生物技术通报 ›› 2024, Vol. 40 ›› Issue (2): 233-244.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0874
收稿日期:
2023-09-11
出版日期:
2024-02-26
发布日期:
2024-03-13
通讯作者:
伍国强,男,博士,教授,博士生导师,研究方向:植物逆境生理与基因工程;E-mail: gqwu@lut.edu.cn作者简介:
李昊,男,硕士研究生,研究方向:植物逆境生理与基因工程;E-mail: 1044624636@qq.com
基金资助:
LI Hao(), WU Guo-qiang(), WEI Ming, HAN Yue-xin
Received:
2023-09-11
Published:
2024-02-26
Online:
2024-03-13
摘要:
【目的】甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase, BADH)是参与甘氨酸甜菜碱(glycine betaine, GB)合成的关键酶之一,在植物响应逆境胁迫过程中发挥重要作用。【方法】为探究甜菜(Beta vulgaris)BvBADH 基因家族成员生物学功能及基因表达模式,本研究采用生物信息学方法,分析了蛋白质的理化性质、系统进化、基因结构、染色体定位、保守基序、蛋白结构、启动子顺式作用调控元件,并通过RT-qPCR对其在盐胁迫下的表达模式进行分析。【结果】共鉴定出9个BvBADH 基因家族成员,含有9-15个外显子,8-14个内含子,平均氨基酸个数为516,平均分子量为55.84 kD,等电点为5.24-6.98。系统进化分析发现,高等植物BADH可分为3个簇,分别为簇I、II和III,其中簇III成员进一步分为簇IIIa和IIIb两个亚簇。BvBADH基因家族在3个簇中均有分布,分别含有3、1和5个成员。甜菜BvBADH基因家族主要含有胁迫响应元件、激素响应元件和生长发育响应元件,每个成员的顺式作用调控元件数量为13-21个。进一步对 BvBADH在盐处理下甜菜叶片中的表达模式分析发现,100和150 mmol/L NaCl不同程度地诱导BvBADH基因家族成员的表达;但相比之下,它们在100 mmol/L NaCl处理下的表达量高于150 mmol/L NaCl。【结论】BvBADH基因家族在甜菜响应盐胁迫过程中扮演着重要角色,为我国北方地区农作物抗逆性遗传改良提供优异基因资源。
李昊, 伍国强, 魏明, 韩悦欣. 甜菜BvBADH基因家族全基因组鉴定及其高盐胁迫下的表达分析[J]. 生物技术通报, 2024, 40(2): 233-244.
LI Hao, WU Guo-qiang, WEI Ming, HAN Yue-xin. Genome-wide Identification of the BvBADH Gene Family in Sugar Beet(Beta vulgaris)and Their Expression Analysis Under High Salt Stress[J]. Biotechnology Bulletin, 2024, 40(2): 233-244.
基因Gene | 正向引物序列Forward primer sequence(5'-3') | 反向引物序列Reverse primer sequence(5'-3') |
---|---|---|
BvBADH1 | GCAGTGCAACATCCAGACTC | CCCCCTCACTCTTTGCTGTT |
BvBADH2 | GTCCTGTTGTCAGCAAGGGA | GCATGGACGTGGAGACATCA |
BvBADH3 | CCAAATTGGGGGAAGTTTGTGT | TGGATCACCAACAACCCAAGA |
BvBADH4 | ACGGGGAATTTGTGGATGCT | TCATCACCCTTCCCCTTTCA |
BvBADH5 | ATTGAGCATGGCAAGCGAGA | TGCCATTCGCTCGCTCTATC |
BvBADH6 | GCCGATTTAGCACCACTTCT | GCAGAAGTGGTGCTAAATCGG |
BvBADH7 | GCATCTGATTATGGGCTGGC | CCACTCATTTTGTACCCGCC |
BvBADH8 | TCGTCGAGCCAATAACACCC | CCACCAAATGGAATGCCTGC |
BvBADH9 | GGCCAGTTCAGAGGTGAGGA | GAACTTGGACCCAAGCCTTTT |
BvACTIN | ACTGGTATTGTGCTTGACTC | ATGAGATAATCAGTGAGATC |
表1 本研究所用RT-qPCR引物序列
Table 1 Sequences of primers used for RT-qPCR in this study
基因Gene | 正向引物序列Forward primer sequence(5'-3') | 反向引物序列Reverse primer sequence(5'-3') |
---|---|---|
BvBADH1 | GCAGTGCAACATCCAGACTC | CCCCCTCACTCTTTGCTGTT |
BvBADH2 | GTCCTGTTGTCAGCAAGGGA | GCATGGACGTGGAGACATCA |
BvBADH3 | CCAAATTGGGGGAAGTTTGTGT | TGGATCACCAACAACCCAAGA |
