Analysis Of Differentially Expressed Genes In Functional Male Sterile Eggplant And Functional Identification Of SmARF

Eggplant(Solanum melongena L.)originated in the tropics and is cultivated all over the world.It is one of the most crucial vegetable crops in the world,and its’ planting area and yield of China are on the top in the world.Eggplant has obvious heterosis,but artificial emasculation and pollination are still needed in eggplant hybrids.Using male sterile line as female parent to produce hybrid can effectively simplify the process of seed production,improve the purity of seeds,and reduce the cost at the same time,which has been paid more attention by breeders.However,compared with other vegetable crops,the research on male sterility of eggplant is still in the preliminary stage.Functional male sterility is a type of male sterility,which is characterized by anther indehiscence and viable pollen.At present,the molecular mechanism of eggplant abortion caused by anthers indehiscence is still unclear.Therefore,exploring the high-quality male sterile resources of eggplant and elucidating its abortion mechanism can improve the utilization of male sterile lines,so as to provide theoretical basis for heterosis utilization and molecular assisted breeding of eggplant.In this study,Using functional male sterile line S12(anthers indehiscence)and fertile line F142 as test materials,flower buds of 8 days before flowering,5 days before flowering and the day of flowering were selected for transcriptome sequencing to analyze the main regulatory pathways of differentially expressed genes in functional male sterile lines and explore the candidate genes related to anther indehiscence.Previous studies have shown that auxin can regulate the development of stamens by mediating the synthesis of jasmonic acid.Therefore,the auxin responsive factors SmARF5,SmARF6 and SmARF8 were focused on to reveal the regulation of auxin on anther dehiscence of eggplant by analyzing the role of ARF gene in auxin signal transduction.The main results are as follows:1.Through the analysis of the full-length transcriptome of eggplant flower buds,797,945 circular consensus reads were obtained,including 652,921 full-length reads non-chimeric(FLNC)sequences.We cluster the full-length non-chimeric sequences to obtain 204,100 consensus sequences,and polish the consensus sequences to obtain high-quality consensus sequences totaling 198,594.Then we used the second-generation transcriptome data to correct the low-quality consensus sequences,which were consistent with high quality after correction.The sequences were merged and subjected to de-redundancy analysis to obtain 85,211 transcript sequences.In these de-redundant transcript sequences,a total of 78,936 sequences were annotated.At the same time we predicted 32,187 SSRs,66,135 complete CDS regions,and 6,838 lnc RNAs.2.By comparing the transcripts of the fertile line F142 and the functional male sterile line S12 at three stages of flower bud development(8 days before flowering,5days before flowering,and day of flowering),8,493 DEGs(differentially expressed genes)were obtained.Among them,1,300 genes were common DEGs in three bud development stages of the two materials,including 557 up-regulated genes and 743down-regulated genes.These DEGs were mainly enriched in "protein processing in the endoplasmic reticulum","amino acid biosynthesis","carbon metabolism","purine metabolism","splicesomes","amino sugar and nucleotide sugar metabolism" and "Plant hormone signal transductionpathway".In addition,nine DEGs were screened to be related to anther development,59 DEGs were involved in phytohormone signal transduction.The DEGs analysis of starch and sucrose metabolism pathways revealed that 4 DEGs were up-regulated in the development of male sterile line flower buds,and 3 DEGs down-regulated expression in the development of male sterile flower buds.263 transcription factors were differentially expressed in functional male sterile flower buds and fertile flower buds,of which 56 TFs were common differentially expressed in three periods.3.The auxin-responsive genes SmARF5,SmARF6 and SmARF8 were cloned,and they all contained highly conserved domains.The transcriptome data indicated that SmARF5 was only expressed in functional male sterile eggplant,while the expression of SmARF6 and SmARF8 between fertile lines and sterile lines at different stages showed no significant difference.However,SmARF6 and SmARF8 was down-regulated in the anthers of S12 on the day of flowering.Plant subcellular localization based on the transient expression of green fluorescent protein showed that SmARF5,SmARF6 and SmARF8 proteins were all located in the nucleus.The self-activation test results showed that SmARF5,SmARF6 and SmARF8 all have transcriptional activation ability,and SmARF5-674,SmARF6-674 and SmARF8-674 which intercepted the III and IV domains still had self-activation.4.Through yeast two-hybrid system,we found that SmARF5,SmARF6 and SmARF8 could interact with IAA16 and IAA26 proteins to form a SmARF-Aux/IAA protein complex.Further analysis of the functional domain of ARF protein showed that the C-terminal dimer structure was indispensable for the formation of the ARF-Aux/IAA protein complex.In addition,it was found that the auxin receptor protein SmTIR1 interacts with SmIAA16 and SmIAA26 depending on auxin,and the F-box domain in SmTIR1 was necessary for the formation of the SmTIR1/AFBs-Aux/IAA protein complex.Further studies showed that in the presence of auxin SmTIR1 could interact with SmTi FY4,a negative regulator of jasmonic acid.5.Yeast one-hybrid experiment and dual luciferase reporter system showed that SmARF6 and SmARF8 bind to the SmDAD1 promoter and activate the expression of SmDAD1,but SmARF5 could not.It was speculated that SmARF6 and SmARF8 can promote the biosynthesis of JA by activating the expression of SmDAD1.6.The SmARF5 gene overexpression vector p CAMBIA-2301G-SmARF5 was constructed and transformed into tobacco,and 16 transgenic tobacco plants were obtained.Compared with wild-type tobacco,transgenic tobacco showed obvious branching,increased stem diameter and premature leaf aging.However,the floral organs of transgenic tobacco did not change at all.The anthers dehisced normally and released active pollens.7.The VIGS expression vector was constructed,and the eggplant cotyledons were infected by injection to silence SmARF6 and SmARF8.Obvious virus spots appeared in the infected plants,and the expression of target genes was significantly reduced.Among them,the eggplant anthers still dehisced normally in the plants that silenced SmARF6 and SmARF8 alone,even together.However,when infecting the young flower buds of eggplant to silence SmARF6 and SmARF8,the anthers of the plants silencing SmARF6 and SmARF8 respectively still dehiscence normally,while the plants that silenced SmARF6 and SmARF8 at the same time showed delayed or no dehiscence in the anthers of the eggplant.