【作者】 郭志富；

【导师】 郑有良； 魏育明；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 类大麦属作为小麦族中较为少见的单种属,在其系统进化方面研究较多,而对其种子贮藏蛋白的研究尚未见报道。为了深入了解类大麦属物种贮藏蛋白及其编码基因,对类大麦属材料毛节草(Crithopsis delileana)的种子贮藏蛋白分别进行了分析与鉴定,并且对部分编码基因进行了克隆和测序,对其高分子量谷蛋白亚基(how-molecular-weight glutenin subunits,HMW-GS)Kx基因进行了表达分析。本研究所获主要结果如下:1.毛节草种子贮藏蛋白SDS-PAGE和A-PAGE分析对2份毛节草材料种子贮藏蛋白进行了SDS-PAGE和A-PAGE分析。利用三种不同的提取方法进行SDS-PAGE分析结果表明,普通提取方法中2份材料在小麦的高分子量谷蛋白区域均仅有一条带,电泳迁移率介于普通小麦By8和Dy10亚基之间,推测为一个高分子量谷蛋白亚基。在普通小麦的低分子量谷蛋白区域,出现若干条分辨率不高的蛋白条带。通过丙酮沉淀法提取分析,发现HMW-GS沉淀中只有电泳迁移率介于普通小麦By8和Dy10亚基之间的带存在,初步确认毛节草中只有一种高分子量谷蛋白亚基。A-PAGE分析表明毛节草种子醇溶蛋白存在着一些变异类型,与普通小麦中国春相应的α、γ和ω醇溶蛋白区域的电泳迁移率有一定差异。2、HMW-GS基因克隆与序列分析利用已知HMW-GS基因序列设计特异性引物,对1份毛节草材料的基因组DNA进行扩增得到两个长度分别为2.1和1.7kb的片段,经过序列分析表明2.1kb片段为Kx亚基编码基因,而1.7kb片段为Ky亚基(未表达)的编码基因。Genebank登录号分别为AY804128和AY834230。Kx基因全长编码区长度为2046bp,由其推导的氨基酸序列含682个氨基酸残基,具有x型HMW-GS的典型特征,包括21个氨基酸残基组成的信号肽,86个氨基酸残基组成的N-端保守区,533个氨基酸残基组成的重复区,42个氨基酸残基组成的C-端保守区。N和C-端保守区内分别含3个和1个半胱氨酸(C)。重复区内,含有9个三肽(GQQ)、56个六肽(PGQGQQ)、16个九肽(GYYPTSLQQ),以及位于重复区起始端的十二肽(RYYPSVTSSQQA)和十一肽(SYYPGQASPQR)和重复区末端的三肽(GYD)。与其它亚基序列进行比较发现它与已知高分子量谷蛋白亚基间的主要差异在重复区内,且集中表现为可重复短肽类型和数目的增加或减少,以及可重复单元内个别座位氨基酸的替换和少量部位的缺失。目前,在已知x型高分子量谷蛋白亚基中,Kx的长度是最短的。Ky亚基的全长序列长度为1725bp,由其推导的氨基酸序列同样具有y型高分子量谷蛋白序列的典型特征。但在核苷酸序列中的1558位置上存在一个提前终止密码子,它的存在可能导致Ky编码基因沉默,从而导致Ky亚基不能得到表达。3、LMW-GS基因克隆与序列分析利用已知LMW-GS基因序列保守区设计一系列引物对毛节草基因组DNA进行扩增,成功克隆了4个LMW-GS基因LMW-GSK1、LMW-GSK2、LMW-GSK3和LMW-GSK4,Genbank序列号依次为EU283813、EU283814、EU283815和EU283816。其中LMW-GSK3和LMW-GSK4为部分基因序列。同小麦族其它LMW-GS基因进行比对分析后发现,其5’侧翼序列和氨基酸序列同其它亚基存在较高的一致性,同样具备LMW-GS的典型特征,同时也存在着一定的结构差异,这些差异主要来源于氨基酸残基的替换以及重复区结构单元的缺失或插入。在LMW-GSK1基因序列5’侧翼区发现了2个顺式作用元件EM(5’-TGTAAAGT-3’)和GLM(5’-GGCGAGTCAT-3’)、2个TATA box、1个CAAT box和1个AGGA box。其推导的氨基酸序列具有一个由20个氨基酸残基组成的信号肽,一个15个氨基酸残基组成的N端保守区,一个由80个氨基酸组成的重复区和一个由182个氨基酸残基组成的C端保守区。LMW-GSK2和LMW-GSK4编码区内部均发现了提前终止密码子,故推测它们为假基因。根据所克隆基因的5’侧翼序列和保守区氨基酸序列同小麦族其他LMW-GS相应序列的比对结果进行分子进化分析表明,来源于K染色体组的LMW-GS可以和小麦A、D染色体组以及黑麦的R和大麦的H染色体组分别聚为关系相对较近的类群。4.醇溶蛋白基因分子克隆与序列分析根据小麦族已知α和γ醇溶蛋白基因保守区设计引物,对毛节草进行PCR扩增,成功获得了3个α醇溶蛋白基因gli-Kal、gli-Ka2和gli-Ka3,Genbank登录号分别为EU283817、EU283819和EU283820;2个γ醇溶蛋白基因gli-KrI和gli-Kr2,Genbank登录号分别为EU283818和EU283821。因在gli-Ka3编码区内发现了1个提前终止密码子,故推测其为假基因。毛节草的3个α醇溶蛋白基因与来源于小麦属普通小麦、乌拉尔图小麦和野生二粒小麦,以及具有S和H染色体组的披碱草属老芒麦、具有D染色体组的粗山羊草、具有W染色体组的澳冰草、具有V染色体组的簇毛麦和具有Ee染色体组的偃麦草属的相应基因进行比较发现它们在结构上有着较高的相似性,同时也存在着一定的差异,这些差异主要来源于氨基酸残基以及重复区内重复单元的的替换、缺失和插入。一致性分析表明3个基因推导的氨基酸序列同其它小麦族相应基因氨基酸序列的相似性在57.59%-88.18%范围内变化。2个γ醇溶蛋白基因与来源于小麦属不同品种相应基因之间同样具有较高的相似性,其一致性在62.41%-85.53%范围内变化。保守区进化分析表明,3个α醇溶蛋白单独聚为一类,其同时与具有Ee染色体组的偃麦草属的α醇溶蛋白基因亲缘关系最近,其次与普通小麦的α醇溶蛋白基因亲缘关系最近;2个γ醇溶蛋白同样单独聚为一类,同时与来自于普通小麦品种Bobwhite的γ醇溶蛋白基因亲缘关系最近。5.HMW-GS基因Kx体外表达与植物转基因载体构建根据Kx基因编码区序列设计表达引物,扩增其切除信号肽部分的全长编码区后连接入pET-30a表达专用载体后,转化大肠杆菌BL21(DE3),经诱导后Kx基因在细菌中获得表达,且表达产物电泳迁移率同种子蛋白中唯一的高分子量谷蛋白亚基相一致。进一步设计遗传转化载体构建所需引物,扩增其全长编码区后连接入遗传转化专用载体pCMBIA1302中,经PCR、双酶切和测序验证了载体构建成功,为进一步利用转基因手段研究Kx与小麦品质的关系奠定了基础。更多还原

【Abstract】 Crithopsis delileana(Schult)Roshev(2n=2x=14,KK)is the only member of the genus Crithopsis Jaub.Et Spach.in the Triticeae group.The classification and evolution about this species had been extensively investigated,whereas the storage protein of this species was unknown.For the better understanding of storage protein of C.delileana,the composition of several storage protein in C.delileana were characterized,and some coding genes were isolated and sequenced.1.SDS-PAGE and A-PAGE analysis of the storage protein in C.delileanaThe storage proteins in C.delileana were analyzed by the SDS-PAGE and A-PAGE.In SDS-PAGE analysis by general extraction method of total seed protein,two candidate HMW glutenin subunits were detected in 2 accessions of C.delileana from Demark.One