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利用同源克隆及RACE结合方法,首次从刺槐(Robinia pseudoacacia)茎部cDNA中获得纤维素合酶基因RpCesA2并获取基因组DNA序列。通过生物信息学分析,发现该基因内部含有3 291 bp完整编码区,编码1 097个氨基酸残基的蛋白;包含纤维素合成酶蛋白典型结构域:如1个锌指结构(zinc finger)及8个跨膜区(Transmembrane domain),与拟南芥纤维素合成酶基因CesA1至CesA10蛋白序列相似性达56.27%(At CesA8)~79.67%(AtCesA2)。通过构建系统进化树,发现RpCesA2基因与CesA5、CesA6、CesA9基因亲缘关系较近。组织特异性Realtime-PCR结果表明,RpCesA2基因在刺槐根、茎、叶片、叶柄4个组织部位具有不同的表达模式:第一时期、第三时期均在叶片中表达量最高,第二个时期茎中表达量最高。在此基础上,对10个刺槐无性系单株RpCesA2序列进行测序比对分析,检测到50个单核苷酸多态性(SNP)位点,其出现频率为1/130。CDS区域共有22个SNPs,其中15个是同义突变,7个为错义突变,非同义突变与同义突变比率为0.47。该研究结果为RpCesA2基因进一步连锁不平衡作图与关联研究提供理论依据,并最终有助于刺槐纤维素合成途径研究、标记辅助分子育种。
Using homologous cloning and RACE method, we first obtained the RpCesA2 gene from the stem cDNA of Robinia pseudoacacia and obtained the genomic DNA sequence. Bioinformatics analysis revealed that the gene contains a 3 291 bp complete coding region and encodes a protein of 1 097 amino acid residues; a typical domain containing a cellulose synthase protein such as a zinc finger and 8 The transmembrane domain shared 56.27% (At CesA8) ~ 79.67% (AtCesA2) similarity with Arabidopsis thaliana cellulose synthase gene CesA1 to CesA10. By constructing phylogenetic tree, it was found that RpCesA2 gene was closely related to CesA5, CesA6 and CesA9 genes. Tissue-specific Realtime-PCR results showed that RpCesA2 gene had different expression patterns in the four tissues of Robinia pseudoacacia roots, stems, leaves and petioles. The first and third periods all showed the highest expression level in leaves, and the second stage stem In the highest expression. On this basis, we sequenced and compared the RpCesA2 sequences of 10 clones of Robinia pseudoacacia clones. Fifty single nucleotide polymorphisms (SNPs) were detected and the frequency of occurrence was 1/130. There are 22 SNPs in the CDS region, of which 15 are synonymous mutations and 7 are missense mutations. The ratio of non-synonymous mutations to synonymous mutations is 0.47. The results provide a theoretical basis for the further linkage disequilibrium mapping and correlation studies of RpCesA2 gene, and ultimately contribute to the research on the pathway of cellulose synthesis in R. pseudoacacia and marker-assisted molecular breeding.