【作者】 蔡华；

【导师】 马传喜；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 长期以来,我国人民对面粉及面制食品的色泽要求很高。普通小麦(Ttiticumaestivum L.2n=6x=AABBDD)籽粒黄色素含量(Yellow Pigment Content,YPC)是造成面粉色泽降低的重要因素之一,而且YPC对蒸煮面食品的色泽以及小麦的营养品质等均具有显著影响。虽然YPC受环境因素的影响,但基因型是其主要决定因素。黄色素是由多种物质组成的混合物,其主要成分是类胡萝卜素,类胡萝卜素的生物合成是在八氢番茄红素合成酶(Phytoene synthase,Psy)的作用下进行的,此酶为类胡萝卜素生物合成途径中的首要限速酶(Primary Rate-limiting Enzyme,PRH),其基因也是类胡萝卜素基因操作的首选基因,其他作物(尤其是玉米)中对Pay基因的研究已较为深入,但对普通小麦Psy基因的研究还未开展。本研究以已经发表的玉米Psy基因为基础,从YPC差异显著的中国小麦品种(系)中克隆普通小麦Psy基因,并进行分子特性和YPC分析;同时选择YPC差异显著的小麦品种组配F1与玉米远缘杂交,诱导可进行黄色素含量遗传分析的小麦双单倍体(Double Haploid,DH)群体,旨在明确Psy基因对小麦YPC的影响、开发小麦YPC的分子标记,同时为小麦YPC的遗传分析、高(低)YPC小麦新品种的选育提供遗传材料和新种质。主要结果如下:1.小麦黄色素合成途径中Pay基因的克隆及分子特性利用玉米Pay基因和小麦EST序列设计引物克隆小麦Pay基因并测序,同时和玉米Pay基因进行序列比对分析。结果表明,5对引物Psy01、Psy03～Psy06在YPC不同的小麦样品中分别扩增出989bp、785 bp、1192 bp、690 bp和302 bp的5条带,同一引物在不同YPC品种中的PCR产物序列一致,经比对分析,5条序列均为Pay基因(片段),并由此组装成一条全长3437bp、包括6个外显子和5个内含子的小麦Psy基因。文中以引物Psy04为例对其扩增的PCR产物进行了详细的分子特性分析。2.小麦Pay基因的等位变异及其对黄色素含量的影响根据已发表的麦族植物Pay基因序列的保守区设计引物Psy02,克隆小麦Psy基因。结果表明,该引物下PCR产物出现3种带型:196bp、233bp和489bp,其中196bp和233bp条带涵盖了小麦Psy基因第2外显子全部序列,相差的37bp为Psy基因第2内含子中的一段插入序列,可反映不同YPC,属小麦Pay基因的等位变异。验证试验表明,248份试材(包括229份中国小麦微核心种质和19份品质性状典型的小麦品种)中有153份材料(占样品数的65.7%)扩增出196bp条带,群体内YPC均值7.314mg/kg,属高YPC范畴;另有95份材料(占样品总数的38.3%)扩增出233bp条带,群体内YPC均值为5.207mg/kg,属低YPC范畴,方差分析表明二者YPC差异达1%极显著水平差异,说明上述37bp的插入序列是导致小麦品种间YPC产生差异的原因之一,因此该引物扩增的Psy基因对小麦YPC具有显著影响,引物Psy02是对小麦YPC进行分子鉴定的重要标记。序列分析表明,196bp和233bp与设计引物的探针序列100%同源,489bp序列的BLAST搜索显示,与序列EU096094(未定位)的同源率最高,达97%,与7A染色体上序列EF600063和7B染色体上序列DQ642439的同源率均为85%,表明该基因片段可能为7A和7B染色体以外的另一Psy基因。结合该序列对应小麦品种的高YPC,推测该基因和7A染色体的Psy基因对黄色素含量存在积加效应。3.普通小麦DD染色体上Psy基因的发现及其等位变异以扩增小麦Psy基因的相应引物Psy01～Psy06,在节节麦(2X=DD=14)中进行PCR反应。结果表明,仅引物Psy02和Psy06能扩增出特异条带。引物Psy02的扩增产物长206bp,与普通小麦中扩增的196bp序列同源率为93.0%,对应的第2外显子区域内仅有1SNP;引物Psy06的PCR产物长305bp,与普通小麦中扩增的302bp序列同源率达95.77%,对应的第6外显子区域内无SNP,说明在小麦DD染色组中存在Psy基因,同时也排除了将引物Psy01、Psy03、Psy04、Psy05的扩增产物和引物Psy02的489bp扩增产物定位于D染色体的可能。4.小麦黄色素含量DH群体的构建为获得一定规模的小麦单倍体以构建小麦黄色素含量的DH群体,用高/低YPC小麦品种组配F1,F1再和玉米远缘杂交诱导小麦单倍体,研究了环境温度对单倍体成胚率、幼胚取材时期、幼胚大小、4℃处理时间、暗处理时间对单倍体成苗率的影响。结果表明,获得最高成胚率的环境温度为21～23℃,授粉后12～16天取材的幼胚成苗率无明显差异;0.5～1.0mm大小的幼胚成苗率显著高于0～0.5mm和1.0～1.5mm大小的幼胚成苗率;1～3天短期4℃处理对胚萌发具有一定促进作用,但处理3天后,出愈率和成苗率都将降低;胚培养过程中12天左右的24h暗处理能有效提高成苗率,在此基础上建立了一套高效、可靠、重复性好的小麦×玉米单倍体诱导系统。更多还原

【Abstract】 For a long time,color of wheat flour and flour foods have been required highly for people in China.Yellow pigment content(YPC) of wheat(Ttiticum aestivum L. 2n=6x=AABBDD) is one of important factors reducing flour color,and yellow pigment content is significant influence to cooked products and nutritional quality in wheat. Although environmental factors have significant effect on yellow pigment content,gene type is main decisive factors.Yellow pigment content is mixture composed of various substances,and its main components is carotenoids,biosynthesis of carotenoids need Phytoene synthase(Psy),the enzyme is the primary rate-limiting enzyme in biosynthetic pathway of carotenoids,and the gene is first selected gene to carotenoids genetic manipulation.Psy gene has been further studied in many crops(especially maize),but the research has not been developed in wheat.In this paper,wheat Psy gene with different significant YPC was cloned in 248 Chinese wheat cultivars based on maize Psy gene registered in GenBank,and the molecular characteristics of Psy gene and YPC of different wheat cultivars were analyzed too.At the same time,wheat F1 with high and low YPC parents were crossed by maize pollen in order to obtain wheat haploid embryos,then induced wheat double haploid for genetic analysis of yellow pigment content through embryo rescue and haploid chromosome doubling,the purposes are clearing the effect of Psy gene to wheat YPC,developing molecular markers of YPC in wheat,and creating genetic materials and new germplasm for wheat YPC genetic analysis and wheat YPC breeding.The results were as follows:1.Cloning and molecular characterization of Psy gene in wheat yellow pigment biosynthesi Phytoene synthase(Psy) gene,a primary rate-limiting enzyme in the biosynthesis pathway of wheat yellow pigment(YP),was cloned and sequenced through Psy gene of maize and wheat ESTs in this study,at the same time,the Psy gene sequences between wheat and maize were compared.The results showed that,5 DNA fragments with 981bp,751bp,1192bp,690bp