毛竹微管蛋白的分子特征及PeTUA3的功能

doi:10.11707/j.1001-7488.20200705

摘要/Abstract

摘要：

目的: 微管蛋白是植物细胞骨架的重要组成部分，在植物细胞分裂与生长、细胞形态维持、细胞内物质运输和细胞壁的生物合成等过程中均发挥着重要作用。为全面了解毛竹微管蛋白的分子特征，开展了毛竹微管蛋白全基因组鉴定与分析，并初步研究了PeTUA3基因的功能，以期为揭示微管蛋白在毛竹生长发育过程的作用提供参考依据。方法: 采用生物信息学的方法开展毛竹中微管蛋白基因的全基因组分析，利用转录组数据和实时荧光定量PCR技术分别分析微管蛋白基因的组织表达模式及其随笋高度增加的表达变化，通过在拟南芥中异位表达及表型观察初步研究PeTUA3基因的功能。结果: 在毛竹基因组中共获得18条微管蛋白基因序列，编码蛋白具有完整的保守结构域，蛋白长度为430~634 aa，分子量为48.31~69.89 kDa。在与水稻、拟南芥微管蛋白构建的系统进化树中，被聚类到2个亚家族(TUA和TUB)，且毛竹各微管蛋白成员与水稻的亲缘关系更近，这些基因分别命名为PeTUA1-PeTUA6和PeTUB1-PeTUB12。亚细胞定位预测显示PeTUAs均定位在细胞质中，而PeTUBs均定位于细胞核中。基于转录组数据的基因组织特异性表达分析表明，不同微管蛋白基因在毛竹7个不同组织中的表达量存在着一定的差异，如PeTUA3、PeTUA6、PeTUB2和PeTUB3在各个组织中均有较高的表达，而PeTUA2和PeTUB12在各组织中表达量均较低。实时荧光定量PCR结果显示，随着毛竹笋高度的增加，6个TUA亚家族成员基因的表达均呈现上调趋势，而TUB亚家族成员基因中有8个呈现上调趋势，4个表现为波动变化，表明它们在笋的不同发育阶段可能发挥着不同的作用。在拟南芥中正义表达PeTUA3促进了转基因植株的生长，尤其是对主根的伸长有明显促进作用，而转反义PeTUA3则抑制转基因植株的生长，主根生长明显受到抑制。结论: 从毛竹中鉴定了6个TUA基因和12个TUB基因，各基因的分子特征存在着一定的差异，并且各基因在毛竹不同组织以及不同发育阶段笋中呈现出不同的模式，说明它们在不同组织以及同一组织的不同生长发育阶段可能发挥着不同的功能；过量表达PeTUA3能够影响转基因拟南芥的生长，说明PeTUA3对毛竹的快速生长可能具有重要的作用。

关键词: 毛竹, 微管蛋白基因, 分子特征, 异位表达

Abstract:

Objective: Tubulin is an important component of plant cytoskeleton and plays an important role in plant cell division and growth,cell morphology maintenance,intracellular material transport and cell wall biosynthesis. In order to comprehensively understand the molecular characteristics of tubulin in Moso bamboo(Phyllostachys edulis),the genome-wide identification and analysis of tubulin were carried out,and the gene function of PeTUA3 was preliminarily studied,intending to provide evidences for revealing the role of tubulin involved in the growth and development of Moso bamboo. Method: A genome-wide analysis of tubulin genes in Moso bamboo was conducted using bioinformatics method. The tissue expression patterns of tubulin genes and their expression changes with increasing shoot height were analyzed by transcriptome data and real-time fluorescence quantitative PCR(qPCR) respectively. The function of PeTUA3 was preliminarily studied by ectopic expression in Arabidopsis thaliana followed by phenotypic observation. Result: A total of 18 tubulin gene sequences were obtained from the Moso bamboo genome,which encoded proteins with complete conserved domains of tubulin. The length of these proteins ranged from 430 aa to 634 aa with the molecular weight of 48.31-69.89 kDa. In the phylogenetic tree constructed with tubulins of Oryza sativa and A. thaliana, they were clustered into two subfamilies(TUA and TUB),and those members of Moso bamboo were closer to those of rice,which were named as PeTUA1-PeTUA6 and PeTUB1-PeTUB12, respectively. Subcellular localization prediction showed that all the PeTUAs were localized in the cytoplasm,while all the PeTUBs were localized in the nucleus. Gene-specific expression analysis based on transcriptome data showed that there were some differences in the expression levels of different tubulin genes in seven different tissues of Moso bamboo. For example,the expression levels of PeTUA3, PeTUA6, PeTUB2 and PeTUB3 were higher in each tissue,while those of PeTUA2 and PeTUB12 were lower in all the tissues. The qPCR demonstrated that with the increasing height of bamboo shoots,all the expression of the members in TUA subfamily was upregulated,while 8 genes in TUB subfamily showed upregulation trend,and 4 genes with fluctuations,indicating they may play different roles in different developmental stages of bamboo shoots. The expression of sense PeTUA3 in A. thaliana promoted the growth of transgenic plants,especially the elongation of the main roots,while that of antisense PeTUA3 inhibited the growth of transgenic plants,and the growth of the main roots was inhibited significantly. Conclusion: Six TUA genes and 12 TUB genes were identified in Moso bamboo. The molecular characteristics of each tubulin gene were different,and the expression of each gene in different bamboo tissues and shoots at different developmental stages showed different patterns,indicating that they may play different functions in different tissues and different growth stages of the same tissue. Overexpression of PeTUA3 could affect the growth of transgenic A. thaliana plants,suggesting that PeTUA3 may play an important role in the rapid growth of bamboo.

