沙冬青干旱诱导表达基因的筛选及AmCASP基因的初步分析
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摘要
干旱是影响植物生长发育的重要环境影响因子之一，严重影响植物生长及产量，现有基因资源的短缺限制了抗旱育种的发展。挖掘有效的抗旱基因是大幅度提高植物抗旱能力的有效手段。沙冬青是我国温带荒漠的惟一珍贵常绿灌木，耐旱性强、抗贫瘠，在长期的逆境适应中形成了独特的耐受机理，是开展植物干旱、盐碱等逆境胁迫研究的理想材料。本文以蒙古沙冬青为材料，在本实验室已有的沙冬青逆境转录表达谱的基础上，筛选抗旱性相关基因并开展功能鉴定研究。为抗旱育种提供基因资源。取得以下进展：
1.沙冬青干旱诱导表达基因的筛选。对已经建立的沙冬青逆境转录表达谱进行生物信息学分析，从中选择45条对沙冬青干旱高温诱导表达条带开展表达谱分析验证。以内源基因β-actin为对照半定量PCR分析，共确定干旱高温诱导表达基因34条。其中细胞代谢相关的4条，逆境相关的3条，信号传递相关的3条，转录表达调控4条，光合作用相关6条，细胞结构2条，物质运输2条，功能未知10条。利用cDNA末端快速扩增（RACR）技术克隆获得其中5个基因全长序列。分别是：片段TDF36：与大豆Rubisco核酮糖羧化酶/加氧酶具有84%同源性；片段TDF179与葡萄叶绿体基因组DNA ATP-β亚基合酶90%同源性；片段TDF466：热激蛋白基因家族同源基因；高度保守功能未知同源基因片段TDF229；同源序列比对选择拟南芥凯氏带膜蛋白（CASP）42%氨基酸序列同源基因TDF445为目标基因进一步研究，初步命名为AmCASP。
2. AmCASP基因特征分析。AmCASP基因全长525bp，编码106个氨基酸，与拟南芥的拟南芥凯氏带膜蛋白（CASP）42%氨基酸序列同源性，其中保守区域C末端约有20aa高度同源。染色体步移技术克隆AmCASP基因启动子长度为2300bp，生物信息学分析明确转录起始位点位于基因上游1443bp处，转录起始位点上游20bp处有典型ATAT区，符合真核生物启动子序列特征，明确AmCASP基因的序列特征并有效验证基因序列全长的完整性。利用Southern杂交技术以同位素标记探针确定AmCASP基因在沙冬青中为单一拷贝。
3. AmCASP基因的功能分析。在pCAMBIA1302基础上构建植物融合表达载体，以绿色荧光蛋白为标记基因进行洋葱亚细胞定位，确定基因AmCASP功能部位为核蛋白，初步预定为干旱胁迫调节蛋白。具体调节通路有待进一步实验验证。
利用具有同源性的拟南芥凯氏带膜蛋白CASP突变体根长度、萌发时间、表性分析、干旱处理等验证AmCASP基因功能。结果表明拟南芥CASP基因功能缺失突变体萌发迟缓，转入生殖生长迅速，初步预测与突变体植株干旱不耐性相关。
此外，构建植物表达载体分别转化野生型和突变体植株，分别获得T0代植株并做初步生理指标检测和根长度分析为下一步功能验证提供了实验材料。
Drought is one of the most important environmental factor which is affecting plant growth anddevelopment, and seriously impact the plant growth and yield. The shortage existing of geneticresources limits the development of drought resistence breeding. Investigating effective drought gene isan effective way to give the potentially possibility to improve plant drought tolerance. Ammopiptanthusmongolicus which is the only temperate desert valuable evergreen shrubs, drought tolerance, anti-barren,and the unique mechanism of tolerance in the long-term adaptation of adversity, is possible to carry outan ideal material for plant drought, salt, etc Stress. In his paper Ammopiptanthus mongolicus was usedas the experiment material, basis on the Ammopiptanthus adversity transcriptional expression profilingof A.mmopiptanthus which was established in our laboratory, drought induced expression genes wereselected and AmCASP gene’s function was identified. The detail results are as follow:
1. Cloning of full-length cDNA drought induced expression genes in A. mongolicus.By usingbioinformatics analysis method,45fragments of adversity transcriptional expression genes in A.mongolicus, were choosed to classify under high temperature and drought stress as the candidate tocarry out expression analysis validation. With the gene β-actin as the control, the semi-quantitative PCRanalysis