Preliminary Study On Multiple Stress Response Gene BnaANN1C-2 In Brassica Napus L.

Plants are often affected by various kind of stress in the process of growth,causing varying degrees of damage to the plant,resulting in the normal growth and development of plants blocked,resulting in crop yield reduction.A large number of studies have shown that plant calcium signaling,as a second messenger in response to plant growth and development and environment,plays a crucial role in plant regulation under stress.Annexin（ANN）is an evolutionarily conserved calcium-dependent phospholipid binding protein that plays an important role in signal transduction in plant growth and development mediated by Ca²⁺and in response to stress.In this study,the Annexin gene family of Brassica napus were identified and their expression patterns were analyzed according to the transcriptome data under different abiotic/biotic stress treatments and phytohormones.Among them,a multiple stress response Annexin gene BnaANN1C-2（Bna C05g27530D）was identified.The expression pattern of BnaANN1C-2 was performed transcriptional level expression analysis and q RT-PCR,the over-expression of BnaANN1C-2 rapeseed lines were generated and the function of BnaANN1C-2 in the tolerance to sclerotinia sclerotiorum were analyzed.The main results are as follows:1.Bioinformatics analysis of Annexin gene family in Brassica napus:in this study,26Annexin genes in Brassica napus were identified based on 8 Arabidopsis thaliana Annexin gene sequences.The molecular properties,phylogenetic tree,gene chromosome distribution,gene structure and motif,cis-element,expression patterns under different stress and hormone treatment of Bna ANN genes were analyzed.The results showed that the molecular characteristics of Bna ANN protein sequences were varied,the number of amino acids ranged from 157 to 320,the molecular weight（Mw）ranged from 17.84 to 36.91 k Da,and the predicted isoelectric point（p I）ranged from 5.16 to 9.56 Subcellular localization prediction showed that all 16 typical Bna ANN proteins were located in cytoplasm.Bna ANN gene can be divided into 6 subgroups（I-VI）according to phylogenetic relationship.All the 8Arabidopsis At ANN genes have 2-5 homologous genes in Brassica napus,and each subgroup gene is distributed on different chromosomes.The distribution of each subgroup chromosome shows the characteristics of A subgroup and C subgroup in pairs,and about half of the genes are tandem repeats.Most Bna ANN genes have conserved domains,with similar gene structures,but some members have obvious mutation characteristics.Only 17 Bna ANN genes contain 4 ANNEXIN（Annexin Repeat）domains.Among 608 cis-elements at 2000bp upstream of Bna ANN gene promoter,Me JA-and ABA-related elements were top 2phytohormone-response cis-elements,Low-temperature（LOW-T）and Light response elements weretop 2 environmental stress response cis-elements.The expression patterns of Bna ANN gene were tissue specific,which may be related to their functional differentiation.Most Bna ANN genes were strongly induced by Me JA and SA in leaves,and induced by Na Cl and PEG in roots.In different low temperature treatments,whether cold acclimation or not,only the subgroup I Bna ANN were induced by low temperature stress,and the up-regulated expression under freezing stress were higher than cold stress;while the other subgroups did not respond to low temperature stress.Vaccination sclerotinia sclerotiorum after 24 hours,the expression of subgroup I（Bna C05g27530D,Bna Anng04520D and Bna A08g06260D）genes in susceptible genotype（Westar）and resistant genotypes（ZY821）were both significantly reduced,and the expression of relatively high resistance materials in susceptible materials.These results suggest that Bna ANNs may play important roles in the response of to various abiotic stresses and sclerotinia sclerotiorum in Brassica napus.2.Genetic transformation and biological function of BnaANN1C-2 gene:The BnaANN1C-2（Bna C05g27530D）gene was cloned from Westar based on the reference genome of Brassica napus.The full-length BnaANN1C-2 gene was 2174 bp with a 954bp open reading frame（ORF）,encoding a protein containing 317 amino acids,molecular weight 36.10 KD,isoelectric point 5.27;The gene structure was conservative and stable,with 5 exons and 4introns.Their subcellular localization is in the cytoplasm;Alignment of homologous sequences revealed that BnaANN1C-2（Bna C05g27530D）features contained four Annexin Repeat,and had high homology with Arabidopsis AT1G35720（At ANN1）protein.Transcriptomic data and q RT-PCR results showed that BnaANN1C-2 was an multiple stress response ANN gene,induced by low-T,freezing,Na Cl、PEG、ABA、Me JA、ET、SA.An overexpression vector（PC1300S）was constructed,and 7 overexpression plants selected from 19 T0 BnaANN1C-2 trangenic rapeseed plants.Vaccination sclerotinia sclerotiorum experiment showed that the Leaf sclerotinia spot of the overexpressed plants was smaller than that of the control plants,indicating that the overexpression of BnaANN1C-2 could improve the tolerance to sclerotinia sclerotiorum in Brassica napus.In conclusion,the evolution of Bna ANN gene in Brassica napus was relatively conserved,but there were some functional differentiation,different expression patterns of different members were different,tissue specificity and response to different biological and abiotic stresses.As a Bna ANN gene responding to multiple stress responses,BnaANN1C-2gene may play an important role in regulating stress tolerance in Brassica napus and is a candidate gene worthy of further study.This study provides a reference for the physiological mechanism of multiple stress responses in Brassica napus.