【作者】 王晓波； 马传喜； 司红起； 乔玉强； 何贤芳； 夏云祥； 蔡华；

【Author】 WANG Xiao-bo MA Chuan-xi SI Hong-qi QIAO Yu-qiang HE Xian-fang XIA Yun-Xiang CAI Hua (Agromomy School of Anhui Agricultural University,Hefei,Anhui 230036,China)

【机构】 安徽农业大学农学院；

【摘要】 小麦籽粒多酚氧化酶（PPO）活性是影响面团褐变的主要原因,本研究通过对 NCBI 上注册的小麦 PPO 基因序列的搜索与比对后发现,现有的小麦 PPO 基因按表达方式可分为两大类（Ⅰ、Ⅱ）,其中第Ⅱ大类的 PPO 基因与小麦籽粒 PPO 活性密切相关,根据其中一条由小麦2D 染色体上 PPO 基因表达的多酚氧化酶 mRNA 序列（GenBank：AY515506）,利用 Primer premier 5．0软件在该基因的不同位置设计了8对引物,对7个高 PPO 活性小麦品种和7个低 PPO 活性小麦品种进行 PCR 扩增,结果表明其中一对引物（STS01）在高、低 PPO 活性材料中出现了差异。该引物在7个低 PPO 活性的材料中能扩增出560bp 的目标片段,在7个高 PPO 活性的材料中则不能扩增出目标片段,利用中国春缺体、四体将该标记定位在小麦2D 染色体长臂上。利用此对引物,对130份已测得连续两年 PPO 活性的小麦品种资源进行 PCR 扩增,结果表明在75个品种中可以扩增出560 bp 的目标片段,这些品种的 PPO 活性均值为221．08 A475/min g×10^-3；55个品种没有扩增出目标片段,PPO 活性均值为309．98 A475/min g×10^-3,方差分析表明不同扩增带型品种的 PPO 活性均值差异达极显著水平（P＜0．01）。通过对具有完整开放阅读框（ORF）的4条 PPO 基因比对后发现,位于小麦2D 染色体长臂上的 PPO 基因（PPO-2D）存在更为丰富的等位变异,等位基因间有94个单核苷酸变异（SNP）,并在非同义 cSNP 处设计了一对引物（STS-H）,对上述130份小麦品种进行 PCR 扩增,结果发现 STS-H 在大部分低 PPO 活性品种中没有扩增出目标片段,而大部分高 PPO 活性品种可以扩增出460 bp 的目标片段。与 STS01比较后发现,STS-H 与 STS01是一对互补标记。根据 STS01和 STS-H 引物各自的特点,研究了能同时扩增两对引物的多重 PCR 反应体系。在与位于小麦2A 染色体长臂上 PPO 基因分子标记（PP018）的联合分析后发现,130份品种中,有37个品种的两个标记（STS01和 PP018）扩增结果均为高活性扩增带型（HIH2）,这些品种 PPO 活性的均值为337．82 A475/min g×10^-3,极显著高于其它几种扩增带型品种的 PPO 活性均值（P＜0．01）。32个两个标记扩增结果均表现为低活性带型（L1L2）的小麦品种 PPO 活性值普遍偏低,可作为改良面制食品外观品质的候选亲本。更多还原

【Abstract】 Polyphenol oxidases（PPOs）present in mature wheat kernerals have been implicated in the unde- sirable darkening of dough.To accelerate the functional characterization of wheat PPOs,allow the identification of those PPO genes that are primarily involved in food biochemistry,and improve the appearance quality of cere- al products of china,basic local alignment search tool（BLAST）searches of expressed sequence tag（EST） databases were performed,and the sequences were aligned using software DNAMAN.Results from this study suggest that the presence of at least 13 PPO sequences in hexapolid wheat fell into two clusters（Ⅰ、Ⅱ）.Genes in ’Ⅱ’ cluster are expressed during kernel development and may therefore influence cereal product quality. Based on the mRNA sequence（GenBank Accession Number AY515506）,8 pairs of primers were designed using the software ’Primer premier 5.0’ and one of the markers designated as STS01 can amplify a 560 bp fragment in cultivars with 7 low PPO activity,while no PCR product was detected in 7 cultivars with high PPO activity. The marker STS01 was then mapped to chromosome 2DL using a set of nulli-tetrasomic lines and ditelosomic line of Chinese Spring.130 cultivars,whose PPO activity was measured in each of two cropping seasons,were used to test the validation of the marker STS01,and the results showed that 75 genotypes amplified 560 bp fragment showed PPO activity 221.08A475/min g×10^-3on average,which were significantly（P＜0.01） lower than the PPO activity of other 55 genotypes with 309.98 A475/min g×10^-3on average without any PCR amplification.The allelic variations of PPO genes on chromosome 2D（PPO-2Da、b）were richer than PPO genes on chromosome 2A（PPO-2Aa、b）according the sequence alignment between four PPO genes with open reading frame（ORF）.94 single nucleotide polymorphisms（SNP）were detected between PPO-2Da and PPO -2Db and 80 SNPs were found in coding region（coding SNP）while 36 SNPs,which affect the PPO amino acid sequece were non-synonymous cSNPs,and one pair of primer were designed as STS-H at some non-syn- onymous cSNPs site.Of the 130 cultivars tested,the STS-H can amplify a 460 bp fragment in most cultivars with high PPO activity（b）,while no PCR product was detected in most cultivars with low PPO activity（a）, and the statistical analysis showed that STS-H is the complementary marker of STS01 and thus the multiplex PCR was developed between STS-H and STS01.To test the effect of the primers in wheat breeding for low grain PPO activity,one STS marker,PPO18,which was located on chromosome of 2AL,was used.The re- sults showed that 37 cultivars with H1H2,with grain PPO activity 337.82 A475 nm/min g×10^-3on average were significantly higher（P＜0.01）than cultivars with L1H1,H1L1 and L1L2.Most of 32 wheat cultivars with L1L2 showed very low grain PPO activity,which can be used as parents in wheat breeding for low grain PPO activity.更多还原