沙冬青种质资源与抗旱基因研究
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摘要
蒙古沙冬青(Ammopiptanthus mongolicus)主要分布在我国西北地区的沙漠、荒漠区,是该区唯一的常绿阔叶灌木,具有很强的抗旱、抗寒等抗逆性。本研究以蒙古沙冬青(A. mongolicus)幼苗为实验材料,测定干旱胁迫下沙冬青幼苗叶片和根生理生化指标；采用454高通量测序技术建立了沙冬青根转录本数据库,并进行了相关分析；采用qRT-PCR技术检测沙冬青抗旱相关基因的表达模式,以揭示沙冬青抗旱生理基础和分子机制。结果如下：
采用20%PEG6000胁迫处理1h-72h,沙冬青幼苗叶片的RWC、MDA含量和REL与对照相比没有变化；经过24h胁迫处理,沙冬青幼苗根的RWC与对照相比显著降低,而MDA含量和REL显著增加。胁迫处理6h,沙冬青幼苗叶片和根的脯氨酸含量均有大量积累。胁迫条件下沙冬青幼苗叶片的SOD活性表现为先降低后上升,POD活性略有增加,处理6h后CAT活性降低；沙冬青幼苗根中的SOD、CAT和POD活性随胁迫时间加长而增加,72h处理SOD和POD活性水平最高。上述研究结果说明,采用灌根法进行模拟干旱胁迫处理,叶片和根中的相关生理响应特征不尽相同,根的反应较强。
应用454高通量测序技术得到沙冬青根转录组共672002条高质量(HQ)序列,平均长度279bp。拼接序列27366条,包括14912个序列重叠群(contig)和12454条singleton,总长约14Mb。共9771个蛋白序列获得了注释,采用GO功能富集分析分别从生物学过程、细胞组分和分子功能进行分类。并根据GO功能富集分析,选择了响应渗透胁迫、氧化胁迫、激素刺激和光信号刺激等4类共计27个抗旱相关基因,采用qRT-PCR技术,以20%PEG6000胁迫1h和72h的沙冬青根为材料分析了表达模式,各自响应的方式不同。以1h和72h胁迫处理皆为上调表达且相对表达量为2以上为标准确定为干旱胁迫诱导的基因,共计获得了4个基因,分别编码糖苷水解酶家族蛋白、过氧化物酶超家族蛋白、谷胱廿肽过氧化物酶(GPX3)和向光反应性NPH3家族蛋白；以1h和72h胁迫处理皆为下调表达且相对农达量为0.8以下为标准确定为干旱胁迫抑制的基因,共计获得了4个基因,分别编码脱水素家族蛋白(ERD14)、硫酸盐转运子(SULTR1:3)、硫氧还蛋白和向光反应性NPH3家族蛋白。本研究为深入开展沙冬青抗旱机理和抗旱基因的研究奠定了基础。
As the only evergreen broad-leaf shrub in the northwestern desert of China, Ammopiptanthus mongolicus shows very strong resistance to drought and cold. In order to explore the physical and molecular mechanisms underlying the drought tolerance in A. mongolicus, we analyzed physiological and biochemical characteristics in A. mongolicus seedling under drought stress. We performed large-scale transcriptome sequencing of A. mongolicus root using Roche/454next-generation sequencing technology; and the drought responsive genes of A. mongolicus differentially expressed under drought treatment were identified by further quantitative real-time PCR analysis. The results are discussed as follow:
The RWC, MDA and REL in the leaf of A. mongolicus seedling did not change compared with control under treatment of20%PEG6000for1h-72h. After24h drought stress, RWC of the root of A. mongolicus seedling decreased significantly, while MDA and REL increased. The proline accumulated abundantly in both leaves and roots of A. mongolicus after6h drought stress. In the drought condition, the activities of SOD, CAT and POD in A. mongolicus seedling were distinctly regulated. In leaves of A. mongolicus seedling, the activity of SOD was down regulated at first then increased; the activity of CAT decreased gradually, but the POD increased a little at6h drought treatment. In the root, the activities SOD, CAT and POD were all up regulated, among which SOD and POD increased the most. These results indicated that the physical responses were different in leaves and roots of A. mongolicus and the response of root was more intense, after watering root with20%PEG6000.
The transcriptome of A. mongolicus root obtained672,002HQ reads from a454GS XLR70Titanium pyrosequencer, and the average length was279bp. The672,002preproeessed sequencing reads were assembled into27,366unique sequences including14,912contig and12,454singlets, which total length was approximately14Mb. Gene ontology assignments were used to classify the functions of the A. mongolicus transcripts, and the9,771annotated sequences can be categorized into3functional groups:biological process, cellular component, and molecular function. To identify drought responsive genes,27unigenes were selected from the unique sequences classified in GO categories by qRT-PCR technology. When the expression models were analyzed with the root of the A. mongolicus treated by20%PEG6000,27genes were identified and categorized into4groups by the stress they responsed to, namely osmotic stress, oxidative stress, hormone stimulus and light stimulus. A gene was difined as the drought stress induced gene if it was up-regulated under1h and72h stress and the relative expression exceeded2-fold change.4 genes we obtained met such a definition, coding glycoside hydrolase family2protein, peroxidase superfamily protein, GPX3and phototropic-responsive NPH3family protein; A gene was difined as the drought stress inhibited gene if it was down-regulated under1h and72h stress and the relative expression did not exceed0.8-fold change.4genes we obtained met such a definition, coding ERD14, SULTR1;3, thioredoxin family protein and phototropic-responsive NPH3family protein. This study facilitated the research on drought resistance mechanism and related genes in A. mongolicus.
