Studies On Cloning Of Genes Encoding Some Key Enzymes Related To Saccharide Metabolism And Their Expression Under Low Temperature Stress In Musa Spp. Leaves

Banana(Musa spp.) is a typical tropical and subtropical fruit crop. Low temperatureinfluences its growth and development, and limits its planting range. Low temperature stress hasalways been one of major environmental stress factors for banana production and it is increasinglybecoming an obstacle to the development of the banana industry. In plants, saccharide metabolismis involved in low temperature stress response. Sugars not only fuel cellular carbon and energymetabolism during the growth and development of plants but also play pivotal roles as signalingmolecules. Different sugar signals are generated by photosynthesis and carbon metabolism insource and sink tissues to modulate growth, development, and stress responses. These effectscould be due to sugar signaling or metabolism or both. In addition, sorbitol as a compatible solute,is closely associated with stress resistance of plants. In this experiment, the genes encoding somekey enzymes related to saccharide metabolism in leaves of Musa spp. cv. Tianbao were cloned.Then the expression of those genes cloned were analyzed by relative quantification using real timePCR, combined with the changes of soluble sugar content under low temperature stress in bananaleaves, in order to reveal the molecular mechanism of genes encoding the key enzymes related tosaccharide metabolism and its relationship with the changes of soluble sugars in response to lowtemperature stress, and to provide reference for studying the fruit saccharide metabolism andrelated gene expression under low temperature stress in banana. Besides, Musa spp. cv. Tianbaowas transformed with S6PDH gene via Agrobacterium-mediated thin cross-sections (TCSs)transformation system and the Rosaceae-specific sorbitol metabolic pathway was introduced intobanana, which would lay important foundation for approaching the effects of the sorbitol on thechanges of soluble sugars in response to low temperature stress. The main results were as follows:1Gene cloning of some key enzymes related to saccharide metabolism in bananaleaves11complete cDNA and several partial cDNA of genes encoding some key enzymes related tosaccharide metabolism in banana were cloned by the methods of homologous cloning, combinedby RT-PCR and RACE:①The complete cDNA of GBSSⅠ(Ma-GBSSⅠ, accession numberHQ646360in GenBank)was2277bp, encoding616amino acids, and consisted of a5' UTR of 121bp, an ORF of1851bp(122-1972), a3' UTR of291bp, and a poly (A) tail of14bp. Besides,6fragments of GBSSⅠ cDNA via3' RACE were obtained, which fell into3different members ofGBSSⅠ gene family.②The cDNA of SSⅢ (Ma-SSⅢ, accession number HQ646361in GenBank)was3011bp, encoding1002amino acids, and consisted of an ORF of3009bp (3-3011), a3' UTRof240bp and a poly (A) tail of17bp. Besides,3fragments of SSⅢ cDNA via3' RACE wereobtained, which fell into2different members of SSⅢ gene family.③Thecomplete cDNA ofAMY (Ma-AMY, accession number JF682522in GenBank) was1565bp, encoding416aminoacids, and consisted of a5' UTR of61bp, an ORF of1275bp (62-1336), a3' UTR of214bp and apoly (A) tail of15bp. Besides,1fragment3' RACE cDNA which contained both intron missingand exon missing simultaneously was obtained.④The complete cDNA of BMY (Ma-BMY,accession number JF501023in GenBank) was1953bp, encoding532amino acids, and consistedof a5' UTR of137bp, an ORF of1599bp (138-1736), a3' UTR of197bp and a poly (A) tailof.20bp.⑤The complete cDNA ofSPS (Ma-SPS, accession number HQ453900in GenBank)was3691bp, encoding1082amino acids, and consisted of a5' UTR of253bp, a ORF of3249bp(254-3502), a3' UTR of173bp and a poly (A) tail of16bp.⑥The complete cDNA of SuSy(Ma-SuSy, accession number JF682523in GenBank) was2787bp, encoding816amino acids,and consisted of a5' UTR of85bp, an ORF of2541bp (86-2536), a3' UTR of235bp and a poly(A) tail of16bp.