镉逆境胁迫下甜菜BvHIPP24基因在大肠杆菌的功能分析
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摘要
重金属关联异戊二烯化植物蛋白(Heavy metal-associated isoprenylated plant proteins,HIPPs)由于其独特的重金属结合域(heavy metal associated domains,HMA)和异戊二烯序列(isoprenylation motif)的结构特点,使其成为一类重要的金属分子伴侣(Metallochaperones)。本研究利用RT-PCR的方法,从甜菜体内克隆获得了Beta vulgaris heavy metal-associated isoprenylated plant protein24(BvHIPP24)基因。该基因CDS全长为444 bp,编码了147个氨基酸,分子量为16.38 kDa。利用大肠杆菌表达载体构建pEASY-Blunt E1-BvHIPP24重组质粒,并转化到BL21中。通过1 mmol/L IPTG诱导发现:在0.5 mmol/L Cd2+逆境胁迫下,表达BvHIPP24重组菌的生长趋势要明显优于对照菌株,并具有更高的Cd耐受性。因此可以推断,甜菜BvHIPP24基因在镉逆境胁迫过程中发挥着重要的作用,并有可能参与到甜菜细胞重金属离子吸收、转运、区隔以及代谢平衡调控过程中。
Because of unique heavy metal associated domains(HMA) and isoprenylation motif, heavy metalassociated isoprenylated plant proteins(HIPPs) are important members in Metallochaperones. In this study, Beta vulgaris heavy metal-associated isoprenylated plant protein24(BvHIPP24) was cloned by RT-PCR in sugar beet. Its full length of CDS was 444 bp, encoding 147 amino acids with a molecular weight of 16.38 kDa. The recombinant plasmid of pEASY-Blunt E1-BvHIPP24 was constructed and transformed into BL21. The results showed that, by 1 mmol/L IPTG induction, reconstructed E. coli expressing BvHIPP24 with superior Cd tolerance grew better than the control under 0.5 mmol/L CdCl2 stress. Hence, it could be concluded that BvHIPP24 played a vital role in cadmium stress process in sugar beet, and might be involved in cell heavy metal ion absorption, transportation,compartmentalization and metabolic homeostasis.
Because of unique heavy metal associated domains(HMA) and isoprenylation motif, heavy metalassociated isoprenylated plant proteins(HIPPs) are important members in Metallochaperones. In this study, Beta vulgaris heavy metal-associated isoprenylated plant protein24(BvHIPP24) was cloned by RT-PCR in sugar beet. Its full length of CDS was 444 bp, encoding 147 amino acids with a molecular weight of 16.38 kDa. The recombinant plasmid of pEASY-Blunt E1-BvHIPP24 was constructed and transformed into BL21. The results showed that, by 1 mmol/L IPTG induction, reconstructed E. coli expressing BvHIPP24 with superior Cd tolerance grew better than the control under 0.5 mmol/L CdCl2 stress. Hence, it could be concluded that BvHIPP24 played a vital role in cadmium stress process in sugar beet, and might be involved in cell heavy metal ion absorption, transportation,compartmentalization and metabolic homeostasis.
引文
[1] Abreu-Neto J B, Turchetto-Zolet A C, Oliveira L F V, et al. Heavy metal-associated isoprenylated plant protein(HIPP):characterization of a family of proteins exclusive to plants[J]. FEBS Journal, 2013, 280(7):1604-1616.
[2] Turchetto-Zolet A C, Pinheiro F, Salgueiro F, et al. Phylogeographical patterns shed light on evolutionary process in South America[J]. Molecular Ecology, 2013, 22:1193-1213.
[3] Barth O, Vogt S, Uhlemann R, et al. Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29[J]. Plant Molecular Biology,2009, 69(1):213-226.
[4] Tehseen M, Cairns N, Sherson S, et al. Metallochaperone-like genes in Arabidopsis thaliana[J]. Metallomics, 2010, 2(8):556-564.
[5] Gao W, Xiao S, Li H Y, et al. Arabidopsis thaliana acyl-CoA-binding protein ACBP2 interacts with heavy-metal-binding farnesylated protein AtFP6[J]. New Phytologist, 2009, 181(1):89-102.
[6] Dykema P E, Sipes P R, Marie A, et al. A new class of proteins capable of biding transition metals[J]. Plant Molecular Biology,1999, 41(1):139-150.
[7] Hung I H, Casareno R L B, Labesse G, et al. HAH1 is a copper binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense[J]. Journal of Biological Chemistry, 1998, 273(3):1749-1754.
[8] Suzuki N, Yamaguchi Y, Koizumi N, et al. Functional characterization of a heavy metal binding protein Cdl19 from Arabidopsis[J]. The Plant Journal, 2002, 32(2):165-173.
[9] Crowell D N. Functional implications of protein isoprenylation in plants[J]. Progress in lipid research, 2000, 39(5):393-408.
[10] Chandran D, Sharopova N, Ivashuta S, et al. Transcriptome profiling identified novel genes associated with aluminum toxicity,resistance and tolerance in Medicago truncatula[J]. Planta, 2008, 228(1):151-166.
[11]刘大丽,马龙彪,郭爱英,等.镉逆境胁迫下甜菜谷胱甘肽合成酶(BvGS)基因的克隆[J].中国糖料, 2017, 39(6):23-25.
[12] Hasan M K, Cheng Y, Kanwar M K, et al. Responses of plant proteins to heavy metal stress-a review[J]. Frontiers in Plant Science, 2017, 8:1492.
[2] Turchetto-Zolet A C, Pinheiro F, Salgueiro F, et al. Phylogeographical patterns shed light on evolutionary process in South America[J]. Molecular Ecology, 2013, 22:1193-1213.
[3] Barth O, Vogt S, Uhlemann R, et al. Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29[J]. Plant Molecular Biology,2009, 69(1):213-226.
[4] Tehseen M, Cairns N, Sherson S, et al. Metallochaperone-like genes in Arabidopsis thaliana[J]. Metallomics, 2010, 2(8):556-564.
[5] Gao W, Xiao S, Li H Y, et al. Arabidopsis thaliana acyl-CoA-binding protein ACBP2 interacts with heavy-metal-binding farnesylated protein AtFP6[J]. New Phytologist, 2009, 181(1):89-102.
[6] Dykema P E, Sipes P R, Marie A, et al. A new class of proteins capable of biding transition metals[J]. Plant Molecular Biology,1999, 41(1):139-150.
[7] Hung I H, Casareno R L B, Labesse G, et al. HAH1 is a copper binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense[J]. Journal of Biological Chemistry, 1998, 273(3):1749-1754.
[8] Suzuki N, Yamaguchi Y, Koizumi N, et al. Functional characterization of a heavy metal binding protein Cdl19 from Arabidopsis[J]. The Plant Journal, 2002, 32(2):165-173.
[9] Crowell D N. Functional implications of protein isoprenylation in plants[J]. Progress in lipid research, 2000, 39(5):393-408.
[10] Chandran D, Sharopova N, Ivashuta S, et al. Transcriptome profiling identified novel genes associated with aluminum toxicity,resistance and tolerance in Medicago truncatula[J]. Planta, 2008, 228(1):151-166.
[11]刘大丽,马龙彪,郭爱英,等.镉逆境胁迫下甜菜谷胱甘肽合成酶(BvGS)基因的克隆[J].中国糖料, 2017, 39(6):23-25.
[12] Hasan M K, Cheng Y, Kanwar M K, et al. Responses of plant proteins to heavy metal stress-a review[J]. Frontiers in Plant Science, 2017, 8:1492.