putative HMW glutenin subunit has the similar electrophoresis mobility between the subunits By8 and By10 in hexaploid wheat,and the other band has abnormal faster electrophoresis mobility than any of the y-type glutenin in wheat.A number of bands were detected in the LMW glutenins region of common wheat.These bands likely were LMW prolamines or gliadins present in C.delileana.Using acetone extraction method,it was found that only one band with electrophoresis mobility between subunits By8 and By10 in hexaploid wheat was detected.A-PAGE analysis indicated that the electrophoresis mobility of gliadins in C.delileana was different from the electrophoresis mobility ofα,γandωgliadins form common wheat Chinese spring.2.Isolation and sequence analysis of HMW-GS genes from C.delileanaOne C.delileana accession was selected for the further studying in the isolation of HMW-GS coding genes.In genomic PCR reactions using the primer pairs designed from published sequences of HMW-GS,two target fragments(2.1kb and 1.7kb)were specifically amplified.It was confirmed that the two target fragments were the coding genes of subunit Kx and Ky(not expressed)by sequence analysis.The sequences of HMW glutenin genes Kx and Ky had been deposited in the GenBank under accession numbers AY804128 and AY834230,respectively.The full coding region of Kx was 2,046bp,and could encode a mature protein with 661 amino acids residues.The coding sequence of Kx has the typical primary structures of HMW glutenin subunits.In the deduced amino acid sequence of Kx,a conserved signal peptide with 21 residues,a N-terminal region with 86 residues,a central repetitive domain with 533 residues and a C-terminal region with 42 resisdues were observed.The repetitive domain consists of interspersed repeats based on 9 tripeptide(consensus GQQ),56 hexapeptide(consensus PGQGQQ)and 16 nonapeptide(consensus GYYPTSPQQ) motifs.Two longer repeat units(11 followed by 12 residues)and a tripeptide(GYD)were adjacent to each other at the beginning and end of the repetitive central domain, respectively.It was indicated that the amino acid sequence of Kx were different from the HMW glutenin representatives by substitutions,insertions and/or deletions involving single amino acid residues or motifs.The size of the repetitive domain of the Kx was substantially smaller than those of the known subunits.The coding sequence of HMW glutenin subunit Ky was 1,725 bp,and had the similar typical primary structure to those of known y-type HMW glutenin.Due to an in-frame stop codon in the coding regions at position 1558 bp,the HMW glutenin Ky could not to be expressed as an active mature protein.3.Isolation and sequence analysis of LMW-GS genes from C.delileanaUsing homologous primers designed from published sequences of LMW-GS,four LMW prolamine gene sequences,designated as LMW-GSK1(EU283813),LMW-GSK2 (EU283814),LMW-GSK3(EU283815)and LMW-GSK4(EU283816),were isolated from C.delileana,among which LMW-GSK3 and LMW-GSK4 were partial gene sequences. These LMW prolamine gene sequences had the similar typical primary structures to the LMW-GS genes previously described in Triticeae.The deduced amino acid sequences of the LMW prolamines of C.delileana were different from the LMW glutenin representatives by substitutions,insertions and/or deletions involving single amino acid residues or motifs.Sequence analysis showed that 5’ flanking region of the LMW-GSK1 contained two cis-elements EM(5’-TGTAAAGT-3’)and GLM(5’-GGCGAGTCAT-3’) besides four general cis-elements with two TATA box,one CAAT box and one AGGA box.The deduced amino-acid