非洲菊GjPAL的克隆及表达分析

为了解非洲菊PAL基因编码蛋白的理化性质和表达模式,以非洲菊品种"玲珑"为材料,利用RACE和RT-PCR技术克隆获得4个PAL基因(命名为GjPAL1-GjPAL4),利用生物信息学方法研究它们的结构及其编码蛋白的特性.利用qPCR检测GjPALs在不同组织部位和不同逆境胁迫处理下的表达模式.序列分析显示:4个GjP...     

独一味苯丙氨酸解氨酶基因的克隆与表达分析

以独一味叶片为材料,采用RT-PCR和RACE方法克隆了独一味苯丙氨酸解氨酶基因（PAL）的全长cDNA,命名为LrPAL基因。测序结果表明,LrPA L基因全长2 298 bp,含有1个2 145 bp的完整开放阅读框（ORF）,编码714个氨基酸。蛋白序列分析表明,其包含典型的PAL活性中心序列（GTITASGDLVPLSYIA）,与其他植物的PAL蛋白有很高的同源性。系统进化树分析表明,独一味LrPAL与唇形科植物的PAL蛋白聚为一类,说明两者的亲缘关系较近。用 Real-Time PCR方法检测发现,LrPAL基因在独一味的叶中表达量最高,茎中表达量最少。研究结果推测,从独一味中克隆获得的苯丙氨酸解氨酶基因（LrPAL）是典型的PAL家族成员,在独一味各组织发育过程中具有重要功能。     

鸭梨PAL基因的反义遗传转化及表达分析

苯丙氨酸解氨酶(phenylalanin ammonia-lyase,PAL,EC4.3.1.5)是植物通过苯丙烷代谢途径合成木质素的关键酶和限速酶,其通过影响木质素的合成而与果实中石细胞的分化、发育及果实品质密切相关。为了降低鸭梨中苯丙氨酸解氨酶的含量,该研究利用反义PAL基因遗传转化鸭梨、降低鸭梨内源PAL基因的表达。结果表明:(1)采用RT-PCR技术,利用根据Gen Bank中西洋梨PAL基因序列设计特异性引物,扩增得到496 bp的鸭梨PAL基因片段。(2)将扩增片段反向插入载体p BI121的MCS区域,构建植物PAL基因反义表达载体p BI121-As PAL。接着采用电转化法将反义表达载体转入农杆菌EHA105中,并制备出农杆菌工程菌液。(3)利用农杆菌介导法对鸭梨组培苗叶片外植体进行遗传转化,得到23株转基因鸭梨苗。PCR检测证实PAL反义基因片段转入鸭梨中,实时定量PCR检测表明转基因鸭梨苗体内PAL基因表达量均有所降低,为非转基因苗的65%~75%。该研究结果表明利用反义RNA技术获得了抑制内源性PAL基因表达的转基因鸭梨植株,为改善鸭梨果实品质、改良品种奠定了基础。     

茉莉花JsPAL基因及其启动子克隆与表达分析

苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)为茉莉花(Jasminum sambac)花香物质苯丙烷类合成的限速酶。为了解茉莉花花香形成的分子机理,以双瓣茉莉花瓣为材料,采用RT-PCR和RACE技术相结合的方法,克隆获得茉莉花苯丙氨酸解氨酶基因的全长cDNA(GenBank:KM406501.1),命名为JsPAL,其全长cDNA为2 220 bp,开放阅读框(ORF)为2 140 bp,编码712个氨基酸,含有lyase_I_like超家族蛋白保守域、活性位点及多肽结合位点。采用染色体步移技术,获得该基因的启动子序列。JsPAL基因上游调控序列为1 201 bp,其含有开花相关元件(CCAATBOX1)、PAL相关元件(BOXLCOREDCPAL、PALBOXPPC、PALBOXLPC、TATABOXOSPAL)以及花特异苯丙烷类相关元件(MYBPLANT)等与花香形成相关的重要顺式作用元件。实时荧光定量PCR检测结果表明,在不同组织和花瓣发育过程中,JsPAL基因在茉莉花的花蕾、花瓣中表达量较高,并且在花瓣开放过程中的22:00前表达量较高,而后呈下调趋势。     

