盐碱胁迫下羊草相关基因及蛋白质的表达谱研究
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摘要
羊草(Leymus chinensis)是一种广布于东北盐碱地区的优良牧草，对盐碱胁迫具有较强的耐受性，其中蕴藏着大量的抗逆基因和相关蛋白质。大规模分离、鉴定cDNA克隆及获得蛋白质表达谱，对进一步丰富抗逆功能基因资源库、探明植物耐受盐碱分子机理具有重要意义。本文以羊草为试验材料，对羊草盐碱胁迫耐受相关基因群及蛋白质表达谱进行了以下研究：
1．盐碱胁迫下羊草消减文库的构建及差异基因片段的筛选分析
以黑龙江省青冈县天然盐碱地(pH 9.5)草场生长的羊草地上部分cDNA为试验方(Tester)，同期日光温室培养土(pH 5-6)条件下生长的8周龄非盐碱条件下生长的羊草地上部分cDNA为消减方(Driver)，利用抑制性消减杂交技术(suppression subtractive hybridization，SSH)构建了盐碱胁迫下羊草消减文库。从消减文库中共筛选1920个阳性克隆，PCR鉴定插入片段大部分集中在300-1000 bp之间。去冗余后得到的552个非重复序列，将这些序列与GenBank数据库中的序列进行比对，发现其中有548条与数据库中的序列有同源性，其中功能已知序列339个，功能未知序列209条；另外有4条序列在数据库中未发现其同源序列。功能已知的基因种类涉及代谢、能量、细胞生长及分化、转录、蛋白质合成、蛋白质定位及贮存、细胞运输、细胞结构、转座子、信号转导、细胞胁迫及防御。消减文库中获得的基因其中包括已报道的在其他植物体中获得的盐碱胁迫相关的基因，如单脱氢抗坏血酸还原酶基因(monodehydroascorbate reductase，MDAR)、γ-谷氨酰半胱氨酸合成酶基因(gamma-glutamylcysteine synthetase，γ-ECS)、铁蛋白基因(ferrtin，FER)、水孔蛋白基因(aquaporin，PIP)、脱水素基因(dehydrin，DHN)、硫氧还蛋白还原酶基因(thioredoxin reductase，TR)、14-3-3蛋白基因等，其功能涉及活性氧清除、渗透调节、离子转运、基因调控等。
2．利用cDNA微阵列检测羊草盐碱胁迫下基因的表达
消减文库获得的552个非重复序列经PCR扩增后，制备cDNA微阵列。在反转录过程中，利用引物序列中包含的Cy3、Cy5荧光染料捕获序列标记盐碱胁迫下生长的羊草和温室培养羊草(非盐碱胁迫)的cDNA，按照3DNA Array900~(?)试剂盒中相应步骤完成芯片杂交，用ScanArray 4000~(?)芯片扫描仪扫描芯片。芯片的扫描根据添加的外标调整Cy3与Cy5的信号强度，使信号均一化后通过SAM软件分析上述样品基因表达差异的显著性。芯片杂交进行了三次生物学重复，杂交结果显示出的变化倍数超过2倍的认为是具有显著表达差异的基因。三次获得的差异表达基因个数分别为198、197、192个。三次杂交显著上调表达的基因个数分别为195、144、192个，显著下调的基因个数分别为3、53、0个。上调基因中部分基因为已报道与盐碱胁迫相关基因，如脱水素基因、单脱氢抗坏血酸还原酶基因、硫氧还蛋白基因、铁蛋白基因、水孔蛋白基因、14-3-3蛋白基因等。另外一些上调基因并没有其与盐碱胁迫相关的报告。
3．盐碱胁迫下上调基因PIP、MDAR、FER、14-3-3在羊草植株中表达分析
羊草植株用浓度为200 mmol／L的Na_2CO_3溶液进行胁迫处理，处理时间分别为2、6、12、24、48和72 h，提取各处理样品的总RNA，利用特异探针进行Northern杂交，分析水孔蛋白基因、单脱氢抗坏血酸还原酶基因、铁蛋白基因、14-3-3蛋白基因在不同胁迫处理时间段内的表达情况。结果表明：在盐碱胁迫条件下，4个基因均被诱导表达，且随盐碱胁迫处理时间的延长而呈上升表达的趋势，但表达特性并不完全一致。PIP、MDAR、FER基因的表达量在测定范围呈递增趋势，而14-3-3蛋白基因从2h开始至72 h表达量明显增加，12h、24h达到最高值，随后表达量开始下降。从这些结果中可以看出，这些基因都受盐碱胁迫诱导，在盐碱胁迫条件下表达量增加，功能基因的表达与调控基因的表达方式有所不同。
