Sequence Analysis of MeSDH Protein and Its Relationship with MeH1.2 in Cassava

doi:10.13560/j.cnki.biotech.bull.1985.2024-0441

Abstract

Abstract:

【Objective】Sorbitol dehydrogenase（SDH）plays an important role in regulating the conversion between fructose and sorbitol in Roseviaceae plants, and also participates in plant resistance to stress. The study on the function of MeSDH gene may provide a theoretical basis for cultivating cassava germplasm with high yield and resistance to stress. 【Method】MeSDH gene was cloned using ‘SC124’ cDNA as template, and the tissue specificity of MeSDH gene and its response pattern to drought, low temperature, PEG and ABA were analyzed by quantitative real-time PCR（RT-qPCR）. MeH1.2 was used as bait to screen the mixed yeast cDNA library of cassava under drought and low temperature. Yeast two hybridization（Y2H）point-to-point and bimolecular fluorescence complementation（BiFC）experiments were further used to confirm the relationship between target proteins. 【Result】The CDS length of MeSDH gene cloned from ‘SC124’ cDNA is 1 092 bp, encoding 364 amino acids, and the sequence shows no difference with it in the database. MeSDH protein contains catalytic Zn binding site, NADP binding site and structural Zn binding site, belonging to the MDR superfamily. MeSDH protein is localized in the nucleus by transforming tobacco leaves. The expression of MeSDH gene is decreasing in the order of functional leaves, young leaves, fibrous roots and stems. The expression of MeSDH is up-regulated in the cassava leaves treated with drought，low temperature and PEG, and significantly up-regulated in the cassava leaves and roots treated with ABA. Yeast library screening, Y2H point-to-point and BiFC experiments confirm the interaction between MeH1.2 and MeSDH. 【Conclusion】 MeSDH gene is up-regulated in response to various stresses, and probably MeSDH and MeH1.2 work together.

Key words: cassava, SDH gene, MeH1.2 protein, resistance to stress, protein interaction

ZHAO Ping-juan, LIN Chen-yu, WANG Meng-yue, ZHANG Xiu-chun, LI Shu-xia, RUAN Meng-bin. Sequence Analysis of MeSDH Protein and Its Relationship with MeH1.2 in Cassava[J]. Biotechnology Bulletin, 2024, 40(9): 74-81.

Figures/Tables 9

Table 1 Primers’ sequences used in the study

引物名称Primer name	引物序列Primer sequence（5'-3'）	目的Purpose
MeSDH-F	atgggtaaaggagggatgtctc	基因克隆 Gene cloning
MeSDH-R	cagattaaacatgaccttaatg	基因克隆 Gene cloning
1300-MeSDH-F	tgatacatatgcccgtcgac atgggtaaaggagggatgtctc	植物表达载体 Plant expression vector
1300-MeSDH-R	ctcaccatggatccggtacc cagattaaacatgaccttaatg	植物表达载体 Plant expression vector
Actin1-F	tggattctggtgatggtgtgagt	内参基因 Reference gene
Actin1-R	ccgttcagcagtggtggtga	内参基因 Reference gene
MeSDH-Q-F	gtgaaggagccgatggtga	MeSDH定量PCR MeSDH RT-qPCR
MeSDH-Q-R	ccacacggtctccaggtaaaa	MeSDH定量PCR MeSDH RT-qPCR
H1.2-PGBKT7-X	tctagaatggccgactctgaagttcaggct	酵母文库筛选 Yeast library screening
H1.2-PGBKT7-B	ggatcctttcttcgccttcttcgctgtc	酵母文库筛选 Yeast library screening
MeSDH-AD-F	ccatggaggccagtgaattcatgggtaaaggagggatgtc	Y2H载体 Y2H vector
MeSDH-AD-R	agctcgagctcgatggatccttacagattaaacatgacctt	Y2H载体 Y2H vector
MeSDH-C-F	tctagaatggccgactctgaagttcaggct	BiFC载体 BiFC vector
MeSDH-C-R	ggatcctttcttcgccttcttcgctgtc	BiFC载体 BiFC vector

