【作者】 刘洋；

【导师】 路明；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2009， 博士

【摘要】 茎秆含糖量是甜高粱品质评价的重要指标,对燃料乙醇的生产有着重要的影响,研究甜高粱茎秆糖分积累的机理对甜高粱品质改良具有重要的意义。本研究首先选用3个早熟、4个中熟和3个晚熟材料,测定其成熟期茎秆6个节间的果糖、葡萄糖、蔗糖和总糖含量以及中性转化酶(Neutralinvertase,NI)、可溶性酸性转化酶(Soluble acid invertase,SAI)、蔗糖磷酸合成酶(Sucrose phosphatesynthase,SPS)和蔗糖合成酶(Sucrose synthase,SS)活性,并对茎秆不同节间的糖分分布情况和糖分与酶活的相关性进行了研究。随后,利用电子克隆结合PCR扩增的方法,克隆了在糖分代谢的中的关键酶SPS、SS和SAI基因,并进行了结构和功能分析。最后,利用SAI等位基因之间的序列差异开发了一个功能标记SBX1,并利用171份甜高粱材料对标记的有效性进行了验证。主要结果如下:1.早、中、晚熟材料间果糖、葡萄糖、蔗糖和总糖含量差异明显。果糖变化范围为1.77-12.43mg/g,葡萄糖变化范围为1.44-14.63 mg/g,蔗糖变化范围为35.92-95.92 mg/g,总糖变化范围为42.65-100.54 mg/g。蔗糖是成熟期茎秆中的主要糖分,大约占总糖的84%,果糖和葡萄糖大约占16%。早熟和中、晚熟材料的糖分分布方式不同,随着节间的变化(1-11),早熟材料己糖(果糖+葡萄糖)含量呈现下降的趋势,蔗糖含量呈现上升的趋势,而中、晚熟品种己糖含量呈现“∪”型分布,蔗糖含量则呈现“∩”型分布。葡萄糖和果糖含量存在显著的正相关性(r=0.98,P＜0.01),己糖和蔗糖含量之间也存在显著负相关性(r=-0.54,P＜0.01)。2.在成熟期茎秆中,不同节间的NI、SAI、SPS和SS酶活性普遍较低,都在35mg/g以下。随着节位的变化(1-11),NI、SAI、SPS和SS酶活性均呈现下降的趋势,但是SPS和SS在某些节间存在较大的波动。3.以不同节间计算,己糖含量与四种酶活性都无任何相关性;蔗糖含量与SAI和SPS-SAI酶活性具有显著的负相关性(r=-0.54,P＜0.01,r=-0.61,P＜0.01);总糖含量与SAI和SPS-SAI酶活性也具有显著的负相关性(r=-0.52,P＜0.01);r=-0.57,P＜0.01)。以整株茎秆计算,己糖含量与SAI酶活性呈现显著的正相关性(r=0.73,P＜0.01);蔗糖含量与SAI和SPS-SAI酶活性具有显著的负相关性(r=-0.77,P＜0.01;r=-0.82,P＜0.01);总糖含量只与SAI存在负相关趋势,与SPS有正相关的趋势,没有达到显著水平,而与SPS-SAI酶活性具有显著的正相关性(r=0.75,P＜0.05)。4.获得了甜高粱SPS基因DNA序列(Sps3-1,NCBI登录号为FJ750250)。此基因属于SPS基因D家族。Sps3-1长6325 bp,包含13个外显子和12个内含子。内含子序列均以GT开始,以AG结尾,其外显子-内含子结构与甘蔗SPSⅢ-2非常相似。SPS3-1 cDNA序列长为2910 bp,包含一个2895bp的开放读码框(ORF),编码的蛋白序列含有964个氨基酸,分子量大小为108.0kDa,等电点pI为6.15。此蛋白包含GT1蔗糖合成活性位点(158-650),能催化6-磷酸果糖和UDPG形成6-磷酸蔗糖,推测此蛋白具有合成蔗糖功能。SPS3-1编码的蛋白与甘蔗、水稻、小麦等作物具有较高的同源性(90-98%)。将Sps3-1序列与GRAMENE网站上的高粱基因组DNA进行Blastn检索,发现Sps3-1基因位于高粱第4染色体上,而且只发现一个拷贝数。5.获得高粱SS基因DNA序列(Susy2,NCBI登陆号为FJ513325)。Susy2长为4587 bp,包含15个外显子和14个内含子。其14个内含子的剪接方式都为GT-AG模式。其外显子-内含子结构与甘蔗Susy2结构非常相似。Susy2 cDNA序列长为2646 bp,包含一个2409bp的ORF。Susy2编码的蛋白含有802个氨基酸,分子量大小为91.7 kDa,等电点pI为6.15。结构域分析表明,Susy2编码蛋白含有有一个475个氨基酸的糖基化酶家族GT1保守结构域(275～759),催化6-磷酸果糖和UDPG形成6-磷酸蔗糖。Susy2编码的蛋白与甘蔗、玉米、竹子、水稻、小麦、黑麦草等植物的SuSy同源性非常高(92-99%)。将Susy2序列与GRAMENE网站上的高粱基因组DNA进行Blastn检索,发现Susy2基因位于高粱第10染色体上,而且只发现一个拷贝数。6.获得高粱SAI基因DNA序列(Sai-1,NCBI登录号为FJ768685)。Sai-1长为3409 bp,包括3个外显子和2个内含子。Sai-1序列与甘蔗ShinvA序列相似性很高,同源性为96%。Sai-1与水稻INV3基因结构相似。Sai-1 cDNA序列长为1683 bp,含有一个558个氨基酸的多肽,分子量大小为61.9kDa。此多肽含有一个完整的糖基水解酶(Glycosyl hydrolases family 32)结构域,推测其具有水解蔗糖的功能。Sai-1编码的蛋白与甘蔗、水稻、玉米等作物的SAI具有很高的同源性(84-96%)。将Sai-1序列与GRAMENE网站上的高粱基因组DNA进行Blastn检索,发现Sai-1基因位于高粱第4染色体上,而且只发现一个拷贝数。7.克隆了甜高粱SAI基因的4个等位变异类型Sai-1a、Sai-1b、Sai-1c和Sai-1d DNA序列。4种等位变异包含35个SNP位点和13个In/Del位点,其中A/T类型有10个,A/G类型有10个,C/T类型有9个,C/G类型有6个。根据Sai-1a、Sai-1b、Sai-1c和Sai-1d第二内含子的一段插入缺失开发了一个功能标记SBX1,分别能扩增出132bp、136bp、141bp和122bp的条带。通过对171份甜高粱材料的检测,证实了132bp的条带与低含糖量有关,而136bp、141bp条带与高含糖量有关。更多还原

【Abstract】 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.更多还原