identified34genes responded to drought and heat induced expression. Those genes related tothe cell metabolism (4genes), the adversity related (3genes), the signaling related (3genes), thetranscriptional expression regulation (4genes), photosynthesis (6genes), the cell structure (2genes) andwater and nourishment transport (2genes), function unknown (10genes). With the method of cDNAends rapid amplification (RACR)5full length cDNA sequence were cloned, they are: TDF36,84%homology with Rubisco ribulose carboxylase/oxygenase enzyme fragment in soybean; TDF179,90%homology with chloroplast genome DNA in, the ATP synthase-β subunit fragment; TDF466, heat shockprotein homologous fragment in Grapes; TDF229, highly conserved homologous fragment whilefunction unknown genes, amino acid sequences42%homologous with Arabidopsis thalianaCasparian strip membrane protein (CASP) gene and TDF445was selected as the target gene to makerfurther study, tentatively named AmCASP.
2. Profile detecting of AmCASP gene. AmCASP gene is525bp in length, encoding106aminoacids,42%amino acid sequence homology with A.thaliana Casparian strip membrane protein (CASP)including conserved region C-terminal about20aa. Throught genome walking the promote of AmCASPgene was cloned and the length of the sequence is2300bp, bioinformatics analysis showed a cleartranscription start site locats at-1443bp upstream the target gene, bsieds at about20bp upstream thetranscription start site there has a typical ATAT box, in line with the sequences of eukaryotic promoterscharacteristics, which conformed AmCASP gene sequence characteristics and verified the integrity ofthe gene sequence. Southern blot hybridization result showed that the AmCASP gene in A. mongolicuswas a single copy.
3. AmCASP gene’s functional analysis. AmCASP gene’s plant expression vector was constructed to pCAMBIA1302, which had a green fluorescent protein as marker gene. AmCASP gene was located inthe nucleoprotein by using the onion sub-cellular localization method, presumed it is related withdrought stress Regulatory pathway verification needs to be further experiment.
To verificate AmCASP gene’s fuction we tested root length, germination time, physiologicalindicators of drought resistance of Arabidopsis CASP gene’s mutant, it’s CASP arian strip membraneprotein gene homology with target gene. The results showed that Arabidopsis without CASP geneinduced the germination delayed, but the reproductive growth rapiddly, initially predict this gene relatedwith drought intolerance of the mutant plants.
In the further, plant expression vectors will be transformed into wild type and mutant plants toverify next function.