香石竹DcPAL1基因的克隆及其原核表达

苯丙氨酸解氨酶(PAL)是植物苯丙烷类代谢途径的关键酶。利用RT-PCR及RACE技术从香石竹茎中克隆到苯丙氨酸解氨酶基因的全长cDNA,命名为DcPAL1(GenBank登录号为FJ864719)。DcPAL1全长2 397bp,预测其开放阅读框长2 121bp,编码一个含有706个氨基酸的蛋白质,其分子量为76.8kD,等电点5.84,且含有PAL的特征序列和活性位点。构建pET-DcPAL1原核表达载体,并转入大肠杆菌(Escherichia coli)中表达,获得1个与预测大小一致的外源蛋白,主要以包涵体形式存在。用Ni2+-NTA螯合琼脂糖层析柱亲和层析得到了纯化的表达蛋白。     

大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析

为了研究苯丙氨酸解氨酶基因与大蕉(Musa ABB cv. Dongguandajiao)抗枯萎病的关系,利用 RT-PCR 和 RACE技术克隆了大蕉苯丙氨酸解氨酶基因全长 cDNA。此 cDNA 长 1 300 bp,包含一个长为 1 191 bp,编码 397 个氨基酸的完整开放阅读框(ORF),推导的氨基酸序列与水稻 PAL 基因氨基酸序列同源性达 89%,将此基因命名为 M-PAL。Southern杂交结果表明大蕉中存在一个包含 4-5 个 PAL基因的基因家族,将此基因克隆到大肠杆菌表达载体 pET32(a )中,表达的蛋白质分子量大小与推导的相一致,并且表达的蛋白质表现出 PAL 酶活性。对接种香蕉枯萎病菌 4 号生理小种(Fusarium oxysporumf. sp. cubense (FOC) race 4 )后大蕉叶片中 M-PAL基因的转录谱进行研究表明,在接种枯萎病菌后,M-PAL基因在叶片中的转录水平提高,因此推测 M-PAL基因的表达可能与香蕉枯萎病抗性相关。     

油木奈果实苯丙氨酸解氨酶基因的分离与表达分析

该试验从(木奈)褐变果实均一化全长cDNA文库中获得一个苯丙氨酸解氨酶全长基因,命名为PsPAL,并对该基因进行了生物信息学分析和表达模式的研究.结果表明:(1) PsPAL基因全长2 497 bp,开放阅读框为2 154bp,编码718个氨基酸,蛋白质分子量为78 kD,理论等电点为6.6.(2)系统进化树比对分析表明,PsPAL蛋白与蔷薇科甜樱桃PaPAL属于同簇,具有苯丙氨酸解氨酶-组氨酸解氨酶(PAL-HAL)和苯丙氨酸解氨酶(PAL)保守区域.(3) P.sPAL在(木奈)果实发育的前期表达量较高,在花后50 d表达量最高,随后开始下降,在成熟果中表达较弱.(4)荧光定量PCR分析表明,在响应机械损伤和低温处理后,与对照相比,PsPAL呈明显的上调表达趋势;高温和无氧处理后PsPAL呈先上升后下降的趋势;乙烯处理后,PsPAL呈上调-下调-上调的变化趋势.     

