巴西橡胶树HbWRKY1基因的克隆及转化烟草的初步研究

利用RACE结合RT-PCR技术,从巴西橡胶树(Hevea brasiliensis)总RNA中扩增得到长度为1234 bp的WRKY基因cDNA全长编码序列。通过氨基酸同源性比对,该序列推导的氨基酸序列与蓖麻、白杨的WRKY同源性分别为79%和73%,表明分离的cDNA序列为橡胶树WRKY基因,命名为HbWRKY1。通过构建pCAMBIA1304-HbWRKY1植物表达载体,经农杆菌GV3101介导,将HbWRKY1基因导入烟草(Nicotiana tabacum)中,对所获得的潮霉素抗性烟草株系进行PCR鉴定。结果表明,HbWRKY1基因已整合到65株转基因植株中。干旱胁迫试验表明,HbWRKY1的过量表达可以明显提高转基因烟草对干旱胁迫的耐受能力。这说明WRKY基因与橡胶树抗旱能力之间存在一定的关系。     

玉米转录因子WRKY76克隆及抗纹枯病表达模式分析

玉米纹枯病是影响玉米产量和品质的重要病害之一。转录因子WRKY家族部分成员能够调控水杨酸和茉莉酸甲酯信号传递方式来激发防卫反应基因的表达。在NCBI上检索玉米中WRKY家族成员及拟南芥中抗病相关的WRKY家族成员,利用CLUSTAL X和MEGA5.05构建系统进化树,发现转录因子WRKY76可能参与玉米抗纹枯病的调控途径。该研究以玉米抗纹枯病材料R15和感病材料Ye478为对象,在玉米拔节期接种立枯丝核菌AG1-IA,首先分别于接菌前(对照)和接菌后1、2、4、6、12、24 h取叶鞘;然后分别进行水杨酸和茉莉酸甲酯胁迫处理,分别于处理前(对照)和处理后1、2、4、6、12 h取叶鞘,提取RNA,实时荧光定量PCR分析WRKY76转录因子基因在玉米叶鞘组织中不同胁迫条件下的差异表达。结果表明:在立枯丝核菌AG1-IA胁迫下,WRKY76转录因子基因在胁迫后1 h表达量达最大值,抗病材料R15的相对表达量高于感病材料Ye478且差异显著(P≤0.05);经水杨酸(Salicylic Acid,SA)处理,WRKY76在抗感材料中表达趋势相似,在感病材料掖478中,WRKY76被诱导而显著地上调表达,在抗病材料中,相对表达量峰值出现在胁迫后4 h,且相对表达量低于感病材料掖478。经茉莉酸甲酯(Methyl jasmine,Me JA)处理,WRKY76基因在感病材料中呈现下调表达趋势。WRKY76基因在1 h表达量为对照的0.6倍,其他调查时间点基本都在0.1~0.3之间。在抗病材料R15中,WRKY76基因表达呈现上升趋势,变化趋势不明显。这表明WRKY76转录因子基因能够被病原物、SA、Me JA诱导表达,可能参与植物抗纹枯病调控途径。     

枸杞WRKY_3基因克隆及组织表达分析

以枸杞为材料,采用PCR及RACE方法,克隆了枸杞WRKY转录因子基因cDNA序列,命名为Lb WRKY3,GenBank登录号为KX196192。在生物信息学分析的基础上,进行亚细胞定位、基因表达分析。结果显示:(1)Lb WRKY3开放阅读框ORF长度为1 068bp,编码356个氨基酸。(2)生物信息学分析显示,Lb WRKY3编码蛋白具有一个WRKY结构域,二级结构中不规则卷曲结构所占比例最大(58.67%),延伸链结构次之(18.88%),α螺旋比例为15.82%,β转角最少,仅为6.63%;Lb WRKY3蛋白与案头菊WRKY蛋白、黄花蒿WRKY蛋白相似性较高。(3)亚细胞定位显示,Lb WRKY3蛋白定位于细胞核。(4)实时定量PCR分析表明,Lb WRKY3在根中表达量最高,在花中表达量最低;在枸杞果实发育过程中Lb WRKY3均有表达,表达量随果实成熟逐渐升高,并于35d达到峰值;Lb WRKY3基因在果实中的表达具有组织特异性表达特性(果肉果皮种子)。研究表明,Lb WRKY3基因参与了枸杞果实生长发育调控。     

辣椒CaWRKY8基因克隆及胁迫下的表达分析

为了揭示辣椒WRKY基因功能,以辣椒PI201234为实验材料,克隆得到WRKY基因全长1 647bp的cDNA序列,命名为CaWRKY8。生物信息学分析表明,该基因含有一个1 647bp完整开放阅读框(ORF),编码548个氨基酸残基。氨基酸序列分析显示,CaWRKY8编码的蛋白含有2个WRKY结构域,属于Group I。氨基酸序列比对结果表明,CaWRKY8与辣椒WRKY25、马铃薯WRKY、番茄基因组中预测的WRKY26、烟草基因组中预测的WRKY33和猕猴桃WRKY的氨基酸序列之间均具有高度的保守性。实时荧光定量分析表明,CaWRKY8受盐、高温、干旱和辣椒疫霉菌诱导表达;其中CaWRKY8的表达量在盐和干旱处理下3h达到峰值,分别是对照的2.38倍和121.10倍,在高温和疫霉菌处理下12h达到峰值,分别是对照的6.12和6.81倍。以上研究结果表明,CaWRKY8基因在辣椒响应胁迫进程中发挥着重要作用。     

