Cloning and Expression Analysis of Invertase Inhibitor Gene Families from Dendrobium officinale

doi:10.13560/j.cnki.biotech.bull.1985.2017-0746

Abstract

Abstract: Invertase inhibitor（InvInh）controls the activity of invertase and plays a significant role in the sucrose metabolic processes. This study aims to clone member of InvInh gene in a rare and endangered medicinal plant Dendrobium officinale,and to quantitatively detect the expression in different organs and to conduct bioinformatics analysis. As results,three InvInh genes were cloned,named as DoInvInh1,DoInvInh2,and DoInvInh3,and they encoded 176,177,and 177 amino acids,respectively. Bioinformatics analysis of 3 DoInvInh proteins showed that there were 1 signal peptide,2 transmembrane structures,and the conservative domain structure of PMEI protein. Though the homology of InvInh protein amino acid sequence was very low among different species,there were 4 conservative Cys and SP sites in all of them. The results of quantitative real-time PCR demonstrated that the expressions of all 3 DoInvInh genes were detected in the roots,stems,leaves,and flowers. The DoInvInh2 and DoInvInh3 genes expressed the lowest in roots,while the highest in flowers. The DoInvInh1 and DoInvInh3 presented a similar gene expression pattern in stems at different growth years of D. officinale,i.e.,the highest expression in the stems of one year,the medium in the stems of two years,and the lowest in stems of three years. The DoInvInh2 gene was at the highest expression in the stems of two years. The expressions of DoInvInh1 and DoInvInh3 genes were significantly in negative correlation with CIN activities,speculated that two genes probably played regulatory roles in CIN activities.

Key words: Dendrobium officinale, invertase inhibitor, gene cloning, gene expression

MIAO Xiao-rong, NIU Jun-qi, MO Zhao-zhan, WANG Ai-qin, HE Long-fei. Cloning and Expression Analysis of Invertase Inhibitor Gene Families from Dendrobium officinale[J]. Biotechnology Bulletin, 2018, 34(1): 129-136.

References

[1] Sturm A. Invertases：primary structures, functions and roles in plant development and sucrose partitioning [J]. Plant Physiol, 1999, 121（1）：1-8.
[2] Jin Y, Ni DA, Ruan YL. Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weight and fruit hexose level [J]. Plant Cell, 2009, 21（7）：2072-2089.
[3] 成善汉, 柳俊, 谢从华, 等. 植物转化酶抑制子及其分子生物学的研究进展 [J]. 海南大学学报：自然科学版, 2007, 25（1）：83-87.
[4] Rausch T, Greiner S. Plant protein inhibitors of invertases [J]. Biochim Et Biophys Acta, 2004, 1696（2）：253-261.
[5] Krausgrill S, Greiner S, Köster U, et al. In transformed tobacco cells the apoplasmic invertase inhibitor operates as a regulatory switch of cell wall invertase [J]. Plant J, 1998, 13（2）：275-280.
[6] Palmer W, Ru L, Jin Y, et al. Tomato ovary-to-fruit transition is characterized by a spatial shift of mRNAs for cell wall invertase and its Inhibitor with the encoded proteins localized to sieve elements [J]. Mol Plant, 2015, 8（2）：315-328.
[7] Hothorna M, Ende WVD, Lammens WL, et al. Structural insights into the pH-controlled targeting of plant cell-wall invertase by a specific inhibitor protein [J]. Proc Nati Acad Sci USA, 2010, 107（40）：17427-17432.
[8] Huang GJ, Sheu MJ, Chang YS, et al. Isolation and characterisation of invertase inhibitor from sweet potato storage roots [J]. J Sci Food Agr, 2008, 88（15）：2615-2621.
[9] Liu X, Song BT, Zhang HL, et al. Cloning and molecular characterization of putative invertase inhibitor genes and their possible contributions to cold-induced sweetening of potato tubers [J]. Molecular Genetics Genomics, 2010, 284（3）：147-159.
[10] Mckenzie MJ, Chen RKY, Harris JC, et al. Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers [J]. Plant Cell Environ, 2013, 36（1）：176-185.
[11] 蒋园, 朱玉球, 高燕会, 等. 铁皮石斛WRKY5基因的克隆与表达分析 [J]. 中草药, 2016, 47（2）：301-308.
[12] 牛俊奇, Phan Thi Thu, 邵敏, 等. 甘蔗工艺成熟期转化酶及其抑制子与蔗糖积累的相关性研究 [J]. 西南农业学报, 2015, 28（4）：1606-1611.
[13] Greiner S, Krausgrill S, Rausch T. Cloning of a tobacco apoplasmic invertase inhibitor. Proof of function of the recombinant protein and expression analysis during plant development [J]. Plant Physiol, 1998, 116（2）：733-742.
[14] 牛俊奇, 黄静丽, 张琨琨, 等. 甘蔗转化酶抑制子（SoInvInh1）基因克隆和表达分析 [J]. 中国农业大学学报, 2015, 20（3）：38-45.
[15] Hothorn M, Wolf S, Aloy P, et al. Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins [J]. Plant Cell, 2014, 16（12）：3437-3447.
[16] Tang X, Tao S, Mei H, et al. Suppression of extracellular invertase inhibitor gene expression improves seed weight in soybean（Glycine max） [J]. J Exp Bot, 2017, 68（3）：469-482.
[17] Qin G, Zhu Z, Wang W, et al. A tomato vacuolar invertase inhibitor mediates sucrose metabolism and influences fruit ripening [J]. Plant Physiol, 2016, 172（3）：1596-1611.
[18] Su T, Wolf S, Han M, et al. Reassessment of an Arabidopsis cell wall invertase inhibitor AtCIF1 reveals its role in seed germination and early seedling growth [J]. Plant Mol Biol, 2016, 90（1）：137-155.
[19] Chen SF, Liang K, Yin DM, et al. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis [J]. J Enzyme Inhib Med Ch, 2016, 31（6）：1381-1385.
[20] Ferrieri AP, Arce CC, Machado RA, et al. A Nicotiana attenuata cell wall invertase inhibitor（NaCWII）reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants [J]. New Phytol, 2015, 208（2）：519-530.