马铃薯光响应StR2R3–MYB1基因的克隆与表达分析

以马铃薯原始栽培种Yan(S.tuberosum subsp.andigena var.yanacochense)为材料,采用同源克隆技术,得到1条800 bp的c DNA片段。该c DNA片段包含1个完整的开放阅读框,编码265个氨基酸长度的蛋白质,定位在马铃薯10号染色体上。在线氨基酸序列分析、原生质体转化核定位分析和定量PCR表达特异性分析结果表明,该蛋白与番茄Sl CMYB1、茄子Sm MYB的同源性达99%,与拟南芥At MYB113的同源性在70%以上;该蛋白含有2个DNA–结合结构域,是2个串联的myb–功能域,属于R2R3类MYB转录因子,命名为StR2R3–MYB1。StR2R3–MYB1基因的调控区域存在G–Box、Box4、BoxⅡ、I–Box、MNF1等光相关元件和脱落酸诱导相关元件ABRE、生物钟相关元件Circadian、茉莉酸甲酯相关元件CGTCA。StR2R3–MYB1氨基酸序列没有跨膜信号,不存在卷曲螺旋,其N端存在蛋白信号肽,具有明显的亲水区域;StR2R3–MYB1基因在细胞核内表达;StR2R3–MYB1在根、茎、叶中的表达量依次升高,同时受白光、远红光和蓝光的诱导表达。综合分析结果表明,马铃薯StR2R3–MYB1基因是1个光响应MYB类转录因子。     

胡杨中两个新DREB类基因的克隆、序列分析及转录激活功能研究

DREB转录因子(干旱应答元件结合因子)是逆境适应中的关键调节因子。该类转录因子的特点是拥有保守的AP2/EREBP(APETALA2/乙烯应答元件结合蛋白)结构域,能够特异性地与抗逆基因启动子上的DRE元件相结合,在低温、干旱和盐碱等条件下调节一系列下游逆境应答基因的表达。本研究从胡杨中分离出2个编码DREB2类蛋白的基因PeDREB3和PeDREB4。PeDREB3和PeDREB4在胡杨根、茎、叶中都有表达。序列分析显示,PeDREB3和PeDREB4均含有非典型性AP2/ERF结构域,即在AP2/EREBP结构域的第2个β折叠和第3个β折叠处多出8个氨基酸残基,用改进的酵母单杂交技术发现PeDREB3和PeDREB4能够在酵母中激活下游报告基因的表达,具有转录活性。     

Isolation and characterization of the secondary wall-related SND1 gene in hawthorn

Secondary wall-associated NAC domain protein1 (SND1) is a key regulator directly regulating the expression levels of MYB46 and MYB83 in the regulation network for secondary wall synthesis, especially in plant fibres. In this study, a SND1 gene was isolated from hawthorn (Crataegus pinnatifida) and named as CpSND1 because it has a conservative N-terminal DNA-binding domain with AtSND1. Arabidopsis plants overexpressing CpSND1 had similar phenotypes as plants overexpressing AtSND1, including inhibited growth, upward-curling leaves, sepal dysplasia and sterility. In addition, overexpressing CpSND1 in Arabidopsis also induced the expression of downstream genes, including lignin, cellulose and xylan biosynthesis genes as well as MYB genes. Our results provided functional information of CpSND1 for future genetic engineering in hawthorn.     

OsMas1, a novel maspardin protein gene,confers tolerance to salt and drought stresses by regulating ABA signaling in rice

Drought and salt stresses, the major environmental abiotic stresses in agriculture worldwide, affect plant growth, crop productivity, and quality. Therefore, developing crops with higher drought and salt tolerance is highly desirable. This study reported the isolation, biological function, and molecular characterization of a novel maspardin gene, OsMas1, from rice. The OsMas1 protein was localized to the cytoplasm. The expression levels of OsMas1 were up-regulated under mannitol, PEG6000, NaCl, ...     

