草业学报 ›› 2024, Vol. 33 ›› Issue (1): 61-74.DOI: 10.11686/cyxb2023076
胡尚钦1,2(), 汪军成1,3, 姚立蓉1,3, 司二静1,3, 马小乐1,3, 杨轲1,3, 张宏1,3, 孟亚雄1,3, 王化俊1,3, 李葆春1,2()
收稿日期:
2023-03-13
修回日期:
2023-05-15
出版日期:
2024-01-20
发布日期:
2023-11-23
通讯作者:
李葆春
作者简介:
E-mail: libc@gsau.edu.cn基金资助:
Shang-qin HU1,2(), Jun-cheng WANG1,3, Li-rong YAO1,3, Er-jing SI1,3, Xiao-le MA1,3, Ke YANG1,3, Hong ZHANG1,3, Ya-xiong MENG1,3, Hua-jun WANG1,3, Bao-chun LI1,2()
Received:
2023-03-13
Revised:
2023-05-15
Online:
2024-01-20
Published:
2023-11-23
Contact:
Bao-chun LI
摘要:
醛酮还原酶(AKR)是构成Shaker型K+通道蛋白的保守核心结构域,在植物应对非生物胁迫时起到关键作用。本研究采用西北旱区典型盐生植物盐生草作为研究材料,基于课题组前期盐生草根系盐胁迫转录组学数据分析结果,筛选并克隆得到耐盐基因HgAKR6C。HgAKR6C基因蛋白质编码区(CDS)全长951 bp,共编码氨基酸317个。系统发育进化树分析表明,HgAKR6C与拟南芥中AtAKR6C1基因亲缘关系最近。亚细胞定位表明该基因可能主要定位于细胞质和细胞核中。qRT-PCR结果表明HgAKR6C在盐处理24 h时表达量达到峰值。构建酵母异源表达载体转化缺陷型菌株发现,HgAKR6C基因可能参与Na+的外排和介导K+的吸收。综上所述,HgAKR6C具有调节盐生草耐盐性的功能,而盐生草根系耐盐基因HgAKR6C的调控机制还需进一步的研究验证。
胡尚钦, 汪军成, 姚立蓉, 司二静, 马小乐, 杨轲, 张宏, 孟亚雄, 王化俊, 李葆春. 盐生草根系基因HgAKR6C的克隆与初步功能分析[J]. 草业学报, 2024, 33(1): 61-74.
Shang-qin HU, Jun-cheng WANG, Li-rong YAO, Er-jing SI, Xiao-le MA, Ke YANG, Hong ZHANG, Ya-xiong MENG, Hua-jun WANG, Bao-chun LI. Cloning and preliminary functional analysis of the root gene HgAKR6C of Halogeton glomeratus[J]. Acta Prataculturae Sinica, 2024, 33(1): 61-74.
引物名称 Name of primers | 引物序列 Sequences of primers (5′→3′) |
---|---|
HgAKR6C-F1 | GC |
HgAKR6C-R1 | TC |
HgAKR6C-F2 | CC |
HgAKR6C-R2 | GC |
HgAKR6C-q1F | AGGAAGCACATCGTTGAGGG |
HgAKR6C-q1R | TTCATTGCCCGGACAGTCTC |
HgActin-F | TGTTCTCAGTGGTGGTACAA |
HgActin-R | GTGCCACCACCTTAATCTTC |
表 1 试验所用引物
Table 1 Primers used in the study
引物名称 Name of primers | 引物序列 Sequences of primers (5′→3′) |
---|---|
HgAKR6C-F1 | GC |
HgAKR6C-R1 | TC |
HgAKR6C-F2 | CC |
HgAKR6C-R2 | GC |
HgAKR6C-q1F | AGGAAGCACATCGTTGAGGG |
HgAKR6C-q1R | TTCATTGCCCGGACAGTCTC |
HgActin-F | TGTTCTCAGTGGTGGTACAA |
HgActin-R | GTGCCACCACCTTAATCTTC |
图1 HgAKR6C基因克隆PCR验证M: D15000+2000 DNA分子量标准D15000+2000 DNA marker; 1~4: 扩增HgAKR6C的片段Amplified HgAKR6C fragments. a用于后续构建亚细胞定位表达载体,b用于后续构建酵母异源表达载体。a was used for the subsequent construction of subcellular localization expression vectors, and b was used for the subsequent construction of yeast heterologous expression vectors.
