摘 要：Wuschel（Wus）转录因子基因在维持干细胞群数量上具有关键性的调控作用。以模式植物拟南芥Wus为信息探针，在杨树基因组中共检测到2个Wus候选基因，并设计了基因特异的引物，从毛白杨形成层cDNA中分离得到长度分别为922 bp和956 bp的2个cDNA，其分别含有编码258个和264个氨基酸残基的完整开放阅读框。所推导的蛋白质氨基酸序列在结构功能域区域分别与拟南芥Wus蛋白的同源性为76.0%和74.9%，故将其命名为PtWus-1和PtWus-2。在此基础上，组合利用MEGA3.1和DnaSP4.0软件对毛白杨36株基因型个体的PtWus-1和PtWus-2序列进行比对和分析，分别检测到58个和51个单核苷酸多态性（SNP）位点，多样性分别为1/27 bp和1/30 bp。对于PtWus-1，共检测到24个常见SNPs和34个罕见SNPs，其中43个属于转换， 15个属于颠换；在外显子区域，共检测到21个SNP位点，其中16个为同义突变，4个为错义突变，1个为无义突变。而对于PtWus-2，基因内部含有28个常见SNPs和23个罕见SNP，其中36个属于转换，15个属于颠换；在编码区域检测到的26个SNPs中，同义突变和错义突变均为13个。对PtWus-1和PtWus-2基因内SNPs进行的连锁不平衡分析结果显示，随着核苷酸序列长度的增加，SNPs的连锁不平衡在基因内部迅速衰退，因此，在毛白杨中，基于候选基因的连锁不平衡作图是可行的，而基于整个杨树基因组的连锁不平衡作图是不可行的，也是不必要的。本研究为毛白杨Wus蛋白转录因子基因的连锁不平衡作图及其基因辅助毛白杨木材纤维性状的分子育种提供了理论依据。
Abstract: The Wuschel (Wus) encoding a homeodomain protein which presumably acts as a transcriptional factor plays a key role in maintaining a pool of pluripotent stem cells. In this study, 2 putative sequences of Wus were identified in the whole genome of Populus by underlying the electronic cloning technique, based on the Populus genome sequence using Arabidopsis Wus gene sequence information. Two cDNA clones encoding Wus were isolated from the cDNA prepared from cambium of Populus tomentosa by the gene specific RT-PCR amplification. The two cDNAs were 922 bp and 956 bp in length with corresponding open reading frames (ORFs) which are capable of encoding the protein of 258 and 264 amino acids (aa), respectively. The deduced aa sequence of them shared 76.0% and 74.9% identity with functional domain of A.thaliana Wus, respectively. They were, therefore, named as PtWus-1 and PtWus-2. The genomic sequences of PtWus-1 and PtWus-2 in 36 individuals were aligned, compared and analyzed using the software MEGA3.1 and DnaSP4.0. A total of 58 and 51 single nucleotide polymorphisms (SNPs) were detected and the diversity of them was 1/27 bp and 1/30 bp, respectively. As for PtWus-1, 24 of 58 were common SNPs and 34 were rare SNPs. There were 43 transitions and 15 transversions of mutation types. In total, 21 SNPs were detected in the coding regions of PtWus-1, of which 16 were silent mutations and 4 were missense mutations and 1 was nonsense mutation. In PtWus-2, 28 were common SNPs and 23 were rare SNPs, of which 36 were transitions and 15 transversions of mutation types. In total, 26 SNPs were detected in coding regions of PtWus-2, in which the numbers of silent mutations and missense mutations were 13. The linkage disequilibrium of SNPs in the PtWus-1 and PtWus-2 was detected and the result showed that LD declined rapidly within the gene regions of PtWus-1 and PtWus-2. It suggested that in Populus genome wide LD mapping may not be feasible and not be necessary, but candidate gene based LD mapping could be particularly useful in breeding programs of forest trees. The results, therefore, provided the important foundation for association genetics of Wus genes and gene assisted breeding of new germplasms with desirable wood fiber traits in P. tomentosa.
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