⑦The complete cDNA of different members of Inv-V gene family. Inv-V(Ma-Inv-V, accession number JN417603in GenBank) was2303bp, encoding645amino acids,and consisted of a5' UTR of66bp,an ORF of1938bp (67-2004), a3' UTR of282bp and a poly(A) tail of17bp. Inv-CW1(Ma-Inv-CW1, accession number JN417601in GenBank) was2116bp,encoding586amino acid, and consisted of a5' UTR of104bp, an ORF of1761bp (105-1865), a3' UTR of228bp and a poly (A) tail of23bp. Inv-CW2(Ma-Inv-CW2, accession numberJN417602in GenBank) was1941bp. encoding583amino acids, and consisted of a5' UTR of62bp, an ORF of1752bp (63-1814), a3' UTR of109bp and a poly (A) tail of18bp. Inv-N1(Ma-Inv-N1, accession number JN794581in GenBank) was2006bp, encoding556amino acids,consist of a5' UTR of198bp, an ORF (199-1869) of1671bp, a3' UTR of180bp and a poly (A)tail of17bp. Inv-N2(Ma-Inv-N2, accession number JN794582in GenBank) was2094bp,encoding547amino acids, and consisted of a5' UTR of283bp, a ORF of1644bp (284-1927), a3' UTR of150bp and a poly (A) tail of17bp.2Bioinformatics analysis of the genes encoding some key enzymes related tosaccharide metabolism in banana leavesBased on the genes encoding some key enzymes related to saccharide metabolism in banana leaves being cloned, the deduced amino acid sequences of proteins encoding by those genes werepredicted and analyzed by bioinformatics, and the results were as follows:①GBSS Ⅰfrombanana leaves was a stable hydrophilic protein located in the chloroplast; there was a chloroplasttransit peptide presented in its N-terminal amino acid sequence; containing conserved domainsincluding ADP binding pocket and homodimer interface; its secondary structure included randomcoils mainly, without the coiled coil; containing a wealth of potential phosphorylation sites andserine sites mainly; containing protein functional sites including the N-glycosylation sites, proteinkinase C phosphorylation sites, casein kinase Ⅱ phosphorylation sites etc.; GBSS Ⅰ frombananaleaves showed higher amino acid sequence identity to GBSSⅠ from monocots, however,phylogenetic analysis showed that it had a relatively closer evolutionary distance with GBSSⅠfrom dicotyledonous species.②SSⅢ from banana leaves was an unstable hydrophilic proteinlocated in the chloroplast; there was a chloroplast transit peptide presented in its N-terminal aminoacid sequence; containing conserved domains including ADP binding pocket and the homodimerinterface; its secondary structure included random coils mainly, with coiled coils possibly;containing a wealth of potential phosphorylation sites and serine sites mainly; containing proteinfunctional sites including N-glycosylation sites, protein kinase C phosphorylation sites and caseinkinase Ⅱ phosphorylation etc.; SSⅢ from banana leaves showed high homology to SSⅢ from avariety of plants, and phylogenetic analysis showed that it had a closer evolutionary distance withmonocots and a remoter evolutionary distance with dicotyledonous species.③AMY from bananaleaves was a stable hydrophilic protein which located outside the cell; there was a signal peptidepresented in its N-terminal amino acid sequence, containing conserved domains including AmyAc_arch_bac_plant_AmyA and Alpha-amylase C-terminal beta-sheet; its secondary structureincluded random coils mainly, without coiled coil; containing a small number of phosphorylationsites (17), tyrosine sites mostly; containing protein functional sites including protein kinase Cphosphorylation, casein kinase Ⅱ phosphorylation sites and N-myristoylation sites etc.; AMYsfrom different species were of higher homology. Phylogenetic analysis placed AMYs from allmonocots in one group, AMY from the banana leaves had a closer evolutionary distance withAMY from banana fruit.④BMY from banana leaves was an unstable hydrophilic protein locatedin the chloroplast; there was a chloroplast transit peptide presented in its N-terminal amino acidsequence; it belonged to the beta-amylase glycosyl hydrolase family14superfamily; its secondarystructure included random coils mainly, and did not contain the coiled coil; containing a wealth ofpotential phosphorylation sites, serine sites mostly; containing protein functional sites includingN-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites etc.; BMYs from different species were of low homology, andphylogenetic analysis showed that BMY from banana leaves had a closer evolutionary distancewith AMY from banana fruit.⑤SPS from banana leaves was an unstable hydrophilic proteinlocated in the nucleus with the most possibility; containing GT1_Sucrose_synthase multi domain;its secondary structure included random coil mainly, with coiled coils possibly; containing awealth of potential phosphorylation sites and serine sites mainly; containing a variety of proteinfunctional sites including cAMP and cGMP-dependent protein kinase phosphorylation sites,protein kinase C phosphorylation sites, casein kinase Ⅱ phosphorylation sites, N-glycosylationsites etc.; SPSs from different species were of highly homology and phylogenetic analysis showedthat SPS from banana leaves had a closer evolutionary distance with SPS from Xerophyta humilis.