油橄榄AP2/ERF转录因子鉴定及水胁迫表达分析

为探究油橄榄AP2/ERF基因家族对水胁迫的响应机制,该研究对受干旱及水淹胁迫的‘佛奥’和‘TYZ-1号’2个品种的根和叶进行转录组测序,并对油橄榄中AP2/ERF转录因子的蛋白理化性质、基因结构及系统进化进行分析,同时分析与水胁迫相关的AP2/ERF转录因子在2个油橄榄品种中的基因表达差异且进行RT-qPCR验证。结果表明：(1)在油橄榄中鉴定得到110个AP2/ERF基因家族成员,该110个蛋白质所含氨基酸大小为173～717 bp,均不存在信号肽,为非分泌蛋白。将油橄榄AP2/ERF与模式植物拟南芥AP2/ERF蛋白构建进化树发现,油橄榄AP2/ERF蛋白分为AP2、RAV、ERF和Solosist 4大类,其中ERF分为ERF和DREB 2个亚类,ERF包含ERF B1～ERF B6 6个子亚类,DREB包含DREB A1～DREB A6 6个子亚类,这与模式植物拟南芥AP2/ERF的分类一致,每个亚家族同时包含了油橄榄和拟南芥AP2/ERF蛋白,说明拟南芥和油橄榄的AP2/ERF家族在进化水平上有一定的相似性。(2)油橄榄AP2/ERF同一亚家族蛋白具有相同的基因结构及保守元...     

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。     

The glycoflavonoid pigments of wheat, Triticum aestivum, leaves

The C-glycosylflavone pigments found in wheat leaves are a varied mixture of compounds in the luteolin and apigenin series; iso-orientin, lutonarin, lucenin-1, lucenin-3, vicenin-2, and an unreported C-glycoside, wyomin, an iso-orientin derivative with rutinose at the 7-position. In addition, a 4′-O-glucosyl derivative of iso-swertisin was identified along with the previously reported flavone, tricin.     

Comparison of genetic and physical maps of group 7 chromosomes from Triticum aestivum L.

We present a high density physical map of homoeologous group 7 chromosomes from Triticum aestivum L. using a series of 54 deletion lines, 6 random amplified polymorphic DNA (RAPD) markers and 91 cDNA or genomic DNA clones from wheat, barley and oat. So far, 51 chromosome segments have been distinguished by molecular markers, and 54 homoeoloci have been allocated among chromosomes 7A, 7B and 7D. The linear order of molecular markers along the chromosomes is almost identical in the A- B- and D-genome of wheat. In addition, there is colinearity between the physical and genetic maps of chromosomes 7A, 7B and 7D from T. aestivum, indicating gene synteny among the Triticeae. However, comparison of the physical map of chromosome 7D from T. aestivum with the genetic map from Triticum tauschii some markers have been shown to be physically allocated with distortion in more distal chromosome regions. The integration of genetic and physical maps could assist in estimating the frequency and distribution of recombination in defined regions along the chromosome. Physical distance did not correlate with genetic distance. A dense map facilitates the detection of multiple rearrangements. We present the first evidence for an interstitial inversion either on chromosome arm 7AS or 7DS of Chinese Spring. Molecularly tagged chromosome regions (MTCRs) provide landmarks for long-range mapping of DNA fragments.     

小麦TaCO9基因的克隆及功能分析

为了明确TaCO9-1A基因在小麦生长发育中的具体调控机理,该研究以同源克隆的方法成功获得了大麦(Hordeum vulgare)光周期基因HvCO9在小麦(Triticum aestivum)中的直系同源基因TaCO9,并对其进行生物信息学分析、亚细胞定位、转录激活以及表达模式分析;利用农杆菌侵染法转化拟南芥,并对过表达株系进行表型分析。结果表明:(1)TaCO9包含2个外显子和1个内含子,CDS区全长876 bp,编码291个氨基酸,蛋白序列含有特有的CCT结构域,且在不同物种间高度保守。(2)生物信息学分析表明,TaCO9基因编码的蛋白质分子量约为30.7 kD,等电点为6.24;TaCO9的启动子区含有光响应、激素应答和胁迫应答等多种顺式作用元件。(3)亚细胞定位和转录活性分析表明,TaCO9主要定位于细胞核中,且具有转录激活活性。(4)qRT-PCR结果表明,TaCO9基因在各个组织中均有表达,叶片中的表达量最高;在光照14 h条件下TaCO9的表达量显著高于光照10 h和12 h条件下的表达量,且TaCO9在大粒小麦‘西农817’子房与籽粒中的表达量显著高于‘中国春’。(5)经潮霉素筛选成功获得3个转基因拟南芥株系;进一步功能鉴定结果表明,过表达转TaCO9基因拟南芥植株的开花期迟于野生型(对照)3~4 d,但角果和籽粒较野生型大。     

Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

Background  

Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened.     

