一个苹果β-微管蛋白基因及其启动子序列特征分析

doi:10.7668/hbnxb.2018.03.013

摘要/Abstract

摘要： 为了探讨来自苹果的β-微管蛋白基因家族成员之一（转录本号：MDP0000749824.1）对茎生长的影响，以普通型苹果品种烟富3号和柱型苹果品种舞姿的茎尖为材料，克隆到了该基因的编码序列，该序列全长1 332 bp，编码443个氨基酸。DNAMAN比对结果表明，烟富3号和舞姿上该基因的编码序列完全一致。以舞姿及烟富3号的DNA为模板，用启动子特异性引物PCR扩增后，均获得一段1 661 bp的核苷酸序列，二者有6处碱基差异。启动子序列分析发现，其中具有赤霉素、脱落酸、茉莉酸甲酯和水杨酸等激素响应元件。构建Cam35S-MDP0000749824-GFP重组载体，在烟草叶片中完成瞬时表达，激光共聚焦显微镜观察结果表明，MDP0000749824.1编码蛋白位于细胞膜和微管上。以普通型苹果品种烟富3号和柱型苹果品种舞姿的茎尖组织为试材，实时荧光定量PCR （qRT-PCR）分析表明，该基因在柱型苹果舞姿茎尖中的转录水平显著低于普通型的烟富3号。因此，推测该基因的表达水平可能受激素调控的作用，进而对茎的伸长生长造成影响。

关键词: 苹果, 微管蛋白基因, 亚细胞定位, 基因表达, 启动子

Abstract: As the basic component of plant microtubules,tubulins are of great significance to the formation of cytoskeleton. To investigate the effect of one member of the β-tubulin gene family with the transcript number of MDP0000749824.1 on the stem growth,the coding sequence of this gene was cloned from the apple young shoot tips of standard apple variety Yanfu 3 and the columnar variety Telemon,respectively. This sequence was 1 332 bp in length which encoded a peptide with 443 amino acids. The results of DNAMAN alignment showed that the coding sequence of the gene was identical between Yanfu 3 and Telemon.Using DNA of Yanfu 3 and Telemon as templates,1 661 bp nucleotide sequences was obtained by PCR amplification with promoter specific primers. There were six bases differences between them. Promoter sequence analysis revealed that some hormone responsive elements of gibberellin,abscisic acid,methyl jasmonate and salicylic acid existed in it. Cam35S-MDP0000749824-GFP recombinant vector was constructed. Subcellular localization analysis with tobacco leaves indicated that the protein encoded by MDP0000749824.1 was on the cell membrane and microtubules. qRT-PCR detected that the transcript level of this gene in apical stems of columnar apple Telemon was significantly lower than that of standard apple Yanfu 3. It was speculated that the gene could affect the stem elongation be cause its expression level was regulated by hormones.

Key words: Apple, Tubulin gene, Subcellular localization, Gene expression, Promoter

中图分类号:

S661.03
Q78

张海月, 肖玉雄, 田义轲, 王彩虹, 杨绍兰, 李昱, 李玉琪. 一个苹果β-微管蛋白基因及其启动子序列特征分析[J]. 华北农学报, 2018, 33(3): 78-85. doi: 10.7668/hbnxb.2018.03.013.

ZHANG Haiyue, XIAO Yuxiong, TIAN Yike, WANG Caihong, YANG Shaolan, LI Yu, LI Yuqi. Sequence Characters Analysis of a β-Tubulin Gene and Its Promoter in Apple[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(3): 78-85. doi: 10.7668/hbnxb.2018.03.013.

