【作者】 刘千；

【导师】 郑有良；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2007， 硕士

【摘要】 本文根据已报道的小麦α-、γ-和ω-醇溶蛋白基因保守区序列，分别设计特异引物，采用PCR克隆的方法，对小麦新品种“川农16”、“良麦2号”和“良麦3号”的相应序列进行克隆测序与分析，其主要结果如下：1．从3个供试材料中克隆到11条不同的α-醇溶蛋白基因序列。其中从“川农16”中所得序列α-CN16-6（GenBank登录号：DQ246449）、α-CN16-9（DQ246446）、α-CN16-12（DQ246447）和α-CN16-14（DQ246448），其长度分别为852bp、861bp、870bp和900bp；从“良麦2号”中得到α-LM2-12（DQ417343）、α-LM2-14（DQ417344）和α-LM2-17（DQ417345），其长度分别为942、852和921bp；从“良麦3号”中得到α-LM3-5、α-LM3-11、α-LM3-12和α-LM3-16（暂未登录），其长度分别为861bp、876bp、858bp和861bp。其中序列α-CN16-6和α-LM2-14为假基因。进一步分析表明，所有序列均具有典型的α-醇溶蛋白基因结构，其重复区开头可分出5个氨基酸残基的N-末端，根据密码子可推导出较为一致的重复单元PF／YPQPQ，重复区主要差异在于重复单元PFPQPQL在不同基因出现频率不同。所有序列的两个多聚谷氨酰胺区除谷氨酰胺外还有其余氨基酸出现，密码子大多是第1个碱基的替换。在特征区6个保守的半胱氨酸残基中，α-CN16-6的第4个半胱氨酸残基被精氨酸（R）所替代，α-CN16-6、α-CN16-12、α-CN16-14和α-LM2-14在特征区Ⅱ的前端均有额外的半胱氨酸残基代替了原来的丝氨酸（S），使半胱氨酸残基数变为奇数。相对其它两个品种，“川农16”中所得序列出现多余半胱氨酸的频率较高，表现出独特性。2．从“川农16”中克隆到6条不同的γ-醇溶蛋白基因序列，分别命名为γ-CN16-1、γ-CN16-3、γ-CN16-4、γ-CN16-5、γ-CN16-20和γ-CN16-24，其长度分别为1335bp、1360bp、1403bp、1359bp、1359bp和1360bp。所有序列包含了完整编码区以及部分5’、3’基因旁侧序列。其中序列γ-CN16-1和γ-CN16-3含有提前终止密码子，均为假基因。序列分析表明，5’旁侧序列上有一致的几个“TATA”元件，3’旁侧序列有一个可能的多聚腺嘌呤信号，在基因编码区具有典型γ-醇溶蛋白基因的结构特征，重复区可划分出较为一致的重复单元PFPQ_1-2（PQQ）_1-2，且重复单元长度变化较大。在多聚谷氨酰胺区各序列长度差异也较大。序列γ-CN16-3在重复区有1个多余半胱氨酸残基，使半胱氨酸总数变为奇数，而其它序列只有8个保守的半胱氨酸残基。序列比对可以看出，所得序列与小麦属各物种γ-醇溶蛋白基因的相似性较高，而与来源于黑麦γ-seealin基因的相似性只有50％左右。3．从“川农16”中克隆到1条完整的ω-醇溶蛋白序列ω-CN16-63（GenBank登录号：EF116277），其长度为1080bp，为假基因。同时获得4条基因片段，分别为ω-CN16-3、ω-CN16-60、ω-CN16-64和ω-CN16-75，其中ω-CN16-3有提前终止密码子，ω-CN16-60发生了移码突变。从“良麦2号”获得1条完整序列和1条基因片段，分别命名为ω-LM2-7（GenBank登录号：EF116278）和ω-LM2-14。ω-LM2-7基因全长1158bp，为假基因。序列分析表明，所得基因和基因片段都属ω-2型，其重复区的脯氨酸含量约30％，谷氨酰胺含量超过40％，其中基因ω-LM2-7谷氨酰胺含量高达44.8％。序列重复单元长短不一，氨基酸变化丰富，但都有较为一致的重复单元PFPQ_1-2（PQQ）_1-2以不同频率连续出现。序列均没有可形成分子内或分子间二硫键的半胱氨酸残基。进一步序列比对表明，它们与来源于普通小麦和山羊草的ω-醇溶蛋白基因相似性很高，与来源于大麦的C-hordein基因和来源于黑麦的ω-secalin基因的相似性也达到70％。更多还原