⑥SuSy from banana leaves was a stable hydrophilic protein, there was a mitochondrial transitpeptide presented in its N-terminal amino acid sequence; containing GT1_Sucrose_synthase multidomain; its secondary structure composed of α-helix mainly, without the coiled coil; containing awealth of potential phosphorylation sites; containing protein functional sites including cAMP andcGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites,casein kinase Ⅱ phosphorylation sites, N-glycosylation sites etc.; SuSy from banana leaves was ofhigher homology with SuSy from a variety of monocots, and phylogenetic analysis palced SuSyfrom monocots in one group.⑦Potential phosphorylation sites of different members of Invprotein family from banana leaves were mainly serine sites, supplemented with threonine andtyrosine sites. Different type of Inv had a different subcellular localization, Inv-V, Inv-CW proteinsubfamily and Inv-N subfamily existed differences on the composition of protein secondarystructure, three-dimensional structure, and conserved domain. There also existed an obvioussequencing in the evolution of protein amino acid sequences. Different type of Inv might havedifferent functional mechanisms.3Analysis of quantitative expression of the genes encoding some key enzymesrelated to saccharide metabolism in banana leaves under low temperature stressBased on the genes encoding some key enzymes related to saccharide metabolism in bananaleaves being cloned, the expression of those genes under low temperature stress was analyzed byreal time PCR, taking18S rRNA as an internal gene. qPCR results showed that:①GBSSⅠ genewas expressed differentially under different low temperature conditions. Its expression wasupregulated slightly at20℃and significantly at13℃, and tended to be downregulated at4℃.GBSSⅠ gene expression increased within a narrow range under gradual cooling condition.②SSⅢgene was expressed differentially under different low temperature conditions, which was dramatically upregulated at20℃and13℃. SSⅢ gene expressed stablely and affected less by thedays, and its expression showed no significant variation compared with the control at4℃. SSⅢgene expression increased within a narrow range under gradual cooling condition.③AMYexpression remained unchanged under different low temperature conditions except a significantdecrease after5days at4℃.④BMY expression was upregulated significantly and rapidly by lowtemperature. It maintained a certain level but increased to a higher level after5days at4℃. Inaddition, it was upregulated under gradual cooling condition.⑤SPS gene was expresseddifferentially under different low temperature conditions. Its expression was dramaticallyupregulated by20℃and13℃, and showed a slight increase firstly and then kept downregulatingas the days increased at4℃. SPS expression increased within a narrow range under gradualcooling condition.⑥SuSy gene was expressed differentially under different low temperatureconditions. Its expression was dramatically upregulated by20℃and13℃and maintained a higherlevel early at4℃. SuSy expression showed no significant variation under gradual coolingcondition compared with the control.⑦Different types of Inv genes showed different expressionpatterns in response to low temperature. Inv-V expression was upregulated within a wide rangeunder low temperature stress condition. Inv-CW1expression was repressed by low temperature.Inv-CW2expression was upregulated rapidly at low temperature. Inv-N1expression increased asthe temperature decreaed and was positively correlated with the days at4℃. Inv-N2expressionshowed a trend of "up→down→up→down", however, it fluctuated within a narrow range.Among the above11genes, Inv-N1was the only one whose expression kept increasing with thedays at4℃, suggesting that it should be of great importance for the mechanism in response to thelow temperature stress.4Analysis of soluble sugar content changes in banana leaves under lowtemperature stressThe standard curve of the soluble sugars extraction was drawed by the glucose. The solublesugar content in banana leaves in response to low temperature stress was determined byanthracene ketone method. The results showed that, the soluble sugar content was doubled at20℃and13℃. The soluble sugar content changed with days and showed the trend of "up→down" at4℃. The soluble sugar content peaked after2days at4℃, which was4times of the control at thenormal temperature. And