割手密SnRK2基因家族全基因组鉴定及干旱胁迫表达分析

【目的】探究割手密SnRK2基因家族成员在干旱胁迫中的调控机制,为抗旱性甘蔗品种的选育提供侯选基因。【方法】以全基因组数据为基础从割手密中鉴定SnRK2基因,并对其进行生物信息学分析和干旱胁迫下的表达分析。【结果】在割手密基因组中共鉴定出11个SnRK2基因家族成员,命名为SsSnRK2.1-SsSnRK2.11,且这些基因不均匀地分布于8条染色体上。SnRK2蛋白的氨基酸残基数为227～580,分子质量为25 683.53～64 695.8 kD,等电点为4.62～8.94,且均为亲水性蛋白。系统发育树可将其分为3个亚组,且同亚组中的保守基序基本相似,外显子数量以7～9个为主。SsSnRK2基因家族成员的启动子中含有多种激素类和逆境胁迫响应类的作用元件。割手密SsSnRK2基因家族成员的表达具有组织特异性。所有的SsSnRK2基因均能不同程度地响应干旱胁迫。【结论】割手密SnRK2基因家族在响应干旱胁迫过程中发挥重要作用,可为割手密的抗逆性研究提供一定的理论依据。     

小麦盐胁迫相关基因的克隆与表达分析

采用RT-PCR方法,从小麦中克隆获得1个盐诱导小麦MYB类转录因子基因TaSIM（Triticum aestivum salt-induced MYB）,该基因cDNA全长1 213bp,具有1个831bp的开放阅读框,编码276个氨基酸,预测分子量约为29.903kD,等电点为10.12,推测的氨基酸序列中含有2个高度保守的SANT结构域。系统发生树分析表明,TaSIM与二穗短柄草XP₀03576185亲缘关系最近。半定量RT-PCR检测结果显示,TaSIM基因受盐胁迫诱导表达。亚细胞定位结果显示,TaSIM-hGFP融合蛋白定位于细胞核中。研究结果表明,小麦TaSIM基因编码的蛋白可能在细胞核内参与小麦对盐胁迫的应答反应。     

辽宁碱蓬SlCMO基因盐胁迫表达分析及盐诱导启动子鉴定

胆碱单加氧酶（choline monooxygenase, CMO）是合成甜菜碱的关键酶,甜菜碱在植物抵抗渗透胁迫中起着重要的作用。本研究室前期克隆了盐生植物辽宁碱蓬CMO（Suaeda liaotungensis CMO）基因及启动子。本研究对SlCMO基因在盐胁迫下的表达及盐诱导启动子进行分析。qRT-PCR分析SlCMO基因在辽宁碱蓬不同器官及盐胁迫下的表达,结果表明,SlCMO基因在根、茎、叶中均有表达,其中茎、叶中的表达量较高,SlCMO基因在根、茎、叶中的表达均受盐胁迫诱导。5′端缺失分析SlCMO启动子的盐诱导区段,结果表明,pC5（-267~+128 bp）是SlCMO启动子的盐诱导功能区段,推测pC5调控SlCMO 基因的盐诱导表达。本研究为SlCMO 基因表达调控研究奠定基础,也为植物抗盐基因工程提供可用的启动子。     

Cloning and Expression Analysis of Salt Responsive Gene from Kandelia candel

Identification of gene expression patterns in mangroves grown under salinity will help to reveal the molecular mechanisms of salt tolerance. Here, 10 cDNAs of genes were isolated from Kandelia candel and identified by representational difference analysis of cDNA (cDNA RDA) under different NaCl concentrations. Of five genes expressed preferentially under salt condition, two were unknown, three were two kinds of low molecular mass heat-shock proteins (sHSPs) and ADP-ribosylation factor, respectively. The expressions of other five genes were repressed under NaCl stress, two encoded cyclophilins, three were tonoplast intrinsic protein, early light-induced protein and 60S ribosomal protein, respectively.     