美洲黑杨木质素生物合成转录因子PdLim1基因的克隆、表达及植物表达载体构建

在植物体内,Lim1基因与木质素生物合成酶基因启动子区域富含AC的Pal盒结合,转录调控木质素的生物合成过程。该研究组合利用生物信息学和realtime PCR方法,首次从美洲黑杨形成层cDNA中分离出PdLim1cDNA全长,并进行了测序和序列分析。结果表明,克隆的美洲黑杨PdLim1cDNA片段总长为952 bp,基因内部含有完整的开放阅读框架,大小为594 bp,可编码长度为197个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列与拟南芥AtLim1和水稻OsLim1蛋白的同源性分别为82.1％和78.2％。组织特异realtime PCR结果显示, PdLim1基因在杨树根、茎、叶片和顶端分生组织中均有表达,但其表达模式却不同:PdLim1在成熟叶片和成熟木质部中表达丰度最高,在树皮、根部、韧皮部和形成层表达丰度较高,在未成熟木质部有少量表达,在顶端分生组织中表达丰度最低。 在此基础上,以pBI121为表达载体,构建了在CaMV35S启动子驱动下,PdLim1基因的正义（pBI121-CaMV35S-sense PdLim1）和反义（pBI121-CaMV35S-antisense PdLim1）类型的双元植物表达载体。该研究为杨树PdLim1的基因工程改良木材纤维品质性状提供了重要的理论依据,具有潜在的应用价值。      

蒺藜苜蓿低温胁迫响应基因MtbHLH-1的克隆及功能分析

【目的】克隆蒺藜苜蓿（Medicago truncatula）低温胁迫响应基因MtbHLH-1,并对其进行序列特征分析,研究MtbHLH-1对牧草蒺藜苜蓿耐低温能力的影响。【方法】利用RT-PCR技术获得蒺藜苜蓿MtbHLH-1全长的cDNA序列。对该序列进行生物信息学分析,采用半定量RT-PCR研究MtbHLH-1的低温（4℃）表达模式。【结果】测序结果显示,cDNA片段全长778 bp,包含一个741 bp完整的开放阅读框,编码246个氨基酸残基,命名为MtbHLH-1,GenBank登录号为JN800447。氨基酸BLAST分析表明,MtbHLH-1氨基酸序列与已报道的其它植物（大豆、拟南芥、烟草、小麦和玉米等）bHLH氨基酸序列具有45%-73%相似性。半定量RT-PCR研究表明,MtbHLH-1在低温胁迫条件下上调表达。【结论】从蒺藜苜蓿中克隆了低温胁迫碱性/螺旋-环-螺旋转录因子MtbHLH-1,半定量分析表明其在蒺藜苜蓿抗冷过程中起作用。     

Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera(Hübner)

Carbohydrate chains are the principal antigens by which Bacillus thuringiensis(Bt) identify receptor proteins. The interaction between the antigen and Bt causes a pore in the membrane of midgut epithelial cells of insects. Receptor proteins, such as aminopeptidase N and alkaline phosphatase, are glycoproteins. Cadherin is another cell surface receptor protein which has potential glycosylation sites. Glycosyltransferase is very important for the synthesis and modification of receptor proteins. It can indirectly influence the function of Bt. The 1 950 bp full-length c DNA encoding β-1,3-galactosyltransferase was cloned from the the midgut of Helicoverpa armigera by degenerative PCR combined with RACE techniques(GAL-Harm, Gen Bank accession no.: GQ904195.1) with two potential N-glycosylation sites(157NNTI160 and 272NKTL275). Protein sequence alignments revealed that H. armigera β-1,3-galactosyltransferase shared high identity with β-1,3-galactosyltransferase in other insect species. The expression level of the β-1,3-galactosyltransferase gene in Cry1Ac-resistant H. armigera larvae was 9.2-fold higher than that in susceptible strain. The function of β-1,3-galactosyltransferase was investigated using RNAi technique. The result showed Cry1 Ac enhanced the toxicity against the si RNA-treated larvae compared with non-si RNA-treated ones, which indicated β-1,3-galactosyltransferase played an important role for the insecticidal toxicity of Cry1 Ac in H. armigera.     

美洲黑杨木质素生物合成转录因子PdLiml基因的克隆、表达及植物表达载体构建

在植物体内,Liml基因与木质素生物合成酶基因启动子区域富含AC的Pal盒结合,转录调控木质素的生物合成过程.该研究组合利用生物信息学和realtime PCR方法,首次从美洲黑杨形成层cDNA中分离出PdLimlcDNA全长,并进行了测序和序列分析.结果表明,克隆的美洲黑杨PdLimlcDNA片段总长为952 bp,基因内部含有完整的开放阅读框架,大小为594 bp,可编码长度为197个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列与拟南芥AtLiml和水稻OsLiml蛋白的同源性分别为82.1%和78.2%.组织特异性realtime PCR结果显示,PdLiml基因在场树根、茎、叶片和顶端分生组织中均有表达,但其表达模式却不同:PdLiml在成熟叶片和成熟木质部中表达丰度最高,在树皮、根部、韧皮部和形成层表达丰度较高,在未成熟木质部有少量表达,在顶端分生组织中表达丰度最低.在此基础上,以pBI121为表达载体,构建了在CaMV35S启动子驱动下,PdLiml基因的正义(pBI121-CaMV35S-sensePdLiml)和反义(pBI121-CaMV35S-antisense PdLiml)类型的双元植物表达载体.该研究为杨树PdLiml的基因工程改良木材纤维品质性状提供了重要的理论依据,具有潜在的应用价值.     

