生物技术通报 ›› 2021, Vol. 37 ›› Issue (8): 141-151.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0068
马亚男1(), 卢旭2, 魏云春1, 李康1, 魏若男1, 李胜1(), 马绍英3()
收稿日期:
2021-01-17
出版日期:
2021-08-26
发布日期:
2021-09-10
作者简介:
马亚男,女,硕士研究生,研究方向:农艺与种业;E-mail: 基金资助:
MA Ya-nan1(), LU Xu2, WEI Yun-chun1, LI Kang1, WEI Ruo-nan1, LI Sheng1(), MA Shao-ying3()
Received:
2021-01-17
Published:
2021-08-26
Online:
2021-09-10
摘要:
醛酮还原酶(aldo-keto reductase,AKR)是一类NADP-依赖型氧化还原酶,通过对葡萄AKR基因鉴定分析,旨在揭示葡萄AKR基因分子生物学功能和遗传调控机制。基于2020年最新组装的葡萄参考基因组数据,利用生物信息学对葡萄AKR基因进行理化性质、基因结构、Motif分析、顺式作用元件、亚细胞定位、二级结构预测以及组织特异性表达分析,最后用荧光定量分析了葡萄AKR基因在盐胁迫下表达水平变化。从全基因组水平上共鉴定出13个葡萄AKR基因家族成员,分为2个亚族;基因结构分析显示,不同成员编码的氨基酸序列外显子数目及位置存在差异;motif分析表明,AKR蛋白保守结构域高度相似;顺式作用元件分析显示,该基因上游启动子元件不仅存在核心元件和增强元件,同时还存在各种激素及胁迫响应元件,器官特异性元件和光调控元件;二级结构和亚细胞定位预测表明,该基因家族主要在细胞核中表达,并以α-螺旋和不规则卷曲为主。基因芯片表达谱分析显示,AKR家族成员间表达水平差异大且存在组织特异性。荧光定量结果显示,盐胁迫条件下VvAKR02、VvAKR05、VvAKR07、VvAKR11、VvAKR12和VvAKR13表达量显著上调。从葡萄中鉴定了13个AKR家族基因,并分析其盐胁迫下的表达水平,对于挖掘葡萄AKR家族中耐盐基因提供了一定的理论依据和实验基础。
马亚男, 卢旭, 魏云春, 李康, 魏若男, 李胜, 马绍英. 葡萄AKR基因家族的鉴定和组织特异性表达分析[J]. 生物技术通报, 2021, 37(8): 141-151.
MA Ya-nan, LU Xu, WEI Yun-chun, LI Kang, WEI Ruo-nan, LI Sheng, MA Shao-ying. Identification and Tissue Specific Expression Analysis of AKR Gene Family in Grape[J]. Biotechnology Bulletin, 2021, 37(8): 141-151.
基因Gene | 上游引物Forward primer(5'-3') | 下游引物Reverse primer(5'-3') |
---|---|---|
VvAKR01 | AGGAGGCAGCAGTGTCAAAATGG | CCAGCCACCGCATCATCATCTC |
VvAKR02 | ACCATTGAAAGCCTCAGCCAGTG | GGCGTTGTCAGAGGGTTATCGTC |
VvAKR03 | TTGTGCTGAGGAAGCTGGGTAAAC | GCCGCAGAGTGGGAACAAGAAG |
VvAKR04 | AGCGGTGATTTGAATGCCTTTTGC | GGCGGTCAATACGGTGGAGATG |
VvAKR05 | AGCGGGTTCATCTCCACTTGATTG | GGAAGGCATGGAAGAGTGTCAGAG |
VvAKR06 | TCTGCTCTGTCTTGTGCGAAACC | GAACCAACCCCATACCCTAAAGCC |
VvAKR07 | TGCTGCCAAAGTGGGTTCATCTC | GTCAGAAGCTCGGTCTCACCAAA |
VvAKR08 | AAAGCTTTGGTGCACCGACG | TCCCAGGCTTCAAGCTCACC |
VvAKR09 | TAGCCGCCTTAAGAGCCTGTCC | ACGCTGTCTTCACCGGCAATCG |
VvAKR10 | GACACTGTGACCGCTCTGAATACC | CTCTCGGCGTACTCTCCTCTTGG |
VvAKR11 | CTTGGATGACAGCAGGAGGAACAC | AGCACATGGAGAGCAATGGAAGC |
VvAKR12 | AGTGCTTCCATTGCTCTCCATGTG | CTTATCCATTGGCCGGTCCGTATG |
VvAKR13 | CTGCCTGCTCTTGCCAGATGTG | CCGTGTTCCTCCTGCTGTCAATC |
UBI | GCTCGCTGTTTTTGCAGTTCTAC | AACATAGGTGAGGCCGCACTT |
表1 葡萄AKR基因家族实时荧光定量引物
Table 1 Real-time fluorescence quantitative primers of AKR gene family in grape
基因Gene | 上游引物Forward primer(5'-3') | 下游引物Reverse primer(5'-3') |
---|---|---|
VvAKR01 | AGGAGGCAGCAGTGTCAAAATGG | CCAGCCACCGCATCATCATCTC |
VvAKR02 | ACCATTGAAAGCCTCAGCCAGTG | GGCGTTGTCAGAGGGTTATCGTC |
VvAKR03 | TTGTGCTGAGGAAGCTGGGTAAAC | GCCGCAGAGTGGGAACAAGAAG |
VvAKR04 | AGCGGTGATTTGAATGCCTTTTGC | GGCGGTCAATACGGTGGAGATG |