采用20%PEG6000胁迫处理1h-72h,沙冬青幼苗叶片的RWC、MDA含量和REL与对照相比没有变化；经过24h胁迫处理,沙冬青幼苗根的RWC与对照相比显著降低,而MDA含量和REL显著增加。胁迫处理6h,沙冬青幼苗叶片和根的脯氨酸含量均有大量积累。胁迫条件下沙冬青幼苗叶片的SOD活性表现为先降低后上升,POD活性略有增加,处理6h后CAT活性降低；沙冬青幼苗根中的SOD、CAT和POD活性随胁迫时间加长而增加,72h处理SOD和POD活性水平最高。上述研究结果说明,采用灌根法进行模拟干旱胁迫处理,叶片和根中的相关生理响应特征不尽相同,根的反应较强。
应用454高通量测序技术得到沙冬青根转录组共672002条高质量(HQ)序列,平均长度279bp。拼接序列27366条,包括14912个序列重叠群(contig)和12454条singleton,总长约14Mb。共9771个蛋白序列获得了注释,采用GO功能富集分析分别从生物学过程、细胞组分和分子功能进行分类。并根据GO功能富集分析,选择了响应渗透胁迫、氧化胁迫、激素刺激和光信号刺激等4类共计27个抗旱相关基因,采用qRT-PCR技术,以20%PEG6000胁迫1h和72h的沙冬青根为材料分析了表达模式,各自响应的方式不同。以1h和72h胁迫处理皆为上调表达且相对表达量为2以上为标准确定为干旱胁迫诱导的基因,共计获得了4个基因,分别编码糖苷水解酶家族蛋白、过氧化物酶超家族蛋白、谷胱廿肽过氧化物酶(GPX3)和向光反应性NPH3家族蛋白；以1h和72h胁迫处理皆为下调表达且相对农达量为0.8以下为标准确定为干旱胁迫抑制的基因,共计获得了4个基因,分别编码脱水素家族蛋白(ERD14)、硫酸盐转运子(SULTR1:3)、硫氧还蛋白和向光反应性NPH3家族蛋白。本研究为深入开展沙冬青抗旱机理和抗旱基因的研究奠定了基础。
As the only evergreen broad-leaf shrub in the northwestern desert of China, Ammopiptanthus mongolicus shows very strong resistance to drought and cold. In order to explore the physical and molecular mechanisms underlying the drought tolerance in A. mongolicus, we analyzed physiological and biochemical characteristics in A. mongolicus seedling under drought stress. We performed large-scale transcriptome sequencing of A. mongolicus root using Roche/454next-generation sequencing technology; and the drought responsive genes of A. mongolicus differentially expressed under drought treatment were identified by further quantitative real-time PCR analysis. The results are discussed as follow:
The RWC, MDA and REL in the leaf of A. mongolicus seedling did not change compared with control under treatment of20%PEG6000for1h-72h. After24h drought stress, RWC of the root of A. mongolicus seedling decreased significantly, while MDA and REL increased. The proline accumulated abundantly in both leaves and roots of A. mongolicus after6h drought stress. In the drought condition, the activities of SOD, CAT and POD in A. mongolicus seedling were distinctly regulated. In leaves of A. mongolicus seedling, the activity of SOD was down regulated at first then increased; the activity of CAT decreased gradually, but the POD increased a little at6h drought treatment. In the root, the activities SOD, CAT and POD were all up regulated, among which SOD and POD increased the most. These results indicated that the physical responses were different in leaves and roots of A. mongolicus and the response of root was more intense, after watering root with20%PEG6000.
The transcriptome of A. mongolicus root obtained672,002HQ reads from a454GS XLR70Titanium pyrosequencer, and the average length was279bp. The672,002preproeessed sequencing reads were assembled into27,366unique sequences including14,912contig and12,454singlets, which total length was approximately14Mb. Gene ontology assignments were used to classify the functions of the A. mongolicus transcripts, and the9,771annotated sequences can be categorized into3functional groups:biological process, cellular component, and molecular function. To identify drought responsive genes,27unigenes were selected from the unique sequences classified in GO categories by qRT-PCR technology. When the expression models were analyzed with the root of the A. mongolicus treated by20%PEG6000,27genes were identified and categorized into4groups by the stress they responsed to, namely osmotic stress, oxidative stress, hormone stimulus and light stimulus. A gene was difined as the drought stress induced gene if it was up-regulated under1h and72h stress and the relative expression exceeded2-fold change.4 genes we obtained met such a definition, coding glycoside hydrolase family2protein, peroxidase superfamily protein, GPX3and phototropic-responsive NPH3family protein; A gene was difined as the drought stress inhibited gene if it was down-regulated under1h and72h stress and the relative expression did not exceed0.8-fold change.4genes we obtained met such a definition, coding ERD14, SULTR1;3, thioredoxin family protein and phototropic-responsive NPH3family protein. This study facilitated the research on drought resistance mechanism and related genes in A. mongolicus.
引文
[1]Wang D, Pan Y, Zhao X, et al. Genome-wide temporal-spatial gene expression profiling of drought responsiveness in rice[J]. BMC Genomics,2011,12:149.
[2]Mitra J.Genetics and genetic improvement of drought resistance in crop plants[J]. Current Sciences,2001,80:758-763.
[3]Courtois B, Shen L, Petalcorin W, et al. Locating QTLs controlling constitutive root traits in the rice population IAC 165 x Co39[J]. Euphytica,2003,134:335-345.
[4]Tripathy JN, Zhang J, Robin S, et al.QTLs for cellmembrane stability mapped in rice (Oryza sativa L.) under drought stress[J]. Theoretical and Applied Genetics,2000,100:1197-1202.
