野生大豆 GsMIPS2 和 GsSNAP33 基因在盐碱胁迫下的功能分析

发布时间：2016-11-09 16:38

1 Introduction

Soil salinity and alkalinity are major problems hinder agriculture production mainly in aridand semi arid regions of world. Either most of the areas of world developed or developingcountries are facing these problems. Therefore, many countries are running projects to sort outstress-related genes, screening and functional analysis of these genes at commercial scale toovercome this problem. In China, according to the statistics, 15 million hectares of land is undersalt-alkaline stress conditions therefore, to improve cultivated land conditions via creation of sa lt& alkaline tolerant transgenic crops is of great significance. We can ensure sustainable andefficient development of agriculture and food security by using these vast saline land resources,which may results in huge economic, social and ecological benefits. Therefore, how to improvestress tolerance of crops is a major question need to be solved. Plants suffer from salt and alkalistress damages under salinity stress conditions where, alkali stress results in higher pH (>8.0) dueto the presence of basic salts (Na2CO3and NaHCO3) as compared to neutral salts (NaCl andNa2SO4) and exerts serious impacts on plants. Ions that contribute to soil salinityinclude SO42-,HCO3-, Na+, Cl-, Ca2+, Mg2+and rarely K+and NO3+. In recent year’s modern molecular biology,genetic engineering, bioinformatics and other frontiers led towards mining of functionallysignificant genes and provide efficient and scientific techniques for molecular breeding andcreation of transgenic varieties.Soybean is one of the most widely grown legumes in the world due to its rich protein and oilcontents. However, high soil salinity causes negative impacts on agronomical traits and ultimatelyleads to reduction in yield of soybean. Therefore, regulation and activation of specificstress-related genes that participate in whole series of stress responses such as transcriptionalcontrol, signaling, proteins and membranes protection and scavenging of toxic compounds are inswing nowadays. Wild soybean has strong ability to survive under extreme environment ofHeilongjiang province (pH=10.6,-52.3℃) and serve as an ideal donor crop for study of salt andalkali tolerant molecular mechanisms and also serve for mining of salt andalkali responsive genes.Our laboratory has been carrying out the early wild soybean genome resources research.

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2 Materials and Methods

2.1 Materials

LB medium: 10 g/L Peptone，10g/L NaCl，5g/L Yeast extract，，15g/L Agar（solid mediumpH7.0YEB medium (Agrobacterium Growth Medium): Beef extract 5g/L, Tryptone5g/L, 5g/LSucrose 1g/L Yeast extract, MgSO4.7H2O 0.493g/L, pH 7.01/2 MS medium (Murashige & Skoog medium): 1/2 MS Inorganic Salt[80], 30g/L Sucrose8g/L Agar（solid medium), pH5.8 RNA quality detector: Agilent 2100 BioanalyzerNucleotide concentration detector: NanoDrop 2000A Real-Time RT PCR: Agilent Technologies 3000 fluorescent quantitative PCRConfocal Laser Scanning Microscope: CarlZeiss LSM710PCR: Eppendorf MastercyclerLow Temperature Centrifuge: Beckman AllegraTM64R high speed refrigerated centrifugetableVacuum concentration meter: Eppendorf Concentrator plus enrichmentapparatusGene gun: PDS-1000/ He system (Bio Rad)

2.2 Methods

As in previous project of our Lab (2011) transcriptome sequencing of G07256 wild soybeanwas conducted with collaboration of international cooperation in scientificresearch studies fundedby Canada, by using Illumina sequencing technology under salt alkali stressconditions. In thisstudy, we further choose 2 stress responsive genes GsMIPS2 and GsSNAP33for further cloning and gene functional analysis. Seeds of wild soybean G. soja07256 were surface sterilized with 98% sulfuric acid for 10~15mins to break seed dormancy followed by washing with sterilized water 5-6times tocompletely remove acid from seeds before sowing. After that, seeds were transferred on wet filterpaper in Petri dishes and placed in dark for germination. The germinatedseeds were moved ingrowth chamber with1/4 strength Hoagland’s solution after two days. G. soja 07256 seedlings weregrown in 1/4 strength Hoagland’s solution at specific growth environment of issue culture room at24 °C with 60 % relative humidity and 16 h light/8 h dark regime, whereas thelight source SON -TARGO 400 W was set up by persistent illumination of 30,000 lx.