Key words: Phyllostachys edulis, tubulin, molecular characteristics, ectopic expression

中图分类号:

S718.46

朱成磊,李彩丽,李晓佩,史晶晶,高志民. 毛竹微管蛋白的分子特征及PeTUA3的功能[J]. 林业科学, 2020, 56(7): 44-54.

Chenglei Zhu,Caili Li,Xiaopei Li,Jingjing Shi,Zhimin Gao. Molecular Characteristics of Tubulins and Preliminary Function Analysis of PeTUA3 in Phyllostachys edulis[J]. Scientia Silvae Sinicae, 2020, 56(7): 44-54.

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用途 Application	引物名称 Primer name	正向序列 Forward sequence(5′—3′)	反向序列 Reverse sequence(5′—3′)
基因克隆 Gene cloning	ORF-TUA3	ATGAGAGAGTGCATCTCGATCCA	CTAGTACTCGTCTCCCTCATCACCTTC
载体构建 Vector construction	STUA3	tctagaATGAGAGAGTGCATCTCGATCCAC	ggtaccGTACTCGTCTCCCTCATCACCTTC
载体构建 Vector construction	ATUA3	ggtaccATGAGAGAGTGCATCTCGATCCAC	tctagaGTACTCGTCTCCCTCATCACCTTC
毛竹样品实时荧光定量PCR qPCR for samples of Moso bamboo	PeTUA1	ACACCGACGTTTCAATCCTCC	AGCACCATCAAACCTCAGGGA
	PeTUA2	GCAGTGATACCTCTATTGGGGTTG	TGTTAGCAGCGTCTTCCTTGTG
	PeTUA3	CCTTGCCAAGGTCTTCTCACG	CTCAGCACCAACCTCCTCGTA
	PeTUA4	GCGGGATAAACTATCAGCCACC	GCGTACATGAGATCGAACTTGC
	PeTUA5	GCATCAACTACCAGCCACCCT	ACCTCCTTTGTGCTCATCTTTCC
	PeTUA6	ACTGGACCGTATGGGCAGATC	TGGAAACCTTGAAGGCAGTCAC
	PeTUB1	CAAAGAGGTGGACGAGCAAATG	TCCTGGATCGAGGTCGAGTTG
	PeTUB2	CAAGTTCTGGGAGGTGGTCTG	CTCAAGCTGGAGGTCGGAGTC
	PeTUB3	CAAGGAGGTGGACGAGCAGAT	CCTGGATTGAGGTCGAGTTGC
	PeTUB4	CCAATGGCCTGAAGATGTCGT	CACCAGGTCGTTCATGTTGCT
	PeTUB5	ACCTTATCCCCTTCCCTCGTCTC	ACCTCCTTTGTGCTCATCTTTCC
	PeTUB6	ACTGGACCGTATGGGCAGATC	TGGAAACCTTGAAGGCAGTCAC
	PeTUB7	TGAGCCCTACAACGCAACACT	AGGCAGCATGTGACACCACTC
	PeTUB8	ATTGACCCTACCGGGCAGTTC	GGGCTCAAGGTCCATAAGAACA
	PeTUB9	GCACCAAGGAAGTGGACGAAC	AACATCTCCTGGATTGAGGTCG
	PeTUB10	ATGCTGACGTTCTCCGTGTTC	TCTCCAAAGCTAGGGGTGGTA
	PeTUB11	TCTGCCGGTTTCCAAGTATGC	CCAACTGGTGGACAGAAAGAGTG
	PeTUB12	CAACTTCGTCTTCGGCCAGTC	TTGGAGGCAGTCGCAATTCTC
	PeNTB	TCTTGTTTGACACCGAAGAGGAG	AATAGCTGTCCCTGGAGGAGTTT
拟南芥样品实时荧光定量PCR qPCR for samples of Arabidopsis	AtActin-7	GCCCCTGAGGAGCACCCAGTT	CCGGTTGTACGACCACTGGCA

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