4．利用二维液相色谱分析盐碱胁迫条件下羊草特异蛋白质的表达
共设四组试验材料：处理组Ⅰ(盐碱胁迫)为温室内栽培的8周龄羊草，用200 mmol／LNa_2CO_3浇灌72h后的植株，对照组Ⅰ(非盐碱胁迫)材料为温室内栽培的8周龄羊草；处理组Ⅱ(盐碱胁迫)为盐碱地草场自然生长的羊草；对照组Ⅱ(非盐碱胁迫)为同期取回的天然生长的植株重新种植在温室栽培土条件下的羊草。用三氯乙酸—丙酮沉淀法提取总蛋白，蛋白质沉淀裂解后，采用第一维色谱聚焦、第二维反相高效色谱的组合模式进行二维液相色谱分离。第一维分离中将等电点(pI)在8.5-4.0范围内的蛋白质样品分离为每间隔0.3个pI梯度的组分。第一维分离组分接下来根据蛋白质的疏水性进行第二维分离。等电点8.5-4.0范围内的蛋白质样品经过二维分离后利用ProteoVue软件将所有组分按照等电点排序后即可获得蛋白质表达谱。本试验获得了处理组Ⅰ、Ⅱ和对照组Ⅰ、Ⅱ的羊草盐碱胁迫及非盐碱胁迫下植物的总蛋白质表达谱，在整个pH 8.5-4.0范围内条带清晰，蛋白质分布呈现为两个主要部分，pI为6.1-4.0和8.5-7.0两个区段范围内，其中pI为6.2-4.0范围内蛋白质表达量高于pI为8.5-7.2范围内蛋白质，即蛋白质集中在酸性范围内。但是pI为6.2-4.0范围内蛋白质疏水性接近导致蛋白质条带区分并不清晰，pI为7.2-8.5范围内蛋白质的分离效果要好于前者。处理组蛋白质中性、碱性蛋白质的表达量要稍高于对照组中性、碱性端蛋白质表达量。
利用DeltaVue软件将色谱峰比值大于2倍的蛋白质初步定为存在表达差异的蛋白质组分。处理组Ⅰ与对照组Ⅰ比较后获得的总差异表达蛋白质组分90个，等电点为8.5-6.4范围差异表达蛋白质组分58个，其中对照组高于处理组的差异表达蛋白质组分共37个，处理组高于对照组的差异表达蛋白质组分共21个。处理组Ⅱ与对照组Ⅱ比较后获得的总差异表达蛋白质组分46个。等电点为8.5-6.4范围差异表达蛋白质组分23个，其中对照组高于处理组表达量的差异表达蛋白质组分共12个，处理组高于对照组的差异表达蛋白质组分共11个。其中大部分差异表达蛋白质组分的差异倍数均集中在2倍左右，少数蛋白质变化倍数在5倍以上。对差异蛋白质的鉴定工作有待进一步研究。
Leymus chinensis (Trin.) Tzvel., a high quality plant for grazing, adapts to alkaline-sodic soil conditions and distributes widely in the Northeast of China. This species has developed molecular and physiological mechanisms during the course of evolution, which involve lots of anti-stress genes and proteins to adapt stress conditions. To identify and characterize the complexity of this adaptation, it is important to identify, genes by large-scale sequencing of cDNA clones and related proteins. The cDNA clones and related proteins are expected to enrich the stress related molecular resources and reveal the molecular mechanism. In this dissertation, studies were made on the stress-related genes and proteins expression profile of Leymus chinensis shoots under saline-alkaline stress.
