【作者】 李青；

【导师】 江千涛；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2022， 硕士

【摘要】 小麦籽粒主要由蛋白质和淀粉组成,二者的组成和含量直接决定着小麦的产量和品质。硬粒小麦-沙融山羊草双二倍体(Amphidiploid,AABBS^shS^sh,2n=42)是本课题组前期将硬粒小麦（Triticum turgidum subsp.durum,AABB,2n=28）与沙融山羊草(Aegilops Sharonensis,S^shS^sh,2n=14)杂交后经染色体自动加倍获得的异源多倍体。其中,沙融山羊草1S^sh染色体上具有分子量特别大的新型HMW-GSs,有利于提高小麦面团加工品质。本研究对沙融山羊草、硬粒小麦及其双二倍体进行转录组、代谢组分析,探究异源多倍化后差异表达基因（DEGs）、差异代谢物（DEMs）的变化,并对双二倍体衍生后代材料进行鉴定。主要研究结果如下:1.沙融山羊草（R7）、硬粒小麦（Z636）和双二倍体（Z636×R7）的淀粉特性和蛋白含量分析。SDS-PAGE和双向电泳鉴定结果表明沙融山羊草、硬粒小麦的Wx-S^sh1、Wx-A1和Wx-B1蛋白在双二倍体中均表达。克隆了沙融山羊草、硬粒小麦和双二倍体的Wx-1基因并进行进化分析,表明R7的Wx-S^sh与Wx-B1高度同源。淀粉含量测定,结果表明双二倍体的总淀粉含量和直链淀粉含量高于沙融山羊草,低于硬粒小麦。电子扫描电镜（SEM）发现双二倍体A/B型淀粉粒的比例及其形状介于亲本之间,呈中间类型。利用差示热量扫描仪（DSC）测定热力学特性,糊化、老化参数结果表明双二倍体的热焓值高于沙融山羊草,低于硬粒小麦。RP-HPLC结果表明双二倍体的总谷蛋白含量高于两个亲本,醇溶蛋白显著高于沙融山羊草,与硬粒小麦无显著差异。2.沙融山羊草、硬粒小麦、双二倍体转录组分析。在R7 vs Z636,Z636 vs Z636×R7,Z636×R7 vs R7中分别鉴定到21037、2197、15090个差异表达基因。三个比较组的差异表达基因KEGG主要富集在碳代谢、氨基酸的生物合成、淀粉和蔗糖代谢、半乳糖代谢、核糖体等代谢途径。比较分析了参与淀粉生物合成和编码贮藏蛋白差异基因的表达水平,结果表明双二倍体中部分淀粉合成酶基因AGP-L、AGP-S、SSII-2、SSIIIa、SBEII、SBEIII和GBSSI的表达量都高于沙融山羊草,但低于硬粒小麦;双二倍体编码的α/β-、ω-、γ-醇溶蛋白的基因表达量高于沙融山羊草,RNA-seq与q RT-PCR结果一致。3.沙融山羊草、硬粒小麦、双二倍体代谢组分析。在R7 vs Z636,Z636 vs Z636×R7,Z636×R7 vs R7中分别鉴定到871、714、836个差异代谢物,主要是羧酸及其衍生物、脂肪酰基、类固醇及类固醇衍生物、有机氧化合物、苯及其取代衍生物、孕烯醇酮脂类等。KEGG富集分析表明,差异代谢物主要富集在酪氨酸和色氨酸的生物合成、氨酰生物合成、精氨酸生物合成、丙氨酸、天冬氨酸和谷氨酸生物合成,甜菜碱生物合成等。4.沙融山羊草、硬粒小麦、双二倍体转录组-代谢组联合分析。R7 vs Z636,Z636 vs Z636×R7,Z636×R7 vs R7的差异表达基因及差异代谢物在正/负离子模式下分别有67/68、50/53、50/67个共同注释的代谢通路。三个比较组主要富集的通路包括碳代谢、氨基酸的生物合成、氨基糖和核苷酸糖的代谢、淀粉和蔗糖的代谢、半乳糖代谢、丙氨酸、天冬氨酸和谷氨酸代谢等代谢通路。双二倍体中GBE基因表达量和糖原积累量趋势一致,但在沙融山羊草和硬粒小麦中两者趋势相反,推测糖原的合成可能还受其他机制调控。还发现2个负责编码谷氨酸合成酶（GS）的DEGs,GSr1和GSe2,其中双二倍体的GSe2基因表达量显著高于两个亲本。5.硬粒小麦-沙融山羊草衍生后代材料鉴定。利用含有Ph基因突变体材料（SM126ph1b）与携带沙融山羊草特异HMW-GSs的材料（66-9-17）杂交,在BC₂F₃衍生后代中鉴定到2个成功将一对外源染色体1S^sh转移至普通小麦的材料（13-1-22-1、13-1-22-3）,两个材料ND-FISH结果一致,总共有42条染色体,缺失6A和3D染色体,增加一对2D染色体和一对1S^sh染色体。在F3代中,2-7-3-9共有42条染色体,缺失4D染色体,还含有一对外源染色体4S^sh。因此表明2-7-3-9为4S^sh（4D）代换系材料。根据沙融山羊草和中国春（CS）的差异序列,开发了一对能够特异于1S^sh-1A染色体共显性分子标记,可以快速从分子水平上追踪及检测1S^sh染色体存在情况。以上获得的衍生材料,为小麦-沙融山羊草易位系的创制提供了材料基础。更多还原