【Abstract】 According to the conserved regions ofα-,γ-, andω-gliadin gene sequences, the gene-specific primers were designed to amplify the full gene coding regions from three new wheat varieties, including Chuannong 16, Liangmai 2 and Liangmai 3. The PCR amplyfied fragments were sequenced, and investigated. The results were as follows:1. Elevenα-gliadin gene sequences, includedα-CN16-6 （GenBank NO. DQ246449）,α-CN16-9 （DQ246446）,α-CN16-12 （DQ246447） andα-CN16-14 （DQ246448） from Chuannong 16,α-LM2-12 （DQ417343）,α-LM2-14 （DQ417344） andα-LM2-17 （DQ417345） from Liangmai 2, andα-LM3-5,α-LM3-11,α-LM3-12 andα-LM3-16 from Liangmai 3, were obtained. The length of all sequence varied from 852bp to 942bp. Two sequence,α-CN16-6 andα-LM2-14, were considered as pseudogenes, because of the premature stop codons in open-reader-frame （ORF）. The structure characters ofα-gliadins gene were identified in all sequences. N-terminal, included 5 amino acid residues, was confirmed and separated from the repetitive domain. The repetitive domain was deduced the same repetitive motifs PF/YPQPQ, which difference was that variable number of repetitive motif PFPQPQL was involved in different sequences. At the two polyglutamine regions, the extensive different of length among all sequences were found, and some other amino acid residues were also detected, besides glutamines. The mutations were mostly happened at the first base in codons. Mostα-gliadins contained six conserved cysteine residues in two unique domains, but the arginine was detected at the forth conserved cysteine residue position in sequenceα-CN16-6. The substitution of serine residue was observed by an additional cysteine residue at unique-Ⅱregions in sequencesα-CN16-6,α-CN16-12,α-CN16-14 andα-LM2-14. Theα-gliadins gene sequences from Chuannong 16 showed special characters, which’s frequency of addition cysteine residue was higher than others sequences from Liangmai 2 and Liangmai 3.2. Sixγ-gliadin gene sequences, included the full-length of coding regions and part of flanking sequences, which were designated asγ-CN16-1 （1335bp）,γ-CN16-3 （1360bp）,γ-CN16-4 （1403bp）,γ-CN16-5 （1359bp）,γ-CN16-20 （1359bp） andγ-CN16-24 （1360bp）, were obtained from Chuannong 16. Sequencesγ-CN16-1 andγ-CN16-3 were considered as pseudogene, because of the premature stop codons and small insertions/deletions leading to frameshift mutations, was also considered as pseudogene. Sequence analysis indicated that these sequences had some characteristics. The coding sequences had the same structure with knownγ-gliadins. The repetitive domain was composed of repeat units PFPQ_1-2（PQQ）_1-2, and the length of repeat units was strongly different. The difference of sequences length was observed in the polyglutamine regions. Mostγ-gliadins gene sequences contained eight conserved cysteine residues, but an additional cysteine residue was detected at repetitive domain in sequenceγ-CN16-3. Some TATA box in 5’-flanks and one potential polyadenylation signals in 3’-flanks, were also found. Multi-alignment analysis indicated that these sequences had the closest homology with previousγ-gliadins sequences from common wheat, and higher homologous （more than 80%） from closely related species of wheat, and only 50% homologous from rye （Secale sylvestre）.3. One complete coding region sequence ofω-gliadin genes and four partial sequences, which were designated asω-CN16-63 （GenBank NO. EF116277）,ω-CN16-3,ω-CN16-60,ω-CN16-64 andω-CN16-75, were obtained from Chuannong 16. And one complete coding region sequence and one partial sequences, which were designated asω-LM2-7 （GenBank NO. EF116278） andω-LM2-14, were also obtained from Liangmai 2. Sequence length ofω-CN16-63 andω-LM2-7 were 1080bp and 1158bp, respectively. These tow sequences were considered as pseudogenes, because of the premature stop codons. Especially, sequenceω-CN16-60 had the frameshift mutations of small insertions/deletions. Sequence analysis indicated that these complete genes and partial sequences belong to theω-2 type. In the repetitive domains, the percentage of proline was approximately 30%, and the proportion of glutamine was more than 40%. The percentage of glutamine of sequenceω-LM2-7, which was 44.8%, was the highest. The repetitive domain of sequence was composed of repeat units PFPQ_1-2（PQQ）_1-2, and there were strongly different in the length and the amino acid types of repeat units. Because there were not cysteine residues in theseω-gliadins gene sequences, the inter- and intramolecular disulfide bonds were not formed. Multi-alignment analysis suggested that these sequences had closer homology with otherω-gliadins sequences from Triticum aestivum and Aegilops tauschii, while had 70% homologous with C-hordein sequences from barley （Hordeum vulgare） andω-secalins sequences from rye （Secale cereale）.更多还原