刺槐纤维素合酶基因(RpCesA2)克隆、表达及SNP分析  

Cloning,Expression and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene(RpCesA2) from Robinia pseudoacacia

作　　者：张凯权[1,2] 马履一[1,2] 段劼[2] 汪力[1] 杨欣超[1,2] Zhang Kaiquan Ma Ltiyi Duan Jie Wang Li Yang Xinchao(Key Laboratory of Silviculture and Conversation, Ministry of Education, Beijing Forestry University, Beijing,100083 National Energy R＆D Center for Non-food Biomass, Beijing Forestry University, Beijing, 100083)

机构地区：[1]北京林业大学省部共建森林培育与保护教育部重点实验室,北京100083 [2]北京林业大学国家能源非粮生物质原料研发中心,北京100083

出　　处：《分子植物育种》2017年第2期474-482,共9页Molecular Plant Breeding

基　　金：国家国际科技合作专项项目(2014DFA31140);教育部博士点基金优先发展领域项目(20120014130001)共同资助

摘　　要：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。In this study, the RpCesA2 gene was cloned firstly from cDNA of stem of Robinia pseudoacacia by using the method of RACE（rapid-amplification of cDNA ends） and homologous cloning. bioinformation analysis indicated that the cDNA included a whole open reading frame of 3 291 bp, which was capable of encoding 1 097 amino acid, an and it had all the typical motifs of plant cellulose synthase： one Zn finger and eight transmembrane domain. The similarities of deduced protein sequence of RpCesA2 were from 56.27%（ At CesA8） to 79.67%（AtCesA2）, comparing with protein sequence of CesAs of Arabidopsis thaliana. The phylogenetic analysis indicated that CesA2, CesA5, CesA6, CesA9 had a close genetic relationship. Tissue-specific expression profiling showed that RpCesA2 had the different expression profile in the tissues, including root, leaf, stem, and petiole： %RpCesA2 was highly expressed in the leaf during the first and third period, and was highly expressed in the stem during the second period. The genomic sequence of RpCesA2 in 10 individuals were aligned, compared and analyzed. A total of 50 SNPs were detected, with the frequency of 1/130, there were 22 SNPs in the CDS region including 15 Synonymous mutations and 7 missense mutations, the ratio of non-synonymous mutation and synonymous mutation was 0.47. The results of this study will provide a theoretical basis for the study of linkage disequilibrium and association of RpCesA2 and willhelp to the study of cellulose synthase path and marker-assisted breeding in block locust.

关 键 词：刺槐 纤维素合成酶 纤维素 单核苷酸多态性 

分 类 号：S792.27[农业科学—林木遗传育种] Q943.2[农业科学—林学]