鹅掌楸DHHC型锌指蛋白家族基因的克隆及表达分析

棕榈酰化修饰是一种最普遍且唯一可逆的翻译后脂质修饰方式,赋予蛋白质多样化的生理功能。DHHC( Asp-His-His-Cys)蛋白家族是一类与棕榈酰化修饰相关的蛋白,多数DHHC蛋白家族成员具有蛋白质酰基转移酶( protein S-acyltransferase,PAT)活性。该研究以鹅掌楸叶芽为材料,采用RT-PCR和RACE技术,克隆获得了3个鹅掌楸DHHC蛋白家族基因cDNA全长,命名为LcPAT7、LcPAT22、LcPAT23。序列分析结果表明：LcPAT7、LcPAT22、LcPAT23基因全长分别为1933、2592、2217 bp,各包含1332、1839、1662 bp的开放阅读框( Open Reading Frame,ORF),编码433、612、533个氨基酸,预测蛋白分子量分别为40.04、67.3、60.57 kDa,理论等电点为9.15、9.03、7.29。3个基因编码的蛋白均有4个跨膜区,并且都在跨膜域( transmembrane domain, TM) TM2和 TM3之间存在一个 DHHC 蛋白家族典型的 DHHC-CRD 结构域。同源性分析表明：鹅掌楸LcPAT7、LcPAT22、LcPAT23编码的氨基酸序列与其他植物中预测的PAT具有较高的相似性。利用荧光定量PCR技术检测3个基因在鹅掌楸不同组织中的表达特性,发现3个基因在不同组织中均有表达,但表达量具有明显区别。同一家族基因表达模式的变化表明其功能非冗余。该研究结果将为鹅掌楸生长发育与形态建成,以及逆境响应信号传导等相关基因的调控研究提供了参考。     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

甘蓝型油菜中一类AP2/ERF转录因子的克隆和生物信息学分析

AP2/ERF转录因子家族是植物中广泛存在的一类转录因子,AP2/ERF这类转录因子主要参与植物的细胞周期、生长发育以及生物和非生物胁迫相关基因的表达调控。由于拟南芥和油菜同属于芸薹属,具有相似的基因信息,利用油菜UniGene数据库,以拟南芥ERF转录因子保守序列为探针,通过电子克隆从一个UniGene Cluster中分离得到三个同类的AP2/ERF转录因子,从cDNA序列、氨基酸序列的相似性、组成成分、理化性质、疏水性/亲水性分析、序列比对、进化树、功能域、二级结构、三级结构、无序化特性进行了预测和较为全面的分析。结果显示油菜来源的BnaERF1、BnaERF2和BnaERF3属于AP2/ERF转录因子的B-2亚族,是亲水性蛋白,在蛋白质的三级结构上与AtERF1相似。蛋白质无序化分析发现,油菜BnaERF1、BnaERF2和BnaERF3无序化程度小于拟南芥AtERF1。设计引物通过PCR和RT-PCR方法分别从甘蓝型双低油菜沪油15幼苗的DNA和cDNA中扩增了上述基因,初步分析BnaERF2没有内含子,BnaERF1和BnaERF3有内含子。另外,通过EST丰度分析显示,该类转录因子的表达最高峰在种子中,其次为花,在芽、茎和分生组织中没有检测出表达的存在。     

Plant regeneration from intergeneric somatic hybridization between Triticum aestivum L. and Leymus chinensis (Trin.) Tzvel

The suspension derived protoplasts of wheat (Triticum aestivum) cv. Jinan 177 were used as a recipient to fuse with the protoplasts of the ⁶⁰Co gamma-ray irradiated calli of Legmus chinensis. The wheat suspension cells and their protoplasts were not capable of differentiating to whole plants. The irradiated calli of L. chinensis were also the same. The protoplasts originated from the treated or untreated calli were both unable to divide under the conditions of this experiment. However, the fusion products grew and developed to whole plants which were identified as hybrids according to the analysis of chromosome, isozyme and morphology. The above result revealed that the lost regeneration capacity of both parents could be complementarily restored through somatic hybridization. This phenomenon also occurred with our work on Triticum aestivum (+) Haynaldia villosa, T. aestivum (+) Agropyron elongatum and T. aestivum (+) Psathyrostachys juncea.