sequence of the LMW-GSK1 consisted of a conserved signal peptide with 20 residues,a N-terminal region with 15 residues,a repetitive domain with 80 residues and a C-terminal region with 182 residues.Several in-frame stop codons were found in the coding sequences of LMW-GSK2 and LMW-GSK4,which showed that the LMW-GSK2 and LMW-GSK4 could be the putative pseudogenes.Phylogenetic analysis based on the alignment of 5’ flanking regions and deduced amino acid sequences indicated that LMW-GSK1,LMW-GSK2 and LMW-GSK4 from K genome were closely related to the corresponding genes from A,D and R genomes,while LMW-GSK3 could be clustered together with X03103 from H genome.4.Isolation and sequence analysis of gliadin genes from C.delileana Using homologous primers designed from published sequences ofα- andγ-gliadin genes,threeα- and twoγ-gliadin genes were isolated from C.delileana,which were designated as gli-Ka1,gli-Ka2,gli-Ka3,gli-Kr1and gli-Kr2,respectively.These gene sequences had been deposited in the GenBank under accession numbers EU283817, EU283819,EU283820,EU283818 and EU283821,respectively.A in-frame stop codon was found in the coding sequences of gli-Ka3,indicating that the gli-Ka3 could be the putative pseudogenes.Threeα-gliadin genes of C.delileana had the similar but not identical primary structures to the corresponding gene sequences from Triticum aestivum, T.urartu,T.turgidum,Elymus sibiricus,Aegilops tauschii,Australopyrum retrofractum, Dasypyrum breviaristatum and Lophopyrum in Triticeae.The differences were mainly resluted from substitutions,insertions and/or deletions involving single amino acid residues or motifs ofα-gliadins.The identity analysis indicated that the identity of threeα-gliadin genes with other correspondingly genes in Triticeae changed from 57.59%to 88.18%.Twoγ-gliadin genes of C.delileana also had the similar typical primary structures to the corresponding gene sequences in Triticum.The identity of twoγ-gliadin genes with other correspondingly genes of Triticum changed from 62.41%to 85.53%. Phylogenetic analysis based on the alignment of conserved regions indicated that the threeα- and twoγ-gliadin genes were clustered into two separate groups,and the threeα-gliadin genes were closely related to the corresponding genes from Lophopyrum contained Ee genome and common wheat,while the twoγ-gliadin genes could be clustered together with the corresponding gene of Bobwhite from common wheat.5.Expression of the coding sequence of HMW prolamine Kx and construction of transgenic vectorFor bacterial expression,the nucleotide sequence of the signal peptide was removed from the Kx by PCR mutagenesis.After cloning the modified coding sequence into the pET-30a vector,an expression construct pET-Kx was obtained and chosen for expressing the mature proteins of the Kx subunits in bacterial cells BL21(DE3)plysS.In SDS-PAGE analysis of protein extracts prepared from induced bacterial cultures and purified expression protein,the electrophoretic mobility of the protein directed by pET-Kx was similar to that of the subunit from seeds.For construction of transgenic vector,the primers were designed by Kx coding sequence.After cloning the modified coding sequence into transgenic vector pCMBIA1302,the validation of PCR,digestibility analysis of double Enzyme and sequencing indicated that the transgenic vector of Kx was constructed successfully.To estimate the comprehensive effects of the prolamine encoding by Kx gene on the baking quality of wheat,the endosperm specific expression of this gene in transgenic wheat is the desirable method.更多还原