木薯SDH蛋白的序列分析及其与MeH1.2关系的研究

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

摘要/Abstract

摘要：

【目的】山梨糖醇脱氢酶（SDH）在调控蔷薇科植物果糖和山梨醇转化中起重要作用，也参与植物对逆境的应答过程，木薯SDH功能的研究可以为培育优质木薯种质提供理论基础。【方法】以‘SC124’的cDNA为模板克隆木薯SDH基因，并利用实时荧光定量PCR分析木薯SDH基因的组织特异性及其对干旱、低温、PEG和ABA的响应模式。通过筛选木薯干旱和低温混合的酵母cDNA文库，并利用Y2H点对点及其双分子荧光互补（BiFC）实验确认与目标蛋白的关系。【结果】木薯SDH基因CDS全长1 092 bp，编码364个氨基酸，与数据库中的序列无差异。MeSDH蛋白含有催化锌结合位点, NADP结合位点，结构锌结合位点，属于MDR超家族。烟草叶片表皮细胞瞬时表达显示MeSDH蛋白定位于细胞核。MeSDH基因的表达量在功能叶、幼嫩叶、须根和茎中依次降低。MeSDH基因受干旱、低温和PEG胁迫诱导在木薯叶片上调表达，在ABA处理后的木薯叶片和根系中都显著上调表达。文库筛选、Y2H点对点和BiFC实验证实MeH1.2与MeSDH互作。【结论】MeSDH基因可以响应多种胁迫上调表达，并可能在蛋白水平和MeH1.2共同作用。

关键词: 木薯, SDH基因, MeH1.2蛋白, 抗逆性, 蛋白互作

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

赵平娟, 林晨俞, 王梦月, 张秀春, 李淑霞, 阮孟斌. 木薯SDH蛋白的序列分析及其与MeH1.2关系的研究[J]. 生物技术通报, 2024, 40(9): 74-81.

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.

图/表 9

表1 实验用引物序列

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

表2 点样设计

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

图1 MeSDH基因PCR扩增 M: DL2000 DNA marker; SDH: MeSDH基因PCR扩增产物

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

图2 MeSDH蛋白结构域分析

Fig. 2 Conserved domain analysis of MeSDH protein

图3 木薯MeSDH基因的组织特异性表达不同字母表示基因在不同组织中的表达量在P<0.05水平有显著性差异

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

图4 木薯MeSDH蛋白亚细胞定位

Fig. 4 Subcellular localization of MeSDH protein in cassava

图5 叶片和根系MeSDH基因对不同胁迫的响应模式 *,**表示基因在处理和对照中的表达量在P<0.05 和P<0.01水平有显著性差异

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

图6 Y2H验证MeSDH与MeH1.2蛋白间的互作关系 pGADT7-T/pGBKT7-53 为阳性对照；pGADT7-T/pGBKT7-Lam和pGADT7-T/pGBKT7-MeH1.2 为阴性对照；100、10-1、10-2、10-3 表示菌液的稀释梯度

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

图7 BiFC验证MeSDH与MeH1.2蛋白间的互作关系 MeTGA2-nYFP/MeHistone3-cYFP为阳性对照；MeH12-nYFP/cYFP和-nYFP/MeSDH-cYFP为阴性对照

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

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