1.沙冬青干旱诱导表达基因的筛选。对已经建立的沙冬青逆境转录表达谱进行生物信息学分析，从中选择45条对沙冬青干旱高温诱导表达条带开展表达谱分析验证。以内源基因β-actin为对照半定量PCR分析，共确定干旱高温诱导表达基因34条。其中细胞代谢相关的4条，逆境相关的3条，信号传递相关的3条，转录表达调控4条，光合作用相关6条，细胞结构2条，物质运输2条，功能未知10条。利用cDNA末端快速扩增（RACR）技术克隆获得其中5个基因全长序列。分别是：片段TDF36：与大豆Rubisco核酮糖羧化酶/加氧酶具有84%同源性；片段TDF179与葡萄叶绿体基因组DNA ATP-β亚基合酶90%同源性；片段TDF466：热激蛋白基因家族同源基因；高度保守功能未知同源基因片段TDF229；同源序列比对选择拟南芥凯氏带膜蛋白（CASP）42%氨基酸序列同源基因TDF445为目标基因进一步研究，初步命名为AmCASP。
2. AmCASP基因特征分析。AmCASP基因全长525bp，编码106个氨基酸，与拟南芥的拟南芥凯氏带膜蛋白（CASP）42%氨基酸序列同源性，其中保守区域C末端约有20aa高度同源。染色体步移技术克隆AmCASP基因启动子长度为2300bp，生物信息学分析明确转录起始位点位于基因上游1443bp处，转录起始位点上游20bp处有典型ATAT区，符合真核生物启动子序列特征，明确AmCASP基因的序列特征并有效验证基因序列全长的完整性。利用Southern杂交技术以同位素标记探针确定AmCASP基因在沙冬青中为单一拷贝。
3. AmCASP基因的功能分析。在pCAMBIA1302基础上构建植物融合表达载体，以绿色荧光蛋白为标记基因进行洋葱亚细胞定位，确定基因AmCASP功能部位为核蛋白，初步预定为干旱胁迫调节蛋白。具体调节通路有待进一步实验验证。
利用具有同源性的拟南芥凯氏带膜蛋白CASP突变体根长度、萌发时间、表性分析、干旱处理等验证AmCASP基因功能。结果表明拟南芥CASP基因功能缺失突变体萌发迟缓，转入生殖生长迅速，初步预测与突变体植株干旱不耐性相关。
此外，构建植物表达载体分别转化野生型和突变体植株，分别获得T0代植株并做初步生理指标检测和根长度分析为下一步功能验证提供了实验材料。
Drought is one of the most important environmental factor which is affecting plant growth anddevelopment, and seriously impact the plant growth and yield. The shortage existing of geneticresources limits the development of drought resistence breeding. Investigating effective drought gene isan effective way to give the potentially possibility to improve plant drought tolerance. Ammopiptanthusmongolicus which is the only temperate desert valuable evergreen shrubs, drought tolerance, anti-barren,and the unique mechanism of tolerance in the long-term adaptation of adversity, is possible to carry outan ideal material for plant drought, salt, etc Stress. In his paper Ammopiptanthus mongolicus was usedas the experiment material, basis on the Ammopiptanthus adversity transcriptional expression profilingof A.mmopiptanthus which was established in our laboratory, drought induced expression genes wereselected and AmCASP gene’s function was identified. The detail results are as follow:
1. Cloning of full-length cDNA drought induced expression genes in A. mongolicus.By usingbioinformatics analysis method,45fragments of adversity transcriptional expression genes in A.mongolicus, were choosed to classify under high temperature and drought stress as the candidate tocarry out expression analysis validation. With the gene β-actin as the control, the semi-quantitative PCRanalysis identified34genes responded to drought and heat induced expression. Those genes related tothe cell metabolism (4genes), the adversity related (3genes), the signaling related (3genes), thetranscriptional expression regulation (4genes), photosynthesis (6genes), the cell structure (2genes) andwater and nourishment transport (2genes), function unknown (10genes). With the method of cDNAends rapid amplification (RACR)5full length cDNA sequence were cloned, they are: TDF36,84%homology with Rubisco ribulose carboxylase/oxygenase enzyme fragment in soybean; TDF179,90%homology with chloroplast genome DNA in, the ATP synthase-β subunit fragment; TDF466, heat shockprotein homologous fragment in Grapes; TDF229, highly conserved homologous fragment whilefunction unknown genes, amino acid sequences42%homologous with Arabidopsis thalianaCasparian strip membrane protein (CASP) gene and TDF445was selected as the target gene to makerfurther study, tentatively named AmCASP.