甜瓜蔓枯病抗性鉴定及PAL基因表达分析

以甜瓜感病品种‘白皮脆’、单基因抗源(PI140471、PI157082、PI511890、PI482398、PI420145)和聚合抗源(145-082、082-890、082-398、145-471、145-890和890-398)为材料,采用梯度浓度蔓枯病菌孢子液接种鉴定以及RT-PCR技术,研究不同材料蔓枯病抗性表现以及抗蔓枯病基因(苯丙氨酸解氨酶基因,PAL)在不同材料及不同组织中的表达情况。结果显示:当接种蔓枯病菌孢子液浓度为5×109个/mL时,单基因抗源已开始出现感病现象,而聚合基因抗源仍表现为高抗或抗,其中145-471(PI420145×PI140471)抗性显著高于单基因抗源亲本和其它聚合抗源材料,表现为高抗(RI1.0)。抗蔓枯病基因PAL在不同抗性材料根、茎、叶中的表达均呈先上调而后下降并趋于稳定的变化趋势,但变化快慢和幅度均不同。研究表明,甜瓜抗蔓枯病基因的聚合能够提高其对蔓枯病的抗性,但不同抗病基因聚合后的抗性表现存在一定差异;抗蔓枯病基因PAL的表达与甜瓜蔓枯病抗性有密切关系,其表达时间与表达量差异可能是影响不同材料抗病能力差异的重要因素;该研究鉴定筛选的高抗蔓枯病材料145-471可用于甜瓜的抗蔓枯病聚合育种。     

红冬孢酵母苯丙氨酸解氨酶基因pal在大肠杆菌中的克隆与表达

以红冬孢酵母总RNA为模板反转录获得其苯丙氨酸解氨酶基因pal,测序与已公布蛋白序列进行比对,相似度为99％.并以含有T7强启动子的pET - 28a(+)为载体构建重组质粒pET - 28a(+)- pal,通过异丙基硫代-β-D-半乳糖苷(IPTG)诱导实现苯丙氨酸解氨酶(PAL)在大肠杆菌中的表达.对诱导条件进行初步优化后,重组茵PAL比酶活可达到42.99 U/g.转化实验中肉桂酸的转化率为48.52％,L-苯丙氨酸生成量为1.73 g/L.结果表明,红冬孢酵母pal基因通过表达载体pET - 28a(+)在E.coli中获得了高效表达.     

胚乳对幼苗中苯丙氨酸解氨酶(PAL)活性的影响

在小麦、大麦、玉米黄化幼苗的萌发过程中,PAL活性有“消长”现象。当去胚乳后,幼苗中PAL活性普遍下降。亚胺环己酮处理可以阻止去胚乳造成的PAL活性下降。大麦、小麦去胚乳老化12h幼苗的提取液可在体外抑制整体苗的PAL活性。表明胚乳在调节幼苗体内PAL活性方面具有一定的作用。去胚乳后体内有PAL“抑制蛋白”的积累。     

两个紧密连锁的小麦苯丙氨酸解氨酶基因的分离与鉴定

利用一个小麦苯丙氨酸解氨酶基因PCR片段为探针,从小麦核DNA基因库中筛选出一个阳性噬菌体克隆,该克隆含有两个高度同源、紧密连锁、转录方向相同的小麦苯丙氨酸解氨酶基因PAL1与PAL2,它们之间的核酸序列同源性。为93％,相距约7kb,利用PAL1特异片段进行Southern分析,表明该基因在小麦基因组中具有多个拷贝。Northern杂交表明,经秆锈菌接种诱导,苯丙氨酸解氨酶基因在一对小麦抗-感近等基因系中差异表达：抗病等基因系中国春-Sr11携带与接种菌无毒性基因P11相对应的抗病基因Sr11,在接种4d后开始诱导表达,8d后表达量更高;而缺少抗病基因的感病系中国春-sr11接种6d后才开始表达,8d后的表达量与抗病系中6d时相当。用秆锈菌诱导物和几丁质寡聚物处理小麦悬浮细胞,均可在2h内激活苯丙氨酸解氨酶基因表达,但真菌诱导物在早期的诱导活性显著高于几丁质寡聚物。从转录水平证实了小麦苯丙氨酸解氨酶基因在秆锈菌诱导的抗性反应中具有重要作用。     