Table 2 Sample design

A 实验组1	B 实验组2	C 实验组3	D 实验组4
pGADT7-T	pGADT7-T	pGADT7-MeSDH	pGADT7
pGBKT7-53	pGBKT7-lam	pGBKT7-MeH1.2	pGBKT7-MeH1.2

Fig. 1 PCR amplification of MeSDH gene M: DL2000 DNA marker; SDH: PCR amplification product of MeSDH gene

Fig. 2 Conserved domain analysis of MeSDH protein

Fig. 3 Tissue-specific expressions of MeSDH gene in cassava Different letters show that gene expression is significant difference among other tissues on P<0.05

Fig. 4 Subcellular localization of MeSDH protein in cassava

Fig. 5 Expression patterns of MeSDH in the leaves and roots under different treatment *, ** indicate gene expression is significant difference between treatment and control groups in P<0.05 and P<0.01 level

Fig. 6 Relationship between MeSDH and MeH1.2 proteins verified by Y2H pGADT7-T/pGBKT7-53 is positive control; pGADT7-T/pGBKT7-Lam and pGADT7-T/pGBKT7-MeH1.2 are negative control; 100, 10-1, 10-2, and 10-3 refers to the dilution gradients of bacterial solution

Fig. 7 Relationship between MeSDH and MeH1.2 proteins verified by BiFC MeTGA2-nYFP/MeHistone3-cYFP is positive control; MeH12-nYFP/cYFP和-nYFP/MeSDH-cYFP are negative control