2. Profile detecting of AmCASP gene. AmCASP gene is525bp in length, encoding106aminoacids,42%amino acid sequence homology with A.thaliana Casparian strip membrane protein (CASP)including conserved region C-terminal about20aa. Throught genome walking the promote of AmCASPgene was cloned and the length of the sequence is2300bp, bioinformatics analysis showed a cleartranscription start site locats at-1443bp upstream the target gene, bsieds at about20bp upstream thetranscription start site there has a typical ATAT box, in line with the sequences of eukaryotic promoterscharacteristics, which conformed AmCASP gene sequence characteristics and verified the integrity ofthe gene sequence. Southern blot hybridization result showed that the AmCASP gene in A. mongolicuswas a single copy.
3. AmCASP gene’s functional analysis. AmCASP gene’s plant expression vector was constructed to pCAMBIA1302, which had a green fluorescent protein as marker gene. AmCASP gene was located inthe nucleoprotein by using the onion sub-cellular localization method, presumed it is related withdrought stress Regulatory pathway verification needs to be further experiment.
To verificate AmCASP gene’s fuction we tested root length, germination time, physiologicalindicators of drought resistance of Arabidopsis CASP gene’s mutant, it’s CASP arian strip membraneprotein gene homology with target gene. The results showed that Arabidopsis without CASP geneinduced the germination delayed, but the reproductive growth rapiddly, initially predict this gene relatedwith drought intolerance of the mutant plants.
In the further, plant expression vectors will be transformed into wild type and mutant plants toverify next function.
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55. Lei J., Li Z., An Effect of the Extract of Ammopiptanthus mongolicus cheng f.(JA1) onInduction of Apoptosis of HepG2in vitro and Its Molecular Mechanisms. Biomedical andenvironmental sciences2006,19:118-123.
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59. Xiong L., Zhu J.K., Molecular and genetic aspects of plant responses to osmotic stress.Plant Cell and Environment2002,25,131-139.
60. Liu M.Q., Lu C.F., Characterization and function analysis of a cold-induced AmCIP geneencoding a dehydrin-like protein in Ammopiptanthus mongolicus. DNA Sequence2006,17(5):342-349.
61. He M., Xia Y.X., Construction and analysis of a normalized cDNA library fromMetarhizium anisopliae var acridum germinating and dierentiating on Locusta migratoriawings.Fems Microbiology letters2008,12:127-135.
62. Hajheidari M., Abdollahian N.M., Askari H., Proteome analysis of sugar beet leaves underdrought stress. Proteomics2005,5:950-960.
63. Motoaki S., Ayako K., Kazuko Y.S., Molecular responses to drought, salinity and frost:common and different paths for plant protection. Plant biotechnology2003,14:194-199.
64. Cao P.X., Song J., Zhou C.J., Characterization of multiple cold induced genes fromAmmopiptanthus mongolicus and functional analyses of gene AmEBP1. Plant MolecularBiology2009,69:529-539.
65. Hajdukiewicz P., Svab Z., Maliga P., The small, versatile pPZP family of Agrobacteriumbinary vectors for plant transformation. Plant Molecular Biology1994,25:989-994.
66. Pineros M., Tester M., Calcium channels in higher plant cells: selectivity, regulation andpharmacology. Journal of experimental botany1997,48:551-577.
67. Agarwal P.K., Reddy M.K., Sopory S.K., Role of DREB transcription factors in abiotic andbiotic stress tolerance in plants. Plant Cell Reports2006,25:1263-1274.
68. Shen Q.X., Chen C.N., Alex B., The stress and abscisic acid-induced barley gene HVA22:developmentalregulation and homologues in diverse organisms. Plant Molecular Biology2001,45:327-340.
69. Wei Q., Guo Y.J., Cloning and characterization of an AtNHX2-like Na+/H+antiporter genefrom Ammopiptanthus mongolicus (Leguminosae) and its ectopic expression enhanceddrought and salt tolerance in Arabidopsis thaliana. Journal of Plant Biote-chnology2011,105:309-316.
70. Mittler R., Abiotic stress, the field environment and stress combination. Trends in PlantScience2006,1:15-19.
71. Rouhi V.R., Samson R., Stomatal resistance under drought stress conditions induced byPEG6000on wild almond. Journal of Plant Physiology and Breeding2006,71(1):269-24.
