组合利用生物信息学和RT-PCR方法，首次从毛白杨未成熟木质部cDNA中分离出PtCesA4 cDNA全长，并进行测序和序列分析，结果表明克隆的毛白杨PtCesA-4 cDNA片段总长为3 757bp，基因内部含有完整的开放阅读框架，大小为3 129 bp，可编码长度为1 042个氨基酸残基的蛋白质，所推导的蛋白质氨基酸序列与拟南芥AtCesA4、水稻OsCesA1和火炬松PtCesA2的蛋白质氨基酸序列同源性分别为80.3％，78.9％和75.6%。组织特异性RealtimePCR结果显示，PtCesA4基因在杨树根、茎、叶片和顶端分生组织中均有表达，但其表达模式却不同：PtCesA4在成熟叶片、未成熟木质部和成熟木质部中表达丰度最高，在根部和顶端分生组织表达丰度中等，在树皮和韧皮部有少量表达，在形成层中表达丰度最低。在此基础上，组合利用MEGA3-1和DnaSP450.4软件对毛白杨40株基因型个体的PtCesA4序列进行比对和分析，检测到153个单核苷酸多态性（single nucleotide polymorphism，SNP）位点，SNP频率为1/35 bp，多样性指数π为0.00502。其中51个是常见SNPs，102个为罕见SNPs。在这些SNPs中，有118个属于转换，35个属于颠换。在外显子区域，共检测到69个SNP位点，其中59个为同义突变，10个为错义突变。对PtCesA4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PtCesA4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。研究结果为毛白杨PtCesA4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。
The cellulose synthase gene(CesA) plays a key role in regulating cellulose biosynthesis during the wood formation. In this study, a full-length cDNA clone encoding PtCesA4 was isolated from the cDNA prepared from immature xylem zone of Populus tomentosa with the biological informatics and RT-PCR. The cDNA was 3 757 bp in length with an open reading frame (ORF) which would be capable to encode a protein of 1 042 AA. The deduced protein sequence of the PtCesA4 shared 80.3％, 78.9％ and 75.6% identity with Arabidopsis thaliana AtCesA4, Oryza sativa OsCesA1 and Pinus taeda PtCesA2, respectively. Realtime-PCR indicated that PtCesA4 transcripts had their mRNA products expressed in roots, stems, leaves and apical shoot meristems, but their expressions were differential in the different tissues. The PtCesA4 transcripts were the most abundant mRNA products in mature leaf, immature and mature xylem, with medium expression in root and apical shoot meristems, with some expression in the bark and phloem, but a lowest-abundance was detected in cambium. The genomic sequences of PtCesA4 in 40 individuals were aligned, compared and analyzed using the software MEGA3.1 and DnaSP4.50.7. A total of 153 single nucleotide polymorphisms (SNPs) were detected and the frequency and diversity of SNPs were 1/35 bp and 0.005 02, respectively. Among them, 51 were common SNPs and 102 were rare SNPs. There were 118 and 35 mutation types of transition and transversion, respectively. There were 69 SNPs detected in the coding regions of PtCesA4, of which 59 were silent mutations and 10 were missense mutations. The linkage disequilibrium of SNPs in the PtCesA4 was analyzed and the result showed that LD declines rapidly within the gene regions of PtCesA4 with the length increase. It suggested that wide LD mapping in Populus genome might not be feasible and not be necessary, but LD mapping based on PtCesA4 gene could be particularly useful in breeding programs of forest trees. The results, therefore, provided the important genetic foundation for associated with PtCesA4gene and gene-assisted breeding of new germplasms with desirable wood fiber traits in P. tomentosa.
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