甘薯盐胁迫响应基因IbMYB3的表达特征及生物信息学分析

MYB转录因子具有多种生物学功能,在植物响应生物和非生物胁迫中发挥重要作用。该文从盐胁迫后的甘薯(Ipomoea batatas)水培苗转录组数据(RNA-seq)中筛选出2个受盐胁迫显著上调表达的MYB基因,分别命名为IbMYB3和IbMYB4。多种非生物胁迫和植物生长物质处理下的基因表达分析显示,IbMYB3受逆境诱导显著上调表达,暗示其可能参与甘薯非生物胁迫响应。生物信息学分析表明,IbMYB3开放阅读框长度为1059 bp,编码353个氨基酸残基,蛋白分子量为39.41 kDa,理论等电点(PI)为5.26,为酸性带负电的亲水性蛋白。亚细胞定位结果表明,IbMYB3蛋白定位于细胞核,具有较强的转录激活活性。上述结果表明,IbMYB3转录因子可能在甘薯非生物胁迫响应过程中发挥重要调控作用,研究结果为进一步探明IbMYB3基因的功能奠定了基础。     

Gene Expression Analysis of Cold and Freeze Stress in Baker's Yeast

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10°C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.     

Expression of a Late Embryogenesis Abundant Protein Gene,HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.     

杨树MYB转录因子家族基因应答盐胁迫表达特性分析

MYB转录因子家族是植物中数量最多的转录因子家族之一,在植物次生代谢调节、信号转导和抗逆等生物过程起重要作用。根据MYB转录因子结构域组成差异可分4个亚家族：即1R-MYB（MYB-relaed）、R2R3-MYB、3R-MYB和4R-MYB。其中,R2R3-MYB亚家族数量最多,可进一步分为22个亚组;利用生物信息学分析杨树MYB转录因子蛋白序列的保守结构域、系统发生、基因组定位、氨基酸组成和理化性质等;参照拟南芥MYB转录因子功能,预测杨树MYB转录因子功能;基于84K杨转录组测序和RT-qPCR分析,从301个杨树MYB转录因子基因中筛选出69个应答盐胁迫基因（P≤0.05）。其中,上调表达基因32个,下调表达基因37个。该研究可为进一步研究杨树MYB家族基因功能提供参考依据。     

费尔干猪毛菜病程相关蛋白基因（SfPR-1）的克隆及在盐胁迫下的表达分析

利用抑制消减杂交法从藜科猪毛菜属盐生植物费尔干猪毛菜（Salsola ferganica）中分离得到了一个盐胁迫响应的cDNA片段,结合SMARTTMRACE技术获得了费尔干猪毛菜病程相关蛋白基因的cDNA,命名该基因为SfPR-1（GenBank登录号：JQ670917）。序列分析表明,SfPR-1长817 bp,含有501 bp的阅读框、65 bp的5＇-UTR和251 bp的3＇-UTR,编码166个氨基酸,分子质量为18.01 kD,理论等电点为9.37。通过BLAST同源序列比对分析,结果显示该基因编码的蛋白与已知甜菜、拟南芥、烟草及玉米的病程相关蛋白PR-1同源性分别为73.6%、57.8%、55.5%和53.9%,且具有PR-1家族特有的6个半胱氨酸保守结构域。半定量RT-PCR和实时荧光定量RT-qPCR分析表明,该基因在盐胁迫后表达呈明显上调,初步推测病程相关蛋白基因SfPR-1可能与费尔干猪毛菜的耐盐性相关。     

菜豆富含脯氨酸蛋白质基因在生物和非生物胁迫下的表达

植物细胞壁是细胞的机械支持物和保护壳,在植物的生长和发育、细胞内的通讯和代谢交换过程中起重要作用。细胞壁主要由多糖和蛋白质组成,根据蛋白质的氨基酸组成和糖的含量特点可把细胞壁结构蛋白分为５类：富含羟脯氨酸的糖蛋白（ｂｙ一ｄｒｏｘｙｐｒｏｌｉｎｇ－ｒｉｃｈ　ｇｌｙｃｏｐｒｏｔｅｉｎｓ,ＨＲＧＰｓ）、富含脯氨酸蛋白质（ｐｒｏｌｉｎｅ－ｒｉｃｈｐｒｏｒｅｉｎｓ,ＰＲＰｓ）、富含甘氨酸蛋白质（ｇｌｙｃｉｎｅ－ｒｉｃｈｐｒｏｔｅｉｎｓ,ＧＲＰｓ）、茄科凝集素（ｓｏｌａｎａｃｅｏｕｓｌｅｃｔｉｎｓ）和阿拉伯…     

