中国农业科技导报 ›› 2023, Vol. 25 ›› Issue (4): 67-76.DOI: 10.13304/j.nykjdb.2021.0794
收稿日期:
2021-09-08
接受日期:
2021-10-20
出版日期:
2023-04-01
发布日期:
2023-06-26
通讯作者:
田洁
作者简介:
闫艺薇 E-mail: yanyw0102@163.com;
基金资助:
Received:
2021-09-08
Accepted:
2021-10-20
Online:
2023-04-01
Published:
2023-06-26
Contact:
Jie TIAN
摘要:
为挖掘大蒜NAC转录因子家族成员,研究其在低温胁迫下的表达特性,基于大蒜转录组的测序结果,对低温胁迫下大蒜NAC转录因子家族的响应进行分析。结果表明,共鉴定出49个大蒜NAC基因,根据系统发育特征将其分为10个亚族。大蒜NAC蛋白的序列长度为81~619 aa,分子量9 293.66~70 229.04 Da,且均为亲水蛋白。保守结构域分析发现,大部分NAC蛋白在N端具有保守性,其中18个NAC蛋白在N端均有motif 3、motif 4、motif 1、motif 2、motif 5保守域。从中随机挑选6个蛋白(AsNAC001、AsNAC010、AsNAC013、AsNAC014、AsNAC017、AsNAC047)对其进行蛋白二级及三级结构的预测,发现无规则卷曲是构成大蒜NAC蛋白的重要组成部分,其次是α-螺旋和延长链,且这6个NAC蛋白具有高度的相似性。实时荧光定量分析发现,检测的6个NAC基因中有4个(AsNAC001、AsNAC010、AsNAC013、AsNAC014)在低温胁迫12和24 h时显著上调表达;2个(AsNAC017、AsNAC047)在低温胁迫4 h时显著下调表达,其中AsNAC010基因的表达量显著高于其他基因,说明NAC转录因子在大蒜响应低温胁迫的过程中起重要调控作用,为大蒜NAC蛋白的生理调控功能的分析奠定了理论基础。
中图分类号:
闫艺薇, 田洁. 大蒜NAC基因家族的鉴定与低温表达分析[J]. 中国农业科技导报, 2023, 25(4): 67-76.
Yiwei YAN, Jie TIAN. Identification and Expression Analysis of NAC Gene Family Under Low Temperature in Allium sativum L.[J]. Journal of Agricultural Science and Technology, 2023, 25(4): 67-76.
基因名称 Gene name | 引物序列 Primer sequence(5’-3’) | 退火温度 Annealing temperature/℃ |
---|---|---|
AsNAC001 | F: CCGAGAAGGCATTATTTG | 52 |
R: ACCCGTCGCTTTCCAGTA | ||
AsNAC010 | F: TCAACCTCTACAAGCACGACC | 56 |
R: GAACCCTTTGGGCACGAT | ||
AsNAC013 | F: CTTCTACAGCCTTCGTG | 50 |
R: AGTCTTCCTCATCCCTAC | ||
AsNAC014 | F: ATCTTGTGATGCCTGGTT | 49 |
R: AATGGCTGCTAATGCTG | ||
AsNAC017 | F: TCCAGCAGGGTTCAGATT | 54 |
R: ACTCCCAAGGCTCACAAG | ||
AsNAC047 | F: AAAATCCTCATACACCGCCTAA | 57 |
R: TCGTCCCGAAGCATCCAC | ||
CYP | F: AAGGACGAGAACTTCATC | — |
R: TCAATATCTCTCACCACTTC |
表1 AsNAC 基因实时荧光定量PCR引物序列
Table 1 Primer sequences of AsNAC genes for real-time fluorescence quantification
基因名称 Gene name | 引物序列 Primer sequence(5’-3’) | 退火温度 Annealing temperature/℃ |
---|---|---|
AsNAC001 | F: CCGAGAAGGCATTATTTG | 52 |
R: ACCCGTCGCTTTCCAGTA | ||
AsNAC010 | F: TCAACCTCTACAAGCACGACC | 56 |
R: GAACCCTTTGGGCACGAT | ||
AsNAC013 | F: CTTCTACAGCCTTCGTG | 50 |
R: AGTCTTCCTCATCCCTAC | ||
AsNAC014 | F: ATCTTGTGATGCCTGGTT | 49 |
R: AATGGCTGCTAATGCTG | ||
AsNAC017 | F: TCCAGCAGGGTTCAGATT | 54 |
R: ACTCCCAAGGCTCACAAG | ||