VvAKR05 | AGCGGGTTCATCTCCACTTGATTG | GGAAGGCATGGAAGAGTGTCAGAG |
VvAKR06 | TCTGCTCTGTCTTGTGCGAAACC | GAACCAACCCCATACCCTAAAGCC |
VvAKR07 | TGCTGCCAAAGTGGGTTCATCTC | GTCAGAAGCTCGGTCTCACCAAA |
VvAKR08 | AAAGCTTTGGTGCACCGACG | TCCCAGGCTTCAAGCTCACC |
VvAKR09 | TAGCCGCCTTAAGAGCCTGTCC | ACGCTGTCTTCACCGGCAATCG |
VvAKR10 | GACACTGTGACCGCTCTGAATACC | CTCTCGGCGTACTCTCCTCTTGG |
VvAKR11 | CTTGGATGACAGCAGGAGGAACAC | AGCACATGGAGAGCAATGGAAGC |
VvAKR12 | AGTGCTTCCATTGCTCTCCATGTG | CTTATCCATTGGCCGGTCCGTATG |
VvAKR13 | CTGCCTGCTCTTGCCAGATGTG | CCGTGTTCCTCCTGCTGTCAATC |
UBI | GCTCGCTGTTTTTGCAGTTCTAC | AACATAGGTGAGGCCGCACTT |
基因Gene | 登录号 Gene accession No. | 染色体定位 Chromosomal location | 全长Full length/bp | CDS/bp | 氨基酸 Amino acid | 分子量 Molecular weight/bp | 等电点 pI | 不稳定系数 Instability index | 脂肪系数Aliphatic index | 平均亲水性Hydropathicity |
---|---|---|---|---|---|---|---|---|---|---|
VvAKR01 | GSVIVT01009396001 | chr18:7983438..7986889 | 3452 | 969 | 322 | 36192.49 | 5.9 | 38.98 | 89.66 | -0.238 |
VvAKR02 | GSVIVT01010238001 | chr1:18082562..18106134 | 23573 | 1002 | 333 | 36674.09 | 7.07 | 34.21 | 90.75 | -0.213 |
VvAKR03 | GSVIVT01011576001 | chr1:6306239..6307845 | 1607 | 867 | 288 | 32083.49 | 9.01 | 42.94 | 96.88 | -0.086 |
VvAKR04 | GSVIVT01011577001 | chr1:6298552..6305704 | 7153 | 912 | 303 | 34017.51 | 6.54 | 47.47 | 89.8 | -0.164 |
VvAKR05 | GSVIVT01011579001 | chr1:6289243..6290820 | 1578 | 951 | 316 | 35455.01 | 5.85 | 51.55 | 95.73 | -0.206 |
VvAKR06 | GSVIVT01011582001 | chr1:6274423..6285766 | 11344 | 1293 | 430 | 48655.51 | 7.1 | 43.48 | 94.12 | -0.184 |
VvAKR07 | GSVIVT01011583001 | chr1:6264420..6272529 | 8110 | 909 | 302 | 34322.71 | 6.34 | 27.01 | 92.02 | -0.274 |
VvAKR08 | GSVIVT01011585001 | chr1:6241008..6248273 | 7266 | 1092 | 363 | 40730.95 | 5.98 | 49.92 | 95.92 | -0.228 |
VvAKR09 | GSVIVT01030263001 | chr8:9745143..9747339 | 2197 | 969 | 322 | 35975.24 | 6.17 | 45.07 | 83.57 | -0.319 |
VvAKR10 | GSVIVT01034098001 | chr8:15157629..15161115 | 3487 | 948 | 315 | 35068.99 | 5.83 | 50.57 | 89.46 | -0.243 |
VvAKR11 | GSVIVT01034099001 | chr8:15153012..15157096 | 4085 | 828 | 275 | 30440 | 6.45 | 46.14 | 87.56 | -0.204 |
VvAKR12 | GSVIVT01034101001 | chr8:15144714..15149849 | 5136 | 903 | 300 | 33663.65 | 6.76 | 42.71 | 88.37 | -0.317 |
表2 葡萄AKR基因理化性质
Table 2 Physical and chemical property of AKR genes in grape
基因Gene | 登录号 Gene accession No. | 染色体定位 Chromosomal location | 全长Full length/bp | CDS/bp | 氨基酸 Amino acid | 分子量 Molecular weight/bp | 等电点 pI | 不稳定系数 Instability index | 脂肪系数Aliphatic index | 平均亲水性Hydropathicity |
---|---|---|---|---|---|---|---|---|---|---|
