芜菁UF3GT基因的克隆及表达特性

目的:克隆‘津田’芜菁和‘赤丸’芜菁的UDP-葡萄糖∶类黄酮3-O-葡萄糖基转移酶(UF3GT)基因并研究其表达特性。方法:利用RT-PCR方法克隆‘津田’芜菁BrUF3GT1基因和‘赤丸’芜菁BrUF3GT2基因,并进行生物信息学分析;通过Northern杂交检测BrUF3GT1和BrUF3GT2基因的UV-A诱导表达特性;对BrUF3GT1和BrUF3GT2基因进行原核诱导表达。结果:BrUF3GT1和BrUF3GT2的开放读框为1407 bp,编码468个氨基酸残基;氨基酸序列分析显示,BrUF3GT1和BrUF3GT2与拟南芥UF3GT的同源性为87%,从第16~453位氨基酸残基的肽段具有糖基转移酶家族成员的结构域;BrUF3GT1和BrUF3GT2基因具有高度同源性,核苷酸序列在7个位点存在差异,推导的氨基酸序列在1个位点存在差异;Northern杂交结果显示,UV-A可以诱导BrUF3GT1和BrUF3GT2基因表达,基因的表达量与处理时间相关;原核诱导表达及纯化后可以获得相对分子质量分别约为51.88×103和51.89×103的BrUF3GT1和BrUF3GT2蛋白。结论:克隆了‘津田’芜菁和‘赤丸’芜菁的UF3GT基因,为初步阐明2种芜菁的花青素生物合成机理奠定了实验基础。     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

芜菁花青素合成酶基因的克隆、序列分析及表达

目的：克隆津田芜菁和赤丸芜菁花青素合成酶（ANS）基因并研究其表达特性。方法：用UV-A处理津田芜菁和赤丸芜菁块根24h后提取总RNA,通过RT-PCR方法克隆BrANS1和BrANS2基因并进行序列分析,通过Northern杂交检测BrANS1和BrANS2基因的表达。结果：BrANS1和BrANS2的开放读码框为1077bp,编码358个氨基酸残基;BRANS1和BRANS2与甘蓝ANS的同源性达97％,第211-307肽段具有20G-Fe（Ⅱ）加氧酶家族基因的结构域;BrANS1和BRANS2基因具有高度同源性,核苷酸序列在5个位置上存在差异,推导的氨基酸序列完全相同;uv-A可以诱导BrANS1和BrANS2表达,基因的表达量与处理时间相关。结论：克隆了津田芜菁和赤丸芜菁的BRANS1和BrANS2基因,这将为筛选依光型和非依光型花青素生物合成催化酶基因奠定研究基础。     

抱茎独行菜种皮粘液质相关基因TTG1的克隆、表达分析及功能鉴定

抱茎独行菜（Lepidium perfoliatum L.）为十字花科具典型粘液质繁殖体植物,而TTG1基因（Transpa-rent testa glabra 1）所编码的蛋白是调控种皮细胞分化并影响粘液质释放的转录因子。目前关于TTG1基因在粘液质繁殖体植物中的研究报道较少,为探究TTG1基因在抱茎独行菜粘液质发育中的作用,本研究利用同源克隆技术获得抱茎独行菜TTG1基因cDNA开放阅读框（ORF）序列,命名为LpTTG1。序列分析表明,该基因ORF全长为1032 bp,编码343个氨基酸,含有WD40基序;qRT-PCR分析结果显示,该基因在抱茎独行菜各组织中均有表达,反映了该基因功能的多样性;免疫组织化学定位结果表明,LpTTG1在种子发育过程中内珠被和外珠被的表达水平变化与外珠被粘液质的合成过程相一致,推测该基因可能参与调控抱茎独行菜种皮的发育及粘液质的形成。将LpTTG1基因转化拟南芥,该基因的过量表达显著促进了粘液质合成途径下游基因AtMUM4在角果中的表达,表明该基因有可能参与粘液质合成途径调控,并促进下游产物MUM4的产生。然而,对LpTTG1转基因拟南芥与野生型植株表型的比较发现,两者种子形态及粘液质分泌与释放方式均无显著差异,这可能是因为抱茎独行菜种皮发育和粘液质形成是一个多基因调控的复杂过程,某一基因的过量表达也许不会引起明显的表型变化。     

