【作者】 陈雪燕；

【导师】 吉万全；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2016， 博士

【摘要】 小麦作为全球主要粮食作物之一,随着市场需求的变化,人们在追求高产的情况下,对其品质的要求也越来越高。小麦品质是由蛋白质、脂质、淀粉等多种因素共同作用决定的,其中蛋白质的含量、质量以及相互作用尤为重要。小麦储藏蛋白包括麦谷蛋白(glutenin)和麦醇蛋白(gliadin),其与面筋流变学特性相关,分别决定面团的粘弹性。麦谷蛋白分为高分子量麦谷蛋白(HMW-GS)和低分子量麦谷蛋白(LMW-GS);醇溶蛋白包括α-、β-、γ-及ω-gliadin等类型。然而大量研究证实,目前研究的传统面筋蛋白仅能解释小麦品质变异的30%-70%。在传统面筋蛋白的氨基酸中半胱氨酸只占一小部分(约2%),它们可形成分子间二硫键,使蛋白质结构变成超高分子聚合物,为面筋提供黏弹性,而avenin-like蛋白(ALPs)作为一种新发现的小麦贮藏蛋白因其含有高冗余的半胱氨酸残基,引起了研究者们的关注。avenin-like b蛋白含有18-19个半胱氨酸残基,可形成分子内或分子间二硫键,进而影响小麦面粉的加工品质。但在该蛋白的研究上仍然缺乏关于该蛋白基因的染色体定位、等位基因的数量以及等位基因的影响等遗传信息。野生型NAM-B1基因主要来源于野生二粒小麦中,其控制小麦中铁、锌以及籽粒蛋白质含量,加快植株衰老,促进叶片中的营养物质向籽粒中的运输。在栽培小麦中很少被发现,尤其在中国栽培小麦中的分布仍不清楚。本研究主要围绕在小麦品质中影响蛋白质量和含量的avenin-like b和NAM-B1基因进行研究。主要研究结果如下:1.选取中国春和野生二粒小麦Bethlehem代换系的材料L16-3BS进行蛋白质组学研究,获得47个差异蛋白点,2个是和对照品种BL相比较,在材料L16-3BS新出现的蛋白点,35个表达量上调的点,10个表达量下调的点。结合质谱鉴定技术,47个差异蛋白点被鉴定为:球蛋白(Globulin)5个,α-醇溶蛋白(Alpha-gliadin)2个,γ-醇溶蛋白(gamma gliadin)2个,MYB转录因子(MYB transcription)1个,延长因子(elongation factor)1个,磷酸丙糖异构酶(Triosephosphate isomeras)2个,丝氨酸蛋白酶抑制剂(Serpin)7个,苹果酸脱氢酶(malate dehydrogenase)1个,类燕麦蛋白(avenin-like)2个,小麦果聚糖(triticin)3个,超氧化物歧化酶(Superoxide dismutase)1个,热激蛋白(Heat shock protein)2个,18个未知蛋白(unknown protein)。确定与品质相关基因avenin-like b为候选基因进行深入研究。2、对avenin-like b基因进行克隆、测序分析,结果表明avenin-like b基因全长855-858bp,无内含子,并且与数据库中的中国春参考序列分别匹配在染色体7DS(99%),4AL(98%)和7AS(97%)上。用中国春缺体四体系进行基因的染色体定位,首次明晰了该基因属于多基因家族,有三位基因位点,并分别位于7A、4A和7D上,依次命名为TaALPb-7A,TaALPb-4A和TaALPb-7D。序列比对分析发现:TaALPb-7A基因有3个等位基因,分别命名为TaALPb-7A1、TaALPb-7A2、TaALPb-7A3。TaALPb-4A基因有4个等位基因,分别命名为TaALPb-4A1、TaALPb-4 A2、TaALPb-4A3、TaALPb-4A4,而在TaALPb-7D位点上没有发现新的变异类型。系统进化分析表明:相同染色体上avenin-like b基因克隆序列分别聚在一起,形成一小类;与已报道的avenin-like蛋白序列在图中构成一大类,彼此间相似度较高,与低分子量谷蛋白(LMW-GS)、高分子量谷蛋白(HMW-GS)亲缘关系较近,与ω-醇溶蛋白亲缘关系最远。3、dd-PCR表达分析:有功能的TaALPb-7A等位基因(TaALPb-7A1、TaALPb-7A2)可正常表达,与内参基因表达拷贝数比例在1:2.54到1:3.36之间。而无功能的TaALPb-7A等位基因(TaALPb-7A3)不能正常表达。4、构建可正常表达的TaALPb-7A1、TaALPb-7A2体外表达载体,进行体外诱导表达、并纯化TaALPb-7A1、TaALPb-7A2目标蛋白,掺粉试验表明,这2种等位基因均对小麦的加工品质有正相关作用。5、针对TaALPb-7A两种类型等位基因(正常表达和沉默)序列开发了ASP功能标记,功能验证表明:正常表达的等位基因和揉混特性中的和面时间(P<0.0443)及8分钟带宽(P<0.0096)呈显著相关。与高分子量麦谷蛋白亚基、籽粒蛋白含量和谷蛋白含量关联分析发现,可表达的TaALPb-7A等位基因及沉默的TaALPb-7A等位基因与这些参数相关性不显著。表明可表达的TaALPb-7A对品质的贡献并未受到这些因素的影响。6、对小麦近缘种属12个基因组(W、E^e、E^b、St、Q、Ns、Ta、U、C、N、V、R)的avenin-like b基因进行研究,结果表明该基因在一些基因组中存在丰富的遗传变异,并且在一些基因组中发现一些新的变异类型,为小麦的品质改良提供了丰富的基因资源和良好的研究基础。7、利用218份中国小麦栽培种,进行NAM-B1等位基因的筛选和分析研究,研究结果表明在参试的中国栽培种中没有发现野生型NAM-B1存在,只有NAM-B1的插入型(24.3%)和缺失型(75.7%)两种类型存在,深入剖析产生这种现象的原因,为今后如何更好利用野生型NAM-B1以及在小麦营养品质改良方面提供理论指导和参考作用。更多还原

【Abstract】 Wheat is one of the major food crops in the world.With the changing of market demand,people pay more and more attention to the quality of the wheat.Wheat quality is determined by the combination effects of many factors such as protein,lipid and starch,among which the protein content,quality,and interaction are especially important.The seed storage proteins consisted of glutenin and gliadin in wheat,which positive correlated closely to dough rheological properties.The glutenins are the major components of wheat storage