VvAKR01 | GSVIVT01009396001 | chr18:7983438..7986889 | 3452 | 969 | 322 | 36192.49 | 5.9 | 38.98 | 89.66 | -0.238 |
VvAKR02 | GSVIVT01010238001 | chr1:18082562..18106134 | 23573 | 1002 | 333 | 36674.09 | 7.07 | 34.21 | 90.75 | -0.213 |
VvAKR03 | GSVIVT01011576001 | chr1:6306239..6307845 | 1607 | 867 | 288 | 32083.49 | 9.01 | 42.94 | 96.88 | -0.086 |
VvAKR04 | GSVIVT01011577001 | chr1:6298552..6305704 | 7153 | 912 | 303 | 34017.51 | 6.54 | 47.47 | 89.8 | -0.164 |
VvAKR05 | GSVIVT01011579001 | chr1:6289243..6290820 | 1578 | 951 | 316 | 35455.01 | 5.85 | 51.55 | 95.73 | -0.206 |
VvAKR06 | GSVIVT01011582001 | chr1:6274423..6285766 | 11344 | 1293 | 430 | 48655.51 | 7.1 | 43.48 | 94.12 | -0.184 |
VvAKR07 | GSVIVT01011583001 | chr1:6264420..6272529 | 8110 | 909 | 302 | 34322.71 | 6.34 | 27.01 | 92.02 | -0.274 |
VvAKR08 | GSVIVT01011585001 | chr1:6241008..6248273 | 7266 | 1092 | 363 | 40730.95 | 5.98 | 49.92 | 95.92 | -0.228 |
VvAKR09 | GSVIVT01030263001 | chr8:9745143..9747339 | 2197 | 969 | 322 | 35975.24 | 6.17 | 45.07 | 83.57 | -0.319 |
VvAKR10 | GSVIVT01034098001 | chr8:15157629..15161115 | 3487 | 948 | 315 | 35068.99 | 5.83 | 50.57 | 89.46 | -0.243 |
VvAKR11 | GSVIVT01034099001 | chr8:15153012..15157096 | 4085 | 828 | 275 | 30440 | 6.45 | 46.14 | 87.56 | -0.204 |
VvAKR12 | GSVIVT01034101001 | chr8:15144714..15149849 | 5136 | 903 | 300 | 33663.65 | 6.76 | 42.71 | 88.37 | -0.317 |
图1 拟南芥、苜蓿和葡萄AKR基因的进化树 粉色正方形代表拟南芥,浅蓝色菱形代表苜蓿,紫色圆圈代表葡萄
Fig.1 Phylogenetic tree of AKR genes in Arabidopsis,alfalfa and grape Pink squares represent Arabidopsis, light blue diamonds represent alfalfa, and purple circles refer to grapes
基因 Gene | 分子式 Formula | α-螺旋 Alpha helix/% | β-转角 Beta turn/% | 扩展链结构 Hydropathicity/% | 不规则卷曲 Random coil/% | 亚细胞定位 Subcellular localization |
---|---|---|---|---|---|---|
VvAKR01 | C1621H2533N437O473S15 | 42.55 | 4.04 | 14.29 | 39.13 | Nucleus,cytosol,cytoskeleton and chloroplast |
VvAKR02 | C1630H2584N452O480S15 | 37.84 | 6.01 | 15.32 | 40.84 | Nucleus,cytosol,cytoskeleton and chloroplast |
VvAKR03 | C1437H2314N388O409S16 | 41.32 | 4.51 | 18.06 | 36.11 | Nucleus,membrane,cytosol,chloroplast and mitochondrion,peroxisome |
VvAKR04 | C1533H2418N402O437S17 | 42.57 | 4.62 | 13.86 | 38.94 | Nucleus,cytosol,cytoskeleton and chloroplast |
VvAKR05 | C1584H2534N424O466S15 | 43.04 | 3.80 | 15.51 | 37.66 | Extracellular,cytosol,chloroplast,mitochondrion and endoplasmic reticulum |
VvAKR06 | C2207H3481N571O627S19 | 34.42 | 3.95 | 17.44 | 44.19 | Nucleus,membrane,cytosol,cytoskeleton,chloroplast and mitochondrion |
