Functional Analysis Of LhSorMYB2 And LhSorMYB11 Genes In Lily ’Sorbonne’

Lily ’Sorbonne’ is a perennial herbaceous bulb flower of Lilium in Liliaceae.It is an important variety in cut-flower industry of lily.However,the biotic and abiotic stress in its growth process seriously limits the quality of cut flowers.In order to cope with the above adversity,plants have evolved a series of response regulation mechanisms,and a large part of these responses are realized by regulating the expression of related genes.MYB transcription factor is one of the most numerous transcription factor families in plants.Its family members play an important role in regulating plant stress response.Salt,drought,extreme temperature,hormones,nutrient deficiency,mechanical damage,biotic stress and other stresses can induce the specific binding of MYB protein with cis-elements in the promoter region of downstream related target genes,inhibit or activate the expression of target genes,and then regulate the stress resistance of plants.In this study,LhSorMYB2 and LhSorMYB11 genes,members of Lily MYB transcription factor family,were cloned.Through bioinformatics analysis,gene expression characteristics analysis,subcellular localization,transcriptional activation verification,and transgenic research of tobacco and Lily,the function of LhSorMYB2 gene was preliminarily explored.The main results are as follows:1.Cloning and sequence analysis of LhSorMYB2 and LhSorMYB11 genes.By analyzing the transcriptome data of Lily ’Sorbonne’,two MYB genes were selected and named LhSorMYB2 and LhSorMYB11.According to the results of transcriptome sequencing,primers were designed to clone the two genes.LhSorMYB2 gene has an open reading frame of 462 bp and encodes 153 amino acids.BLAST analysis on NCBI showed that it belongs to R3 MYB subfamily and has the closest genetic relationship with lily Lh MYB2 protein.The open reading frame of LhSorMYB11 gene is942 bp and encodes 313 amino acids.BLAST analysis on NCBI found that it belongs to R2R3 MYB subfamily and has the closest genetic relationship with lily Lh MYB11 protein.2.Expression characteristics of LhSorMYB2 and LhSorMYB11 genes in LilyThe expression of LhSorMYB2 and LhSorMYB11 genes in different tissues,pathogens,hormones and stress treatments of lily were studied.The results of real-time fluorescence quantitative PCR showed that LhSorMYB2 gene was mainly expressed in leaves,and LhSorMYB11 gene was mainly expressed in scales and leaves.LhSorMYB2 and LhSorMYB11 genes were up-regulated in varying degrees induced by F.oxysporum,B.cinerea,salt,drought,abscisic acid(ABA),salicylic acid(SA)and methyl jasmonate(Me JA).The change trends of the two genes induced by different stresses were similar.It is speculated that the two genes may have similar regulatory pathways in stress response.3.Subcellular localization and transcriptional activation verificationZm Ubi2 promoter:: LhSorMYB2::e GFP and Zm Ubi2 promoter::LhSorMYB11::e GFP subcellular localization vectors were constructed by seamless cloning technology.The subcellular localization results of onion epidermis showed that the two genes were mainly localized in the nucleus;p GBKT7-LhSorMYB2 and p GBKT7-LhSorMYB11 transcriptional self activation vectors were constructed.Transcriptional self activation experiments in yeast Y2 H Gold strain showed that only LhSorMYB2 gene had transcriptional self activation activity.4.Stress resistance study of LhSorMYB2-transgenic tobaccoTobacco overexpression vectors p GMF500-LhSorMYB2 and p GMF500-LhSorMYB11 were constructed.Stable tobacco plants overexpressing LhSorMYB2 gene were obtained by Agrobacterium mediated method.Through q-PCR identification,two strains with high and medium expression were selected for abiotic stress experiment.In terms of drought stress,in the MS plate mannitol simulated drought stress experiment,the root length of LhSorMYB2 transgenic tobacco seedlings was significantly longer than wild type;In the soil PEG simulated drought experiment,the integrity of cell membrane and SOD activity of LhSorMYB2 transgenic tobacco were significantly higher than wild type.In the MS plate salt stress experiment,the root length of LhSorMYB2 transgenic tobacco seedlings was significantly longer than wild type;In the experiment of soil salt stress,the integrity of cell membrane and SOD activity of LhSorMYB2 transgenic tobacco were significantly higher than wild type.This experiment preliminarily believes that LhSorMYB2 gene can play a role in drought and salt tolerance of plants.5.The establishment of Lily genetic transformation systemConstructed the transgenic Lily overexpression vectors p VM01-LhSorMYB2 and p VM01-LhSorMYB11,and preliminarily established the lily genetic transformation system.The test results of some materials showed that two resistant buds of lily with LhSorMYB2 and LhSorMYB11 genes were obtained.