AsNAC047 | F: AAAATCCTCATACACCGCCTAA | 57 |
R: TCGTCCCGAAGCATCCAC | ||
CYP | F: AAGGACGAGAACTTCATC | — |
R: TCAATATCTCTCACCACTTC |
基因名称 Gene name | 氨基酸长度 Number of amino acid/aa | 编码区长度 Length of cDNA/bp | 等电点 Theoretical pI | 分子量 Molecular weight/Da | 不稳定指数 Instability index | 脂溶系数 Aliphatic index | 总平均疏水指数Grand average of hydropathicity |
---|---|---|---|---|---|---|---|
AsNAC001 | 317 | 966 | 5.48 | 36 739.14 | 40.50 | 60.60 | -0.939 |
AsNAC002 | 227 | 692 | 9.18 | 25 415.27 | 48.38 | 56.65 | -0.654 |
AsNAC003 | 314 | 966 | 6.22 | 36 414.20 | 35.74 | 67.36 | -0.662 |
AsNAC004 | 299 | 911 | 6.34 | 34 235.70 | 29.90 | 57.06 | -0.716 |
AsNAC005 | 297 | 905 | 4.98 | 34 391.81 | 44.56 | 64.31 | -0.904 |
AsNAC006 | 181 | 555 | 9.86 | 20 697.99 | 25.75 | 77.07 | -0.480 |
AsNAC007 | 284 | 866 | 5.93 | 32 876.85 | 38.61 | 63.84 | -0.816 |
AsNAC008 | 174 | 533 | 9.83 | 19 776.87 | 33.91 | 74.08 | -0.466 |
AsNAC009 | 295 | 899 | 8.61 | 33 523.98 | 39.65 | 65.15 | -0.637 |
AsNAC010 | 220 | 670 | 7.17 | 25 613.94 | 35.54 | 66.45 | -0.780 |
AsNAC011 | 81 | 247 | 9.79 | 9 293.66 | 29.75 | 53.09 | -0.793 |
AsNAC012 | 350 | 1 067 | 4.95 | 40 488.80 | 43.51 | 69.63 | -0.827 |
AsNAC013 | 299 | 914 | 8.07 | 34 120.71 | 37.43 | 63.24 | -0.581 |
AsNAC014 | 311 | 948 | 6.83 | 35 302.75 | 45.96 | 74.63 | -0.591 |
AsNAC015 | 153 | 466 | 9.60 | 17 763.48 | 36.73 | 72.61 | -0.460 |
AsNAC016 | 142 | 433 | 9.54 | 16 467.63 | 30.22 | 68.59 | -0.706 |
AsNAC017 | 241 | 735 | 6.17 | 27 870.55 | 40.27 | 59.83 | -0.651 |
AsNAC018 | 234 | 713 | 5.74 | 27 139.67 | 41.73 | 55.77 | -0.631 |
AsNAC019 | 196 | 597 | 8.62 | 22 554.44 | 21.80 | 61.68 | -0.671 |
AsNAC020 | 256 | 780 | 6.34 | 28 965.66 | 40.18 | 63.52 | -0.550 |
AsNAC021 | 205 | 625 | 8.75 | 23 361.40 | 38.21 | 60.78 | -0.573 |
AsNAC022 | 185 | 567 | 9.45 | 21 445.02 | 20.73 | 72.16 | -0.374 |
AsNAC023 | 159 | 484 | 9.47 | 18 236.02 | 23.81 | 61.95 | -0.664 |
AsNAC024 | 151 | 460 | 9.40 | 17 500.04 | 36.29 | 57.48 | -0.788 |
AsNAC025 | 147 | 448 | 9.70 | 16 631.15 | 31.41 | 69.12 | -0.589 |
AsNAC026 | 322 | 985 | 8.94 | 36 669.70 | 40.31 | 68.17 | -0.567 |
AsNAC027 | 256 | 780 | 8.57 | 29 357.41 | 30.19 | 61.37 | -0.656 |
AsNAC028 | 144 | 439 | 9.10 | 16 917.46 | 34.03 | 56.25 | -0.816 |
