Cloning And Functional Identification Of Transcription Factor LoUDT1 Related To Lily Anther Development

Lilium is a perennial herbaceous bulbous flower of the lily family,which has both ornamental,edible and medicinal value.Lily is loved by people because of its beautiful meaning,bright colors,and strong floral fragrance.However,due to the huge anthers of lilies,once the anthers are dehiscence and scattered,they will seriously pollute the petals and reduce their ornamental value.Moreover,because the pollen is highly viscous,it is easy to contaminate the clothes of viewers.At the same time,pollen floating in the air can cause allergic reactions in pollen allergies.Artificial emasculation is often used in the production of lily cut flowers to solve this problem.Manual emasculation is not only time-consuming and laborious,but also not suitable for large areas of Lily Tourism Park.Therefore,studying the molecular mechanism of lily anther development and breeding pollen-free lilies through genetic engineering is an effective way to solve this problem.In order to study the molecular mechanism of lily anther development,this study cloned the differentially expressed gene LoUDT1 before and after pollen abortion from the transcriptome database,and analyzed its biochemical characteristics.The characteristics and functions during the development of anthers were analyzed,and the main results are as follows:The LoUDT1 gene was cloned from the cDNA of the anthers of ’Siberia’ lily.The largest open reading frame(ORF)of LoUDT1 was 609 bp,which was predicted to encode a protein of 202 amino acids.Amino acid homologous sequence alignment and protein structure analysis revealed that it contains bHLH domain,BIF domain;through phylogenetic analysis,it was found that LoUDT1 belongs to the bHLH family,and was closely related to AtbHLH021 and AtbHLH022 of Arabidopsis in evolutionary relationship;the results of transcription activation activity analysis showed that LoUDT1 has no transcriptional activation activity in yeast cells;protein subcellular localization result showed that LoUDT1 protein was localized in the cytoplasm and nucleus.Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that LoUDT1 can interact with another bHLH transcription factor LoAMS,and the protein truncation indicated that their interaction depends on their respective BIF domains.The subcellular localization and transcriptional activation activity analysis of LoAMS found that LoAMS has transcriptional activation activity,and the transcriptional activation domain at its N-terminal;the subcellular location was in the nucleus.Real-time quantitative PCR showed that LoUDT1 showed continuous and fluctuating expression throughout the developmental stages of anthers;the expression was highest in anthers of 12 cm flower buds,followed by anthers from 10 cm flower buds.The expression of LoAMS was mainly concentrated in the early stage of anther development,and decreased in the later stage of anther development.Through heterologous transformation of Arabidopsis thaliana,it was found that two of the six transgenic lines obtained were male sterile.The gene expression level of LoUDT1 was detected by quantitative PCR and it was found that the expression level of LoUDT1 in the two sterile lines was other fertile 2～3 times,indicated that the excessive accumulation of LoUDT1 in anthers will affect the normal development of anthers.By complementing Arabidopsis dyt1-3 mutants,it was found that LoUDT1 can partially restore the sterile phenotype of dyt1-3 and produce viable pollen.