1. The construction of subtraetive library of Leymus chinesis under saline-alkaline stress
Using cDNAs from Leymus chinensis seedlings grown on the natural habitat of saline-alkaline soil was regarded as tester and cDNA from seedlings in normal growth as driver, suppression subtractive hybridization (SSH) was employed to construct cDNA subtracted library. Of the 1920 cDNA clones showed high quality sequences. The size of inserts was 300-1000 base pairs as it was determined by PCR. In the subtractive library, 552 clones were non- redundant, of which 339 had homology to the known transcripts, and 209 were unknown or unclassified. The remaining 4 ESTs had no homology in the databases searched. The suggested functions ESTs which have the annotation in the database include metabolism, energy, protein synthesis, protein storage, disease/defense, signal transduction, transporter and transpon. Sequence comparison of these clones using BlastN led to the identification of several putative saline-alkaline stress related genes, such as monodehydroascorbate reductase (MDAR), gamma-glutamylcysteine synthetase (γ-ECS), thioredoxin reductase (TR), Ferrtin (FER), Aquaporin (PIP), Dehydrin (DHN) and 14-3-3 protein. The suggested function of the ESTs involved active oxygen elimination, osmotic adjustment, ion transport and regulation of the genes. The putative ESTs will serve as useful resource to demonstrate the molecular basis of saline-alkaline stress related genes and for the further study of gene expression in Leymus chinensis. We consider that some of these genes and their metabolisms are possibly involved in the process of salt tolerance in Leymus chinensis.
2. Gene expression profile of Leymus chinensis under saline-alkaline stress detected by cDNA microarray
The inserts amplified from 552 sequences of Leymus chinensis SSH library clones were spotted on slides to get cDNA microarray. Saline-alkaline stress and non-stress mRNA were labeled with Cy3- or Cy5- fluorescence dye respectively during reverse transcription and then mixed as targets. Microarray hybridization was accomplished following the direction of user manual of 3DNA 900~(?). The array was scanned by ScanArray 4000~(?) microarray scanner. For standardization, the Cy3 and Cy5 signal intensities were adjusted to fit external controls. The microarry was hybrid for three times. Using SAM software to analyze the significance of difference of the microarray genes, 198, 197, and 192 significant regulated genes were detected in the three hybridizations, respectively. Among the significant regulated genes there were 3, 53, 0 down-regulated genes, respectively. In function-known genes included monodehydro-ascorbate reductase, gamma-glutamylcysteine synthetase, ferrtin, aquaporin, dehydrin, thioredoxin reductas, and 14-3-3 proteins, all of which were known to be related with stress tolerance. There is no report for other up-regulated genes.
3. Expression of up-regulated genes, MDAR, FER, PIP, and 14-3-3s investigated by Northern blotting.
Firstly, to blast the sequence of MDAR, FER, PIP, 14-3-3s with the related genes of other plants in database to design the primer. The special sequences of the four genes were labeled by the dig- mark to get the probe. The seedlings of treated with 200 mmol/L Na_2CO_3 for 2, 6,12, 24, 48 and 72h were used for extraction of RNA for Northern blot. The expression of four genes was detected in treatments. MDAR, FER, PIP, and 14-3-3s transcriptome increased with the duration of the treatment. But the genes showed different expression patterns. In 14-3-3s expression pattern, the largest amount was observed in seedlings treated for 12h and 24h, and then the expression amount decreased. The results indicated the genes induced by saline-alkaline stress. Yet the function genes and regulation genes showed different expression patterns.