【Abstract】 Wheat grain is mainly composed of protein and starch,their composition and content determine the yield and processing quality of wheat.The amphidiploid(Amphidiploid,AABBS^shS^sh,2n=42)between durum wheat（Triticum turgidum subsp.durum,AABB,2n=28）and Aegilops sharonensis(Ae.sharonensis,S^shS^sh,2n=14)were obtained by distant hybridization.Ae.sharonensis has a novel HMW-GSs with a particularly large molecular weight,which is beneficial to improve the quality of wheat dough processing.In this study,transcriptome and metabolome analysis were conducted on Ae.sharonensis,durum wheat and amphidiploid,to explore the changes of DEGs and DEMs after polyploidization,and to identify offspring materials derived from amphidiploid.The main results are as follows:1.Analysis of starch properties and protein content in R7,Z636,Z636×R7.SDS-PAGE and 2D-PAGE identification showed that Wx-S^sh1,Wx-A1 and Wx-B1proteins of R7 and Z636 were expressed in Z636×R7.Wx-1 genes of R7,Z636 and Z636×R7 were cloned and analyzed,it showed that Wx-S^sh1gene of R7 was highly homologous with Wx-B1.The results showed that the contents of total starch and amylose in Z636×R7 were higher than those in R7,but lower than that in Z636.Scanning electron microscopy（SEM）showed that the Z636×R7 has intermediate starch grain shape,that is,the proportion and shape of A/B starch grains were in the middle of it’s parents.Differential thermal scanner（DSC）was used to determine the thermodynamic characteristics,gelatinization and aging parameters indicated that the enthalpy value of Z636×R7 was higher than that of R7,but lower than that of Z636.RP-HPLC showed that the glutenin content of Z636×R7 was higher than that of two parents,and the gliadin was significantly higher than that of R7,which had no significant difference with Z636.2.Transcriptome analysis of amphidiploid and its parents.A total of 21037,2197 and 15090 differentially expressed genes were identified in R7 vs Z636,Z636 vs Z636×R7,and Z636×R7 vs R7,respectively.KEGG analysis indicated that the differentially expressed genes in the three comparison groups were mainly enriched in carbon metabolism,amino acid biosynthesis,starch and sucrose metabolism,galactose metabolism,ribosome and other metabolic pathways.The expression levels of differentially expressed genes involved in starch biosynthesis and encoding storage proteins were compared.The results showed that the expression levels of starch synthase genes AGP-L,AGP-S,SSII-2,SSIIIa,SBEII,SBEIII and GBSSI in Z636×R7were higher than those in R7,but lower than Z636.The gene expression levels ofα/β-,ω-andγ-gliadin encoded by Z636×R7 were higher than those of R7,RNA-seq result was consistent with q RT-PCR.3.Metabolome analysis of amphidiploid and its parents.In R7 vs Z636,Z636 vs Z636×R7,and Z636×R7 vs R7,871,714,836 differential metabolites were identified,including carboxylic acids and their derivatives,aliphatic acylates,steroids and their derivatives,organic oxygen compounds,benzene and its substituted derivatives,and progeenolone lipids.KEGG analysis showed that the differential metabolites were mainly enriched in tyrosine and tryptophan biosynthesis,aminoacyl biosynthesis,arginine biosynthesis,alanine,aspartic acid and glutamic acid biosynthesis,and betaine biosynthesis.4.Combined analysis of transcriptome and metabolome of amphidiploid and its parents.The differentially expressed genes and metabolites of R7 vs Z636,Z636 vs Z636×R7,and Z636×R7 vs R7 had 67/68,50/53 and 50/67 metabolic pathways annotated in positive/negative ion mode,respectively.The main enrichment pathways in the three comparison groups included carbon metabolism,amino acid biosynthesis,amino sugar and nucleotide sugar metabolism,starch and sucrose metabolism,galactose metabolism,alanine,aspartic acid and glutamate metabolism.The GBE gene expression and glycogen accumulation trend in amphidiploid were consistent,but the trends were opposite in R7 and Z636,suggesting that glycogen synthesis might be regulated by other mechanisms.Two DEGs,GSr1 and GSe2 encoding glutamate synthase（GS）were also characterized.The expression level of GSe2 gene in diploid was significantly higher than that of the two parents.5.Identification of T.durum-Ae.sharonensis derived progeny material.Hybridization of Ph mutated material（SM126ph1b）with material（66-9-17）carrying specific HMW-GS of Ae.sharonensis.In BC₂F₃derived progenies,two lines（13-1-22-1 and 13-1-22-3）that successfully transferred a pair of exogenous 1S^shchromosomes to common wheat were identified.The ND-FISH results of the two lines were consistent,with a total of 42 chromosomes,a with a pair of 1Ssh chromosomes,missing 6A and 3D chromosomes,and adding a pair of 2D chromosomes.In addition,the line 2-7-3-9 has a total of 42 chromosomes,missing4D chromosome and containing a pair of exogenous chromosome 4S^sh,was charcterized as a 4S^sh（4D）substitution line.A pair of codominant molecular markers specific to chromosome 1S^sh-1A were developed based on the sequences of Ae.sharonensis and Chinese spring（CS）,which can be used to quickly track and detect the presence of chromosome 1S^shat the molecular level.The derived materials provided the material basis for the establishment of the translocation line of wheat-Ae.sharonensis.更多还原