小麦PPO基因的分类及非同义cSNP对籽粒PPO活性的影响

小麦籽粒多酚氧化酶（PPO）活性是影响面团褐变的主要原因，检索NCBI网站上注册的小麦PPO基因并对其进行分类及相关变异的研究，对于弄清PPO基因与籽粒PPO活性的关系，开发分子标记选育出含有低PPO活性基因的品种，改良我国面制食品的外观品质有重要作用。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，第II大类第i小类中的PPO基因可能位于小麦2A、2D以外的染色体上，可做为改良面团色泽的侯选基因。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）存在丰富的等位变异，等位基因间有94个单核苷酸变异（SNP），其中发生在编码区的有80个（cSNP），这些cSNP中有36个影响到基因编码的氨基酸序列，属非同义cSNP。在非同义cSNP处，设计引物（STS-H），对130个已连续测得两年PPO活性的小麦品种进行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（a），而大部分高PPO活性品种可以扩增出460bp的目标片段（b）。方差分析表明，a、b两种类型品种的PPO活性均值差异达极显著水平（p<0.01），说明非同义cSNP对小麦籽粒PPO活性有重要影响。与STS01（低PPO活性显性标记）比较后发现，STS-H与STS01是一对互补标记。为提高单显性分子标记的使用效率，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。

The Classification of Wheat PPO Genes and The Effect of Non-synonymous cSNP on Kernel PPO Activity

Polyphenol oxidases (PPOs) present in mature wheat kernerals have been implicated in the undesirable 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 cereal 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, and the genes in ‘i’ class of II cluster which can be used in the further molecular marker related research seemed not on the wheat chromosome 2A and 2D. 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. To understand the effect of non-synonymous cSNPs in PPO gene on grain PPO activity, primer was designed as STS-H at some non-synonymous cSNPs site. 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). A total of 130 wheat cultivars were used to validate the correlation between the polymorphic fragments of STS-H and grain PPO activity. The results showed that PPO alleles ‘a’ and ‘b’ at the STS-H locus gave significantly different effect on grain PPO activity. The cultivars with allele ‘b’ had greater (P<0.01) PPO activity than the cultivars with allele ‘a’. To enhance the selection efficiency of single dominance molecular marker, the multiplex PCR was developed between STS-H and STS01, which is a STS molecular marker for low PPO activity and complementary with STS-H tested in this study.