72. Liu R.L., Liu M.Q., Heterologous expression of an Ammopiptanthus mongolicus lateembryogenesis abundant protein gene (AmLEA) enhances Escherichia coli viability undercold and heat stress. Plant Growth Regulation2010,60:163-168
73. Xu S.J., An L.Z., The seasonal effects of water stress on Ammopiptanthus mongolicus in adesert environment. Journal of Arid Environments2002,51:437-447.
74. Kenta S., Tomoko T., Tetsuko T., et al., Accumulation of glycinebetaine in rice plants thatoverexpress choline monooxygenase from spinach and evaluation of their tolerance to abioticstress. Annales Botanici Fennici2006,98(3):565-571.
75. Shusei S., Yasukazu N., Takakazu K., Structural Analysis of Arabidopsis thaliana Chromosome3Ⅱ.Sequence Features of the Regions of4,504,864bp Covered by Sixty PI and TAC Clones. DNAresearch2000,7:131-13.
76. Kurkela S., Franck M., Cloning and characterization of a cold and ABA inducible Arabidopsisgene. Plant Molecular Biology1990,15:137-144.
77. Guo S.J., Ajay P., Characterization of an Oxidative Stress Inducible Nonspecific Lipid TransferProtein Coding cDNA and its Promoter from Drought Tolerant Plant Prosopis juliflora. PlantMolecular Biology2010,28:32-40.
78. Umezawa T.S., Sakurai T., Totoki Y., Sequencing and Analysis of Approximately40000SoybeancDNA Clones from a Full-Length-Enriched cDNA Library. DNA Research2008,15:333-346.
79. Gao T.P., Chen T., Feng H.Y., Seasonal and annual variation of osmotic solute and stable carbonisotope composition in leaves of endangered desert evergreen shrub Ammopiptanthus mongolicus.South African Journal of Botany2006,72:570-578.
80. Tran L.S., Nakashima K., Sakuma Y., Isolation and functional analysis of Arabidopsisstress-inducible NAC transcription factors that bind to a drought-responsive cis-element in theearly responsive to dehydration stress promoter. Plant Cell2004,16:2481-2498.
81. Babu V., Henry T.N., Understanding regulatory networks and engineering for enhanced droughttolerance in plants. Plant Biology2006,9(2):189-195.
82. Chinnusamy V., Karen S., Zhu J.K., Molecular genetic perspectives on cross-talk and specicity inabiotic stress signalling in plants. Journal of Experimental Botany2004,1:225-236.
83. Wang W.X., Basia V.A., Plant responses to drought, salinity and extreme temperaturestowards genetic engineering for stress tolerance. Planta2003,218:1-14.
84. Wang W., Chen J.J., Extraordinary accumulations of antioxidants in A. mongolicus andTetraena mongolica distributed in extremely stressful environments. Botanical Studies2007,48:55-61.
85. Ge X.J., Yu Y., Yuan Y.M., Genetic Diversity and Geographic Differentiat-ion inEndangered Ammopiptanthus (Leguminosae) Populations in Desert Regions of NorthwestChina asRevealed by ISSR Analysis. Annals of Botany2005,95:843-851.
86. Fei Y.B., Cao P.X., Gao S.Q., et al., Purification and Structure Analysis of AntifreezeProteins from Ammopiptanthus mongolicus. Preparative Biochemistry and Biotechnology2008,7(38):172-183.
87. Wang Z.L., An X.M., Li B., Identification and characterization of CBF/DR EB1relatedgenes in Populus hopeiensis. Forestry Studies in China2006,10(3):143-148.
88. Hong Z., Lakkineni K., Zhang Z., Removal of Feedback Inhibition of△1–Pyrro-line-5-Carboxylate Synthetase Results in Increased ProlineAccumulation and Protection ofPlants from Osmotic Stress. Plant Physiology2000,4(122):1129-1136.
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