Transcriptome Sequencing Analysis Of Apple Rootstocks Under Waterlogging Stress And Functional Identification Of MdSHN1

Apple(Malus × domestica Borkh)is one of the most cultivated fruit crops in China,while apple tree frequently encounters waterlogging stress mainly due to excess rainfall,soil compaction,or poor soil drainage,resulting in yellowing leaf,declined fruit quality and yield in some region,which has become an urgent problem to be solved in some apple production areas.However,the molecular mechanism of waterlogging tolerance remains unclear.Therefore,it is of great theoretical and practical significance to study the damage mechanism and adaptation mechanism of apple rootstock under waterlogging stress and identify the key genes of waterlogging tolerance.In this study,,the changes of ethylene release,ROS accumulation,stomatal characteristics and leaf microstructure of two apple rootstock were comparatively studied in M.hupenensis Rehd and M.toringoides Hughes under waterlogging stress.Illumina Hi Seq2500 transcriptome sequencing technology was used to compare the transcriptome differences between the two apple rootstocks under waterlogging stress.An ERF transcription factor MdSHN1,which may be related to waterlogging tolerance,was screened out.Apple gene MdSHN1 was isolated by homology cloning and PCR,and the overexpression vector of MdSHN1 was constructed using c DNA of M.hupenensis Rehd as template.Transgenic Arabidopsis thaliana plants were obtained by agrobacterium-mediated method,and their functions were verified by waterlogging treatment.The main results obtained are as follows:1.Physiological responses of M.hupenensis Rehd and M.toringoides Hughes to waterlogging stress were different.The waterlogging tolerance of M.hupenensis Rehd was stronger than that of M.toringoides Hughes.On the second day of waterlogging,the leaves of M.toringoides Hughes were yellowing and wilting,while the symptoms of M.hupenensis Rehd appeared on the seventh day of waterlogging.Diaminobenzidine(DAB)and azblue tetrazole(NBT)staining showed that the leaves of M.toringoides Hughes were stained more deeply,indicating that waterlogging stress caused accumulation of reactive oxygen species(ROS)in leaves,resulting in cell and plant damage,and the damage of M.toringoides Hughes was more severe.Ethylene release quantity was different in M.hupenensis Rehd and M.toringoides Hughes under waterlogging stress.The ethylene content increased to about 5 times in M.hupenensis Rehd after 3days’ waterlogging stress,and kept stable with the prolongation of waterlogging time.However,a significant rise in ethylene content in M.toringoides Hughes,and reached its maximum value at 9 h and increased to about20 times.There were also differences in stomatal characteristics of two apple rootstocks under waterlogging stress.After waterlogging treatment,stomatal length and stomatal width of both rootstocks showed a decreasing trend,and stomatal closure degree of M.toringoides Hughes was significantly greater than that of M.hupenensis Rehd.In addition,the leaf thickness,mesophyll thickness,palisade tissue thickness and sponge tissue thickness of the two apple stocks decreased under waterlogging stress.2.There were differences in transcriptome level between M.hupenensis Rehd and M.toringoides Hughes under waterlogging stress.Transcriptome analysis was carried out on leaves of two apple rootstocks after waterloging stress.Compared with the control,the number of up-regulated genes was 5110 in M.hupenensis Rehd,and the number of down-regulated genes was 3732.The number of up-regulated genes and down-regulated genes was 3204 and 4321,respectively.The number of differentially expressed genes in M.hupenensis Rehd was more than that in M.toringoides Hughes.GO enrichment analysis of two apple rootstocks showed that the differentially expressed genes enriched by M.hupenensis Rehd were significantly higher than those of M.toringoides Rehd under waterlooging stress.KEGG enrichment analysis showed that the differentially expressed genes of both apple rootstocks were enriched in the plant hormone transduction pathway,and the genes enriched in M.hupenensis Rehd were more abundant than those enriched in M.toringoides Hughes.The genes of plant hormone transduction pathway and those of Md ERFs family were analyzed by heat map,and the differentially expressed genes were screened out.The differentially expressed genes were verified by qRT-PCR,and a candidate gene MdSHN1 was seclected for functional verification.3.Sequence analysis of MdSHN1 gene showed that the CDS length of MdSHN1 gene was 717 bp,encoding 238 amino acids.Phylogenetic tree analysis showed that the gene was clustered with Arabidopsis waterlogging tolerance gene RAP2.6(At5g13330).The gene may have a similar function.PCAMBIA2300-GFP-MdSHN1 overexpression vector was constructed and transient expression was carried out in tobacco,and it was found that it was located in the nucleus.4.The overexpressed MdSHN1 vector was transferred into Arabidopsis thaliana by agrobacterium-mediated method,and the transgenic lines were used to waterlogging treatment.The results showed that the transgenic and wild-type Arabidopsis thaliana leaves became red,and leaves in wild-type was much redder than the transgenic lines.Transgenic lines could flower normally,while wild-type Arabidopsis thaliana was severely inhibited in growth and development,resulting in inability to flower.After 10 days of recover,leaves in transgenic lines turned green and grew normally,while some wild-type plants died.The content of chlorophyll and anthocyanin in transgenic lines under waterlogging stress was higher than that of wild-type Arabidopsis thaliana,while the content of relative electrical conductivity was lower than that of wild-type Arabidopsis thaliana,which indicated that the overexpression of MdSHN1 improved its waterlogging tolerance.