and 302bp was amplified by polymerase chain reaction in DNA samples extracted from the high YP and low YP content wheat cultivars,the sequences of high YPC wheat are same as those of low YPC with same primer.Sequences alignment showed that all the 5 DNA fragments were Psy gene(fragment) of wheat,and the full length gene with 6 exons and 5 introns,3437bp was assembled.Detailed molecular characterization of PCR products was analyzed with a case study of primer Psy04,and preliminary locating 5 Psy gene fragments on 7A chromosome in wheat.2.Allelic variation of wheat Psy gene and effect on yellow pigment content Primer Psy02 was designed according to sequence conservative areas of Psy gene in Ttiticum,cloning Psy gene of wheat.The results showed that,3 kinds of PCR products were amplified with 196bp,233bp and 489bp,among them,196bp and 233bp bands cover all sequences of the second exon of wheat Psy gene,37bp difference sequence is a insertion sequence in the second exon of Psy gene,which display different YPC in wheat. Verification test showed,153 samples of 248 wheat cultivars(including 229 micro core germplasms and 19 cultivars with typical quality characters) amplified 196bp band, account for 61.7%,the YPC average value is 7.314mg/kg belonging to high YPC rang,and 95 samples amplified 233bp band,account for 38.3%,the YPC average value is 5.207mg/kg belonging to low YPC rang.Variance analysis showed that the YPC difference reached 1%significant level,which proved the 37bp insertion sequence is one of the reasons of different YPC in cultivars,therefore,primer Psy02 is a important molecular marker identifying YPC,and the gene effects on wheat YPC significantly.Sequence analysis showed that 196bp and 233bp were 100%homologous to primer probe sequence, and the two sequences were located on 7A chromosome.489bp sequence was high homologous to Psy-B1 of EU096094 with 97%identity,and 85%identity to the Psy-A1 of EF600063 on 7A chromosome and DQ642439 on 7B,thus the gene can not lacate on 7A or 7B chromosome.Combining with high YPC of 489bp,conjecturing the Psy genes on 7A and other chromosomes have additive effect to YPC.3.Identifying Psy gene with Aegilops tauschii(Triticum tauschii L.2x=DD=14) and allelic variation Psy gene were cloned by primer Psy01-Psy06 in Aegilops tauschii (Triticum tauschii L.),the results showed that only primer Psy02 and Psy06 amplified specific bands,the PCR product of primer Psy was 206bp,with 93.0%sequence homologous to wheat PCR product,there was only 1SNP in the second exon.And the PCR product of primer Psy06 in Aegilops tauschii was 305bp,with 95.77%sequence homologous to wheat PCR product,there was no SNP in the 6th exon,which showed that there were Psy genes in DD genome of wheat,and the possibilities of locating primer Psy01,Psy03,Psy04,Psy05 and 489bp gene fragment of Psy02 on the D chromosome were eliminated.4.Establishment of DH groups used to study inheritance laws in wheat yellow pigment In order to obtain a large number of wheat haploids to establish the DH groups of wheat yellow pigment content,wheat and maize cross were used to induce wheat haploid embryos and then the haploid plants were produced via young embryo culture.The factors affecting wheat embryos and the regeneration of young plants from young embryos, such as environmental temperature,different period of peeling embryo,size and development stage of young embryos,the treatment time at 4℃and dark treatment time were studied.The results showed:the embryos frequency was the highest when environmental temperature was 21～23℃,there was not significant difference on the frequency of young plants production when haploid embryos was peeled in different time from 12d to 16d;the frequency of young plants production of the size of 0.5～1.0mm is much higher than that of the size of 0～0.5mm and 1.0～1.5mm;the short time treatment between 1～3 days at 4℃would accelerate the regeneration of young embryo,but after 3 days the frequency of callus induction and plants improve the frequency of plants production during the process of young embryo culture.Production would decrease;dark treatment of all 24 hours for 12 days could effectively.And on this condition,a high efficiency and repetitive regeneration system of wheat haploid from wheat×maize was established.更多还原