proteins,which consist of high-molecular-weight glutenin subunits(HMW-GS)and low-molecular-weight glutenin subunits(LMW-GS).Gliadin including α-、 β-、 γ-andω-gliadin.However,30%-70% of the wheat quality variation can be explained in the traditional wheat gluten.Although cysteine content is very low in amino acids of traditional gluten protein,the effect of cysteine residues can be to make the protein structure into polymers through intermolecular disulfide bond.A new found storage protein,avenin-like,is caught a great attention for its number of cysteine residues in recent years.It is apt to form inter-disulfide bonds or intra-disulfide bonds with neighboring storage proteins,which may broaden the protein-nets and develop elasticity in a dough,resulted changing the processing quality.In fact,research in this field has just been started not long ago.There still remained a paucity of genetic information about the chromosomal location,number of loci and alleles,and allelic effects.The ancestral NAM-B1 allele has been discovered in many tetraploid wild emmer(T.turgidum ssp.dicoccoides)accessions and contributed to the rate of senescence and allowed more effective translocation of nutrients to the grain,resulting in shorter grain filling and higher concentrations of protein,Fe,Zn,and Mn in the grain but reduced yield.To date,there are few studies on the distribution of the ancestral NAM-B1 allele in Chinese wheat cultivars.The goal of this study was to identify the NAM-B1 alleles(wild-type,1-bp insertion and deletion)in Chinese cultivars from different geographical areas and provide some information for improving wheat quality in China and other countries.The genes of avenin-like b and NAM-B1 are the primary focus in our study due to affecting the protein quality and content,respectively.The major results of this study are listed as the following:1.Using two-demensional electrophoresis(2-DE)method,we analyzed the proteinsprofiles of L16-3BS which were chromosome segment substitution lines.50 differentially expressed protein spots with higher than 2-fold spot thicknesses were found in gel image.These protein spots were identified with MALDI-TOF/TOF and 47 spots were identified successfully.Compared to control,the abundance of 35 proteins increased and of 10 decreased.As follow,Globulin(5),Alpha-gliadin(2),gamma gliadin(2),MYB transcription(1),elongation factor(1),Triosephosphate isomeras(2),Serpin(7),malate dehydrogenase(1),avenin-like(2),triticin(3),Superoxide dismutase(1),Heat shock protein(2)and unknown protein(18).Avenin-like b genes was choosed as candidate genes to study.2.The avenin-like genes were cloned by gene specific primers,followed by sequencing and bioinformatic anayzing.The coding gene sequences were 855-858 bp long and no introns.Chinese Spring nulli-tetrasomic confirmed that type-b ALP genes of wheat belong to a multigenefamily.Three genes of type-b ALPs were identified and mapped to chromosomes7AS(TaALPb-7A),4AL(TaALPb-4A)and 7DS(TaALPb-7D).Further comparison revealed