[5]Lilley JM, Ludlow MM, McCouch SR, et al.Locating QTL for osmotic adjustment and dehydration tolerance in rice[J]. Journal of ExperimentalBotany,1996,47:1427-1436.
[6]8Bartels D, Sunkars R.Drought and salt tolerance in plants[J]. Critical Reviews in Plant Science,2005,24:23-58.
[7]李慧卿,马文元,李慧勇.沙冬青抗逆性研究进展[J].世界林业研究,2000,13(5)：67-71.
[8]陶玲,李新荣,刘新民,等.中国珍惜濒危荒漠植物保护等级的定量研究[J].林业科学,2001,37
(1)：52-57.
[9]林清芳,王茅雁,刘佳杰,等.沙冬青细胞与分子生物学研究进展[J].植物遗传资源学报,2010,11(6)：793-797.
[10]Cheng S. Ammopiptanthus Cheng f. A new genus of Leguminosae from CentralAsian (in Russian) [J]. Bot. Zh.,1959, (44):1381-1386.
[11]马世威,马宝明.沙漠学[M].呼和浩特：内蒙古人民出版社,1998.
[12]夏恩龙,彭祚登,翟明普.沙冬青的分布和保护利用研究进展[J].防护林利·技,2006.7(4)：56-58,70.
[13]周宜君,高飞,冯金朝,等.民族地区沙冬青种质资源保护与利用[J].安徽农业科学,2011,39(10)：5851-5853,5856.
[14]李玉俊.沙坡头地区沙地育苗试验[A].流沙治理研究(二)[C].银川：宁夏人民出版社,1991.
[15]Goldblatt P. Index to plant chromosome numbers for 1975-1978[M]. Missuuri Bot Gard,1981: 220.
[16]Goldblatt P. Index to plant chromosome numbers for 1982-1983[M]. Missuuri Bot Gard,1985: 173.
[17]冯显逵,宋玉霞.沙冬青核型研究[J].宁夏农林科技,1988(3)：29.
[18]潘伯荣,黄少甫.沙冬青属的细胞学研究[J].植物学报,1993,35(4)：314-317.
[19]袁永明,彭泽祥,任廷国.兰州及其邻近地区几种植物的染色体[J].西北师范学院学报,1988(专辑1号)：58-63.
[20]张寿洲,曹瑞.沙冬青染色体数与核型研究[J].植物分类学报,1990,28(2)：133-135.
[21]Chen C J, Zhu X Y, YuanYM. Cytological studies on the tribe Then-nopsideae (Fabaceae)[C] //Reporton karyotypes of eleven species of four genera[J].Cathaya,1992,4:103-116.
[22]刘玉红,王善敏,王荷生.试论沙冬青属的染色体地理[J].地理研究,1996,15(4)：40-47.
[23]宋娟娟,唐源江,廖景平,等.濒危植物矮沙冬青减数分裂期染色体行为的观察[J].热带亚热带植物学报,2003,11(2)：166-168.
[24]吴征镒.中国植被[M]北京：科学出版社,1980.84,585-593.
[25]伊稍,K.种子植物解剖学[M]李正理,译.上海：上海人民出版社,1973 268-271.
[26]刘家琼.我国荒漠特有的常绿植物——沙冬青的生态生理及解剖学特征[J].植物学报,1982,24(6)：570-573.
[27]周宜君,刘春兰,冯金朝,等.沙冬青抗旱、抗寒机理的研究进展[J].中国沙漠,2001,9(3)：312-316.
[28]郭生祥,刘志银,郝听.沙冬于的研究进展[J].甘肃林业科技,2005,30(4)：4-8,12.
[29]蒋志荣.沙冬青抗旱机理的探讨[J].中国沙漠,2000,20(4)：71-74.
[30]段慧荣,李毅,马彦军.PEG胁迫对沙冬青种子萌发过程的影响[J].水土保持研究,2011,18(3)：221-225.
[31]夏晗,黄金生.低温、干旱和盐胁迫下沙冬青幼苗脯氨酸含量的变化[J].吉林林业科技,2007,36(4)：1-2,20.
[32]段慧荣,马彦军,李毅.模拟干旱和盐胁迫下沙冬青种子硬实特性和抗逆性的研究[J].草地学报,2011,19(1)：75-80.
[33]于军,焦培培.聚乙二醇(PEG6000)模拟干旱胁迫抑制矮沙冬青种子的萌发[J].基因组学与应用生物学,2010,29(2)：355-360.
[34]李婧男,刘强,贾志宽,等.盐胁迫对沙冬青幼苗生长与生理特性的影响[J].植物研究,2009,29(5)：553-558.
[35]焦培培,王彦芹,李志军.盐分和贮藏对矮沙冬青种子萌发的影响[J].塔里木大学学报,2010,22(2)：16-21.
[36]林清芳,王茅雁,刘佳杰,等.沙冬青细胞与分子生物学研究进展[J].植物遗传资源学报,2010,11(6)793-797.
[37]Griffith M, Ala P, Yang DSC, et al.Antifreeze protein produced endogenously in winter rye leaves[J].Plant Ph.,1992,100:593-596.
[38]费云标,孙龙华,黄涛,等.沙冬青高活性抗冻蛋白的发现[J].植物学报,1994,36(8)：649-650.
[39]江勇,魏令波,费云标,等.分离和鉴定沙冬青抗冻蛋白质[J].植物学报,1999,41(9)：967-971.
