甘蔗SsCBL4基因的克隆及表达特性分析
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摘要
类钙调磷酸酶B亚基蛋白(calcineurin B-like, CBL)作为一种特殊的钙感受器在植物逆境胁迫响应过程中发挥重要作用。甘蔗(Saccharum spp.)是我国最主要的糖料作物和能源作物。根据甘蔗前期转录组测序结果,采用反转录PCR (reverse transcription-PCR, RT-PCR)从低钾胁迫甘蔗根系中克隆得到CBL基因,命名为SsCBL4 (GenBank No. KY674 987.1)。该基因包含1个长度为636 bp的开放阅读框,可编码211个氨基酸。SsCBL4蛋白的相对分子量为23.97 903 kD,等电点为4.88,含有2个保守结构域,均与Ca2+结合相关。同时,SsCBL4具有CBL家族典型的EF-hand (elongation factor hand)结构域,并且含有N-豆蔻酰化结构域和与CBL互作激酶蛋白(CBL-interacting protein kinase, CIPK)互作的FPSF位点。同源性分析结果显示,SsCBL4与高粱(Sorghum bicolor)、玉米(Zea mays)、水稻(Oryza sativa)和小麦(Triticum aestivum)等作物的CBL4序列有较高一致性。qPCR检测结果显示,在低氮、低磷、低钾、干旱和盐胁迫下SsCBL4基因的表达均有发生改变。其中,低钾、干旱和盐胁迫条件下,该基因表达均为上调。低氮和低磷胁迫条件下,SsCBL4的表达量表现上下波动的变化趋势,在96 h表达量均达到最高点,随后显著下降;低钾处理8～48 h,SsCBL4基因相对表达量随着时间的延长逐渐下降;干旱胁迫8、24和96 h,该基因的相对表达量相对较高,96 h表达量达到最高,约为对照的20.5倍;盐胁迫24～96 h,表达量随着胁迫时间的延长相对表达量逐渐升高,96 h该基因表达量达到最高,约为对照的9.1倍。这些结果说明SsCBL4基因可能在不同逆境胁迫中发挥重要作用,该研究为深入研究SsCBL4抗逆功能和相关分子机制提供参考。
The calcineurin B-like protein(CBL), which is a unique group of calcium sensors in plant, play a key role in stress response. Sugarcane(Saccharum spp.) is an important crop for sugar and bio-energy in China. According to the datasets which are transcription sequencing of sugarcane genes under abiotic stress, a calcineurin B-like gene named SsCBL4(GenBank No. KY674 987.1), was cloned from root in sugarcane under low potassium stress by RT-PCR(reverse transcription-PCR). SsCBL4 contained a 636 bp complete open reading frame that encodes 211 amino acids. Its molecular weight was 23. 97 903 kD. And the isoelectric point of SsCBL4 was 4.88. The deduced polypeptide of SsCBL2 had 2 transmembrane domains which could bind calcium ions. The SsCBL4 protein contain 3 EF-hand domains(elongation factor hand) that are the typical domain of CBL family, a conserved N-myristoylation motif and FPSF motif which interacted with CBL-interacting protein kinase(CIPK) kinases. The homology analysis indicated that SsCBL4 has high homology to the CBL4 from Sorghum bicolor, Zea mays, Oryza sativa and Triticum aestivum. The changes in expression of SsCBL4 under low nitrogen, low phosphorus, low potassium, drought or salt stresses were detected by qPCR analysis. The expressions of SsCBL4 were commonly up-regulated under low potassium,drought or salt stresses. Under low nitrogen or low phosphorus stress, the fluctuations in SsCBL4 gene expression levels were observed, with the highest level at 96 h, followed by a significant decrease. Under low potassium stress from 8 to 48 h, the expression of SsCBL4 gradually decreased. SsCBL4 was induced to express at relatively high levels at 8, 24 and 96 h after drought treatment, and reached the highest level at 96 h,which was 20.5-fold as high as that of control. Under salt stress from 24 h to 96 h, the expression of SsCBL4 gradually increased, and reached the highest inducible expression level at 96 h, which was 9.1-fold as high as control. These results suggest that SsCBL4 gene may play an important role in different stresses. This study provides a reference for further study of SsCBL4 stress resistance and related molecular mechanisms.
