Cloning And Functional Analysis Of Genes Related To Salt Stress In Mulberry

Soil salinization is one of the main problems in agricultural production.The effects of salt stress on plants include physiological drought,membrane system destruction,photosynthesis inhibition and metabolic disorder,which seriously affect plant growth.Planting plants with salt tolerance,economic value and ornamental value in salinized soil can not only effectively use land resources,but also improve the ecological environment.Mulberry is the only natural food of bombyx mori.It has the characteristics of salt tolerance,drought tolerance,strong root force and rapid growth.It is an important ecological tree species for environmental restoration and improvement,and has very strong economic and social benefits.In this thesis,transcriptome sequencing,physiological indexes,photosynthesis and stomata detection data of Mulberry under salt stress were analyzed,and the key genes of salt tolerance were cloned and identified.The results provide important molecular basis for mining key response genes of salt tolerance of Mulberry and screening individual Mulberry plants for salt tolerance,which is conducive to the screening and application of salt tolerance varieties of Mulberry.The main research results are as follows:1.Analysis of physiological,biochemical and photosynthetic characteristics in Mulberry under salt stress:The physiological and biochemical indexes of mulberry seedlings under salt stress were determined,and the content of Malondialdehyde（MDA）and Soluble protein increased significantly after salt treatment.The activity of PRO,Superoxide dismutase（SOD）and Peroxidase（POD）increased.The results showed that mulberry seedlings controlled the oxidative damage of MDA and H₂O₂by regulating the activity of antioxidant enzymes under salt stress,thus providing a feedback mechanism to the salt-induced environment.After high Na Cl treatment,the net photosynthetic rate（A）,stomatal conductance（GS）,transpiration rate（E）and water use efficiency（WUE）of mulberry seedlings decreased,while the intercellular CO₂concentration（Ci）fluctuated.The results indicated that salt stress had a great influence on photosynthetic characteristics of mulberry leaves.Under salt stress,the high concentration of salt ions in soil and the accumulation of Na⁺in plants would cause ionic stress and osmotic stress on plants,which would destroy the water absorption and chlorophyll synthesis of mulberry leaves and inhibit photosynthesis.Within 24h after Na Cl solution treatment,the stomatal opening degree of mulberry leaves gradually decreased with the increase of stress time until it closed.The decrease of stomatal opening degree of leaves would prevent the further development of leaf water transpiration,resulting in the inability of a large amount of CO₂required for photosynthesis to enter the leaves through stomata,resulting in the decrease of photosynthetic rate of mulberry leaves.2.Transcriptome analysis of Mulberry under salt stress:Transcriptome sequencing was performed on mulberry leaves and diffused tissue samples.The average yield of mulberry leaf transcriptome was 6.24 Gb,and 20,867 genes were detectable,19,826 were known genes,1,041 were predicted new genes,and a total of11,857 new transcripts were detected.Through data filtering,reference genome alignment and differential gene analysis,the number of differentially expressed genes in c K-SY VS SY-1,CK-SY VS SY-3,CK-SY VS SY-5 and CK-SY VS SY-7 groups was 236,1421,1678and 5334,respectively.The average yield of each sample in the dredging tissue transcriptome was 6.42 Gb,and 20,497 genes could be detected,among which 19,561 were known genes,936 were predicted new genes,and 9,447 new transcripts were detected.The number of differential genes in the four groups was 241,444,1757 and 3853,respectively.Comparative analysis of the two transcriptome data showed that the differentially expressed genes in GO annotation were mainly involved in cell,metabolic process,organelle assembly,membrane structure,catalytic activity and other processes.KEGG Pathway enrichment found that flavonoid biosynthesis,starch and sucrose metabolism,mitogen-activated protein kinase（MAPK）signaling Pathway,and plant hormone signal transduction were related to salt stress in Mulberry trees,among which the number of differentially expressed genes in MAPK signaling Pathway and plant hormone signaling Pathway was the largest.3.Cloning,expression and functional identification of Mm WRKY33 gene in Mulberry:Mm WRKY33 gene（Gen Bank:ON437586）was cloned from Mulberry.The cds region was 1656 bp,encoding 551 amino acids.The predicted molecular size of the protein was61.1KD,and the isoelectric point size was 4.71,which belonged to the WRKY transcription factor family.Bioinformatics software was used to predict its tertiary structure and analyze its homology and genetic relationship with other plant species.The result of q PCR indicated that the expression levels of Mm WRKY33 and its downstream gene CYP94B1 were increased under salt stress.VIGS successfully silenced the target fragment of Mm WRKY33gene.The expression levels of Mm WRKY33 and CYP94B1 in mulberry leaf samples in the experimental group were significantly lower than those in the blank and no-load control groups.It is speculated that Mm WRKY33 and its downstream gene CYP94B1 in MAPK signaling pathway are positive regulatory factors of mulberry under salt stress.4.Cloning,expression and functional identification of Mm EIL1 gene in Mulberry:Mm EIL1 gene was cloned from Mulberry（Gen Bank:ON437587）,with a cds region of1842 bp and encoding 613 amino acids.The predicted molecular size of the protein is 69.72k D and the isoelectric point size is 5.96.Mm EIL1 gene belongs to the EIN3 transcription factor family and is a transcription factor in ethylene signal transduction pathway,which positively regulates ERF1 gene and improves salt tolerance in plants.Bioinformatics software was used to predict its tertiary structure and analyze its homology and genetic relationship with other plant species.The result of q PCR indicated that Mm EIL1 and its downstream gene ERF1B expression increased under salt treatment.After Mm EIL1 gene was silenced by VIGS,the expression levels of Mm EIL1 and ERF1B in mulberry leaves of the experimental group were significantly lower than those of the control group,suggesting that both Mm EIL1 and its downstream gene ERF1B in ethylene signaling pathway were involved in salt stress response of Mulberry.