BvBADH4 | ACGGGGAATTTGTGGATGCT | TCATCACCCTTCCCCTTTCA |
BvBADH5 | ATTGAGCATGGCAAGCGAGA | TGCCATTCGCTCGCTCTATC |
BvBADH6 | GCCGATTTAGCACCACTTCT | GCAGAAGTGGTGCTAAATCGG |
BvBADH7 | GCATCTGATTATGGGCTGGC | CCACTCATTTTGTACCCGCC |
BvBADH8 | TCGTCGAGCCAATAACACCC | CCACCAAATGGAATGCCTGC |
BvBADH9 | GGCCAGTTCAGAGGTGAGGA | GAACTTGGACCCAAGCCTTTT |
BvACTIN | ACTGGTATTGTGCTTGACTC | ATGAGATAATCAGTGAGATC |
基因名称 Gene name | 基因登录号 Gene ID | CDS /bp | 相对分子量 Mw/kD | 亲水性平均值 GRAVY | 等电点 pI | 脂肪指数 Aliphatic index | 氨基酸数目 Protein length/aa | 不稳定指数 Instability index |
---|---|---|---|---|---|---|---|---|
BvBADH1 | Bv5_ 116250_aodi | 1 500 | 54.72 | -0.077 | 5.45 | 88.78 | 500 | 32.48 |
BvBADH2 | Bv5_ 116230_ntjn | 1 512 | 54.78 | -0.062 | 5.37 | 88.51 | 503 | 31.62 |
BvBADH3 | Bv8_ 195870_rfyd | 1 509 | 54.69 | -0.081 | 5.24 | 87.45 | 502 | 28.42 |
BvBADH4 | Bv8_ 195860_yiyp | 1 506 | 54.25 | -0.079 | 5.60 | 89.00 | 501 | 22.07 |
BvBADH5 | Bv8_ 195850_giqy | 1 509 | 54.34 | -0.039 | 6.01 | 88.05 | 502 | 25.97 |
BvBADH6 | Bv5_ 116750_zqhy | 1 605 | 58.11 | -0.111 | 6.65 | 85.90 | 534 | 31.08 |
BvBADH7 | Bv6_ 149040_dgrz | 1 605 | 57.84 | -0.076 | 6.53 | 89.55 | 534 | 32.99 |
BvBADH8 | Bv6_ 149060_fmfu | 1 608 | 58.71 | -0.109 | 6.54 | 85.74 | 535 | 32.04 |
BvBADH9 | Bv2_047750_ktxe | 1 602 | 57.11 | 0.066 | 6.98 | 95.72 | 533 | 38.12 |
表2 甜菜BvBADH基因家族成员鉴定
Table 2 Identification of BvBADH gene family members in sugar beet
基因名称 Gene name | 基因登录号 Gene ID | CDS /bp | 相对分子量 Mw/kD | 亲水性平均值 GRAVY | 等电点 pI | 脂肪指数 Aliphatic index | 氨基酸数目 Protein length/aa | 不稳定指数 Instability index |
---|---|---|---|---|---|---|---|---|
BvBADH1 | Bv5_ 116250_aodi | 1 500 | 54.72 | -0.077 | 5.45 | 88.78 | 500 | 32.48 |
BvBADH2 | Bv5_ 116230_ntjn | 1 512 | 54.78 | -0.062 | 5.37 | 88.51 | 503 | 31.62 |
BvBADH3 | Bv8_ 195870_rfyd | 1 509 | 54.69 | -0.081 | 5.24 | 87.45 | 502 | 28.42 |
BvBADH4 | Bv8_ 195860_yiyp | 1 506 | 54.25 | -0.079 | 5.60 | 89.00 | 501 | 22.07 |
BvBADH5 | Bv8_ 195850_giqy | 1 509 | 54.34 | -0.039 | 6.01 | 88.05 | 502 | 25.97 |
BvBADH6 | Bv5_ 116750_zqhy | 1 605 | 58.11 | -0.111 | 6.65 | 85.90 | 534 | 31.08 |
BvBADH7 | Bv6_ 149040_dgrz | 1 605 | 57.84 | -0.076 | 6.53 | 89.55 | 534 | 32.99 |
BvBADH8 | Bv6_ 149060_fmfu | 1 608 | 58.71 | -0.109 | 6.54 | 85.74 | 535 | 32.04 |
BvBADH9 | Bv2_047750_ktxe | 1 602 | 57.11 | 0.066 | 6.98 | 95.72 | 533 | 38.12 |