VvAKR07 | C1560H2445N401O445S12 | 41.39 | 5.30 | 13.58 | 39.74 | Nucleus,extracellular,cytosol and cytoskeleton |
VvAKR08 | C1829H2900N490O533S14 | 42.15 | 4.41 | 13.50 | 39.94 | Nucleus,membrane,cytosol and cytoskeleton |
VvAKR09 | C1601H2518N442O469S16 | 40.37 | 8.39 | 14.60 | 36.65 | Nucleus,membrane,cytosol,cytoskeleton and chloroplast |
VvAKR10 | C1582H2451N419O463S10 | 42.54 | 6.67 | 13.97 | 36.83 | Nucleus,cytosol and chloroplast |
VvAKR11 | C1378H2147N363O395S10 | 43.27 | 5.09 | 14.18 | 37.45 | Nucleus,cytosol,cytoskeleton,endoplasmic reticulum and vacuole |
VvAKR12 | C1518H2375N411O435S10 | 43.00 | 6.67 | 12.00 | 38.33 | Nucleus,membrane,cytosol,cytoskeleton and vacuole |
VvAKR013 | C1872H2916N492O531S11 | 48.91 | 6.25 | 11.41 | 33.42 | Nucleus,cytosol and chloroplast |
表3 葡萄AKR基因二级结构及亚细胞定位预测
Table 3 Secondary structure and Subcellular location prediction of AKR genes in grape
基因 Gene | 分子式 Formula | α-螺旋 Alpha helix/% | β-转角 Beta turn/% | 扩展链结构 Hydropathicity/% | 不规则卷曲 Random coil/% | 亚细胞定位 Subcellular localization |
---|---|---|---|---|---|---|
VvAKR01 | C1621H2533N437O473S15 | 42.55 | 4.04 | 14.29 | 39.13 | Nucleus,cytosol,cytoskeleton and chloroplast |
VvAKR02 | C1630H2584N452O480S15 | 37.84 | 6.01 | 15.32 | 40.84 | Nucleus,cytosol,cytoskeleton and chloroplast |
VvAKR03 | C1437H2314N388O409S16 | 41.32 | 4.51 | 18.06 | 36.11 | Nucleus,membrane,cytosol,chloroplast and mitochondrion,peroxisome |
VvAKR04 | C1533H2418N402O437S17 | 42.57 | 4.62 | 13.86 | 38.94 | Nucleus,cytosol,cytoskeleton and chloroplast |
VvAKR05 | C1584H2534N424O466S15 | 43.04 | 3.80 | 15.51 | 37.66 | Extracellular,cytosol,chloroplast,mitochondrion and endoplasmic reticulum |
VvAKR06 | C2207H3481N571O627S19 | 34.42 | 3.95 | 17.44 | 44.19 | Nucleus,membrane,cytosol,cytoskeleton,chloroplast and mitochondrion |
VvAKR07 | C1560H2445N401O445S12 | 41.39 | 5.30 | 13.58 | 39.74 | Nucleus,extracellular,cytosol and cytoskeleton |
VvAKR08 | C1829H2900N490O533S14 | 42.15 | 4.41 | 13.50 | 39.94 | Nucleus,membrane,cytosol and cytoskeleton |
VvAKR09 | C1601H2518N442O469S16 | 40.37 | 8.39 | 14.60 | 36.65 | Nucleus,membrane,cytosol,cytoskeleton and chloroplast |
VvAKR10 | C1582H2451N419O463S10 | 42.54 | 6.67 | 13.97 | 36.83 | Nucleus,cytosol and chloroplast |
VvAKR11 | C1378H2147N363O395S10 | 43.27 | 5.09 | 14.18 | 37.45 | Nucleus,cytosol,cytoskeleton,endoplasmic reticulum and vacuole |
VvAKR12 | C1518H2375N411O435S10 | 43.00 | 6.67 | 12.00 | 38.33 | Nucleus,membrane,cytosol,cytoskeleton and vacuole |
VvAKR013 | C1872H2916N492O531S11 | 48.91 | 6.25 | 11.41 | 33.42 | Nucleus,cytosol and chloroplast |
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