References 37

[1]	Khumaida N, Ardie S, Dianasari M, et al. Cassava(Manihot esculenta crantz.) improvement through gamma irradiation[J]. Procedia Food Sci, 2015, 3: 27-34.
[2]	付丹丹, 韩昕儒, 问锦尚, 等. 基于Rotterdam模型的中国热带农产品进口市场格局研究[J]. 中国农业资源与区划, 2022, 43(11):168-177.
	Fu DD, Han XR, Wen JS, et al. China's tropical agricultural product imports: A rotterdam model anlysis[J]. Chinese Journal of Agricultural Resources and Rrgional Planning, 2022, 43(11):168-177.
[3]	Muiruri SK, Ntui VO, Tripathi L, et al. Mechanisms and approaches towards enhanced drought tolerance in cassava(Manihot esculenta)[J]. Curr Plant Biol, 2021, 28: 100227.
[4]	Jia Y, Wong DCJ, Sweetman C, et al. New insights into the evolutionary history of plant sorbitol dehydrogenase[J]. BMC Plant Biol, 2015, 15: 101. doi: 10.1186/s12870-015-0478-5 pmid: 25879735
[5]	Chen T, Zhang ZQ, Li BQ, et al. Molecular basis for optimizing sugar metabolism and transport during fruit development[J]. aBIOTECH, 2021, 2(3): 330-340. doi: 10.1007/s42994-021-00061-2 pmid: 36303881
[6]	Dai MS, Shi ZB, Xu CJ. Genome-wide analysis of sorbitol dehydrogenase(SDH)genes and their differential expression in two sand pear(Pyrus pyrifolia)fruits[J]. Int J Mol Sci, 2015, 16(6): 13065-13083.
[7]	Nosarzewski M, Archbold DD. Tissue-specific expression of SORBITOL DEHYDROGENASE in apple fruit during early development[J]. J Exp Bot, 2007, 58(7): 1863-1872. pmid: 17404378
[8]	Wanek W, Richter A. L-Iditol: NAD+5-oxidoreductase in Viscum album: utilization of host-derived sorbitol[J]. Plant Physiol Biochem, 1993, 31: 205-211.
[9]	Kuo TM, Doehlert DC, Crawford CG. Sugar metabolism in germinating soybean seeds: evidence for the sorbitol pathway in soybean axes[J]. Plant Physiol, 1990, 93(4): 1514-1520. doi: 10.1104/pp.93.4.1514 pmid: 16667649
[10]	Nosarzewski M, Downie AB, Wu BH, et al. The role of SORBITOL DEHYDROGENASE in Arabidopsis thaliana[J]. Funct Plant Biol, 2012, 39(6): 462-470. doi: 10.1071/FP12008 pmid: 32480797
[11]	Ohta K, Moriguchi R, Kanahama K, et al. Molecular evidence of sorbitol dehydrogenase in tomato, a Non-Rosaceae plant[J]. Phytochemistry, 2005, 66(24): 2822-2828. doi: 10.1016/j.phytochem.2005.09.033 pmid: 16289145
[12]	Nadwodnik J, Lohaus G. Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens[J]. Planta, 2008, 227(5): 1079-1089. doi: 10.1007/s00425-007-0682-0 pmid: 18188589
[13]	刘政, 安莉园, 林世华, 等. 梨树山梨醇代谢及其调控因子研究进展[J]. 中国南方果树, 2018, 47(4): 165-168.
	Liu Z, An LY, Lin SH, et al. Research progress on sorbitol metabolism and its regulatory factors in pear trees[J]. South China Fruits, 2018, 47(4): 165-168.
[14]	聂佩显, 王来平, 韩雪平, 等. 杏山梨醇脱氢酶基因克隆及其在果实发育过程中的表达与酶活性分析[J]. 核农学报, 2021, 35(2): 291-297. doi: 10.11869/j.issn.100-8551.2021.02.0291
	Nie PX, Wang LP, Han XP, et al. Cloning of NAD-dependent sorbitol dehydrogenase gene from apricot fruit and analysis of its expression and enzyme activity[J]. J Nucl Agric Sci, 2021, 35(2): 291-297. doi: 10.11869/j.issn.100-8551.2021.02.0291
[15]	Yamada K, Oura Y, Mori H, et al. Cloning of NAD-dependent sorbitol dehydrogenase from apple fruit and gene expression[J]. Plant Cell Physiol, 1998, 39(12): 1375-1379. pmid: 10050321
[16]	任秋萍, 周淑梅, 王秀玲. 苹果山梨醇脱氢酶的基因表达、组织分布和活性调控[J]. 中国细胞生物学学报, 2010, 32(4): 668-672.
	Ren QP, Zhou SM, Wang XL. Gene expression, tissue distribution and activity regulation of NAD+-dependent sorbitol dehydrogenase in apple[J]. Chin J Cell Biol, 2010, 32(4): 668-672.
[17]	Bantog NA, Yamada K, Niwa N, et al. Gene expression of NAD+-dependent sorbitol dehydrogenase and NADP+-dependent sorbitol-6-phosphate dehydrogenase during development of loquat(Eriobotrya japonica lindl.) fruit[J]. Engei Gakkai Zasshi, 2000, 69(3): 231-236.
[18]	Kelker NE, Anderson RL. Sorbitol metabolism in Aerobacter aerogenes[J]. J Bacteriol, 1971, 105(1): 160-164. doi: 10.1128/jb.105.1.160-164.1971 pmid: 5541002
