| Sugarcane is one of the most important sugar and energy crops in the world,which is characterized having high photosynthetic efficiency,good ratooning ability and high yield potential.Improving the cane yield and sugar production is beneficial to increase the income of farmers,enhance the economic returns of sugar mills,and promote the sustainable development of sugar industry.Currently,it is the challenges to select the new sugarcane varieties with high yield,high sugar and stress resistance in sugarcane breeding.Therefore,the present study focuses on sucrose synthesis and transport in leaf and the key enzyme activities related sucrose metabolism in stalk at different growth stages of the sugarcane,and the correlation between the enzyme activities and sucrose accumulation in sugarcane in order to provide theoretical basis for sugarcane variety breeding.The full-length cDNA sequences of INV,InvInh and SUT family genes were amplified from sugarcane cultivar GT28 using rapid amplification of cDNA ends(RACE)and reverse transcription-polymerase chain reaction(RT-PCT).The functions of SoInvInh2 and SoSUT5 genes were identified with transgenic technology.The contents of sucrose,glucose and fructose in sugarcane leaves and stalks were determinate with high performance liquid chromatography(HPLC).The relationships between sugar contents and enzyme activities involved in sucrose metabolism were analyzed using sugarcane varieties ROC22 and GT28 grown in field at technical maturing stage,and sugarcane varieties GT35 with high sucrose and B8 with low sucrose grown in pots at seedling,elongation and maturing stages..The main results are as follows.1.Three invertase genes were cloned:soluble acid invertase gene(SoSAI1),cell wall invertase gene(SoCIN1)and neutral/alkaline invertase gene(SoNIN1)from sugarcane,and they encode 685 amino acids,576 amino acids and 603 amino acids,respectively.SoSAI1 and SoCIN1 are glycosylated proteins and belong to GH32 gene family,while SoNIN1 is a non-glycosylated protein which is classified in GH100 gene family.The DNA sequence of SoNIN1 contains six exons and five introns,which has 268 bp intron at 5’UTR(Untranslated regions).SoCINl gene is consisted of seven exons and six introns.Using genome walking approach,the promoter sequences of the three new invertase genes were isolated and they have several key cis-regulatory elements which involve in various biotic and abiotic stresses.The results of quantitative real-time fluorescent quantitative PCR(qRT-PCR)analysis showed that the three invertase genes were constitutively expressed,and induced under the stresses of 15%PEG,100 mmol·L-1 NaCl and 6 ℃ in roots and leaves with different expressed patterns at seedling stage.2.Two Invertase inhibitors were cloned from sugarcane.The SoInvInh1 is 678 bp in length,encoding 176 amino acids,and SoInvInh2 is 927 bp in length,encoding 216 aa.in the DNA sequences of the two invertase inhibitors.They are high in GC genes as the GC contents of SoInvInh1 and SoInvInh2 are 66.8%and 70.05%,respectively.Plant InvInhs proteins can be divided into four types(InvInh Ⅰ to Ⅳ)based on phylogenetic analysis,while SoInvInhl and SoInvInh2 in this study are classified to type III and type I,respectively.The amino acid sequence of InvInh protein showed very low in different species,however,there are four conservative Cys sites.qRT-PCR showed that the two SoInvlnhs genes expressed in the stalks,leaves,flowers and rachis.Compared with wild-type Arabidopsis,SoInvInh2 transgenic Arabidopsis showed shorter root and smaller leaf blade at seedling stage,and shorter plant height,smaller blade,and less inflorescence branch at flowering stage.The analysis of enzyme activity related sucrose metabolism and sugar content in SoInvInh1 transgenic Arabidopsis showed that SoInvInh2 gene may inhibit CIN activity,which consequently affects plant phenotype.3.The full-length cDNA of three new SUT genes i.e.SoSUT2,SoSUT3 and SoSUT5 were cloned from sugarcane.There were two DNA sequences encoding the SoSUT3 gene with six exons and five introns.The molecular mass of predicted sugarcane sucrose transporter proteins was between 53.44 to 61.80 kD,and the pIs were between 5.94 to 10.68.There are 12 typical trans-membrane domains and a conserved histidine residue in all the sugarcane sucrose transporter proteins.Only the protein encoded by SoSUT5 shows sucrose transporting activity.There are 5 sub-families in plant SUTs,and SoSUT2,SoSUT4 and SoSUT5 belong to the clades of SUT2,SUT4 and SUT5,respectively,while other SoSUT proteins belong to the clade of SUT3.The gene expression detected with qRT-PCR analyses at physiological maturity stage showed that SoSUT1,SoSUT4 and SoSUT5 were highly abundant expressed in inflorescence,and SoSUT2 and SoSUT3 highly expressed in buds and inflorescence.The results indicated that SoSUTs may play an important role in sugarcane inflorescence development and seed formation,and the different expressed patterns of 5 SoSUT genes may involve in sucrose accumulation process in cane stalks at technical maturing stage.4.The relationships between sucrose accumulation and enzyme activities related to sucrose metabolism in internodes were analyzed using early maturing cultivar GT28 and early-intermediate cultivar ROC22 at maturing stage in field planting sugarcane.The results showed that the activites of SPS and CIN were significantly positively and correlated with sucrose content,on the contrary,the activites of SS-s and SAI were significantly and negatively correlated with sucrose content.It suggested that the activities of SPS and SS-s might promote the sucrose accumulation in the immature internodes.With the internode maturing,sucrose content might be an important regulatory factor to convert the SS activity from synthesis to cleavage direction.SoInvInh2 gene espression was significantly negative correlation with CIN activities,which can inhibit the CIN activities.The results for sugarcane grown in pot showed that at seedling stage the activities of SPS and SS-s in leaves were significantly and positively correlated with sucrose content,and,the activities of SS-c,SAI and CIN in sugarcane stalks were significantly and negatively correlated with sucrose content while the SPS activity were significantly and positively correlated with sucrose content.Sucrose content in stalks was significantly and positively correlated with the activities of SPS and CIN,and significantly and negatively correlated with activities of SAI,SS-c and NI at elongation stage and maturing stage of sugarcane.The SS-s activity was significantly and negatively correlated with the sucrose content in stalks at maturing stage.The NI activity was significantly and positively correlated with hexose content in leaves at elongation stage,but it was significantly and negotively correlated with hexose content in leaves at atechnical maturing stage of sugarcane. |
PDF Full Text Request