Cloning And Analysis Of The Transcription Factor LILBD8 Related To Bulbil Formation In Lilium Lancifolium

Bulbil propagation is an important asexual propagation strategy for lily.Analyzing the regulatory mechanism of bulbil formation can lay a theoretical foundation for improving the reproductive efficiency of lily.Lilium lancifolium is an excellent experimental material for studying the formation of bulbils.They do not produce seeds,but can form bulbils in the leaf axils.Mature bulbils will take root and grow into independent plants,with a very high reproductive coefficient and the ability to maintain excellent maternal characteristics.LBD is a unique transcription factor family in higher plants,which plays an important role in regulating plant lateral organs.In the early stage,the research team carried out transcriptome sequencing on bulbils at different development stages of L.lancifolium,and found a large number of Ll LBD transcripts.However,the key Ll LBD transcription factor that regulates bulbil formation in L.lancifolium is still unknown.Find out relevant transcripts from the transcriptome library,and combine q PCR assay and previous conservative biological function studies on LBD to screen out the key Ll LBD factor significantly related to bulbil formation.The full length of the transcription factor ORF was obtained by RT-PCR and bioinformatics analyzed.Analysis of tissue specificity by q PCR assay.Subcellular localization vector was constructed to transform tobacco and the expression position of Ll LBD protein in cells was analyzed.The overexpression vector and TRV-VIGS vector were constructed,and the stem segments of L.lancifolium were transformed instantaneously,and the function of the transcription factor was preliminarily studied.The main results are as follows:(1)All relevant Ll LBD transcripts were screened from the transcriptome of bulbils of L.lancifolium,including c80484＿g1、c84377＿g1、c112613＿g1、c114216＿g3、c114216＿g5、c118861＿g2、c120163＿g1、c123467＿g6、c124509＿g1 and c126102＿g4。Combining previous gene function tests and q PCR assay,c112613＿g1 and c124509＿g1(all belong to LBD18 fragments)was determined as the research object.(2)The 684 bp ORF sequence of Ll LBD18(Gen Bank number ON455210)encoding 227 amino acids was sequenced by RT-PCR.It contains a typical LOB domain and belongs to the LBD member of IB class.With a molecular weight of 23.713 KD and an isoelectric point of 7.63,this protein is an unstable hydrophilic protein with no transmembrane structure and signal peptide,and contains 1 glycosylation site and 21 phosphorylation sites.Protein structure prediction found that Ll LBD18 protein was the most similar to the three-dimensional structure of Ramosa2.1protein in wheat.Cluster analysis found that Ll LBD18 protein was closely related to monocot ginger Zo LBD18 protein.(3)RNA was extracted from the roots,scales,stems,leaves,mature bulbils and primary bulbils of L.lancifolium,and q PCR analysis was conducted using c DNA after reverse transcription as template.The results showed that Ll LBD18 was expressed in all tissues,but the expression level was different.Among them,the highest expression level was found in primary bulbils,the second in the roots,and the lowest in the leaves.(4)The Ll LBD18 was constructed onto the GFP-labeled p CAMBIA-2300 vector,and the no-load and recombinant vectors were transformed into tobacco by injection.The localization of Ll LBD18 protein in cytoplasm and nucleus was observed by laser confocal microscope.(5)The overexpression vector of Ll LBD18 was constructed by homologous recombination method,which was transformed into the stem segment by vacuum-assisted agrobacterium infiltration.Verify the positive material from the RNA level,regularly observe the development process of bulbils under microscopy,and the bulbil induction rate was counted on the 12 th day of culture.It was observed that there was no significant difference in the development process,but the bulbil induction rate in the control group was 47.0% after 12 days of cultivation,while the bulbil induction rate in the overexpression Ll LBD18 treatment group increased to 73.5%.(6)Avoiding the conserved LOB domain,a specific 300 bp fragment of Ll LBD18C-terminal was selected to construct a TRV-VIGS vector.The bacterial solution containing the auxiliary vector p TRV1 was mixed with the bacterial solution containing p TRV2 no-load and p TRV2 recombinant vector in a ratio of 1:1,and the stem segment of L.lancifolium was transformed by vacuum infiltration.The successful infection and stable expression of the vector were detected from the DNA level and RNA level,regular microscopic observation of the development process of bulbils,and the rate of bulbil induction was counted on the 12 th day of culture.The results showed that silencing Ll LBD18 reduced the number of bulbils,resulting in a decrease in bulbil induction rate from 37.8% to 18.9%,but there was no significant difference in developmental stages.In this study,we screened a transcription factor Ll LBD18 that is significantly related to bulbil formation.The full-length ORF was cloned and the preliminary functional analysis showed that Ll LBD18 positively regulated the formation of axillary bulbils.