AsNAC029 | 156 | 475 | 9.67 | 17 769.38 | 43.29 | 73.78 | -0.574 |
AsNAC030 | 198 | 603 | 5.25 | 22 305.54 | 44.75 | 62.47 | -0.819 |
AsNAC031 | 619 | 1 887 | 4.78 | 70 229.04 | 46.44 | 70.39 | -0.709 |
AsNAC032 | 176 | 542 | 9.19 | 20 419.75 | 38.67 | 73.64 | -0.274 |
AsNAC033 | 238 | 728 | 9.17 | 27 182.77 | 34.76 | 60.21 | -0.607 |
AsNAC034 | 272 | 835 | 7.80 | 31 081.00 | 43.57 | 64.89 | -0.604 |
AsNAC035 | 209 | 637 | 7.00 | 23 813.71 | 30.07 | 73.68 | -0.678 |
AsNAC036 | 276 | 841 | 4.95 | 31 051.17 | 32.52 | 67.50 | -0.791 |
表2 大蒜NAC蛋白的理化性质
Table 2 Physical and chemical properties of NAC proteins in Allium sativum L.
基因名称 Gene name | 氨基酸长度 Number of amino acid/aa | 编码区长度 Length of cDNA/bp | 等电点 Theoretical pI | 分子量 Molecular weight/Da | 不稳定指数 Instability index | 脂溶系数 Aliphatic index | 总平均疏水指数Grand average of hydropathicity |
---|---|---|---|---|---|---|---|
AsNAC001 | 317 | 966 | 5.48 | 36 739.14 | 40.50 | 60.60 | -0.939 |
AsNAC002 | 227 | 692 | 9.18 | 25 415.27 | 48.38 | 56.65 | -0.654 |
AsNAC003 | 314 | 966 | 6.22 | 36 414.20 | 35.74 | 67.36 | -0.662 |
AsNAC004 | 299 | 911 | 6.34 | 34 235.70 | 29.90 | 57.06 | -0.716 |
AsNAC005 | 297 | 905 | 4.98 | 34 391.81 | 44.56 | 64.31 | -0.904 |
AsNAC006 | 181 | 555 | 9.86 | 20 697.99 | 25.75 | 77.07 | -0.480 |
AsNAC007 | 284 | 866 | 5.93 | 32 876.85 | 38.61 | 63.84 | -0.816 |
AsNAC008 | 174 | 533 | 9.83 | 19 776.87 | 33.91 | 74.08 | -0.466 |
AsNAC009 | 295 | 899 | 8.61 | 33 523.98 | 39.65 | 65.15 | -0.637 |
AsNAC010 | 220 | 670 | 7.17 | 25 613.94 | 35.54 | 66.45 | -0.780 |
AsNAC011 | 81 | 247 | 9.79 | 9 293.66 | 29.75 | 53.09 | -0.793 |
AsNAC012 | 350 | 1 067 | 4.95 | 40 488.80 | 43.51 | 69.63 | -0.827 |
AsNAC013 | 299 | 914 | 8.07 | 34 120.71 | 37.43 | 63.24 | -0.581 |
AsNAC014 | 311 | 948 | 6.83 | 35 302.75 | 45.96 | 74.63 | -0.591 |
AsNAC015 | 153 | 466 | 9.60 | 17 763.48 | 36.73 | 72.61 | -0.460 |
AsNAC016 | 142 | 433 | 9.54 | 16 467.63 | 30.22 | 68.59 | -0.706 |
AsNAC017 | 241 | 735 | 6.17 | 27 870.55 | 40.27 | 59.83 | -0.651 |
AsNAC018 | 234 | 713 | 5.74 | 27 139.67 | 41.73 | 55.77 | -0.631 |
AsNAC019 | 196 | 597 | 8.62 | 22 554.44 | 21.80 | 61.68 | -0.671 |
AsNAC020 | 256 | 780 | 6.34 | 28 965.66 | 40.18 | 63.52 | -0.550 |
AsNAC021 | 205 | 625 | 8.75 | 23 361.40 | 38.21 | 60.78 | -0.573 |