[40]魏令波,江勇,舒念红,等.沙冬青叶片热稳定抗冻蛋白特性分析[J].植物学报,1999,41(8)：837-841.
[41]费云标,魏令波,高素琴,等.沙冬青抗冻蛋白的分离、纯化及其理化特性分析[J].科学通报,2000,45(20)：2185-2189.
[42]舒念红,魏令波,高素琴,等.植物抗冻蛋白纯化、活性与二级结构测定[J].中国医学研究与临床,2003,1(12)：45-47.
[43]Fei YB,Cao PX, Gao SQ, et al.Discovery of a Homolog of Siderophilin in a Plan[J]. Journal of integrative plant biology,2005,47(12):1409-1411.
[44]Fei YB,Cao PX, Gao SQ, et al.Purification and structure analysis of antifreeze proteins from Ammopiptanthus mongolicus[J].Prep Biochem Biotechnol,2008,38(2):172-183.
[45]李召春.沙冬青逆境转录表达谱的建立及分析[D].北京,中国农业科学院,2010.
[46]乔慧蕾,五瑞刚.郭九峰,等.沙冬青脱水素基因的克隆及表达载体的构建[J].华北农(?)报,2009,24(4)：88-91.
[47]李晓东,自晨,张玉金,等.沙冬青脱水素基因的克隆、重组及对糖料作物的遗转化[J].华北农学,2010,25(1)：68-74.
[48]刘佳杰.沙冬青低温胁迫全长cDNA文库构建及AmDHN和AmRD22基因的克隆[D].呼和浩特,内蒙古农业大学,2011.
[49]宋健.沙冬青耐寒丛因AmGolS的克隆和遗传转化研究[D].泰安,山东农业大学,2007.
[50]史军娜,刘美芹,师静,等.沙冬青GATA型锌指蛋自基因序列及表达分析[J].北京林业大学学报,2011,33(3)：21-25.
[51]孙燕.沙冬青CBL1基因的克隆及表达特性研究[D].北京,北京林业大学,2005.
[52]孙芳.少冬青CBL1基因的功能研究[D].北京,北京林业大学,2009.
[53]周自去,梁宗锁,李硕,等.干旱-复水对酸枣相对含水量、保护酶及光合特征的影响[J].中国生态农业学报,2011,19(1)：93-97.
[54]杨冬艳.干旱胁迫下沙芥(P. cormutum)与斧翅沙芥(P. dolabratum)幼苗生理生化应答反应及机理探讨[D].呼和浩特,内蒙古农业大学,2005.
[55]陈少瑜,郎南军,李吉跃,等.干旱胁迫下3树种苗木叶片相对含水量、质膜相对透性和脯氨酸含量的变化[J].西部林业科学,2004,33(3)：30-33.
[56]邵艳军,山仑.植物耐旱机制研究进展[J].中国生态农业学报,2006,14(4)：16-20.
[57]白志英,李存东,刘渊.干旱胁迫下小麦叶片脯氨酸和蛋白质含量变化与染色的关系[J].植物遗传资源学报,2007,8(3)：325-330.
[58]刘娥娥,宗会,郭振飞,等.干旱、盐和低温胁迫对水稻幼苗脯氨酸含量的影响[J].热带亚热带植物学报,2000,8(3)：235-238.
[59]董喜存,李岩,李文建,等.聚乙二醇模拟干旱对甜高粱幼苗丙二醛、脯氨酸含量的影响[J].云南农业大学学报,2010,25(4)：726-730.
[60]李波,贾秀峰,白庆武,等.干旱胁迫对首蓓脯氨酸累积的影响[J].植物研究,2003,23(2)：189-191.
[61]袁有波,李继新,丁福章,等.干早胁迫对烤烟叶片脯氨酸和可溶性蛋白质含量的影响[J].安徽农业科学,2008,36(21)：8891-8892.
[62]蒋明义.水分胁迫与植物膜脂过氧化[J].西北农业大学学报,1991,19(2)：84-94.
[63]田国忠,李怀方,裘维番.植物过氧化物酶研究进展[J].武汉植物学研究,2001,19(4)：332-344.
[64]米海莉,许兴,李树华,等.干旱胁迫下牛心朴子幼苗相对含水量、质膜透性及保护酶活性变化[J].西北植物学报2003,23(11)：1871-1876.
[65]Trick M, Long Y, Meng J, et al. Single nucleotide polymorphism (SNP) discovery in the polyploid Brassica napus using Solexa transcriptome sequencing[J]. Plant Biotechnol J,2009, 7:334-346.
[66]Wang W, Wang Y, Zhang Q, et al. Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing[J]. BMC Genomics,2009,10:465.
[67]Novaes E, Drost DR, Farmerie WG, et al. High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome[J]. BMC Genomics,2008,9:312.
[68]Alagna F, D'Agostino N, Torchia L, et al. Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development[J]. BMC Genomics,2009,10:399.
[69]Jones-Rhoades MW, Borevitz JO, Preuss D. Genome-wide expression profiling of the Arabidopsis female gametophyte identifies families of small, secreted proteins[J]. PLoS Genet,2007,3:1848-1861.
[70]Weber AP, Weber KL, Carr K, et al. Sampling the Arabidopsis transcriptome with massive parallel pyrosequencing[J]. Plant Ph.,2007,144:32-42.
[71]Hsiao YY, Jeng MF, Tsai WC, et al. A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif[J]. Plant J.2008,55:719-733.
[72]Varshney RK, Nayak SN, May GD, et al.Next-generation sequencing technologies and their implications for crop genetics and breeding[J]. Trends Biotechnol,2009,27:522-530.