then it decreased gradually. It was2times of the control, which was thelowest point after5days at4℃. The soluble sugar content increased nearly twice under gradualcooling condition (T8), indicating that the accumulation of soluble sugar played an important rolein low temperature acclimation. The combination of the expression analysis of the genes encoding some key enzymes related to saccharide metabolism in with the dynamic changes of soluble sugarcontent under different low temperature conditions laid an important foundation for discussing themolecular mechanism of the soluble sugars changes in response to low temperature. Themolecular mechanism of soluble sugars changes varied under different low temperature conditions.①At20℃and13℃, the upregulated expression of GBSSⅠ and SSⅢ indicated that starch contentcould increase to provide carbon source for the accumulation of soluble sugars. The upregulatedexpression of SPS suggested that the level of sucrose be elevated. The upregulated expression ofSuSy was thought to be involved in meeting the increased glycolytic demand during stress. Theupregulated expression of BMY could cause the amylolysis and then maltose increased. And theupregulated expression of Inv-V and Inv-N2suggested that some sucrose be supposedly brokeninto hexoses and hexose levels increased.②At4℃, though the expression of GBSSⅠ, SSⅢ andSPS was repressed, indicating that the synthesis of starch and sucrose was suppressed, while thelevel of soluble sugars was higher at4℃than at20℃and13℃. It was probably due to the reasonas follows: the expression levels of Inv-CW2and Inv-N1were higher at4℃than at13℃and20℃. And the exprssion of Inv-V maintained a high level. As the result, hexose levels could behigher. Secondly, the upregulated expression of BMY could cause the amylolysis and then maltoseincreased. Finally, the upregulated expression of SuSy was thought to be involved in meeting theincreased glycolytic demand during stress.③Under gradual cooling condition, a slight increaseof GBSSⅠ and SSⅢ expression indicated that the content of starch and sucrose could increaseto provide carbon source for the accumulation of soluble sugars. The upregulated expression ofBMY could cause the amylolysis and then maltose increased. The upregulated expression of Inv-V,Inv-CW2and Inv-N1could cause the elevated level of hexose.5Transformation mediated by Agrobacterium with cDNA encoding S6PDH fromPrunus salicina var. Cordata in Musa spp. cv. TianbaoSorbitol is a polyol, which is the main photosynthetic product, the transport form ofassimilates and the soluble storage carbohydrate in the woody Rosaceae. Musa spp. cv. Tianbaowas transformed with cDNA encoding S6PDH (NADP-dependent sorbitol-6-phosphatedehydrogenase)isolated from Prunus salicina var. cordata by an Agrobacterium-mediated thincross-sections (TCSs) transformation system. The growth of the buds improved effectivelywhen the TCSs were transferred onto the medium M4adding5%-7%(V/V) coconut water. Andthe highest GUS transient expression occurred while2mm thin TCSs from the healthy and strongbuds were used as the receptor material. Total37putative transformants were selected via thetwo-step method and31putative transformants survived after transplanting. Finally, four transgenic lines were conformed by PCR analysis of S6PDH gene and GUS gene. Sorbitolsynthesis pathway which was unique to the Rosaceae plants had been introduced into banana,laying the groundwork to discuss the impact of the sorbitol on soluble sugars changes in responseto low temperature stress.In conclusion,11complete cDNA of genes encoding some key enzymes related to saccharidemetabolism were cloned by the methods of homologous cloning, combined by RT-PCR and RACEfrom leaves of Musa spp. cv. Tianbao. And the deduced amino acid sequences of the proteinsencoding by the11genes were analyzed by bioinformatics, which laid an important foundation forthe molecular mechanism of the saccharide metabolism. Besides, the expression of the11genesunder the low temperature stress was analyzed by real time PCR, combined by the dynamicchanges of soluble sugar content in banana leaves, which preliminarily uncovered the molecularmechanism of soluble sugars changes in response to low temperature stress and provided scientificevidences for improving the cold resistance of banana. In addition, sorbitol synthesis pathwaywhich was unique to the Rosaceae plants had been introduced into banana, which created a newpossible way for breeding of cold tolerant banana.