4. Protein expression profile of Leymus chinensis with two-dimensional liquid chromatography
There were four groups of Leymus chinensis plants seedlings collected from the natural habitat (treatmentⅠ), seedlings poured with 200 mmol/L Na_2CO_3 after 72h (treatmentⅡ), seedlings in the natural habitat planted in the non-saline-alkaline soil (controlⅠ), and eight- week-old seedlings grown in the greenhouse without any stress (controlⅡ). Total proteins were extracted by TCA-acetone precipitation method. Proteins were fractionated in the first dimension using chromatofocusing (CF). Subsequently the fractions with pH value between 8.5 and 4.0 collected after first dimension separation were further fractionated by high-performance reverse phase liquid chromatography (HPRP). The pI/UV map showed that the protein expression profiling of Leymus chinensis was generated by ProteoVue software. In the experiment, four protein express profiles were got from samples of each of treatmentsⅠ,Ⅱ, controlⅠ, andⅡ.
The protein expression profile maps showed clear bands and good-qulity fraction. The contribution of the protein fraction focused on pI range 6.1-4.0 and 8.5-7.0. The amount of protein in pI 6.2-4.0 was higher than that in 8.5-7.2, indicating mostly acidic proteins. But the proteins with pI in 6.2-4.0 had the similar hydrophilicity. The expression amount of neutral and basic proteins in treatments was higher than that of the controls.
To get the differential proteins under the saline-alkaline stress, protein expression profiles of controls and treatments were compared by Deltavue software. Proteins which the peak height of controls and treatments was above two was decided the differential ones.There were 90 differential proteins, in each of controlⅠand treatmentⅠ, and 46 in each of controlⅡand treatmentⅡ. In pI 8.5-6.4, there were 58 differential proteins in controlⅠand treatmentⅠ. The higher amounts of differential proteins in control were 37, and that in treatment were 21. And in controlⅡand treatmentⅡthere are 23 differential proteins. The higher amount of differential proteins in control were 12, and that in treatment were 11.The peak height ratio of mostly differential proteins were about two, with some observations showing higher than five. Further studies remain to be made on such as the identification of differential proteins. The differential expression protein is important in order to understand the mechanism under saline-alkaline stress.
1．盐碱胁迫下羊草消减文库的构建及差异基因片段的筛选分析
