林业科学 ›› 2018, Vol. 54 ›› Issue (8): 79-87.doi: 10.11707/j.1001-7488.20180809

• 论文与研究报告 • 上一篇    下一篇


任健, 殷雨晴, 张慧慧, 陈典, 王可新, 王勇   

  1. 东北农业大学园艺园林学院 农业部东北地区园艺作物生物学与种质创制重点实验室 哈尔滨 150030
  • 收稿日期:2017-10-23 修回日期:2017-12-01 出版日期:2018-08-25 发布日期:2018-08-18
  • 基金资助:

Cloning and Analysis of AmCesA1 Gene in Acacia mangium

Ren Jian, Yin Yuqing, Zhang Huihui, Chen Dian, Wang Kexin, Wang Yong   

  1. Key Laboratory of Biology and Genetic Improvement of Horticultural Crops in Northeast Region, Ministry of Agriculture College of Horticulture and Landscape Architecture, Northeast Agricultural University Harbin 150030
  • Received:2017-10-23 Revised:2017-12-01 Online:2018-08-25 Published:2018-08-18

摘要: [目的]马占相思是我国南方广泛种植的一种造纸用树。纤维素合酶(CesA)在植物纤维素合成途径中发挥主要调节作用,是控制木材纤维品质和产量的重要因素。本研究克隆马占相思的纤维素合酶基因,研究它对激素的应答,为了解马占相思的纤维素合成和获得高纤维素得率的马占相思良种提供帮助。[方法]采用RT-PCR和RACE技术,从马占相思幼苗中获得1个CesA基因,命名为AmCesA1(AY643519)。利用生物信息学软件对该基因进行分析。使用Southern分析确定该基因的拷贝形式。采用实时荧光定量PCR确定该基因在不同组织中的表达量以及基因在赤霉素(GA3)、6-BA、茉莉酸甲酯(MeJA)处理后的表达变化。[结果]AmCesA1的cDNA大小为3 793 bp,其开放读码框为3 249 bp,推测这个基因编码1 082个氨基酸;蛋白分子式为C5495H8491N1457O1579S50,分子质量为121.83 kDa。正电荷氨基酸残基数(Arg+Lys)的数目为121,负电荷氨基酸残基数(Asp+Glu)的数目为125;等电点为6.51,为酸性蛋白质;它的不稳定系数是40.82,属于不稳定蛋白质。AmCesA1的氨基酸一级结构分析显示它具有纤维素合酶保守的D,D,D,QxxRW功能域,具有植物纤维素合酶所特有的P-CR区及HVR区和N端的锌指结构,在C端存在6个跨膜区域,但在N端的2个跨膜区域并不明显。二级结构分析显示它具有较多的α-螺旋、无规则卷曲,β-转角很少,β-折叠数目因算法的不同存在较大差异。聚类分析表明,AmCesA1与大豆GmCesA1和蔓花生AdCesA1相似性较高。但并没有出现预期同木本植物亲缘较近的结果。进一步与拟南芥纤维素合酶家族氨基酸序列比较,发现AmCesA1与拟南芥的AtCesA1和AtCesA10比较相似,其相似性为86%和80%,从而推测它的功能与拟南芥的AtCesA1和AtCesA10相近。Southern分析显示,马占相思基因组中,AmCesA1是以多拷贝形式存在。实时荧光定量PCR表明,AmCesA1广泛表达于根、茎、叶部位,且差异不显著。AmCesA1对GA3、6-BA、MeJA处理均有响应,其中GA3处理响应相对最强。[结论]本研究克隆到的马占相思AmCesA1为植物CesA基因家族中的一员,推测其参与初级细胞壁的形成。该基因对赤霉素、6-BA、茉莉酸甲酯处理均有响应,其中基因的表达量在不同的激素处理中都有不同程度的上调。表明该基因参与马占相思对激素应答的正调控。

关键词: 马占相思, AmCesA1, 基因克隆, 聚类分析, 激素应答

Abstract: [Objective] Acacia mangium is widely cultivated in southern China for paper production. Cellulose synthase (CesA) plays a vital role in the synthesis of cellulose, which is an important factor in controlling the quality and yield of wood fiber. In this study, we cloned a cellulose synthase gene of A.mangium, and studied its response to hormones to help the understanding of the synthesis of cellulose and the high fiber yield of A.mangium.[Method] A CesA was obtained from A.mangium seedlings by RT-PCR and RACE, named AmCesA1 (AY643519). The gene was analyzed by bioinformatics software. The copy form of the gene was determined through using Southern analysis.The expression level of the gene in different tissues and the expression of gibberellin,6-BA and methyl jasmonate were determined by real-time fluorescence quantitative PCR.[Result] The molecular size of AmCesA1 is 3 793 bp, and its ORF is 3 249 bp,suggesting that this gene encodes 1 082 amino acids.Protein molecular formula is C5495H8491N1457O1579S50. The number of positive charge amino acid residues (Arg + Lys) is 121, and the number of negative charge amino acid residues (Asp + Glu) is 125. The isoelectric point is 6.51, which means that it is acidic protein. Its instability coefficient is 40.82, belonging to the unstable protein. The amino acid primary structure analysis of AmCesA1 showed that it had the conserved D, D, D and QxxRW functional domains of cellulose synthase, and had a unique P-CR region and the HVR region and N-terminal zinc finger structure. There were six transmembrane regions at the C-terminus, but the two transmembrane regions at the N-terminus were not significant. Secondary structure analysis showed that it had more α-helix, random coil, but fewer β-turn, while β-sheet number varies greatly due to the different algorithms. Cluster analysis showed that AmCesA1 had similarity with Glycine max GmCesA1 and Arachis duranensis AdCesA1.But it did not appear to be close to the results of woody plants. Further comparison with Arabidopsis thaliana cellulose gene family amino acid sequence indicated a fact that AmCesA1 was similar to AtCesA1 and AtCesA10 in A.thaliana, and its similarity was 86% and 80%, suggesting that it had the same function as A.thaliana AtCesA1 and AtCesA10.Southern analysis showed that AmCesA1 was present in multiple copies of the Acacia tree genome. Real-time quantitative PCR showed that AmCesA1 was widely expressed in roots, stems and leaves, and the differences among them were not significant. AmCesA1 had a response to GA3, 6-BA and MeJA treatments, in which the response to GA3 was relatively strong.[Conclusion] AmCesA1 cloned in this study is a member of the plant CesA family, presumably involved in the formation of primary cell walls. The gene was responsive to gibberellin, 6-BA and methyl jasmonate, and the expression level of the gene was up-regulated in different hormone treatments,which indicates that the gene was involved in the positive regulation of the hormone response.

Key words: Acacia mangium, AmCesA1, gene cloning, cluster analysis, hormone response