图2 植物BADH基因家族系统发育树 BADHs分为3个簇,簇I(黑色)、簇II(红色)和簇III ,其中簇III 又分为簇III a(绿色)和III b(金色);深绿色标记为甜菜BvBADH家族成员
Fig. 2 Phylogenetic tree of BADH gene family in plants BADHs is divided into three clusters. Cluster I(black), II(red)and III. Cluster III is further divided into Cluster III a(green)and III b(gold). The dark green marks in the figure represent the BvBADH family members
图3 甜菜BvBADH基因家族氨基酸序列多重比对 蓝色方框为保守十肽序列
Fig. 3 Amino acid sequence multiple alignment of the BvBADH gene family in sugar beet The blue box is a conserved decapeptide sequence
顺式作用元件Cis-acting element | 功能Function | 序列Sequence | BvBADH | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | ||||
ABRE | Abscisic acid-responsive element | ACGTG | 3 | 2 | 5 | 3 | 3 | 2 | 0 | 4 | 3 | |
TCA-element | Involved in salicylic acid responsiveness | CCATCTTTTT | 0 | 1 | 0 | 1 | 0 | 0 | 2 | 0 | 1 | |
TGA-element | Auxin-responsive element | AACGAC | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 1 | 0 | |
ARE | Anaerobic-responsive element | AAACCA | 4 | 3 | 1 | 2 | 4 | 4 | 5 | 2 | 6 | |
LTR | Low-temperature responsiveness | CCGAAA | 1 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 2 | |
Box 4 | Involved in light responsiveness | ATTAAT | 3 | 3 | 2 | 1 | 0 | 1 | 0 | 3 | 1 | |
GT1-motif | Involved in light responsiveness | GGTTAAT | 2 | 0 | 4 | 3 | 4 | 0 | 2 | 2 | 1 | |
TCT-motif | Involved in light responsiveness | TCTTAC | 6 | 0 | 2 | 1 | 1 | 1 | 1 | 2 | 0 | |
AE-box | Modul for light response | AGAAACTT | 0 | 1 | 0 | 2 | 0 | 1 | 0 | 0 | 0 | |
O2-site | Zein metabolism regulation | GATGACATGA | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | |
G-box | Involved in light responsiveness | TACGTG | 1 | 2 | 7 | 0 | 4 | 4 | 3 | 0 | 0 |
表3 BvBADHs基因启动子顺式作用调控元件分析
Table 3 Analysis of cis-acting regulatory elements in the promoter of BvBADHs genes
顺式作用元件Cis-acting element | 功能Function | 序列Sequence | BvBADH | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | ||||
ABRE | Abscisic acid-responsive element | ACGTG | 3 | 2 | 5 | 3 | 3 | 2 | 0 | 4 | 3 | |
TCA-element | Involved in salicylic acid responsiveness | CCATCTTTTT | 0 | 1 | 0 | 1 | 0 | 0 | 2 | 0 | 1 | |
TGA-element | Auxin-responsive element | AACGAC | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 1 | 0 | |
ARE | Anaerobic-responsive element | AAACCA | 4 | 3 | 1 | 2 | 4 | 4 | 5 | 2 | 6 | |
LTR | Low-temperature responsiveness | CCGAAA | 1 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 2 | |