[73]张蓓,沈立松.实时荧光定量PCR的研究进展及其应用[J].国外医学：临床生物化学与检验学分册,2003,24(6)：327-329.
[74]袁亚男,刘文忠.实时荧光定量PCR技术的类型、特点及应用[J].中国畜牧兽医,2008,35(3)27-30.
[75]吴静,吴茂森,何晨阳.实时定量PCR的原理及其在植物病理学研究中的应用[J].植物保护,2007,33(6)123-128.
[76]马荣群,陈红运,黄文胜,等.实时定量PCR方法检测转基因产品[J].植物免疫,2002,16(1)：61-64.
[77]高东,王云月,何霞红,等TaqMan荧光定量RT-PCR检测水稻RAcl基因mRNA方法的建(?)[J].云南植物研究,200931(1)：75-81.
[78]高东,何霞红,王云月,等.水稻GA200x-2丛因1nRNA的TaqMan荧光定量RT-PCR检测[J]. 中国水稻科学,2009,23(3)：309-314.
[79]向林,李伯钧,秦德辉,等.春兰GLO基因的克隆和实时定量表达分析[J].浙江农业科学,2011,23(3)517-522.
[80]刘雨,刘春霞,李早霞,等.植物同源结构域(PHD)-finger家族转录因子OsPHD1的过量表达可提高水稻的耐逆性能[J].农业生物技术学报,2011,19(3)462-469.
[81]赵晋锋,余爱丽,王寒玉,等.非生物逆境胁迫下ZmCIP10基因表达分析[J].生物技术进展,2011,1(2)：130-134.
[82]任敏,何金环.自然干旱胁迫下紫花苜蓿叶片和根ABA的代谢变化[J],安徽农业科学,2010,38(4)：1771-1772.
[83]汤章城.现代植物生理学实验指南[M].北京：科学出版礼,2004：302-306.
[84]高俊凤.植物生理学实验指南[M].北京：高等教育出版社,2006：211-218.
[85]蒋高明.植物生理生态学[M].北京：高等教育出版社,2004.
[86]李慧卿,马文元.沙生植物抗旱性比较的主要指标及分析方法[J].干旱区研究,1998,15(4)：12-15.
[87]Xu S, An L, Feng H, et al. The seasonal effects of water stress on Ammopiptanthus mongolicus in a desert environment[J]. Arid Environ,2002,51:437-447.
[88]刘家琼,周湘红.几种固沙植物过氧化物酶、过氧化氢酶慨活性及其同工酶胁的初步分析[A].中国科学院(1991-1992)沙坡头沙漠试验研究站年报[C].兰州：甘肃科技出版社,1993.
[89]孙美莲,王云生,杨冬青等.茶树实时荧光定量PCR分析中内参基因的选择[J].植物学报,2010,45(5)：579-587.
[90]Sekalska B., Ciechanowicz A.,Dolegowska B., et al. Optimized RT-PCR method for assaying expression of monocyte chemoisctic protein type 1(MCP-1) in rabbit aorta[J]. Biochem Genet,2006,44:133-143.
[91]Li L,Xu J,Xu ZH, et al. Brassionosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis[J]. Plant Cell,2005,17:2738-2753.
[92]Williams ME, Torabinejad J, Cohick E, et al. Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9 lead to overaccumulation of Ptdlns(4.5) P2 and constitutive expression of the stress-response pathway[J]. Plant Physiol,2005,138:686-700.
[93]Domoki M. Gyorgyey J. Biro J, et al. Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derive alfalfa cells[J]. Biochim Biophys Acta,2006,1759:543-551.
[94]Jeong YM. Mun JH. Lee I., et al. Distinct roles of the first introns on the expression of Arabidopsis profilin gene family mcmbcrs[J]. Plant Physiol,2006,140:196-209.
[95]Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. Mol Endocrinol,2000,25:169-193.
[96]吴宇,高蕾,曹民杰,等.植物硫营养代谢、调控与生物学功能[J].植物学通报,2007,24(6)：735-761.
[97]夏德习.拟南芥硫氧还蛋白基因在逆境胁迫下的功能解析[D].哈尔滨,东北林业大学,2007.
[98]Su K W,Yong J L,HaY L, et al. Cis-element and transcriptome-based screening of root hair-specific genes and their functional characterization in Arabidopsis[J].Plant Physiol,2009, 150:1459-1473.
[99]Cho H T,Cosgrove D J.Regulation of root hair initiation and expansin gene expression in Arabidopsis[J]. Plant Cell.2002,14:3237-3253.
[100]张素维,蒋阿维,孙扬吾,等.基于RHE元件和拟南芥转录组的根毛发育基因筛选[J].核农学报,2010,24(5)：922-925.
[101]孙萍.乙烯对拟南芥耐受干旱胁迫的作用及抗氧化研究[D].哈尔滨,东北林业大学,2010.
[102]Ullas V. Pedmale, EmmanuelLiscum. Regulation of Phototropic Signaling in Arabidopsis via Phosphorylation State Changes in the Phototropin 1-interacting Protein NPH3[J]. Journal of biological chemistry.2007,6(27):19992-20001.
[103]Vij S,Tyagi AK.Emerging trends in the functional genomics of the abiotic stress response in crop plants[J]. Plant Biotechnol.2007,5(3):361-380.
[104]付畅,黄宇.转录组学平台技术及其在植物抗逆分子生物学中的应用[J].生物技术通报,2011.6：40-46.
[2]Mitra J.Genetics and genetic improvement of drought resistance in crop plants[J]. Current Sciences,2001,80:758-763.
