Studies On Salt Tolerance Mechanism And Cloning And Functional Verification Of Salt Stress Responsive Gene In Sunflower

With the increase of population and the area of the saline land,under the decrease of the area of cultivated land and the lack of fresh water resources stress,the development and improvement of wasteland had become a global problem to be solved.Sunflower was known as the pioneer crop on saline soil,so the research on salt-tolerant mechanism of sunflower and the excavation of salt-tolerance genes had important significance for breeding salt-tolerance varieties and improving ecological environment.In this study,salt tolerance of oil sunflower hybrids at seedling stage were identified by two kinds of cultivation methods,soil culture and water culture.The differences of physiological and biochemical indexes,such as soluble protein content,Pro,MDA,SOD and POD,were compared between the salt tolerant hybrid and the salt sensitive hybrid.Ion fluxes of Na~+、K~+、H~+on the roots of the salt-tolerant hybrid and the salt-sensitive hybrid were investigated under NaCl stress by NMT(Non-invasive Micro-test Technology).The data of transcriptome and DGE(Digital Gene Expression Profiling)under NaCl stress in sunflowers were analyzed by bioinformatics.The DEGs(Differential Expression Genes)was screened.The accuracy of DGE was verified by qPCR.The full length cDNA sequence of V-type proton ATPase Subunit a3 gene V-ATPase a3 was cloned by RACE,and ORF of cDNA and gDNA of E3 ubiquitin ligase gene HERC2 were cloned by RT-PCR.Bioinformatics and the genomic structures analysis had been done for V-ATPase a3 and HERC2,and expression analysis were accessed by RT-qPCR.Subcellular localization of HERC2 was carried out by transforming onion epidermal cells with particle bombardment.HERC2 was transferred into tobacco to verify salinity functionality by Agrobacterium-mediated method.The main results are as follows:1.The salt tolerance capacities of five oil sunflower hybrids were P50>P65>P6>S13>P29.The degree of salt tolerance showed:P50 and P65 were very strong salt-tolerant type.S13 and P6 were moderate salt-tolerant type.P29 was salt-sensitive type.The soluble protein content,Pro,SOD and POD of P50 were higher than P29,but MDA was lower than it.The soluble protein content,Pro,MDA,SOD and POD could be regarded as the indexes of salt-tolerance identification on sunflower.1 to 3 day after salt stress was the critical period for sunflower response to salt stress.2.The results of ion fluxes on the roots of sunflower showed that the Na~+and H~+influx,K~+efflux in salt-tolerant hybrid P50 and salt sensitive hybrid P29 both before and after salt stress,and had no difference between P29 and P50 in normal.But under short-term salt stress,the salt-tolerant hybrid P50 had a stronger ability to regulating ion fluxes of Na~+,K~+,H~+than the salt sensitive variety.3.By analyzing the date of the transcriptome and DGE(Digital gene expression profiling)in sunflower roots and leaves in different salt stress time,68 co-expression DEGs(Differentially expressed genes)which were the key DEGs were found.CL5734.Contig2_All played a binding role in the ubiquitin mediated proteolysis,and Unigene32233_All exercised the function of hydrogen ion transmembrane transporter activity through oxidative phosphorylation pathway.Randomly selected 10 key DEGs to verify the accuracy of DGE by qPCR.The compliance reached 83.33%between qPCR and DGE in the roots of sunflower under salt stress;the compliance reached 81.67%between qPCR and DGE in the leaves of sunflower under salt stress.4.The full length cDNA sequence of V-type proton ATPase Subunit a3 gene named V-ATPase a3 was cloned from sunflower by RACE.GenBank accession number was KU315054.The sequencing result showed that full length cDNA was 2873 bp,which contained the 2469bp CDS,encoding 822 amino acids,109 bp 5′-untranslated region and295 bp 3′-untranslated region.The expression of V-ATPase a3 gene was up-regulated between salt-tolerant hybrids and salt-sensitive hybrids in sunflower under NaCl stress,but the expression of the former is higher than the latter.The expression of this gene was up-regulated under different stress conditions in different abiotic stresses,such as NaCl,ABA and PEG.But they had different expression patterns in different stress conditions,and different organs existed specific expression in same stress conditions.5.The cDNA and gDNA ORF(open reading frame)sequence of the E3 ubiquitin ligase gene named HERC2 were cloned from sunflower.Gene accession number of cDNA was KT832066,and gene accession number of gDNA was KT832067.The CDS of HERC2 was 1608bp,encoding 535 amino acids.The predicted molecular weight was 131KD,isoelectric point was 5.03.The ORF of gDNA of HERC2 was 3 409 bp,which contained 5 exon and 4 introns.HERC2 protein was located in the cell membrane,cytoplasm and nucleus.6.The expression of HERC2 gene was up-regulated between salt-tolerant hybrids and salt-sensitive hybrids in sunflower under NaCl stress,but the expression of the former was higher than the latter.The expression of this gene was up-regulated under different stress conditions in different abiotic stresses,such as NaCl,ABA and PEG.But they had different expression patterns in different stress conditions,and different organs existed specific expression in the same stress condition.HERC2 was transferred into tobacco by Agrobacterium-mediated method.The DNA of HERC2 was expression,and RNA was transcriptional expression.The salt tolerant of transgenic tobacco was stronger than that of the wild type tobacco.