Box 4 | Involved in light responsiveness | ATTAAT | 3 | 3 | 2 | 1 | 0 | 1 | 0 | 3 | 1 | |
GT1-motif | Involved in light responsiveness | GGTTAAT | 2 | 0 | 4 | 3 | 4 | 0 | 2 | 2 | 1 | |
TCT-motif | Involved in light responsiveness | TCTTAC | 6 | 0 | 2 | 1 | 1 | 1 | 1 | 2 | 0 | |
AE-box | Modul for light response | AGAAACTT | 0 | 1 | 0 | 2 | 0 | 1 | 0 | 0 | 0 | |
O2-site | Zein metabolism regulation | GATGACATGA | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | |
G-box | Involved in light responsiveness | TACGTG | 1 | 2 | 7 | 0 | 4 | 4 | 3 | 0 | 0 |
蛋白名称 Protein name | α螺旋 α-helix | 伸展主链 Extended strand | β转角 β-turn | 无规卷曲 Random coil |
---|---|---|---|---|
BvBADH1 | 43.40 | 16.40 | 7.80 | 32.40 |
BvBADH2 | 42.94 | 15.90 | 7.55 | 33.60 |
BvBADH3 | 40.84 | 17.53 | 8.17 | 33.47 |
BvBADH4 | 40.32 | 17.56 | 7.98 | 34.13 |
BvBADH5 | 39.44 | 17.73 | 8.37 | 34.46 |
BvBADH6 | 43.07 | 15.54 | 7.49 | 33.90 |
BvBADH7 | 41.76 | 15.54 | 7.87 | 34.83 |
BvBADH8 | 42.80 | 16.07 | 7.85 | 33.27 |
BvBADH9 | 48.22 | 15.01 | 8.07 | 28.71 |
表4 BvBADHs蛋白二级结构组成
Table 4 Secondary structure composition of BvBADHs proteins %
蛋白名称 Protein name | α螺旋 α-helix | 伸展主链 Extended strand | β转角 β-turn | 无规卷曲 Random coil |
---|---|---|---|---|
BvBADH1 | 43.40 | 16.40 | 7.80 | 32.40 |
BvBADH2 | 42.94 | 15.90 | 7.55 | 33.60 |
BvBADH3 | 40.84 | 17.53 | 8.17 | 33.47 |
BvBADH4 | 40.32 | 17.56 | 7.98 | 34.13 |
BvBADH5 | 39.44 | 17.73 | 8.37 | 34.46 |
BvBADH6 | 43.07 | 15.54 | 7.49 | 33.90 |
BvBADH7 | 41.76 | 15.54 | 7.87 | 34.83 |
BvBADH8 | 42.80 | 16.07 | 7.85 | 33.27 |
BvBADH9 | 48.22 | 15.01 | 8.07 | 28.71 |
图6 BvBADHs蛋白质二级结构 蓝色为α螺旋;红色为伸展主链;绿色为β转角;紫色为无规卷曲
Fig. 6 Secondary structure of BvBADHs protein Blue is the α-helix; red is the extended strand; green is the β-turn; purple is the random coil
图7 甜菜BvBADH蛋白质三维结构 a-i:BvBADH1-BvBADH9三维结构示意图
Fig. 7 Three-dimensional structure of BvBADH proteins in sugar beet a-i: Schematic diagram of the three-dimensional structure of BvBADH1-BvBADH9
图8 不同浓度 NaCl 处理下甜菜叶片BvBADHs相对表达水平 竖线表示标准误差(SE),n=3。误差线上小写字母表示在P<0.05水平差异显著
Fig. 8 Relative expressions of BvBADHs in sugar beet leaves under various concentrations of NaCl The vertical bar represents the standard error(SE), n=3. The lowercase letter at the top of each bar indicates a significant deviation at the P<0.05 level
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