以黑龙江省青冈县天然盐碱地(pH 9.5)草场生长的羊草地上部分cDNA为试验方(Tester)，同期日光温室培养土(pH 5-6)条件下生长的8周龄非盐碱条件下生长的羊草地上部分cDNA为消减方(Driver)，利用抑制性消减杂交技术(suppression subtractive hybridization，SSH)构建了盐碱胁迫下羊草消减文库。从消减文库中共筛选1920个阳性克隆，PCR鉴定插入片段大部分集中在300-1000 bp之间。去冗余后得到的552个非重复序列，将这些序列与GenBank数据库中的序列进行比对，发现其中有548条与数据库中的序列有同源性，其中功能已知序列339个，功能未知序列209条；另外有4条序列在数据库中未发现其同源序列。功能已知的基因种类涉及代谢、能量、细胞生长及分化、转录、蛋白质合成、蛋白质定位及贮存、细胞运输、细胞结构、转座子、信号转导、细胞胁迫及防御。消减文库中获得的基因其中包括已报道的在其他植物体中获得的盐碱胁迫相关的基因，如单脱氢抗坏血酸还原酶基因(monodehydroascorbate reductase，MDAR)、γ-谷氨酰半胱氨酸合成酶基因(gamma-glutamylcysteine synthetase，γ-ECS)、铁蛋白基因(ferrtin，FER)、水孔蛋白基因(aquaporin，PIP)、脱水素基因(dehydrin，DHN)、硫氧还蛋白还原酶基因(thioredoxin reductase，TR)、14-3-3蛋白基因等，其功能涉及活性氧清除、渗透调节、离子转运、基因调控等。
2．利用cDNA微阵列检测羊草盐碱胁迫下基因的表达
消减文库获得的552个非重复序列经PCR扩增后，制备cDNA微阵列。在反转录过程中，利用引物序列中包含的Cy3、Cy5荧光染料捕获序列标记盐碱胁迫下生长的羊草和温室培养羊草(非盐碱胁迫)的cDNA，按照3DNA Array900~(?)试剂盒中相应步骤完成芯片杂交，用ScanArray 4000~(?)芯片扫描仪扫描芯片。芯片的扫描根据添加的外标调整Cy3与Cy5的信号强度，使信号均一化后通过SAM软件分析上述样品基因表达差异的显著性。芯片杂交进行了三次生物学重复，杂交结果显示出的变化倍数超过2倍的认为是具有显著表达差异的基因。三次获得的差异表达基因个数分别为198、197、192个。三次杂交显著上调表达的基因个数分别为195、144、192个，显著下调的基因个数分别为3、53、0个。上调基因中部分基因为已报道与盐碱胁迫相关基因，如脱水素基因、单脱氢抗坏血酸还原酶基因、硫氧还蛋白基因、铁蛋白基因、水孔蛋白基因、14-3-3蛋白基因等。另外一些上调基因并没有其与盐碱胁迫相关的报告。
3．盐碱胁迫下上调基因PIP、MDAR、FER、14-3-3在羊草植株中表达分析
羊草植株用浓度为200 mmol／L的Na_2CO_3溶液进行胁迫处理，处理时间分别为2、6、12、24、48和72 h，提取各处理样品的总RNA，利用特异探针进行Northern杂交，分析水孔蛋白基因、单脱氢抗坏血酸还原酶基因、铁蛋白基因、14-3-3蛋白基因在不同胁迫处理时间段内的表达情况。结果表明：在盐碱胁迫条件下，4个基因均被诱导表达，且随盐碱胁迫处理时间的延长而呈上升表达的趋势，但表达特性并不完全一致。PIP、MDAR、FER基因的表达量在测定范围呈递增趋势，而14-3-3蛋白基因从2h开始至72 h表达量明显增加，12h、24h达到最高值，随后表达量开始下降。从这些结果中可以看出，这些基因都受盐碱胁迫诱导，在盐碱胁迫条件下表达量增加，功能基因的表达与调控基因的表达方式有所不同。
4．利用二维液相色谱分析盐碱胁迫条件下羊草特异蛋白质的表达
共设四组试验材料：处理组Ⅰ(盐碱胁迫)为温室内栽培的8周龄羊草，用200 mmol／LNa_2CO_3浇灌72h后的植株，对照组Ⅰ(非盐碱胁迫)材料为温室内栽培的8周龄羊草；处理组Ⅱ(盐碱胁迫)为盐碱地草场自然生长的羊草；对照组Ⅱ(非盐碱胁迫)为同期取回的天然生长的植株重新种植在温室栽培土条件下的羊草。用三氯乙酸—丙酮沉淀法提取总蛋白，蛋白质沉淀裂解后，采用第一维色谱聚焦、第二维反相高效色谱的组合模式进行二维液相色谱分离。第一维分离中将等电点(pI)在8.5-4.0范围内的蛋白质样品分离为每间隔0.3个pI梯度的组分。第一维分离组分接下来根据蛋白质的疏水性进行第二维分离。等电点8.5-4.0范围内的蛋白质样品经过二维分离后利用ProteoVue软件将所有组分按照等电点排序后即可获得蛋白质表达谱。本试验获得了处理组Ⅰ、Ⅱ和对照组Ⅰ、Ⅱ的羊草盐碱胁迫及非盐碱胁迫下植物的总蛋白质表达谱，在整个pH 8.5-4.0范围内条带清晰，蛋白质分布呈现为两个主要部分，pI为6.1-4.0和8.5-7.0两个区段范围内，其中pI为6.2-4.0范围内蛋白质表达量高于pI为8.5-7.2范围内蛋白质，即蛋白质集中在酸性范围内。但是pI为6.2-4.0范围内蛋白质疏水性接近导致蛋白质条带区分并不清晰，pI为7.2-8.5范围内蛋白质的分离效果要好于前者。处理组蛋白质中性、碱性蛋白质的表达量要稍高于对照组中性、碱性端蛋白质表达量。
利用DeltaVue软件将色谱峰比值大于2倍的蛋白质初步定为存在表达差异的蛋白质组分。处理组Ⅰ与对照组Ⅰ比较后获得的总差异表达蛋白质组分90个，等电点为8.5-6.4范围差异表达蛋白质组分58个，其中对照组高于处理组的差异表达蛋白质组分共37个，处理组高于对照组的差异表达蛋白质组分共21个。处理组Ⅱ与对照组Ⅱ比较后获得的总差异表达蛋白质组分46个。等电点为8.5-6.4范围差异表达蛋白质组分23个，其中对照组高于处理组表达量的差异表达蛋白质组分共12个，处理组高于对照组的差异表达蛋白质组分共11个。其中大部分差异表达蛋白质组分的差异倍数均集中在2倍左右，少数蛋白质变化倍数在5倍以上。对差异蛋白质的鉴定工作有待进一步研究。