The calcineurin B-like protein(CBL), which is a unique group of calcium sensors in plant, play a key role in stress response. Sugarcane(Saccharum spp.) is an important crop for sugar and bio-energy in China. According to the datasets which are transcription sequencing of sugarcane genes under abiotic stress, a calcineurin B-like gene named SsCBL4(GenBank No. KY674 987.1), was cloned from root in sugarcane under low potassium stress by RT-PCR(reverse transcription-PCR). SsCBL4 contained a 636 bp complete open reading frame that encodes 211 amino acids. Its molecular weight was 23. 97 903 kD. And the isoelectric point of SsCBL4 was 4.88. The deduced polypeptide of SsCBL2 had 2 transmembrane domains which could bind calcium ions. The SsCBL4 protein contain 3 EF-hand domains(elongation factor hand) that are the typical domain of CBL family, a conserved N-myristoylation motif and FPSF motif which interacted with CBL-interacting protein kinase(CIPK) kinases. The homology analysis indicated that SsCBL4 has high homology to the CBL4 from Sorghum bicolor, Zea mays, Oryza sativa and Triticum aestivum. The changes in expression of SsCBL4 under low nitrogen, low phosphorus, low potassium, drought or salt stresses were detected by qPCR analysis. The expressions of SsCBL4 were commonly up-regulated under low potassium,drought or salt stresses. Under low nitrogen or low phosphorus stress, the fluctuations in SsCBL4 gene expression levels were observed, with the highest level at 96 h, followed by a significant decrease. Under low potassium stress from 8 to 48 h, the expression of SsCBL4 gradually decreased. SsCBL4 was induced to express at relatively high levels at 8, 24 and 96 h after drought treatment, and reached the highest level at 96 h,which was 20.5-fold as high as that of control. Under salt stress from 24 h to 96 h, the expression of SsCBL4 gradually increased, and reached the highest inducible expression level at 96 h, which was 9.1-fold as high as control. These results suggest that SsCBL4 gene may play an important role in different stresses. This study provides a reference for further study of SsCBL4 stress resistance and related molecular mechanisms.
引文
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王毅,武维华,2009,植物钾营养高效分子遗传机制.植物学通报,44(1):27-36.(Wang Y.,Wu W.H.,2009,Molecular genetic mechanism of high efficient potassium uptake in plant,Chinese Bulletin of Botany,44(1):27-36)
Chen L,Ren F,Zhou L,et al.2012.The Brassica napus calcineurin B-Like 1/CBL-interacting protein kinase 6(CBL1/CIPK6)component is involved in the plant response to abiotic stress and ABA signaling[J].Journal of Experimental Botany,63(17):6211-6222.
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Fitter A H.1982.Morphometric analysis of root systems:Application of the technique and influence of soil fertility on root system development in two herbaceous species[J].Plant,Cell&Environment,5(4):313-322.
Fitter A H.1985.Functional significance of root morphology and root system architecture[J].Ecological Interaction in Soil Plant Microbes and Animals,87-106.
Gu Z,Ma B,Jiang Y,et al.2008.Expression analysis of the calcineurin B-like gene family in rice(Oryza sativa L.)under environmental stresses[J].Gene,415(1):1-12.
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Held K,Fran?ois P,Eckert C,et al.2011.Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[J].Cell Research,21(7):1116.
Ho C H,Tsay Y F.2010.Nitrate,ammonium,and potassium sensing and signaling[J].Current Opinion in Plant Biology,13(5):604-610.
Lewit-Bentley A,Réty S.2000.EF-hand calcium-binding proteins[J].Current Opinion in Structural Biology,10(6):637-643.
Luan S.2009.The CBL-CIPK network in plant calcium signaling[J].Trends in Plant Science,14(1):37-42.
Tang R J,Liu H,Bao Y,et al.2010.The woody plant poplar has a functionally conserved salt overly sensitive pathway in response to salinity stress[J].Plant Molecular Biology,74(4-5):367-380.
Wang M,Gu D,Liu T,et al.2007.Overexpression of a putative maize calcineurin B-like protein in Arabidopsis confers salt tolerance[J].Plant Molecular Biology,65(6):733-746.
Wang Y,Wu W H.2013.Potassium transport and signaling in higher plants[J].Annual Review of Plant Biology,64(1):451-476.
Ye J,Zhang W,Guo Y.2013.Arabidopsis SOS3 plays an important role in salt tolerance by mediating calcium-dependent microfilament reorganization[J].Plant Cell Reports,32(1):139-148.
Zeng Q Y,Ling Q P,Fan L N,et al.2015.Transcriptome profiling of sugarcane roots in response to low potassium stress[J].PLOS ONE,10(5):e0126306.
Zhang Y,Linghu J,Wang D,et al.2017.Foxtail millet CBL4(SiCBL4)interacts with SiCIPK24,modulates plant salt stress tolerance[J].Plant Molecular Biology Reporter,35(6):634-646.
Zhang H,Yin W,Xia X.2008,Calcineurin B-Like family in Populus:Comparative genome analysis and expression pattern under cold,drought and salt stress treatment[J].Plant Growth Regulation,56(2):129-140.
董连红,史素娟,Manik S M N,等.2014.植物CBL基因家族的研究进展[J].核农学报,29(5):892-898.(Dong L H,Shi S J,Manik S M N,et al.2014.Research advances in CBLs gene family in plants[J].Journal of Nuclear Agricultural Sciences,29(5):892-898.)