一种嗜热菌Pif1解旋酶的表达纯化及活性分析

【目的】利用大肠杆菌表达纯化嗜热脱铁去硫弧菌(Deferribacter desulfuricans)解旋酶DePif1,并对其结合与解旋DNA的活性进行分析,为Pif1家族解旋酶结构和功能的阐明奠定基础。【方法】将促溶标签SUMO编码序列和人工合成的DePif1解旋酶编码序列依次连入pET15b载体,获得重组融合表达载体pET15b-SUMO-DePif1,然后将其导入E.coli BL21(DE3)菌株进行诱导表达;利用Ni-NTA亲和层析柱获得融合蛋白,SUMO蛋白酶酶切去除融合标签,再经Heparin和Ni-NTA柱分离获得无标签的纯化重组DePif1蛋白;采用荧光各向异性分析,研究pH和NaCl浓度对DePif1与DNA结合的影响及DePif1与不同底物(单链DNA、双链DNA和G4-DNA)的结合特性;使用基于荧光共振能量转移的stopped-flow技术,分析DePif1对不同底物(G4-DNA with 5′26nt tail和dsDNA with 5′26nt tail)的解旋活性。【结果】每升菌液可获得9 mg纯度大于95%的DePif1解旋酶。DePif1结合不同DNA底物的强度依次为G4-DNA单链DNA双链DNA,其对G4-DNA的解旋活力大于双链DNA。【结论】成功表达并纯化了嗜热脱铁去硫弧菌Pif1解旋酶,并证明其具有特异的G4-DNA结合和解旋能力。     

青稞转录因子HvnANT1基因的克隆与表达分析

为探究HvnANT1基因在青稞粒色形成过程中的基因表达模式,以紫粒青稞‘涅如姆扎’和白粒青稞‘昆仑10号’为试材,利用简化基因组GBS(Genotyping-by-Sequencing)对青稞紫粒进行基因定位,克隆到HvnANT1基因,对其进行生物信息学分析。结果表明：1）在7H染色体的84.30—86.00cM获得1个MYB类转录因子,命名为HvnANT1。2）该基因长1 033bp,其完整开放阅读框为762bp,编码253个氨基酸。HvnANT1蛋白分子量为27.14kU,是亲水性的不稳定碱性蛋白且不存在跨膜结构,无信号肽。该蛋白的二级结构主要是由无规卷曲、α-螺旋、延伸链和β-转角组成,具有2个SANT结构域（分别位于第13—第63个;第66—第114个氨基酸）。3）同源比对与系统进化分析表明：青稞HvnANT1蛋白与大麦、乌拉尔图小麦、高梁、玉米、二型花、小米、水稻、土瓶草、车轴草和杨梅10种植物的ANT1蛋白序列相似性分别为100.00%、88.85%、54.64%、60.95%、60.82%、58.46%、57.61%、37.76%、36.05%和36.33%;这些序列都具...     

基于MGM（1，n）模型的中国水产品产量时间序列的实证分析

运用灰色关联度与多变量灰色系统理论对中国1986-2011年水产品产量时间序列进行了实证分析。首先用灰色关联分析方法将水产品产量所包含的5组时间序列即海洋捕捞、远洋渔业、海水养殖、淡水捕捞、淡水养殖产量进行了分类;再将关联度达到0.9以上的海洋捕捞、海水养殖、淡水养殖产量3组时间序列划分为一组并运用多变量灰色MGM (1, n)模型对其进行建模,由于这3组序列关联度高且单个序列累加数据近似服从指数分布,因而拟和结果数据逼近初始数据;对关联度达到0.8的远洋渔业与淡水捕捞两组时间序列,也尝试用MGM (1, n)模型进行建模,结果模型拟合略有欠缺。本研究中的这种建模方法既考虑了序列间的内在联系,又避免了分别使用单一序列建模割裂序列相互制约因素的情况发生,图示与误差分析结果均显示该方法具有效性。     

家蚕糖转运蛋白BmST1基因的克隆与序列分析

糖转运蛋白运输葡萄糖、果糖、半乳糖、木糖等透过细胞膜为机体提供能量.根据GenBank已登陆的其他物种的糖转运蛋白序列与家蚕基因组框架图和EST序列获得家蚕同源糖转运蛋白基因BmST1.基因编码区长1 392 bp(GenBank登录号:FJ373021),编码463个氨基酸,预测蛋白分子质量为51.635 kDa.序列分析显示基因有2个外显子,含有11个跨膜结构域.RT-PCR表明该基因仅在此文所检测的家蚕丝腺中表达.     