http://www.hbnxb.net/CN/Y2018/V33/I3/78

参考文献

[1] 高囡囡,鲍岚. 微管蛋白的翻译后修饰及功能研究[J]. 生命科学,2015,27(3):363-373.
[2] Rasmussen C G,Wright A J,Müller S. The role of the cytoskeleton and associated proteins in determination of the plant cell division plane[J]. The Plant Journal:for Cell and Molecular Biology,2013,75(2):258-269.
[3] Krtková J,Benáková M,Schwarzerová K. Multifunctional microtubule-associated proteins in plants[J]. Frontiers in Plant Science,2016,7(436):474.
[4] Hiwatashi Y,Sato Y,Doonan J H. Kinesins have a dual function in organizing microtubules during both tip growth and cytokinesis in Physcomitrella patens[J]. The Plant Cell,2014,26(3):1256-1266.
[5] Paredez A R,Somerville C R,Ehrhardt D W. Visualization of cellulose synthase demonstrates functional association with microtubules[J]. Science,2006,312(5779):1491-1495.
[6] Lindeboom J,Mulder B M,Vos J W,et al. Cellulose microfibril deposition:coordinated activity at the plant plasma membrane[J]. Journal of Microscopy,2008,231(2):192-200.
[7] Gutierrez R,Lindeboom J J,Paredez A R,et al. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments[J]. Nature Cell Biology,2009,11(7):797-806.
[8] Onelli E,Idilli A I,Moscatelli A. Emerging roles for microtubules in angiosperm pollen tube growth highlight new research cues[J]. Frontiers in Plant Science,2015,6:51.
[9] Catterou M,Dubois F,Schaller H,et al. Brassinosteroids,microtubules and cell elongation in Arabidopsis thaliana. Ⅱ. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant[J]. Planta,2001,212(5/6):673-683.
[10] Hardham A R. Microtubules and biotic interactions[J]. The Plant Journal:for Cell and Molecular Biology,2013,75(2):278-289.
[11] Microtubules N P. Signalling and abiotic stress[J]. Plant Journal,2013,75(2):309.
[12] Wang W Z,Lazareva E,Kyreev I,et al. The role of microtubules in the maintenance of regular localization and arrangement of Golgi apparatus in root cells of Triticum aestivum L.[J]. Process Biochemistry,2012,47(11):1545-1551.
[13] Hamada T,Ueda H,Kawase T,et al. Microtubules contribute to tubule elongation and anchoring of endoplasmic reticulum,resulting in high network complexity in Arabidopsis[J]. Plant Physiology,2014,166(4):1869-1876.
[14] Liaud M F,Brinkmann H,Cerff R. The beta-tubulin gene family of pea:primary structures,genomic organization and intron-dependent evolution of genes[J]. Plant Molecular Biology,1992,18(4):639-651.
[15] Snustad D P,Haas N A,Kopczak S D,et al. The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes[J]. The Plant Cell,1992,4(5):549-556.
[16] Oakley R V,Wang Y S,Ramakrishna W,et al. Differential expansion and expression of alpha-and beta-tubulin gene families in Populus[J]. Plant Physiology,2007,145(3):961-973.
[17] Villemur R,Joyce C M,Haas N A,et al. Alpha-tubulin gene family of maize(Zea mays L.). Evidence for two ancient alpha-tubulin genes in plants[J]. Journal of Molecular Biology,1992,227(1):81-96.
[18] Villemur R,Haas N A,Joyce C M,et al. Characterization of four new beta-tubulin genes and their expression during male flower development in maize(Zea mays L.)[J]. Plant Molecular Biology,1994,24(2):295-315.
[19] Yoshikawa M,Yang G,Kawaguchi K,et al. Expression analyses of beta-tubulin isotype genes in rice[J]. Plant & Cell Physiology,2003,44(11):1202-1207.
[20] 侯董亮,王彩虹,田义轲,等. 梨β-微管蛋白基因及其在矮生型与普通型梨茎尖中的表达差异[J]. 园艺学报,2016,43(2):320-328.
[21] Gavazzi F,Pigna G,Braglia L,et al. Evolutionary characterization and transcript profiling of β-tubulin genes in flax(Linum usitatissimum L.)during plant development[J]. BMC Plant Biology,2017,17(1):237.
[22] 睢金凯,饶国栋,张建国. 柳树β-微管蛋白基因家族的克隆和序列分析[J]. 西北植物学报,2016,36(5):902-909.
[23] Rao G,Zeng Y,He C,et al. Characterization and putative post-translational regulation of α-and β-tubulin gene families in Salix arbutifolia[J]. Scientific Reports,2016,6:19258.
[24] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J]. Methods,2001,25(4):402-408.
[25] Blazek J. Segregation and general evaluation of spur type or compact growth habits in apples[J]. Acta Horticulturae,1992,317(317):71-80.
[26] Fallahi E,Simons B R,Fellman J K,et al. Tree growth and productivity and postharvest fruit-quality in various strains of delicious apple[J]. Journal of the American Society for Horticultural Science,1994,119(3):389-395.
[27] 杨佩芳,郝燕燕,田彩芳. 苹果短枝型品种导管分子的解剖学研究[J]. 园艺学报,2000,27(1):52-54.
[28] Bai T,Zhu Y,Fernández-Fernández F,et al. Fine genetic mapping of the Co locus controlling columnar growth habit in apple[J]. Molecular Genetics and Genomics,2012,287(5):437-450.
[29] Moriya S,Okada K,Haji T,et al. Fine mapping of Co,a gene controlling columnar growth habit located on Apple(Malus×domestica Borkh.)linkage group 10[J]. Plant Breeding,2012,131(5):641-647.
[30] Zhang Y G,Guo S X,Dai H Y. Morphological differences of vessels in the secondary xylem of columnar and standard apple trees[J]. Phyton,International Journal of Experimental Botany,2012,81(1):229-232.
[31] 宋杨,张艳敏,吴树敬,等. 短枝型苹果赤霉素受体基因MdGID1a 及其启动子克隆和表达分析[J]. 园艺学报,2013,40(11):2237-2244.
[32] Talwara S,Grout B,Toldam-Andersen T B. Modification of leaf morphology and anatomy as a consequence of columnar architecture in domestic apple(Malus×domestica Borkh.)trees[J]. Scientia Horticulturae,2013,164(2):310-315.
[33] Wolters P J,Schouten H J,Velasco R,et al. Evidence for regulation of columnar habit in apple by a putative 2OG-Fe(Ⅱ)oxygenase[J]. The New Phytologist,2013,200(4):993-999.
[34] Lee J M,Looney N E. Absisic acid levels and genetic compaction in apple seedlings[J]. Canadian Journal of Plant Science,1977,57(1):81-85.
[35] Looney N E,Lane W D. Spur-type growth mutants of McIntosh apple a review of their genetics,physiology and field performance[J]. Acta Horticulturae,1984,146(146):31-46.
[36] Watanabe M,Suzuki A,Komori S,et al. Comparison of endogenous IAA and cytokinins in shoots of columnar and normal type apple trees[J]. Engei Gakkai Zasshi,2004,73(1):19-24.
[37] Watanabe M,Bessho H,Suzuki A,et al. Seasonal changes of IAA and cytokinin concentration in shoots of columnar type apple trees[J]. Acta Horticulturae,2008,774(774):75-80.
[38] Krost C,Petersen R,Lokan S,et al. Evaluation of the hormonal state of columnar apple trees(Malus×domestica)based on high throughput gene expression studies[J]. Plant Molecular Biology,2013,81(3):211-220.

选择文件类型/文献管理软件名称

选择包含的内容