Preliminary Analysis Of The Functions Of The LhSorPR4b,LhSorPR10a And LhSorPR10b Genes In Oriental Lily ‘Sorbonne’

Lilium spp.Sorbonne is a perennial herbaceous bulbous plant.It is an important economic crop for the cut flower industry,but the disease causes serious losses.Pathogenesis-related Proteins(PR)is a general term for a class of proteins that plants accumulate when they are subjected to biological stress.They are widely involved in the response of plants to biotic and abiotic stresses and are an important part of plant disease resistance.The cloning and functional analysis of lily LhSorPR4b,LhSorPR10a and LhSorPR10b genes will lay the foundation for enhancing the research on disease resistance of lily and cultivating new varieties with strong disease resistance.In this study,the highly expressed LhSorPR4b,LhSorPR10a and LhSorPR10b genes were screened from the transcriptome data of Botrytis elliptica infection in Lilium ‘Sorbonne’,and the full length of these three PR genes were cloned using leaf cDNA as a template.Using bioinformatics analysis,the characteristics of 3 genes and their encoded proteins were analyzed.Real-time fluorescent quantitative PCR was used to analyze the expression specificity of the three genes in each tissue;and the response mode of these genes to the disease after infection by the pathogens of B.elliptica and F.oxysporum Analyze their response to hormones through SA,Me JA,and ETH hormone treatments;analyze their expression in temperature stress through high temperature and low temperature stress treatments.The genes of LhSorPR4b,LhSorPR10a and LhSorPR10b were overexpressed in tobacco,and their disease resistance to B.cinerea and F.oxysporum were analyzed,and physiological indicators of the transgenic tobacco after stress were measured to preliminarily verify its function.The aim is to clarify the functions of LhSorPR4b,LhSorPR10a and LhSorPR10b genes in the disease resistance process of lily,provide a molecular basis for strengthening the resistance of lily to biotic and abiotic stress environments,and for further study the biological functions of lily PR genes and their resistance The mechanism of action during the disease process provides a theoretical basis,and opens up new development ideas and directions for the ‘Sorbonne’ lily to reduce disease loss and germplasm innovation.The results of the study are as follows:1.B.elliptica processed the transcriptome sequencing analysis of the leaves of "Sorbonne" at different times,from which three PR genes were screened.The NCBI amino acid BLAST analysis showed a sequence of 495 bp with an open reading frame of 474 bp,encoding 158 amino acids,The isoelectric point is 5.72,and BLST results show that it has a 95% similarity with the PR107 gene of Lilium longiflorum,so it was named LhSorPR10 b.And referring to previous studies,the gene lengths of LnSorPR4b(APG55503.1)and LnSorPR10a(ALO77720.1)were 432 bp and 471 bp,respectively,by cDNA template amplification and cloning.2.The specific expression of LhSorPR4b,LhSorPR10a and LhSorPR10b genes in‘Sorbonne’ tissues was analyzed,and the results were as follows: LhSorPR4b was the highest expression in the stem;LhSorPR10 a was the highest expression in the root;LhSorPR10b was the highest expression in the leaves.The lily tissue culture seedlings were treated with hormones,pathogenic bacteria and stress.The results showed that LhSorPR4b,LhSorPR10a and LhSorPR10b can be treated by salicylic acid(SA),jasmonic acid(MeJA),ethephon(ETH),and high temperature,Low temperature induces expression;in the treatment of pathogenic bacteria,LhSorPR4b,LhSorPR10a and LhSorPR10b are induced by B.cinerea,and LhSorPR4 b,LhSorPR10a and LhSorPR10b can be induced by F.oxysporum.3.Successfully constructed the plant overexpression vectors pGMF500-LnSorPR4b,pGMF500-LnSorPR10 a,pGMF500-LnSorPR10 b of LnSorPR4b,LnSorPR10a and LnSorPR10b genes,and successfully integrated the plasmids into the genome of common tobacco using Agrobacterium-mediated method and obtained transgenic tobacco positive plants.Fluorescence quantitative results showed that these three genes were stably expressed in transgenic tobacco lines,and high,medium,and low expression lines were screened out at the same time.The plate bacteriostasis experiment proved that the crude protein of tobacco leaves transferred with PR gene inhibited the growth of B.elliptica and F.oxysporum hyphae to varying degrees.The results of in vivo antibacterial experiments showed that the transgenic tobacco plants were more resistant to F.oxysporum than wild-type tobacco.The test results of physiological indicators of genetically modified tobacco leaves showed that the resistance of genetically modified tobacco was significantly enhanced after being infected by F.oxysporum,which preliminarily verified the function of the ‘Sorbonne’PR gene in the process of disease resistance.