Leymus chinensis (Trin.) Tzvel., a high quality plant for grazing, adapts to alkaline-sodic soil conditions and distributes widely in the Northeast of China. This species has developed molecular and physiological mechanisms during the course of evolution, which involve lots of anti-stress genes and proteins to adapt stress conditions. To identify and characterize the complexity of this adaptation, it is important to identify, genes by large-scale sequencing of cDNA clones and related proteins. The cDNA clones and related proteins are expected to enrich the stress related molecular resources and reveal the molecular mechanism. In this dissertation, studies were made on the stress-related genes and proteins expression profile of Leymus chinensis shoots under saline-alkaline stress.
1. The construction of subtraetive library of Leymus chinesis under saline-alkaline stress
Using cDNAs from Leymus chinensis seedlings grown on the natural habitat of saline-alkaline soil was regarded as tester and cDNA from seedlings in normal growth as driver, suppression subtractive hybridization (SSH) was employed to construct cDNA subtracted library. Of the 1920 cDNA clones showed high quality sequences. The size of inserts was 300-1000 base pairs as it was determined by PCR. In the subtractive library, 552 clones were non- redundant, of which 339 had homology to the known transcripts, and 209 were unknown or unclassified. The remaining 4 ESTs had no homology in the databases searched. The suggested functions ESTs which have the annotation in the database include metabolism, energy, protein synthesis, protein storage, disease/defense, signal transduction, transporter and transpon. Sequence comparison of these clones using BlastN led to the identification of several putative saline-alkaline stress related genes, such as monodehydroascorbate reductase (MDAR), gamma-glutamylcysteine synthetase (γ-ECS), thioredoxin reductase (TR), Ferrtin (FER), Aquaporin (PIP), Dehydrin (DHN) and 14-3-3 protein. The suggested function of the ESTs involved active oxygen elimination, osmotic adjustment, ion transport and regulation of the genes. The putative ESTs will serve as useful resource to demonstrate the molecular basis of saline-alkaline stress related genes and for the further study of gene expression in Leymus chinensis. We consider that some of these genes and their metabolisms are possibly involved in the process of salt tolerance in Leymus chinensis.