[3]Courtois B, Shen L, Petalcorin W, et al. Locating QTLs controlling constitutive root traits in the rice population IAC 165 x Co39[J]. Euphytica,2003,134:335-345.
[4]Tripathy JN, Zhang J, Robin S, et al.QTLs for cellmembrane stability mapped in rice (Oryza sativa L.) under drought stress[J]. Theoretical and Applied Genetics,2000,100:1197-1202.
[5]Lilley JM, Ludlow MM, McCouch SR, et al.Locating QTL for osmotic adjustment and dehydration tolerance in rice[J]. Journal of ExperimentalBotany,1996,47:1427-1436.
[6]8Bartels D, Sunkars R.Drought and salt tolerance in plants[J]. Critical Reviews in Plant Science,2005,24:23-58.
[7]李慧卿,马文元,李慧勇.沙冬青抗逆性研究进展[J].世界林业研究,2000,13(5)：67-71.
[8]陶玲,李新荣,刘新民,等.中国珍惜濒危荒漠植物保护等级的定量研究[J].林业科学,2001,37
(1)：52-57.
[9]林清芳,王茅雁,刘佳杰,等.沙冬青细胞与分子生物学研究进展[J].植物遗传资源学报,2010,11(6)：793-797.
[10]Cheng S. Ammopiptanthus Cheng f. A new genus of Leguminosae from CentralAsian (in Russian) [J]. Bot. Zh.,1959, (44):1381-1386.
[11]马世威,马宝明.沙漠学[M].呼和浩特：内蒙古人民出版社,1998.
[12]夏恩龙,彭祚登,翟明普.沙冬青的分布和保护利用研究进展[J].防护林利·技,2006.7(4)：56-58,70.
[13]周宜君,高飞,冯金朝,等.民族地区沙冬青种质资源保护与利用[J].安徽农业科学,2011,39(10)：5851-5853,5856.
[14]李玉俊.沙坡头地区沙地育苗试验[A].流沙治理研究(二)[C].银川：宁夏人民出版社,1991.
[15]Goldblatt P. Index to plant chromosome numbers for 1975-1978[M]. Missuuri Bot Gard,1981: 220.
[16]Goldblatt P. Index to plant chromosome numbers for 1982-1983[M]. Missuuri Bot Gard,1985: 173.
[17]冯显逵,宋玉霞.沙冬青核型研究[J].宁夏农林科技,1988(3)：29.
[18]潘伯荣,黄少甫.沙冬青属的细胞学研究[J].植物学报,1993,35(4)：314-317.
[19]袁永明,彭泽祥,任廷国.兰州及其邻近地区几种植物的染色体[J].西北师范学院学报,1988(专辑1号)：58-63.
[20]张寿洲,曹瑞.沙冬青染色体数与核型研究[J].植物分类学报,1990,28(2)：133-135.
[21]Chen C J, Zhu X Y, YuanYM. Cytological studies on the tribe Then-nopsideae (Fabaceae)[C] //Reporton karyotypes of eleven species of four genera[J].Cathaya,1992,4:103-116.
[22]刘玉红,王善敏,王荷生.试论沙冬青属的染色体地理[J].地理研究,1996,15(4)：40-47.
[23]宋娟娟,唐源江,廖景平,等.濒危植物矮沙冬青减数分裂期染色体行为的观察[J].热带亚热带植物学报,2003,11(2)：166-168.
[24]吴征镒.中国植被[M]北京：科学出版社,1980.84,585-593.
[25]伊稍,K.种子植物解剖学[M]李正理,译.上海：上海人民出版社,1973 268-271.
[26]刘家琼.我国荒漠特有的常绿植物——沙冬青的生态生理及解剖学特征[J].植物学报,1982,24(6)：570-573.
[27]周宜君,刘春兰,冯金朝,等.沙冬青抗旱、抗寒机理的研究进展[J].中国沙漠,2001,9(3)：312-316.
[28]郭生祥,刘志银,郝听.沙冬于的研究进展[J].甘肃林业科技,2005,30(4)：4-8,12.
[29]蒋志荣.沙冬青抗旱机理的探讨[J].中国沙漠,2000,20(4)：71-74.
[30]段慧荣,李毅,马彦军.PEG胁迫对沙冬青种子萌发过程的影响[J].水土保持研究,2011,18(3)：221-225.
[31]夏晗,黄金生.低温、干旱和盐胁迫下沙冬青幼苗脯氨酸含量的变化[J].吉林林业科技,2007,36(4)：1-2,20.
[32]段慧荣,马彦军,李毅.模拟干旱和盐胁迫下沙冬青种子硬实特性和抗逆性的研究[J].草地学报,2011,19(1)：75-80.
[33]于军,焦培培.聚乙二醇(PEG6000)模拟干旱胁迫抑制矮沙冬青种子的萌发[J].基因组学与应用生物学,2010,29(2)：355-360.
[34]李婧男,刘强,贾志宽,等.盐胁迫对沙冬青幼苗生长与生理特性的影响[J].植物研究,2009,29(5)：553-558.
[35]焦培培,王彦芹,李志军.盐分和贮藏对矮沙冬青种子萌发的影响[J].塔里木大学学报,2010,22(2)：16-21.
[36]林清芳,王茅雁,刘佳杰,等.沙冬青细胞与分子生物学研究进展[J].植物遗传资源学报,2010,11(6)793-797.
[37]Griffith M, Ala P, Yang DSC, et al.Antifreeze protein produced endogenously in winter rye leaves[J].Plant Ph.,1992,100:593-596.
[38]费云标,孙龙华,黄涛,等.沙冬青高活性抗冻蛋白的发现[J].植物学报,1994,36(8)：649-650.