十倍体长穗偃麦草雄性育性基因ThMs1的克隆、表达及功能分析

【目的】从小麦隐性核雄性不育突变体ms1与十倍体长穗偃麦草异附加系中,克隆雄性育性恢复基因ThMs1,解析该基因的表达模式、编码蛋白的生物化学活性和生物学功能,鉴定新的小麦ms1育性恢复基因,从而更好地应用于小麦杂交育种。【方法】通过基因组原位杂交（genomic in situ hybridization,GISH）,鉴定ms1与十倍体长穗偃麦草异附加系中小麦外源的染色体类型;通过同源克隆法在小麦-十倍体长穗偃麦草异附加系中克隆雄性育性基因ThMs1,并稳定转化小麦ms1进行基因功能互补验证;利用RT-PCR及实时荧光定量PCR分析检测基因的表达模式;进一步通过RNA原位杂交分析基因的组织细胞特异性表达特性;利用特异性抗体进行Western blot检测ThMs1蛋白在体内的表达情况;经SignalP软件分析预测蛋白结构;通过蛋白-脂质结合活性分析检测ThMs1蛋白是否具有脂类分子结合活性。【结果】证实小麦ms1与十倍体长穗偃麦草异附加系细胞中含有偃麦草4Ag染色体;从小麦-十倍体长穗偃麦草异附加系中克隆了雄性育性基因ThMs1,遗传转化功能互补试验证实ThMs1能够完全恢复小麦ms1突变体的雄性不育表型,基因的聚类分析表明MS1只存在于禾本科植物中;通过RT-PCR和实时荧光定量PCR检测发现ThMs1在减数分裂期的花药中特异表达,RNA原位杂交分析证实ThMs1在小麦小孢子发生过程中特异表达;Western blot检测发现ThMs1在小麦减数分裂期的花药中表达;对ThMs1蛋白氨基酸序列进行结构预测发现,该蛋白具有推测的脂类结合分子结构域,ThMs1蛋白脂类分子结合试验表明ThMs1蛋白特异结合磷脂酸和磷酸化的磷脂酰肌醇。【结论】克隆了一个新的来自十倍体长穗偃麦草、可恢复小麦隐性核雄性不育突变体ms1雄性育性的ThMs1,该基因与小麦Ms1具有相似的分子结构、时空表达模式和脂结合活性,导致2个蛋白在小麦中也具有相似的生物学功能,ThMs1可以替代普通小麦Ms1的功能调控小麦花粉育性,该结果为利用小麦ms1通过分子设计建立小麦杂交育种体系（新一代杂交育种体系）提供了一个新的育性恢复基因。     

Comparative analysis of genomes in Oryza sativa, O. officinalis and O. meyeriana with C 0 t-1 DNA and genomic DNA of cultivated rice

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosome preparations of Oryza sativa, O. officinalis and O. meyeriana with labeled probes of C ₀ t-1 DNA and genomic DNA from cultivated rice. The coverage percentage (%) and size (Mb) of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana were 47.1 ± 0.16, 38.61 ± 0.13, 44.38 ± 0.13 and 212.33 ± 1.21, 269.42 ± 0.89, 532.56 ± 1.68, respectively. The coverage percentage and size of probe signals with genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0%, 93.6% and 634 Mb, 1 123 Mb respectively, in which there were 365 and 591 Mb in O. officinalis and O. meyeriana which came from O. sativa genomic DNA not from repetitive sequences of O. sativa, and the uncovered genome size in O. officinalis and O. meyeriana was 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during the evolution process. The repetitive sequence reduplication might be one of the important causes of genome enlargement in O. officinalis and O. meyeriana; the O. officinalis genome enlarged more slowly compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana formed by reduplication, rearrangement and gene selective loss during the evolution process. Translated from Scientia Agricultura Sinica, 2006, 39(6): 1083–1090 [译自: 中国农业科学]