以10个O2O2基因型的普通玉米自交系和3个o2o2基因型的高赖氨酸玉米自交系为材料，用3对O2基因内SSR引物phi112、umc1066和phi057进行PCR扩增产物电泳图谱分析和特异片段序列比较分析，以期了解O2位点显性等位变异对赖氨酸含量的影响。结果表明，玉米phi112、umc1066和phi057三个SSR位点均有丰富的显性等位变异，phi112位点的变异主要发生在其SSR序列的两侧，umc1066和phi057两位点的变异主要表现为SSR序列内部重复数目的变化和点突变。这些变异均对玉米籽粒的赖氨酸含量有影响。还讨论了这些显性等位变异影响O2基因的转录效率及其翻译产物对激活玉米醇溶蛋白合成能力改变的可能机制。
The recessive allelic variations of SSRs at o2 locus and their effect on lysine level have been studied. The result showed that the variations could affect the lysine content of o2 maize. In this experiment, dominant allelic variations of SSRs at O2 locus were investigated, in order to know the effect of SSRs variations on lysine content. Ten O2O2 lines and three o2o2 lines were selected to perform PCR with three pairs of primers—phi112, umc1066 and phi057. The PCR products were sequenced and the lysine content of kernels of these 13 lines was determined by using dye-binding method. The results showed that the dominant allelic variations were diverse at phi112, umc1066 and phi057 sites. There were four alleles at each of three sites, respectively. Comparing to recessive alleles, one dominant allele had nucleotides insertion, two were same as one of two recessive alleles at phi112 site; three dominant alleles had nucleotides insertion, one were nearly same as recessive allele at umc1066 sites; three dominant alleles were identical with one of three recessive alleles at phi057 site, respectively (Fig.1). The variations of sequences flanking SSR were observed and complex, but sequence and number of SSR were not changed at phi112 site; the variations, i.e. increase or decrease of SSR motif besides point mutations, were observed at umc1066 and phi057 (Fig.2–4). At phi112 site, the bigger the variations of nucleotides flanking SSR, the higher the lysine content of kernels; the effect of nucleotides deletion on lysine content of kernels was bigger than other variations. At umc1066 site, the longer the inserted nucleotides, the higher the lysine content; the effect of nucleotide substitutions on lysine content was bigger than nucleotides insertion. At phi057 site, there was a trend of rising lysine content along with insertions and substitutions of nucleotides (Table 2–4). Our results indicate the difference of lysine content among the O2O2 lines may be resulted from following two reasons: one is that the nucleotides variations of phi112 site located in leading sequence of O2 gene affect transcription of O2 gene; another is that the nucleotides variations of umc1066 and phi057 located in coding sequence of O2 gene result in increase or decrease of prolin in their translated O2 proteins (Fig.5, 6), leading to change of O2 protein conformation, and then affecting amount of Zein synthesis. The fact that some dominant alleles are the same as one of recessive alleles, respectively, indicates it is possible to explain the difference of lysine level among normal maize materials by SSR marker detection. It also suggests that the significant difference of lysine content between O2O2 and o2o2 lines appears to be affected by other nucleotide sequence within O2 gene. These findings may be valuable for improving maize quality.
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