Fig.1 The PCR validation of HgAKR6C
图2 重组载体菌液PCR验证1~5: 扩增重组载体的片段The fragments of amplified recombinant vectors. a为重组亚细胞定位表达载体,b为重组酵母异源表达载体。a is the recombinant subcellular locational expression vector, and b is the recombinant yeast heterologous expression vector.
Fig.2 PCR verification of recombinant vector solution
理化性质Physical and chemical properties | 预测结果Prediction results |
---|---|
编码的氨基酸数Number of amino acids | 317 |
理论等电点Theoretical pI | 6.25 |
蛋白质分子量Molecular weight (Da) | 35151.21 |
分子式Formula | C1575H2473N419O468S12 |
负电荷的残基总数(天冬氨酸+谷氨酸)Total number of negatively charged residues (Asparticacid+glutamicacid, Asp+Glu) | 36 |
正电荷的残基总数(精氨酸+赖氨酸)Total number of positively charged residues (Arginine+lysine, Arg+Lys) | 34 |
脂肪系数Aliphatic index | 90.41 |
亲水性平均值Grand average of hydropathicity | -0.219 |
不稳定系数The instability index (II) | 32.06 |
表2 HgAKR6C基因的理化性质分析
Table 2 Analysis of physical and chemical properties of HgAKR6C
理化性质Physical and chemical properties | 预测结果Prediction results |
---|---|
编码的氨基酸数Number of amino acids | 317 |
理论等电点Theoretical pI | 6.25 |
蛋白质分子量Molecular weight (Da) | 35151.21 |
分子式Formula | C1575H2473N419O468S12 |
负电荷的残基总数(天冬氨酸+谷氨酸)Total number of negatively charged residues (Asparticacid+glutamicacid, Asp+Glu) | 36 |
正电荷的残基总数(精氨酸+赖氨酸)Total number of positively charged residues (Arginine+lysine, Arg+Lys) | 34 |
脂肪系数Aliphatic index | 90.41 |
亲水性平均值Grand average of hydropathicity | -0.219 |
不稳定系数The instability index (II) | 32.06 |
图4 HgAKR6C基因的生物信息学分析预测a: 基因编码蛋白的跨膜区预测Prediction map of transmembrane region of protein encoded by gene; b: 信号肽预测Signal peptide prediction; c: 基因编码蛋白的亲疏水性分析Hydrophobicity analysis of protein encoded by gene; d: 基因编码蛋白的磷酸化分析Phosphorylation analysis of protein encoded by gene; e: 结构域预测Domain prediction.
Fig.4 Bioinformatics analysis and prediction of HgAKR6C genes
定位Positioning | 预测占比Forecast proportion (%) |
---|---|
细胞质Cytoplasm | 60.9 |
细胞核 | 13.0 |
线粒体Mitochondrium | 13.0 |
细胞骨架Cytoskeleton | 4.3 |
表3 亚细胞定位预测
Table 3 Subcellular localization prediction (%)
定位Positioning | 预测占比Forecast proportion (%) |
---|---|
细胞质Cytoplasm | 60.9 |
细胞核 | 13.0 |
线粒体Mitochondrium | 13.0 |
细胞骨架Cytoskeleton | 4.3 |
图 5 HgAKR6C同源蛋白序列比对Hg: 盐生草H. glomeratus; Md: 苹果Malus domestica; Ag: 旱芹Apium graveolens; Gm: 大豆Glycine max; Ms: 苜蓿Medicago sativa; Ge: 刺甘草Glycyrrhiza echinata; Gg: 洋甘草Glycyrrhiza glabra; Sr: 长喙田菁Sesbania rostrata; Ps: 罂粟Papaver somniferum; Fa: 草莓Fragaria ananassa; Zm: 玉米Zea mays; Os: 水稻O. sativa; Hv: 大麦Hordeum vulgare; Ta: 小麦Triticum aestivum; 无芒雀麦Bromus inermis; Af: 野燕麦Avena fatua; Xv: 翡若翠科黑炭木属Xerophyta viscosa; Dp: 毛地黄Digitalis purpurea; At: 拟南芥A. thaliana. 下同The same below.
Fig.5 Sequence alignment of HgAKR6C homologous protein
图 8 200 mmol·L-1 NaCl 胁迫不同时间下盐生草根系 HgAKR6C 的表达量不同小写字母表示差异显著(P<0.05),下同。Different lowercase letters indicate significant difference (P<0.05), the same below.
Fig.8 Expression of HgAKR6C in saltbush root systems under different times of 200 mmol·L-1 NaCl stress
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