[39]江勇,魏令波,费云标,等.分离和鉴定沙冬青抗冻蛋白质[J].植物学报,1999,41(9)：967-971.
[40]魏令波,江勇,舒念红,等.沙冬青叶片热稳定抗冻蛋白特性分析[J].植物学报,1999,41(8)：837-841.
[41]费云标,魏令波,高素琴,等.沙冬青抗冻蛋白的分离、纯化及其理化特性分析[J].科学通报,2000,45(20)：2185-2189.
[42]舒念红,魏令波,高素琴,等.植物抗冻蛋白纯化、活性与二级结构测定[J].中国医学研究与临床,2003,1(12)：45-47.
[43]Fei YB,Cao PX, Gao SQ, et al.Discovery of a Homolog of Siderophilin in a Plan[J]. Journal of integrative plant biology,2005,47(12):1409-1411.
[44]Fei YB,Cao PX, Gao SQ, et al.Purification and structure analysis of antifreeze proteins from Ammopiptanthus mongolicus[J].Prep Biochem Biotechnol,2008,38(2):172-183.
[45]李召春.沙冬青逆境转录表达谱的建立及分析[D].北京,中国农业科学院,2010.
[46]乔慧蕾,五瑞刚.郭九峰,等.沙冬青脱水素基因的克隆及表达载体的构建[J].华北农(?)报,2009,24(4)：88-91.
[47]李晓东,自晨,张玉金,等.沙冬青脱水素基因的克隆、重组及对糖料作物的遗转化[J].华北农学,2010,25(1)：68-74.
[48]刘佳杰.沙冬青低温胁迫全长cDNA文库构建及AmDHN和AmRD22基因的克隆[D].呼和浩特,内蒙古农业大学,2011.
[49]宋健.沙冬青耐寒丛因AmGolS的克隆和遗传转化研究[D].泰安,山东农业大学,2007.
[50]史军娜,刘美芹,师静,等.沙冬青GATA型锌指蛋自基因序列及表达分析[J].北京林业大学学报,2011,33(3)：21-25.
[51]孙燕.沙冬青CBL1基因的克隆及表达特性研究[D].北京,北京林业大学,2005.
[52]孙芳.少冬青CBL1基因的功能研究[D].北京,北京林业大学,2009.
[53]周自去,梁宗锁,李硕,等.干旱-复水对酸枣相对含水量、保护酶及光合特征的影响[J].中国生态农业学报,2011,19(1)：93-97.
[54]杨冬艳.干旱胁迫下沙芥(P. cormutum)与斧翅沙芥(P. dolabratum)幼苗生理生化应答反应及机理探讨[D].呼和浩特,内蒙古农业大学,2005.
[55]陈少瑜,郎南军,李吉跃,等.干旱胁迫下3树种苗木叶片相对含水量、质膜相对透性和脯氨酸含量的变化[J].西部林业科学,2004,33(3)：30-33.
[56]邵艳军,山仑.植物耐旱机制研究进展[J].中国生态农业学报,2006,14(4)：16-20.
[57]白志英,李存东,刘渊.干旱胁迫下小麦叶片脯氨酸和蛋白质含量变化与染色的关系[J].植物遗传资源学报,2007,8(3)：325-330.
[58]刘娥娥,宗会,郭振飞,等.干旱、盐和低温胁迫对水稻幼苗脯氨酸含量的影响[J].热带亚热带植物学报,2000,8(3)：235-238.
[59]董喜存,李岩,李文建,等.聚乙二醇模拟干旱对甜高粱幼苗丙二醛、脯氨酸含量的影响[J].云南农业大学学报,2010,25(4)：726-730.
[60]李波,贾秀峰,白庆武,等.干旱胁迫对首蓓脯氨酸累积的影响[J].植物研究,2003,23(2)：189-191.
[61]袁有波,李继新,丁福章,等.干早胁迫对烤烟叶片脯氨酸和可溶性蛋白质含量的影响[J].安徽农业科学,2008,36(21)：8891-8892.
[62]蒋明义.水分胁迫与植物膜脂过氧化[J].西北农业大学学报,1991,19(2)：84-94.
[63]田国忠,李怀方,裘维番.植物过氧化物酶研究进展[J].武汉植物学研究,2001,19(4)：332-344.
[64]米海莉,许兴,李树华,等.干旱胁迫下牛心朴子幼苗相对含水量、质膜透性及保护酶活性变化[J].西北植物学报2003,23(11)：1871-1876.
[65]Trick M, Long Y, Meng J, et al. Single nucleotide polymorphism (SNP) discovery in the polyploid Brassica napus using Solexa transcriptome sequencing[J]. Plant Biotechnol J,2009, 7:334-346.
[66]Wang W, Wang Y, Zhang Q, et al. Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing[J]. BMC Genomics,2009,10:465.
[67]Novaes E, Drost DR, Farmerie WG, et al. High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome[J]. BMC Genomics,2008,9:312.
[68]Alagna F, D'Agostino N, Torchia L, et al. Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development[J]. BMC Genomics,2009,10:399.
[69]Jones-Rhoades MW, Borevitz JO, Preuss D. Genome-wide expression profiling of the Arabidopsis female gametophyte identifies families of small, secreted proteins[J]. PLoS Genet,2007,3:1848-1861.
[70]Weber AP, Weber KL, Carr K, et al. Sampling the Arabidopsis transcriptome with massive parallel pyrosequencing[J]. Plant Ph.,2007,144:32-42.
[71]Hsiao YY, Jeng MF, Tsai WC, et al. A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif[J]. Plant J.2008,55:719-733.