[19]	Sarthy AV, Schopp C, Idler KB. Cloning and sequence determination of the gene encoding sorbitol dehydrogenase from Saccharomyces cerevisiae[J]. Gene, 1994, 140(1): 121-126. pmid: 8125328
[20]	Yamaki S, Ishikawa K. Roles of four sorbitol related enzymes and invertase in the seasonal alteration of sugar metabolism in apple tissue[J]. J Amer Soc Hort Sci, 1986, 111(1): 134-137.
[21]	Loescher WH. Physiology and metabolism of sugar alcohols in higher plants[J]. Physiol Plant, 1987, 70(3): 553-557.
[22]	Wu BH, Li SH, Nosarzewski M, et al. Sorbitol dehydrogenase gene expression and enzyme activity in apple: tissue specificity during bud development and response to rootstock vigor and growth manipulation[J]. J Amer Soc Hort Sci, 2010, 135(4): 379-387.
[23]	Brown PH, Bellaloui N, Hu H, et al. Transgenically enhanced sorbitol synthesis facilitates phloem boron transport and increases tolerance of tobacco to boron deficiency[J]. Plant Physiol, 1999, 119(1): 17-20. doi: 10.1104/pp.119.1.17 pmid: 9880341
[24]	Bellaloui N, Brown PH, Dandekar AM. Manipulation of in vivo sorbitol production alters boron uptake and transport in tobacco[J]. Plant Physiol, 1999, 119(2): 735-742. doi: 10.1104/pp.119.2.735 pmid: 9952470
[25]	Wang T, Hou M, Zhao N, et al. Cloning and expression of the sorbitol dehydrogenase gene during embryonic development and temperature stress in Artemia sinica[J]. Gene, 2013, 521(2): 296-302.
[26]	El-Kabbani O, Darmanin C, Chung RPT. Sorbitol dehydrogenase: structure, function and ligand design[J]. Curr Med Chem, 2004, 11(4): 465-476. pmid: 14965227
[27]	Wu T, Wang Y, Zheng Y, et al. Suppressing sorbitol synthesis substantially alters the global expression profile of stress response genes in apple(Malus domestica)leaves[J]. Plant Cell Physiol, 2015, 56(9): 1748-1761.
[28]	Aguayo MF, Ampuero D, Mandujano P, et al. Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana[J]. Plant Sci, 2013, 205-206: 63-75.
[29]	Bellaloui N, Yadavc RC, Chern MS, et al. Transgenically enhanced sorbitol synthesis facilitates phloem-boron mobility in rice[J]. Physiol Plant, 2003, 117(1): 79-84.
[30]	Hergeth SP, Schneider R. The H1 linker histones: multifunctional proteins beyond the nucleosomal core particle[J]. EMBO Rep, 2015, 16(11): 1439-1453. doi: 10.15252/embr.201540749 pmid: 26474902
[31]	Andrés M, García-Gomis D, Ponte I, et al. Histone H1 post-translational modifications: update and future perspectives[J]. Int J Mol Sci, 2020, 21(16): 5941.
[32]	Wu X, Xu JN, Meng XN, et al. Linker histone variant HIS1-3 and WRKY1 oppositely regulate salt stress tolerance in Arabidopsis[J]. Plant Physiol, 2022, 189(3): 1833-1847.
[33]	Zhao PJ, Guo X, Wang B, et al. Overexpression of MeH1.2 gene inhibited plant growth and increased branch root differentiation in transgenic cassava[J]. Crop Sci, 2021, 61(4): 2639-2650.
[34]	Zhao PJ, Liu P, Shao JF, et al. Analysis of different strategies adapted by two cassava cultivars in response to drought stress: ensuring survival or continuing growth[J]. J Exp Bot, 2015, 66(5): 1477-1488. doi: 10.1093/jxb/eru507 pmid: 25547914
[35]	薛满德, 赵峰月, 李洁, 等. 组蛋白变体在植物表观遗传调控中的研究进展[J]. 生物技术通报, 2022, 38(7): 1-12. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0071
	Xue MD, Zhao FY, Li J, et al. Advances in histone variants in plant epigenetic regulation[J]. Biotechnol Bull, 2022, 38(7): 1-12.
[36]	Kalashnikova AA, Rogge RA, Hansen JC. Linker histone H1 and protein-protein interactions[J]. Biochimica et Biophysica Acta 2016, 1859(3):455-461. doi: 10.1016/j.bbagrm.2015.10.004 pmid: 26455956
[37]	凡慧明. 砂梨山梨醇脱氢酶基因PpSDH3和PpSDH9调控水心病发生的分子机制研究[D]. 扬州: 扬州大学, 2021.
	Fan HM. Study on the molecular mechanism of sorbitol dehydrogenase genes PpSDH3 and PpSDH9 regulating the occurrence of water heart disease in Pyrus pyrifolia[D]. Yangzhou: Yangzhou University, 2021.