同源四倍体水稻籼型突变体Wx基因序列分析及特异识别分子标记的建立

Wx sequence analysis of a autotetraploid indica mutant rice and establishment of molecular marker for identifying

同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因,根据普通水稻Wx基因设计引物,扩增测序获得了D4063-1Wx基因的全序列,并与已报道的Wx基因进行比对分析;同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生碱基缺失,导致移码突变,在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致其序列上酶切位点的变化,对常用限制性内切酶位点分析结果表明,同源四倍体水稻相对于籼稻和粳稻多了2个sphⅠ酶切位点,相对于粳稻减少了6个AccⅠ,增加了4个XbaⅠ,1个XhoⅠ,1个PstⅠ和1个SalⅠ酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近,由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外,根据D4063-1Wx基因的碱基差异,推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因,并可能与其米饭较软等品质相关。本研究还根据D4063-1和籼稻、粳稻的序列差异及D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与作者设计的扩增普通籼稻、粳稻Wx基因的引物F5配合使用,建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确鉴别低直链淀粉含量突变体D4063-1创造了条件。

The amylose content of the mutant of autotetraploid indica rice D4063-1 is 5,23% anout, which was half of its origin diploid rice Minghui 63. The whole sequence of Waxy gene of D4063-1 was amplified and sequenced. A base was absent on the Wx of D4063-1 in exon sequence, which resulted in frameshift mutation and terminating codon occurred ahead in the 9 exon. The mutation of Wx also led to the change of some mutation in the 9 exon and terminating codon occurred early. The change of Wx also led to changes of the sites of common restriction endonuclease. The results showed that D4063-1 added two sph I sites compared to indica and japonica rice; Compared to japonica rice, D4063-1decreased six Acc I sites, and added 4 Xba I, a Pst I and a Sal I restriction sites. Phylogenic analysis showed that the DNA sequence of Waxy gene of D4063-1 was closer to indica rice. We supposed that the Waxy gene of D4063-1 originated from genotype of Wxa. According to the differences of Wx in D4063-1, we deduced the absent base led to RNA splicing obstacle, which was the main cause of low amylose content and it might be related to the soft rice phenotype. Based on analysis of Wx of D4063-1, indica and japonica and according to the special sites of the three species, primers as markers-AUT4063-I were designed to distinguish D4063-1 from other rice. Combining with primer pair FS, dominant and codominant ways were established for discriminating them, and rapid and correct identification of D4063- l from other rice could be done.