[72]Varshney RK, Nayak SN, May GD, et al.Next-generation sequencing technologies and their implications for crop genetics and breeding[J]. Trends Biotechnol,2009,27:522-530.
[73]张蓓,沈立松.实时荧光定量PCR的研究进展及其应用[J].国外医学：临床生物化学与检验学分册,2003,24(6)：327-329.
[74]袁亚男,刘文忠.实时荧光定量PCR技术的类型、特点及应用[J].中国畜牧兽医,2008,35(3)27-30.
[75]吴静,吴茂森,何晨阳.实时定量PCR的原理及其在植物病理学研究中的应用[J].植物保护,2007,33(6)123-128.
[76]马荣群,陈红运,黄文胜,等.实时定量PCR方法检测转基因产品[J].植物免疫,2002,16(1)：61-64.
[77]高东,王云月,何霞红,等TaqMan荧光定量RT-PCR检测水稻RAcl基因mRNA方法的建(?)[J].云南植物研究,200931(1)：75-81.
[78]高东,何霞红,王云月,等.水稻GA200x-2丛因1nRNA的TaqMan荧光定量RT-PCR检测[J]. 中国水稻科学,2009,23(3)：309-314.
[79]向林,李伯钧,秦德辉,等.春兰GLO基因的克隆和实时定量表达分析[J].浙江农业科学,2011,23(3)517-522.
[80]刘雨,刘春霞,李早霞,等.植物同源结构域(PHD)-finger家族转录因子OsPHD1的过量表达可提高水稻的耐逆性能[J].农业生物技术学报,2011,19(3)462-469.
[81]赵晋锋,余爱丽,王寒玉,等.非生物逆境胁迫下ZmCIP10基因表达分析[J].生物技术进展,2011,1(2)：130-134.
[82]任敏,何金环.自然干旱胁迫下紫花苜蓿叶片和根ABA的代谢变化[J],安徽农业科学,2010,38(4)：1771-1772.
[83]汤章城.现代植物生理学实验指南[M].北京：科学出版礼,2004：302-306.
[84]高俊凤.植物生理学实验指南[M].北京：高等教育出版社,2006：211-218.
[85]蒋高明.植物生理生态学[M].北京：高等教育出版社,2004.
[86]李慧卿,马文元.沙生植物抗旱性比较的主要指标及分析方法[J].干旱区研究,1998,15(4)：12-15.
[87]Xu S, An L, Feng H, et al. The seasonal effects of water stress on Ammopiptanthus mongolicus in a desert environment[J]. Arid Environ,2002,51:437-447.
[88]刘家琼,周湘红.几种固沙植物过氧化物酶、过氧化氢酶慨活性及其同工酶胁的初步分析[A].中国科学院(1991-1992)沙坡头沙漠试验研究站年报[C].兰州：甘肃科技出版社,1993.
[89]孙美莲,王云生,杨冬青等.茶树实时荧光定量PCR分析中内参基因的选择[J].植物学报,2010,45(5)：579-587.
[90]Sekalska B., Ciechanowicz A.,Dolegowska B., et al. Optimized RT-PCR method for assaying expression of monocyte chemoisctic protein type 1(MCP-1) in rabbit aorta[J]. Biochem Genet,2006,44:133-143.
[91]Li L,Xu J,Xu ZH, et al. Brassionosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis[J]. Plant Cell,2005,17:2738-2753.
[92]Williams ME, Torabinejad J, Cohick E, et al. Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9 lead to overaccumulation of Ptdlns(4.5) P2 and constitutive expression of the stress-response pathway[J]. Plant Physiol,2005,138:686-700.
[93]Domoki M. Gyorgyey J. Biro J, et al. Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derive alfalfa cells[J]. Biochim Biophys Acta,2006,1759:543-551.
[94]Jeong YM. Mun JH. Lee I., et al. Distinct roles of the first introns on the expression of Arabidopsis profilin gene family mcmbcrs[J]. Plant Physiol,2006,140:196-209.
[95]Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. Mol Endocrinol,2000,25:169-193.
[96]吴宇,高蕾,曹民杰,等.植物硫营养代谢、调控与生物学功能[J].植物学通报,2007,24(6)：735-761.
[97]夏德习.拟南芥硫氧还蛋白基因在逆境胁迫下的功能解析[D].哈尔滨,东北林业大学,2007.
[98]Su K W,Yong J L,HaY L, et al. Cis-element and transcriptome-based screening of root hair-specific genes and their functional characterization in Arabidopsis[J].Plant Physiol,2009, 150:1459-1473.
[99]Cho H T,Cosgrove D J.Regulation of root hair initiation and expansin gene expression in Arabidopsis[J]. Plant Cell.2002,14:3237-3253.
[100]张素维,蒋阿维,孙扬吾,等.基于RHE元件和拟南芥转录组的根毛发育基因筛选[J].核农学报,2010,24(5)：922-925.
[101]孙萍.乙烯对拟南芥耐受干旱胁迫的作用及抗氧化研究[D].哈尔滨,东北林业大学,2010.
[102]Ullas V. Pedmale, EmmanuelLiscum. Regulation of Phototropic Signaling in Arabidopsis via Phosphorylation State Changes in the Phototropin 1-interacting Protein NPH3[J]. Journal of biological chemistry.2007,6(27):19992-20001.
[103]Vij S,Tyagi AK.Emerging trends in the functional genomics of the abiotic stress response in crop plants[J]. Plant Biotechnol.2007,5(3):361-380.
[104]付畅,黄宇.转录组学平台技术及其在植物抗逆分子生物学中的应用[J].生物技术通报,2011.6：40-46.