3 Results ......23

3.1 Functional Analysis of Wild Soybean Gene GsMIPS2 and its Physiological and Molecular Mechanisms under Salt Stress Conditions .......23
3.1.1 Determination of GsMIPS2 Gene Expression Profile under Salt Stress Conditions.......23
3.1.2 Sequence Analysis of GsMIPS2 Gene ..................24
3.1.3 Identification of the GsMIPS2 Overexpression OX and atmips2 Plants ........................26
3.1.4 Overexpression of GsMIPS2 Conferred Increased Salt Tolerance at Various GrowthStages of Arabidopsis thaliana .........27
3.1.5 GsMIPS2 Overexpression Induced Expression of Stress Responsive Genes ........34
3.2 Functional Analysis of Wild Soybean Gene GsSNAP33 .............36
3.2.1 Cloning and Sequence Analysis of GsSNAP33 Gene ............36
3.2.2 Bioinformatics Analysis of GsSNAP33 Gene Family............37
3.3 Determination of GsSNAP33 Gene Expression Profile under Salt, Alkali, ABA and PEGStress Conditions .......39
3.4 Analysis of Tissue Specific Expression of GsSNAP33 Gene in Glycine soja .......................43
3.5 Generation of GsSNAP33 Overexpression lines .............44
3.5.1 Construction of Plant Expression Vector ..............44
3.5.2 Transformation of pCAMBIA2300-GsSNAP33 Construct into Arabidopsis by Floral DipMethod................45
3.5.3 Selection of GsSNAP33 Overexpressed Lines ...........46
3.6 Subcellular Localization Analysis of GsSNAP33 Protein .................47
4 Discussion.............50
4.1 Diversity of GsMIPS2 and GsSNAP33 Genes in Plant Kingdom..........50
4.2 GsMIPS2 Gene Expression was Identified to be Up-regulated under Salt Stress Conditions........51
4.3 GsMIPS2 Overexpression Confers Enhanced Salt Tolerance at Various Growth Stages ofArabidopsis thaliana .....51
4.4 GsMIPS2 Overexpression Alleviates Stress Injuries at Mature Plant Stage .........................52
4.5 GsMIPS2 Overexpression Results in Up-regulation of Various Abitic Stress-related MarkerGenes............52
4.6 GsSNAP33 Gene can be Induced under Salt, Alkali, PEG and ABA Stress Conditions ........53
4.7 GsSNAP33 is a Plasma Membrane Protein ...................54
5 Conclusion.......55

