Cloning Of Genes Of The Key Enzymes Involved In Sugar Accmulation And Development Of Functional Markers In Sweet Sorghum

Sugar content is an important trait for sweet sorghum improvement,exerting significant influences on fuel ethanol production,so it is important to investigate the mechanisms related to sugar accumluaiton for sweet sorghum.In this study,fructose,glucose,sucrose and total sugar contents of six diffenent intemodes in three ear/y-maturing,four middle-maturing and three late-maturing cultivars were assayed. Then,correlation between sugar content and neutral invertase(NI),soluble acid invertase(SAI),sucrose phosphate synthase(SPS) and sucrose synthase(SS) associated with sugar accumulation were investigated.Sugar contents(Brix) of 171 sweet sorghum cultivars were evaluated.SPS,SS and SAI genes are were cloned by the method of in silico cloning in combination with PCR amplification and functional markers were developed for the SAI genes according to their allelic variants in different sweet sorghum cultivars,and their associations with phenotypes were then analyzed.The main results were obtained as follows:1.Fructose,glucose,sucrose and total sugar contents were diffenent significantly in early,middle and late-maturing cultivars.Range of fructose,glucose,sucrose and total sugar contents were 1.77-12.43 mg/g,1.44-14.63 mg/g,35.92-95.92 mg/g and 42.65-100.54 mg/g respectively.Sucrose was the main sugar in the mature stem,was about 84%of total sugar,fructose and glucose were about 16%.The modes of sugar accumulation in different intemodes of early-maturing and midele,late-maturing cultivars were different.With the changes of intemodes(1-11),there was a downward trend in hexose and upward trend in sucrose content of early-maturing cultivars,and a "∪" trend in hexose content and"∩" trend in sucrose content of middle and late-maturing cultivars.There was a significant positive correlation between glucose and fructose contents(r=0.98,P<0.01),and a significant negative correlation between hexose and sucrose contents(r=-0.54,P<0.01).2.In the mature stem,the NI,SAI,SPS and SS activity were generally low in different internodes, were about 35mg/g below.With the changes of internodes(1～11),there was a downward trend in NI, SAI,SPS and SS activity,but have some greater fluctuation in SPS and SS activity.3.For the individual internodes,there was no correlation between hexose content and NI,SAI,SPS and SS activity;a significant negative correlation between sucrose content and SAI and SPS-SAI activity respectively(r=-0.54,P<0.01,r=-0.61,P<0.01);also a significant negative correlation between total sugar content and SAI and SPS-SAI activity(r=-0.52,P<0.01);r=-0.57,P<0.01).For the whole stem, there was a significant positive correlation between hexose content and SAI activity(r=0.73,P<0.01);a significant negative correlation between sucrose content and SAI activity and SPS-SAI(r=-0.77, P<0.01;r=-0.82,P<0.01);a significant positive correlation between total sugar content and SPS-SAI activity(r=0.75,P<0.05) and a trend of negative correlation and a trend of positive correlation between total sugar content and SAI and SPS,but not significant.4.A novel SPS gene(Sps3-1,GenBank Accession number,FJ750250) full length DNA sequence of sweet sorghum was cloned.This gene was belongs to SPS gene D family.Sps3-1 comprised 6325 bp, including 13 exons and 12 introns and 2 bp of 5 'and 13 bp of 3' flanking sequence.Splice sites of intron conserved GU sequences at 5' donor sites and AG at 3' acceptor sites.Structure of exon/intron of Sps3-1 was very similar to sugar cane SPSⅢ-2.Length of Sps3-1 cDNA sequence was 2910 bp,containing a 2bp of 5 'untranslated region(UTR),a 13bp of 3'UTR,and a 2895bp open reading frame(ORF), encoding a protein containing 964 amino acids.Protien encoded by Sps3-1,molecular weight of 108.0 kDa,pI of 6.15,contained a GT1 sucrose synthase active site(158-650),can catalyze a reaction from 6-phosphate fructose and UDPG-diphosphate to 6 - phosphate sucrose,presumed this protein has a function of synthesis of sucrose.This protein has a high homology with sugar cane,rice,corn and other crops of the SPS(90-98%).SPS3-1 gene was found located on sorghum chromosome No.4,and only one copy by blast between Sai-1 sequences and sorghum genomic DNA in GRAMENE website.5.A novel SS gene(Susy2,GenBank Accession number,FJ513325) full length DNA sequence of sweet sorghum was cloned.Susy2 comprised 4587 bp,including 15 exons,14 introns,and 17 bp of 5 'and 220 bp of 3' flanking sequence.Splice sites of intron conserved GU sequences at 5' donor sites and AG at 3' acceptor sites.Structure of exon/intron of Susy2 was very similar to sugar cane Susy2.Length of Susy2 cDNA sequence was 2646bp,containing a 17 bp of 5'(UTR),a 220 bp of 3'UTR,and a 2409bp open reading frame(ORF),encoding a protein containing 802 amino acids.Protien encoded by Susy2,molecular weight of 91.7 kDa,pI of 6.15,contained a GT1 sucrose synthase active site(275 759),can catalyze a reaction from 6 - phosphate fructose and UDPG-diphosphate to 6 - phosphate sucrose,presumed this protein has a function of synthesis of sucrose.This protein has a high homology with sugar cane,rice,corn and other crops of the SS(92%～99%).Susy2 gene was found located on sorghum chromosome No.10,and only one copy by blast between Sai-1 sequences and sorghum genomic DNA in GRAMENE website.6.A novel SAI gene(Sai-1,GenBank Accession number,FJ768685) DNA sequence of sweet sorghum was cloned by blasting the sorghum bicolor EST database with the sugarcane ShinvA(GenBank Accession,AY302083) cDNA sequence information as a querying probe.Sai-1 comprised 3409 bp, including two introns and a terminal codon.Sai-1 was very similar with sugar cane ShinvA(96%). Structure of Sai-1 gene was similar to rice INV3.Length of Sai-1cDNA sequence was 1683 bp,encoding a polypeptide of 558 amino acids.Protein encoded by Sai-1,molecular weight of 61.9 kDa,contained a Glycosyl hydrolases family 32 conserved domain which catalyzes the hydrolysis of sucrose.This protein has a high homology with sugar cane,rice,corn and other crops of the SAI(84-96%).Sai-1 gene was found located on sorghum chromosome No.4,and only one copy by blast between Sai-1 sequences and sorghum genomic DNA in GRAMENE website. 7.Genomic DNA sequences of Sai-1a,Sai-1b,Sai-1c and Sai-1d,respectively,were cloned from sweet sorghum.35 SNP,containing 10 of A/T type,10 of A/G type,9 of C/T type,and 6 of C/G type,13 In/Del sites wre found in these sequences.For the four allelic variants Sai-1a,Sai-1b,Sai-1c and Sai-1d locus,dominant functional markers SBX1 were developed and 132 bp,136 bp,141 bp and 122 bp product amplified by SBX1.In 171 sweet sorghum cultivars,the 132bp fragment amplified by SBX1 was proven to be associated with lower sugar content,while the 136 bp and 141 bp product amplified by SBX1 was related to higher sugar content.