that the TaALPb-7A gene had three alleles,designated TaALPb-7A1,TaALPb-7A2 and TaALPb-7A3.While TaALPb-4A gene had four alleles,TaALPb-4A1,TaALPb-4A2,TaALPb-4A3 and TaALPb-4A4.The TaALPb-7D locus did not show any allelic variation.The phylogenetic relationship of the 42 cloned type b ALPs sequences was analyzed by applying UPGMA to the aligned complete coding sequences of all clones and wheat storage protein genes,as well as the reported ALPs of wheat-related species available from various databases.The cloned type-b ALP sequences clustered according to their chromosomal origin.The cloned ALP sequences were closest to the reported type-b ALP sequences of related species,followed by sequences corresponding to HMW-GS and LMW-GS,while ω-gliadin were the furthest in evolutionary terms.3.Results of ddPCR revealed that the cultivars with the active allele give a normal gene expression,the ratios of expressed gene copy numbers between TaALPb-7A and actin ranged from 1:2.54 to 1: 3.36,while the four cultivars with the silent allele had no gene expression.4.TaALPb-7A1,TaALPb-7A2 were cloned into the expression vector,and transformed into host bacteria Escherichia coli Rosetta-gami BL21(DE3).After induced by IPTG,the fusion protein was purified and then been integrated into the control flour to determine the quality effect utilization of Mixograph.The results revealed that TaALPb-7A1,TaALPb-7A2 has an important effect on processing quality.5.The fact that the TaALPb-7A locus has two types of alleles,active and silent,allowed us to study its allelic effects.Allele-specific PCR markers were designed to differentiate the two types of TaALPb-7A alleles.Significant allelic effect differences were detected bewteen the active and silent alleles of TaALPb-7A.The active allele was significantly associated withhigher dough strength parameters,including Midline Peak Time(P<0.0443),and Midline Time x Width(P<0.0096).No significant association was detected between the allelic types and grain protein content or gluten content,indicating the high dough strength of the active allelic type is from the expression of TaALPb-7A.6.The avenin-like genes were cloned by gene specific primers,followed by sequencing and bioinformatics analyzing in 12 genomes(W、E^e、E^b、St、Q、Ns、Ta、U、C、N、V 、 R)of the wheat related species.Results suggested that abundant genetic variation and mutation of avenin-like genes were found in some genomes,which is available for incorporation into cultivated wheat by wide crossing.7.This study investigated the NAM-B1 alleles in 218 Chinese cultivars that were collected from five major wheat regions(12 provinces),covering most of the winter wheat growing regions in China.The results showed that the NAM-B1 gene is present in 53(24.3%)cultivars.The remaining 165(75.7%)cultivars do not contain this gene.Thus,exploring the distribution of NAM-B1 allelic variations(1-bp insertion and deletion)can provide some information for improving the quality of winter wheat in China and other countries.更多还原