论文部分内容阅读
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 content of amylose in the autotetraploid rice mutant D4063-1 was reduced by half compared with the diploid Minghui63, that is, the amylose content was 5.23%. In order to study the reason of the decrease of amylose content, the full-length sequence of D4063-1Wx gene was amplified and sequenced according to the Wx gene of common rice and compared with the reported Wx gene. The autotetraploid rice The most significant change in the D4063-1Wx gene was a base deletion in the exonic sequence, resulting in a frameshift mutation that preceded the stop codon at exon 9. The change of the base site of the D4063-1Wx gene also resulted in the change of its sequence restriction site. The analysis of common restriction enzyme sites indicated that the number of autotetraploid rice was two more than that of indica and japonica sph Ⅰ restriction site, compared with japonica rice decreased by 6 Acc Ⅰ, an increase of 4 Xba Ⅰ, 1 Xho Ⅰ, 1 Pst Ⅰ and 1 Sal Ⅰ restriction sites. Cluster analysis showed that the D4063-1Wx gene was closely related to indica, suggesting that the D4063-1Wx gene was derived from the Wxa genotype of indica. In addition, according to the base difference of the D4063-1Wx gene, it is speculated that the RNA processing obstacle caused by the exon base change of the D4063-1Wx gene is the main reason for the amylose reduction, and may be related to the quality of its soft rice. Based on the sequence differences between D4063-1 and indica rice and japonica rice and the characteristic sequence of D4063-1 on this fragment, we designed an oligonucleotide fragment for D4063-1 and named it as AUT4063-1, the primer was designed to amplify the common indica rice, japonica rice Wx gene primer F5 with the establishment of identification D4063-1 dominant and co-dominant two detection methods of molecular markers for rapid and accurate Identifying low amylose content mutant D4063-1 created the conditions.