AsNAC022 | 185 | 567 | 9.45 | 21 445.02 | 20.73 | 72.16 | -0.374 |
AsNAC023 | 159 | 484 | 9.47 | 18 236.02 | 23.81 | 61.95 | -0.664 |
AsNAC024 | 151 | 460 | 9.40 | 17 500.04 | 36.29 | 57.48 | -0.788 |
AsNAC025 | 147 | 448 | 9.70 | 16 631.15 | 31.41 | 69.12 | -0.589 |
AsNAC026 | 322 | 985 | 8.94 | 36 669.70 | 40.31 | 68.17 | -0.567 |
AsNAC027 | 256 | 780 | 8.57 | 29 357.41 | 30.19 | 61.37 | -0.656 |
AsNAC028 | 144 | 439 | 9.10 | 16 917.46 | 34.03 | 56.25 | -0.816 |
AsNAC029 | 156 | 475 | 9.67 | 17 769.38 | 43.29 | 73.78 | -0.574 |
AsNAC030 | 198 | 603 | 5.25 | 22 305.54 | 44.75 | 62.47 | -0.819 |
AsNAC031 | 619 | 1 887 | 4.78 | 70 229.04 | 46.44 | 70.39 | -0.709 |
AsNAC032 | 176 | 542 | 9.19 | 20 419.75 | 38.67 | 73.64 | -0.274 |
AsNAC033 | 238 | 728 | 9.17 | 27 182.77 | 34.76 | 60.21 | -0.607 |
AsNAC034 | 272 | 835 | 7.80 | 31 081.00 | 43.57 | 64.89 | -0.604 |
AsNAC035 | 209 | 637 | 7.00 | 23 813.71 | 30.07 | 73.68 | -0.678 |
AsNAC036 | 276 | 841 | 4.95 | 31 051.17 | 32.52 | 67.50 | -0.791 |
基因名称 Gene name | 氨基酸长度 Number of amino acid/aa | 编码区长度 Length of cDNA/bp | 等电点 Theoretical pI | 分子量 Molecular weight/Da | 不稳定指数 Instability index | 脂溶系数 Aliphatic index | 总平均疏水指数Grand average of hydropathicity |
---|---|---|---|---|---|---|---|
AsNAC037 | 290 | 884 | 6.45 | 33 210.39 | 45.73 | 63.59 | -0.694 |
AsNAC038 | 176 | 548 | 9.74 | 20 481.63 | 39.16 | 79.77 | -0.354 |
AsNAC039 | 147 | 448 | 9.72 | 16 705.32 | 31.72 | 70.41 | -0.582 |
AsNAC040 | 179 | 555 | 9.74 | 21 143.43 | 51.78 | 68.60 | -0.450 |
AsNAC041 | 152 | 463 | 7.78 | 17 151.33 | 42.10 | 56.51 | -0.648 |
AsNAC042 | 145 | 442 | 9.46 | 16 896.43 | 38.54 | 62.48 | -0.587 |
AsNAC043 | 149 | 454 | 9.46 | 17 444.08 | 36.77 | 63.42 | -0.533 |
AsNAC044 | 130 | 396 | 9.80 | 15 114.11 | 16.76 | 50.23 | -0.893 |
AsNAC045 | 149 | 454 | 9.39 | 17 409.75 | 35.18 | 66.04 | -0.799 |
AsNAC046 | 140 | 426 | 9.43 | 16 452.80 | 46.91 | 63.36 | -0.699 |
AsNAC047 | 251 | 765 | 5.24 | 28 836.27 | 31.96 | 54.74 | -0.734 |
AsNAC048 | 260 | 792 | 5.04 | 29 891.58 | 47.35 | 94.42 | -0.202 |
AsNAC049 | 261 | 796 | 8.63 | 29 843.57 | 37.33 | 67.59 | -0.593 |
表2 大蒜NAC蛋白的理化性质 (续表Continued)
Table 2 Physical and chemical properties of NAC proteins in Allium sativum L.