4 Discussion

4.1 Diversity of GsMIPS2 and GsSNAP33 Proteins in PlantKingdom

Plants frequently encounter adverse growth conditions and contamination of soils by high saltconcentration. Tolerance to salt stress in plants is a dynamic coordinated action of various genes atdifferent regulatory levels[112]. Myo-inositol phosphate synthase (MIPS) catalyses the primary stepin inositol biosynthesis and takes part in many physiological processes including abiotic stressesand seed germination[53]. Mutations in MIPS genes lead towards complete elimination of themyo-inositol pathway that not only hinders phytic acid formation butalsodisturb a number ofbiochemical pathways required for germination[113.114]. Furthermore, GsSNAP33 gene belongs toSNAP25-like family of SNARE protein. SNAREs play a key role during vesicle associatedmembrane fusion events at endomembrane system. But recently few reports also hig hlighted roleof this family proteins in abiotic stress responses despite of its housekeeping activates. However,biological functions of MIPS and SNAREs family genes are still unclear specifically under saltand alkali stress conditions. In this study we primarily focused on the characterization ofphysiological and biological functions of wild soybean (Glycine soja 07256) myo-inositol-1-phosphate-synthase gene (GsMIPS2) in response to salt stress. Whereas, expression level analysisunder salt, alkali, ABA and PEG treatments, tissue specific expression analysis, bioinformaticsanalysis and subcellular localization of SNARE domain protein GsSNAP33 were performed toinvestigate functional roles of this protein.Multiple Sequence analysis revealed a high degree of homology among the members ofsoybean MIPS genes family and the other legume species at both protein and nucleotide sequencelevels with four highly conserved motifs as highlighted in red squares (Fig.5). Previously studieson DNA blot analysis revealed multiple copies of the MIPS genes in soybean genome (Hegeman etal., 2001[40]) while, protein sequence analysis revealed presence of four highly conserved motifs:GWGGNNG / LWTANTERY / NGSPQNTFVPG and SYNHLGNNDG that confirmed a highdegree of conservation among the members of MIPS gene family[37]. MIPS2 gene was found tohave 99% coverage and identity on chromosome 18 and 95% identity on chromosome 9 asrevealed through MIPS sequence BLAST with complete genome[115]. Similarly, sequence analysisof GsSNAP33 gene (Fig.22) revealed presence of two conserved motifs, and phylogenetic analysisshows close homology of this gene with angiosperms but this gene also shares homology withgymnosperms and eudicots which prove its abundant occurrence in plant kingdom (Fig.23). Thegenomes of all higher plant species have been investigated so far, and SNAREs stands at largerepertoire and encodes 60 genes in Arabidopsis thaliana (The Arabidopsis Genome Initiative2000),69 in Populus trichocarpa, (Tuskanetal.2006). 57 in Oryza sativa (International Rice GenomeSequencing Project 2005) and similarly, comparable numbers of this subfamily members were encoded by the various other plant genomes which suggest that SNARE domain family is notlinked with any particular biogeographic habitat or plant life style (Lipka et al., 2007).

4.2 GsMIPS2 Gene Expression was Found Up-regulated under

Salt Stress ConditionsEarlier studies have revealed that MIPS genes are differentially expressed in different organsof several plant species such as soybean[116], rice[117], A. thaliana [118], chickpea [119], and P.vulgaris[120-122]. In this study GsMIPS2 transcripts were examined in root tissues under differenttime points after NaCl treatment and the most abundant transcript levels were noted at 3hr. Theseresults suggest that GsMIPS2 gene is differentially expressed in roots of Glycine soja 07256 undersalt stress conditions to coordinate inositol metabolism (Fig.4). These findings corroborate resultsof (Kumari et al., 2012[37]) who reported higher levels of MIPS2 transcript abundance in the rootand seedlings than in developing seeds. Similarly, (Majumder et al., 1997[47]) observed that theexpression of MIPS gene family members is highly organ specific and suchdeviated regulation isdirected to correlative inositol metabolism and cellular growth. Our consecutive experimental dataalso suggested that GsMIPS2 gene was functional in Arabidopsis thalianaplants.
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5 Conclusion

1. In conclusion, the over expression of GsMIPS2, a novel salt tolerant gene identified in G.soja 07256, resulted in improved salt tolerance in transgenic Arabidopsis, likely via theactivation of inositol metabolism at developmental stages, enhanced uptake of Na, protection ofphotosystem , stabilizing stress injuries and increased expression levels of stress-inducible markergenes. Ectopic expression of GsMIPS2 from wild soybean Glycine sojaimproves salt toleranceand can be a candidate gene to contribute in stress adaptation particularly saltstress tolerance andits role in growth and development.2. GsSNAP33 gene is a plasma membrane protein and can play an important role in stresssignaling pathway. Relative expression levels of GsSNAP33 gene were found higher under salt,alkali, ABA and PEG stress conditions in roots or leaves of Glycine soja which clearly indicatethat GsSNAP33 gene is a stress responsive gene. Tissue specific expressionanalysis of GsSNAP33gene showed presence of higher transcripts in pods, roots and seeds than in leaves andinflorescence. However, there is need to further elucidate its molecular and biological functions atcellular levels under salt and alkali stress conditions.

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参考文献（略）

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