2. Gene expression profile of Leymus chinensis under saline-alkaline stress detected by cDNA microarray
The inserts amplified from 552 sequences of Leymus chinensis SSH library clones were spotted on slides to get cDNA microarray. Saline-alkaline stress and non-stress mRNA were labeled with Cy3- or Cy5- fluorescence dye respectively during reverse transcription and then mixed as targets. Microarray hybridization was accomplished following the direction of user manual of 3DNA 900~(?). The array was scanned by ScanArray 4000~(?) microarray scanner. For standardization, the Cy3 and Cy5 signal intensities were adjusted to fit external controls. The microarry was hybrid for three times. Using SAM software to analyze the significance of difference of the microarray genes, 198, 197, and 192 significant regulated genes were detected in the three hybridizations, respectively. Among the significant regulated genes there were 3, 53, 0 down-regulated genes, respectively. In function-known genes included monodehydro-ascorbate reductase, gamma-glutamylcysteine synthetase, ferrtin, aquaporin, dehydrin, thioredoxin reductas, and 14-3-3 proteins, all of which were known to be related with stress tolerance. There is no report for other up-regulated genes.
3. Expression of up-regulated genes, MDAR, FER, PIP, and 14-3-3s investigated by Northern blotting.
Firstly, to blast the sequence of MDAR, FER, PIP, 14-3-3s with the related genes of other plants in database to design the primer. The special sequences of the four genes were labeled by the dig- mark to get the probe. The seedlings of treated with 200 mmol/L Na_2CO_3 for 2, 6,12, 24, 48 and 72h were used for extraction of RNA for Northern blot. The expression of four genes was detected in treatments. MDAR, FER, PIP, and 14-3-3s transcriptome increased with the duration of the treatment. But the genes showed different expression patterns. In 14-3-3s expression pattern, the largest amount was observed in seedlings treated for 12h and 24h, and then the expression amount decreased. The results indicated the genes induced by saline-alkaline stress. Yet the function genes and regulation genes showed different expression patterns.
4. Protein expression profile of Leymus chinensis with two-dimensional liquid chromatography
There were four groups of Leymus chinensis plants seedlings collected from the natural habitat (treatmentⅠ), seedlings poured with 200 mmol/L Na_2CO_3 after 72h (treatmentⅡ), seedlings in the natural habitat planted in the non-saline-alkaline soil (controlⅠ), and eight- week-old seedlings grown in the greenhouse without any stress (controlⅡ). Total proteins were extracted by TCA-acetone precipitation method. Proteins were fractionated in the first dimension using chromatofocusing (CF). Subsequently the fractions with pH value between 8.5 and 4.0 collected after first dimension separation were further fractionated by high-performance reverse phase liquid chromatography (HPRP). The pI/UV map showed that the protein expression profiling of Leymus chinensis was generated by ProteoVue software. In the experiment, four protein express profiles were got from samples of each of treatmentsⅠ,Ⅱ, controlⅠ, andⅡ.
The protein expression profile maps showed clear bands and good-qulity fraction. The contribution of the protein fraction focused on pI range 6.1-4.0 and 8.5-7.0. The amount of protein in pI 6.2-4.0 was higher than that in 8.5-7.2, indicating mostly acidic proteins. But the proteins with pI in 6.2-4.0 had the similar hydrophilicity. The expression amount of neutral and basic proteins in treatments was higher than that of the controls.
To get the differential proteins under the saline-alkaline stress, protein expression profiles of controls and treatments were compared by Deltavue software. Proteins which the peak height of controls and treatments was above two was decided the differential ones.There were 90 differential proteins, in each of controlⅠand treatmentⅠ, and 46 in each of controlⅡand treatmentⅡ. In pI 8.5-6.4, there were 58 differential proteins in controlⅠand treatmentⅠ. The higher amounts of differential proteins in control were 37, and that in treatment were 21. And in controlⅡand treatmentⅡthere are 23 differential proteins. The higher amount of differential proteins in control were 12, and that in treatment were 11.The peak height ratio of mostly differential proteins were about two, with some observations showing higher than five. Further studies remain to be made on such as the identification of differential proteins. The differential expression protein is important in order to understand the mechanism under saline-alkaline stress.
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