基因名称 Gene name | 氨基酸长度 Number of amino acid/aa | 编码区长度 Length of cDNA/bp | 等电点 Theoretical pI | 分子量 Molecular weight/Da | 不稳定指数 Instability index | 脂溶系数 Aliphatic index | 总平均疏水指数Grand average of hydropathicity |
---|---|---|---|---|---|---|---|
AsNAC037 | 290 | 884 | 6.45 | 33 210.39 | 45.73 | 63.59 | -0.694 |
AsNAC038 | 176 | 548 | 9.74 | 20 481.63 | 39.16 | 79.77 | -0.354 |
AsNAC039 | 147 | 448 | 9.72 | 16 705.32 | 31.72 | 70.41 | -0.582 |
AsNAC040 | 179 | 555 | 9.74 | 21 143.43 | 51.78 | 68.60 | -0.450 |
AsNAC041 | 152 | 463 | 7.78 | 17 151.33 | 42.10 | 56.51 | -0.648 |
AsNAC042 | 145 | 442 | 9.46 | 16 896.43 | 38.54 | 62.48 | -0.587 |
AsNAC043 | 149 | 454 | 9.46 | 17 444.08 | 36.77 | 63.42 | -0.533 |
AsNAC044 | 130 | 396 | 9.80 | 15 114.11 | 16.76 | 50.23 | -0.893 |
AsNAC045 | 149 | 454 | 9.39 | 17 409.75 | 35.18 | 66.04 | -0.799 |
AsNAC046 | 140 | 426 | 9.43 | 16 452.80 | 46.91 | 63.36 | -0.699 |
AsNAC047 | 251 | 765 | 5.24 | 28 836.27 | 31.96 | 54.74 | -0.734 |
AsNAC048 | 260 | 792 | 5.04 | 29 891.58 | 47.35 | 94.42 | -0.202 |
AsNAC049 | 261 | 796 | 8.63 | 29 843.57 | 37.33 | 67.59 | -0.593 |
图3 大蒜NAC蛋白二级结构分析注:蓝色为α-螺旋;绿色为β-转角;红色为延长链;紫色为无规则卷曲。
Fig. 3 Secondary structure prediction of NAC proteins in Allium sativum L.Note: Blue indicates α-helix; green indicates β-turn; red indicates extended strand; purple indicates random curl.
编号 Code | α-螺旋 Alpha helix | 延长链 Extended strand | 无规卷曲 Random coil | β-转角 Beta turn |
---|---|---|---|---|
AsNAC001 | 31.55 | 9.46 | 54.89 | 4.10 |
AsNAC010 | 32.73 | 15.00 | 48.18 | 4.09 |
AsNAC013 | 16.05 | 15.38 | 64.88 | 3.68 |
AsNAC014 | 24.44 | 13.83 | 58.20 | 3.54 |
AsNAC017 | 13.69 | 20.33 | 62.24 | 3.73 |
AsNAC047 | 17.53 | 19.52 | 58.96 | 3.98 |
表3 大蒜NAC蛋白二级结构分析 (%)
Table 3 Secondary structure prediction of NAC proteins in Allium sativum L.
编号 Code | α-螺旋 Alpha helix | 延长链 Extended strand | 无规卷曲 Random coil | β-转角 Beta turn |
---|---|---|---|---|
AsNAC001 | 31.55 | 9.46 | 54.89 | 4.10 |
AsNAC010 | 32.73 | 15.00 | 48.18 | 4.09 |
AsNAC013 | 16.05 | 15.38 | 64.88 | 3.68 |
AsNAC014 | 24.44 | 13.83 | 58.20 | 3.54 |
AsNAC017 | 13.69 | 20.33 | 62.24 | 3.73 |
AsNAC047 | 17.53 | 19.52 | 58.96 | 3.98 |
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