Revealing the genetic architecture of FHB resistance in hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) by QTL meta-analysis

Fusarium head blight (FHB) in wheat results in reduced yield and quality and in accumulation of mycotoxins. The objective of this study was to identify genomic regions in wheat involved in the control of FHB resistance applying a QTL meta-analysis approach by combining QTL of 30 mapping populations to propose independent meta-QTL (MQTL). A consensus map was created on which initial QTL were projected. Nineteen MQTL comprising 2–13 initial QTL with widely varying confidence intervals were found on 12 chromosomes. Some of them coincided with genomic regions previously identified (e.g. chromosomes 3BS, 6B), however, some MQTL were newly detected by this study. Separate analysis of populations with the same resistant parent showed a rather high consistency for the Chinese spring wheat donor ‘Sumai 3’, but little consistency for the Chinese donor ‘Wangshuibai’ and the Swiss donor ‘Arina’. According to our results breeders can in future (1) choose parents for crossing not comprising the same resistance loci or QTL intervals, (2) exploit new MQTL, and (3) select markers of some of these MQTL to be used in marker-assisted selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dissecting of the FHB resistance QTL on the short arm of wheat chromosome 2D using a comparative genomic approach: from QTL to candidate gene

Colinearity in gene content and order between rice and closely related cereal crops has been a powerful tool for gene identification. Using a comparative genomic approach, we have identified the rice genomic region syntenous to the region of the short arm of wheat chromosome 2D, on which quantitative trait loci (QTLs) for Fusarium head blight (FHB) resistance and for controlling accumulation of the mycotoxin deoxynivalenol (DON) are closely located. Utilizing markers known to reside near the FHB resistance QTL and data from several wheat genetic maps, we have limited the syntenous region to 6.8 Mb of the short arm of rice chromosome 4. From the 6.8-Mb sequence of rice chromosome 4, we found three putative rice genes that could have a role in detoxification of mycotoxins. DNA sequences of these putative rice genes were used in BLAST searches to identify wheat expressed sequence tags (ESTs) exhibiting significant similarity. Combined data from expression analysis and gene mapping of wheat homologues and results of analysis of DON accumulation using doubled haploid populations revealed that a putative gene for multidrug resistance-associated protein (MRP) is a possible candidate for the FHB resistance and/or DON accumulation controlling QTLs on wheat chromosome 2DS and can be used as a molecular marker to eliminate the susceptible allele when the Chinese wheat variety Sumai 3 is used as a resistance source. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

小麦抗赤霉病品种NBS同源序列克隆与分析

根据二穗短柄草NBS-LRR类基因的保守序列设计同源引物,以小麦抗赤霉病品种苏麦3号、宁7840和望水白基因组DNA为模板,通过PCR扩增,得到43条序列,其中4条为非编码序列或结构域不完整;39条与植物抗病基因同源,其中的7条内部存在终止密码子,可能是假基因,经过比对分析,其余32条具有连续的开放阅读框和保守结构域,推导的氨基酸序列均具有Kinase-1a、Kinase-2和Kinase-3a及GLPL区等几个保守区,在GenBank中均能找到与之高度同源的其他物种的核酸序列,并且Kinase-2的最后一个氨基酸均为色氨酸(W),属于non-TIR类NBS基因。32条序列可分为4大类,它们之间核苷酸同源性为64%-98%,编码氨基酸同源性为22%-98%。根据序列分析随机设计5对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,7-1、s-3、s-4和w-2均能表达,说明这些片段可能是功能性抗病基因的部分序列;7-13不表达,再次证明属于假基因。32条序列在之前未被报道过,这些RGA可以作为筛选赤霉病功能性抗病基因的候选序列。     