水稻中与盐碱适应性相关的VB12不依赖型蛋氨酸合成酶基因的克隆和表达

水稻中与盐碱适应性相关的VB12不依赖型蛋氨酸合成酶广泛在于高等植物中，它可催化高半光氨酸甲基化而生成蛋氨酸，为生物体内的甲基化反应和多胺，乙烯的合成提供中间产物。以水稻品种日本晴为材料，在碱性条件下利用cDNA-RAPD法，在水稻中首次报道了VB12不依赖型蛋氨酸合成酶基因的克隆和表达，结果表明：VB12不依赖型蛋氨酸合成酶cDNA基因全长为2740bp，在水稻基因组中以单或低拷贝存在，编码765个氨基酸，与Mesembryanthemum cystallinum (784889)和Cathararanthus roseus(C83499)的同源性分别为92％和83％，水稻在受到碳酸钠胁迫12h和24h后，其转录较氯化钠明显增强，而到48h后下降，暗示它可能与水稻的盐碱适应性有关。     

一个水稻多逆境响应基因OsMsr3克隆与表达分析

为深入探讨水稻对逆境的反应机理并寻找新的植物耐逆基因,采用Affymetrix水稻表达芯片(含51279个转录本)分析了培矮64S全基因组在不同逆境(高温、干旱、低温)胁迫下、不同生育时期叶片和穗中的表达谱,从中筛选出一个受多种逆境诱导表达的基因OsMsr3(Oryza sativa L.multiple stresses responsive gene3,GenBank登陆号为 FJ383169).定量实时PCR分析结果进一步证实了此基因在逆境条件下的诱导表达模式.用 RT-PCR方法扩增获得了包含其完整开放阅读框的cDNA克隆,序列分析表明,其ORF大小为480 bp,编码一个具有160个氨基酸残基的低分子量热激蛋白,推测分子量约为18.0 kD;pI约为6.8.对其编码的蛋白质进行分析,发现其羧基端存在一个HSP20的蛋白保守结构域,与其他植物中的低分子量热激蛋白的相似性介于33.7%～97.5%.对其可能的启动子序列分析,发现6类与逆境反应有关的顺式作用元件.推测该基因在逆境反应中起着重要的作用,进一步的研究正在进行中.     

离心力和剪应力应答蛋白1是肿瘤坏死因子受体1的新调控因子

离心力和剪应力应答基因1(responsive to centrifugal force and shear stress gene 1,RECS1)被剔除的小鼠易患囊性内侧坏死和动脉扩张症,伴随着血管组织基质金属蛋白酶9表达水平的增强.本室前期研究发现,稳定表达RECS1的小鼠成纤维细胞对肿瘤坏死因子受体2激动性抗体的敏感性被明显弱化,显示RECS1参与肿瘤坏死因子信号的调控.本文研究了RECS1对肿瘤坏死因子受体1（tumor necrosis factor receptor-1, TNFR1）的调控作用.结果显示,RECS1结合TNFR1,并抑制过量表达TNFR1诱导的核转录因子-κB (NF-κB)活化.缺失突变研究发现,RECS1分子上有NPLY和SPEDY两个模体是其抑制TNFR1信号所必需的.免疫共沉淀实验发现,NPLY是RECS1与TNFR1结合所必需的.而SPEDY的缺失不影响RECS1与TNFR1的结合.另外,免疫共染色实验显示,RECS1与TNFR1共定位于细胞内核体.这些实验结果进一步揭示了RECS1负调控肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)信号进而参与调控血管发育与重塑的生物功能及可能机理.     

斑茅割手密复合体(GXAS07-6-1)及其与甘蔗F_1的GISH分析

斑茅割手密复合体(GXAS07-6-1)是广西蔗茅属斑茅和广西甘蔗属割手密的属间杂种,聚集了双亲的优点。本研究利用基于Alu-like的PCR鉴定方法对GXAS07-6-1及甘蔗与GXAS07-6-1的3份F_1材料进行真实性鉴定,基于基因组原位杂交技术(GISH)对父本GXAS07-6-1及其3份F_1染色体组成及核型进行分析。研究结果表明:3份F_1材料为GXAS07-6-1的真杂种;父本GXAS07-6-1的染色体众数为62条,其中30条来自蔗茅属斑茅,32条来自甘蔗属割手密,核型分类属于1B,其染色体按"n+n"方式传递;GXASF_108-2-17、GXASF_108-2-22、GXASF_108-2-32的染色体数目为78~80条,其中69~71条来自甘蔗属,9~11条来自蔗茅属斑茅,3份F_1的核型分类分别属于2B、1B、1B,染色体传递方式均为"n+n"。父本GXAS07-6-1及3份F_1材料中均未发现有染色体的交换或易位现象。甘蔗与斑割复合体杂交,蔗茅属斑茅染色体在亲子间传递过程存在丢失现象。