中间锦鸡儿WRKY75基因对拟南芥耐受盐和ABA能力的影响

该研究从旱生灌木中间锦鸡儿中克隆得到1个CiWRKY75基因。序列分析显示,CiWRKY75开放阅读框长570bp,编码189个氨基酸,含有1个WRKYGQK基序和1个C2H2型锌指结构,属于第二类WRKY转录因子。亚细胞定位显示,CiWRKY75定位于细胞核。实时荧光定量PCR检测表明,CiWRKY75基因的表达受盐胁迫和ABA诱导。在拟南芥中过量表达CiWRKY75后,与野生型拟南芥相比,转基因株系种子的萌发率在盐胁迫下降低,并且对盐胁迫的耐受能力明显减弱;ABA处理下,2个转基因株系的种子萌发率(10.3%、9.6%)较野生型(25.9%)明显降低。研究表明,CiWRKY75是中间锦鸡儿对盐和ABA响应的重要调控因子。     

香蕉果实冷胁迫相关MaWRKY11转录因子的特性、互作蛋白筛选与鉴定

为探讨香蕉(Musa acuminata)响应冷胁迫的分子机制,从香蕉果实冷害的数字基因表达谱中筛选并分离了1 个WRKY转录因子,命名为MaWRKY11。MaWRKY11 具有2 个WRKY 保守结构域,属于I 类WRKY 成员,定位于细胞核,是核蛋白。MaWRKY11 具有转录激活活性,且激活区在N 端。实时荧光定量PCR 分析表明MaWRKY11 受冷胁迫诱导,外源茉莉酸甲酯(MeJA)处理减轻香蕉果实冷害的同时也上调了其表达。另外,酵母双杂交筛选表明,MaWRKY11 可与脱水诱导的早期应答蛋白MaERD 相互作用。这些表明MaWRKY11 可能通过与逆境相关蛋白如MaERD 互作来响应香蕉果实的冷胁迫。     

Cloning and expression of a gene coding for the major light-harvesting chlorophyll a/b protein of photosystem II in the green alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Genes encoding proteins of the major light-harvesting complex of photosystem II (LHCII) in higher plants are well studied. However, little is known about the corresponding genes in the green alga Dunaliella salina, although this knowledge might provide valuable information about the respective roles of each LHCII protein at the molecular level under extreme environmental conditions. Here, we describe an additional LhcII gene from D. salina. An LhcII cDNA cloned by screening a D. salina cDNA library contains an open reading frame encoding a protein of 261 amino acids with a calculated molecular mass of 27.8 kDa. The deduced amino acid sequence shows high homology with other LHCII proteins. Genomic DNA—obtained by PCR using a specific primer set corresponding to the 5′ and 3′ untranslated regions—was used to determine the intron-exon structure. Short-term changes in mRNA levels after a shift from low-light to high-light or dark conditions were analyzed by real-time quantitative PCR, and indicated that this gene expresses different mRNA levels under different light conditions.     

Molecular characterization of the sheep <Emphasis Type="Italic">CIB1</Emphasis> gene

The calcium and integrin binding protein 1(CIB1), is an EF-hand-containing protein that binds many effector proteins including the platelet αIIbβ3 integrin and potentially regulates their functions. Here we report the cloning and characterization of the sheep CIB1 gene. The CIB1 cDNA is 885-bp in size, containing a 45-bp of 5′ untranslated region (UTR), a 264-bp long 3′-UTR and a 576-bp open reading frame that encodes 191 amino acids. The sheep CIB1 cDNA shows 98.3, 92.0, 91.8, 91.3, 90.5 and 90.1% of similarity, at the nucleotide level, to its equivalents in cattle, pigs, rhesus monkey, humans, rats and mice, respectively at the deduced protein level, the corresponding values are more than 94%. The sheep CIB1 gene consisted of seven exons. Quantitative PCR (Q-PCR) showed that CIB1 was widely expressed in different tissues with the highest level in the testis, suggesting that it may play a role in ram fertility. We cloned the sheep CIB2, CIB3 and CIB4 genes and detected their expression patterns in different tissues.     

Molecular Cloning and Characterization of a NAC-like Gene in “Navel” Orange Fruit Response to Postharvest Stresses

A cDNA subtraction library had been constructed to identify differentially expressed genes in peel pitting of citrus fruit. Based on the sequence of a cDNA fragment homologous to NAC gene family, the full-length cDNA of 1,203 nucleotides was cloned from “navel” orange by rapid amplification of cDNA ends. It was designated as CsNAC, encoding a protein of 305 amino acids. The calculated molecular weight of the CsNAC protein was 35.2 kDa, and theoretical isoelectric point was 6.72. Sequence comparison showed that the CsNAC protein had a strikingly conserved region at the N terminus, which is considered as the characteristic of the NAC protein family. CsNAC protein was orthologous to Arabidopsis thaliana ATAF1. Phylogenetic analysis confirmed CsNAC belonged to the ATAF subfamily, which plays an important role in response to stress stimuli. RNA gel blot analysis showed that the expression of CsNAC gene was rapidly and strongly induced by stresses such as wounding and no oxygen. Low temperature (4°C) and exposure to ethylene also increased the expression level of CsNAC gene. However, its expression was suppressed by high temperature (40°C) but not affected by low oxygen (3%). Our results may provide the basis for future research of NAC-like gene’s role in stress-induced citrus peel pitting. Sequence data of CsNAC from this article have been deposited at GenBank under accession number EF596736.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.