小麦抗赤霉病研究现状与展望

小麦是我国最重要的口粮作物之一。在小麦生产所面临的各种病害中,赤霉病的发生具有愈来愈严重的趋势,引起小麦产业界的高度关注。近几十年来,科研人员在小麦抗赤霉病遗传育种以及防控技术领域进行了持续不懈的努力,在赤霉病病原菌致病基因、小麦赤霉病抗性基因定位、克隆及功能研究以及抗赤霉病分子育种等方面取得了重大进展。本文主要从赤霉病抗性基因资源的发掘和鉴定、不同抗源遗传基础解析、小麦赤霉病抗性基因、抗赤霉病分子标记辅助选择育种与基因聚合以及小麦抗赤霉病基因的克隆和功能研究等方面进行了综述,分析了目前小麦抗赤霉病研究中存在的问题,并提出应加强基因克隆、功能分子标记开发以及应用单体型辅助选择(HAS)和标记组辅助选择(MSAS)等小麦抗赤霉病研究的相关建议。     

Identification of a UDP-glucosyltransferase conferring deoxynivalenol resistance in Aegilops tauschii and wheat

Aegilops tauschii is the diploid progenitor of the wheat D subgenome and a valuable resource for wheat breeding, yet, genetic analysis of resistance against Fusarium head blight (FHB) and the major Fusarium mycotoxin deoxynivalenol (DON) is lacking. We treated a panel of 147 Ae. tauschii accessions with either Fusarium graminearum spores or DON solution and recorded the associated disease spread or toxin-induced bleaching. A k-mer-based association mapping pipeline dissected the genetic basis of resistance and identified candidate genes. After DON infiltration nine accessions revealed severe bleaching symptoms concomitant with lower conversion rates of DON into the non-toxic DON-3-O-glucoside. We identified the gene AET5Gv20385300 on chromosome 5D encoding a uridine diphosphate (UDP)-glucosyltransferase (UGT) as the causal variant and the mutant allele resulting in a truncated protein was only found in the nine susceptible accessions. This UGT is also polymorphic in hexaploid wheat and when expressed in Saccharomyces cerevisiae only the full-length gene conferred resistance against DON. Analysing the D subgenome helped to elucidate the genetic control of FHB resistance and identified a UGT involved in DON detoxification in Ae. tauschii and hexaploid wheat. This resistance mechanism is highly conserved since the UGT is orthologous to the barley UGT HvUGT13248 indicating descent from a common ancestor of wheat and barley.     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子。利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因。该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74％的同源性。在多肽链的N-端具有在真核生物中高度保守的UFD1结构域。我们将该基因定位在小麦的第六染色体群并将其命名为了UFD1。Southern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的。无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源。除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域。TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达。     

Minor Differences in Anther Extrusion Affect Resistance to Fusarium Head Blight in Wheat

Fusarium head blight (FHB) remains a serious problem due to yield loss and mycotoxin accumulation in wheat production worldwide. We previously reported that the closed‐flowering (no anther extrusion) characteristic was effective for increasing resistance to FHB infection. In this study, we investigated the relationships between the degree of anther extrusion (AE) and FHB damage using double haploid lines (DHLs), derived from F₁ plants from crosses between closed‐flowering and opened‐flowering varieties. These DHLs exhibited various degrees of AE, and the degree of AE was significantly different among DHLs, regardless of the year and environment (pot‐ or field‐grown). FHB severity was the lowest in closed‐flowering DHLs, and DHLs with partially extruded anthers showed significantly higher FHB symptoms than those with closed‐flowering phenotypes. In general, DHLs with partially extruded anthers also had relatively severe FHB symptoms compared with those exhibiting full anther extrusion. FHB severity was significantly correlated with Fusarium‐damaged kernels and deoxynivalenol concentration. The results of this study showed that partially extruded anthers were considered to be a source of FHB infection. The closed‐flowering phenotype improved resistance to FHB infection. Meanwhile, phenotypes with rapid anther extrusion and ejection also could contribute to the avoidance of FHB infection.