed that the anthers of the positive plants dehisced normally and released vigorous pollen in time,and the filaments became longer,which was no differences from the wild type.5.Using Y2H technology to detect transcriptional activity,it was found that the full-length Sm MYB108 protein had transcriptional activation activity.By truncating the conserved domain,it was found that the N-terminal of Sm MYB108 protein had no transcriptional activation activity,the C-terminal had transcriptional activation activity,and the C-terminal 137～317aa and 262～317aa fragments had self-activation activity.As the distance from the C-terminal decreases,the C-terminal 262～317aa fragments were split into 43aa and 13aa again.Attenuated,indicating that the autoactivation domain of the Sm MYB108 transcription factor was disrupted.Therefore,the self-activation domain of Sm MYB108 transcription factor was located at 262～317aa of the C-terminus.6.The promoter of Sm MYB108 with a sequence length of 2000 bp was cloned,and Plant CARE software analyzed that the promoter contained multiple transcription factor binding sites.Using Y1H technology,it was found that Y1H(pr Sm MYB108+Sm ARF6),Y1H(pr Sm MYB108+Sm ARF8),and Y1H(pr Sm MYB108+Sm MYB21)could grow white plaques on SD/-Leu/Ab A600,while Y1H(pr Sm MYB108+Sm MYB26)could not grow white plaques on SD/-Leu/Ab A600.White plaques growed on Leu/Ab A600,indicating that Sm ARF6,Sm ARF8 and Sm MYB21 could bind to the Sm MYB108promoter,while Sm MYB26 couldn’t bind to the Sm MYB108 promoter.The results obtained by the dual-luciferase system assay were consistent with the Y1H assay.