郭晋隆.2013.PEG胁迫下的甘蔗RNA-Seq定量分析与差异表达基因鉴定[D].博士学位论文,福建农林大学,导师:许莉萍,pp.Ⅰ.(Guo J L.2013.Quantitative RNA-seq analysis of the sugarcane response to PEGand the identification of differentially expressed genes[D].Thesis for Ph.D.,Fujian Agriculture and Forestry University,Supervisor:Xu L P,pp.Ⅰ.)
汤湖斌,闵康康,徐玲玲,等.2015.CBL-CIPKs信号系统的研究进展[J].中国细胞生物学学报,37(1):100-105.(Tang H B,Min K K,Xu L L,et al.2015.Research progress in CBL-CIPKs signaling system[J].Chinese Journal of Cell Biology,37(1):100-105.)
王毅,武维华,2009,植物钾营养高效分子遗传机制.植物学通报,44(1):27-36.(Wang Y.,Wu W.H.,2009,Molecular genetic mechanism of high efficient potassium uptake in plant,Chinese Bulletin of Botany,44(1):27-36)
Chen L,Ren F,Zhou L,et al.2012.The Brassica napus calcineurin B-Like 1/CBL-interacting protein kinase 6(CBL1/CIPK6)component is involved in the plant response to abiotic stress and ABA signaling[J].Journal of Experimental Botany,63(17):6211-6222.
Cheong Y H,Sung S J,Kim B G,et al.2010.Constitutive overexpression of the calcium sensor CBL5 confers osmotic or drought stress tolerance in Arabidopsis[J].Molecules&Cells,29(2):159-165.
Du W M,Lin H X,Chen S,et al.2011.Phosphorylation of SOS3-like calcium-binding proteins by their interacting SOS2-like protein kinases is a common regulatory mechanism in Arabidopsis[J].Plant Physiology,156(4):1-32.
Famoso A N,Clark R T,Shaff J E,et al.2010.Development of a novel aluminum tolerance phenotyping platform used for comparisons of cereal aluminum tolerance and investigations into rice aluminum tolerance mechanisms[J].Plant Physiology,153(4):1678-1691.
Fitter A H.1982.Morphometric analysis of root systems:Application of the technique and influence of soil fertility on root system development in two herbaceous species[J].Plant,Cell&Environment,5(4):313-322.
Fitter A H.1985.Functional significance of root morphology and root system architecture[J].Ecological Interaction in Soil Plant Microbes and Animals,87-106.
Gu Z,Ma B,Jiang Y,et al.2008.Expression analysis of the calcineurin B-like gene family in rice(Oryza sativa L.)under environmental stresses[J].Gene,415(1):1-12.
Hashimoto K,Eckert C,Anschütz U,et al.2012.Phosphorylation of calcineurin B-like(CBL)calcium sensor proteins by their CBL-interacting protein kinases(CIPKs)is required for full activity of CBL-CIPK complexes toward their target proteins[J].Journal of Biological Chemistry,287(11):7956.
Held K,Fran?ois P,Eckert C,et al.2011.Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[J].Cell Research,21(7):1116.
Ho C H,Tsay Y F.2010.Nitrate,ammonium,and potassium sensing and signaling[J].Current Opinion in Plant Biology,13(5):604-610.
Lewit-Bentley A,Réty S.2000.EF-hand calcium-binding proteins[J].Current Opinion in Structural Biology,10(6):637-643.
Luan S.2009.The CBL-CIPK network in plant calcium signaling[J].Trends in Plant Science,14(1):37-42.
Tang R J,Liu H,Bao Y,et al.2010.The woody plant poplar has a functionally conserved salt overly sensitive pathway in response to salinity stress[J].Plant Molecular Biology,74(4-5):367-380.
Wang M,Gu D,Liu T,et al.2007.Overexpression of a putative maize calcineurin B-like protein in Arabidopsis confers salt tolerance[J].Plant Molecular Biology,65(6):733-746.
Wang Y,Wu W H.2013.Potassium transport and signaling in higher plants[J].Annual Review of Plant Biology,64(1):451-476.
Ye J,Zhang W,Guo Y.2013.Arabidopsis SOS3 plays an important role in salt tolerance by mediating calcium-dependent microfilament reorganization[J].Plant Cell Reports,32(1):139-148.
Zeng Q Y,Ling Q P,Fan L N,et al.2015.Transcriptome profiling of sugarcane roots in response to low potassium stress[J].PLOS ONE,10(5):e0126306.
Zhang Y,Linghu J,Wang D,et al.2017.Foxtail millet CBL4(SiCBL4)interacts with SiCIPK24,modulates plant salt stress tolerance[J].Plant Molecular Biology Reporter,35(6):634-646.
Zhang H,Yin W,Xia X.2008,Calcineurin B-Like family in Populus:Comparative genome analysis and expression pattern under cold,drought and salt stress treatment[J].Plant Growth Regulation,56(2):129-140.