光皮桦ACC氧化酶基因BlACO的克隆和表达分析

ACC（1-aminocyclopropane-1-carboxylic acid）氧化酶（ACO）是植物乙烯合成过程中的关键限速酶,对乙烯的合成具有重要的调控作用。以光皮桦茎叶组织提取的RNA为模板,据已报道的ACO同源序列设计简并引物,通过RT-PCR扩增获得部分基因片段,结合5＇,3＇RACE方法从光皮桦中扩增出1个ACO的全长cDNA序列。该基因cDNA全长1262bp,具有一个957bp的完整开放阅读框架,编码含318个氨基酸的蛋白。与其他植物中的ACO基因进行同源性比对的结果显示,BlACO蛋白与欧洲白桦的同源性最高,达到97%。该基因在光皮桦的雄花和雌花中表达量较高,而在茎中的表达量较低。     

津田芜菁和赤丸芜菁查尔酮异构酶基因的克隆及表达特性

目的：克隆津田芜菁和赤丸芜菁查尔酮异构酶（CHI）基因并研究其表达特性。方法：利用UV-A处理2种芜菁未见光块根24h,提取总RNA后通过RT-PCR方法克隆津田芜菁和赤丸芜菁的BrCH11和BrCH12基因,通过Northern杂交检测BrCH11和BrCH12基因的UV-A诱导表达特性。结果：BrCH11和BrCH12的开放读码框为756bp,编码251个氨基酸残基;氨基酸序列分析显示,BrCH11和BrCH12与萝卜CHI的同源性达91％,第11-222的肽段具有CHI结构域;BrCH11和BrCH12,2的核苷酸序列和推导的氨基酸序列分别在3个位点存在差异;BrCH11和BrCH12基因具有高度同源性;BrCH11和BrCH12基因的表达量与UV-A处理时间相关。结论：克隆了津田芜菁和赤丸芜菁的BrCH11和BrCH12基因,这2个基因的表达受UV-A诱导。     

巴西橡胶树（Hevea brasiliensis）微管相关蛋白全长cDNA克隆及表达分析

从巴西橡胶树Hevea brasiliensis差减cDNA文库中分离到微管相关蛋白（Microtubule-associated protein,MAPs）基因片段,根据该基因片段序列信息,设计特异引物,采用cDNA末端快速扩增技术RACE（Rapid Amplification ofcDNA Ends）进行差异片段的5＇和3＇端的扩增,获得了长度为788bp的全长cDNA,该基因在GenBank中的登录号为AY461412.序列分析表明该基因包含完整的开放阅读框,编码144个氨基酸,与微管相关蛋白基因家族具有很高的同源性,推测该基因是微管相关蛋白基因.半定量RT-PCR检测证实它在胶乳中的表达强于叶中,胁迫处理（伤害及乙烯处理）使其表达上调.     

烟草磷转运蛋白基因NtPHT2;1的克隆和表达分析

植物对磷酸盐的吸收与利用主要依靠磷转运蛋白,其中PHT2家族编码的低亲和磷转运蛋白主要负责植物在正常供磷条件下磷酸盐的吸收、转运与再利用。为了探究低亲和磷转运蛋白基因NtPHT2;1在烟草转运磷酸盐中的作用和表达模式,本研究以普通烟草K326的cDNA为模板,克隆得到NtPHT2;1,对该基因进行生物信息学分析和蛋白质的亚细胞定位,并通过荧光定量PCR技术对该基因在低磷等非生物胁迫下的基因表达模式进行分析。结果表明：（1）NtPHT2;1基因的全长为1 764 bp,编码587个氨基酸。（2）亚细胞定位结果表明,NtPHT2;1蛋白定位于叶绿体上。（3）同源性比对发现,NtPHT2;1蛋白与辣椒CaPHT2;1蛋白的同源性最高达到91.00%。（4）启动子分析表明,NtPHT2;1启动子含有参与调控植物衰老、逆境胁迫相关的顺式作用元件。（5）组织表达模式分析表明,NtPHT2;1在叶片中的表达量最高,新叶中的表达量比老叶中的高;在低磷诱导条件下,该基因的表达量与正常条件相比差异不显著。（6）不同非生物胁迫下的表达模式表明,在盐胁迫和干旱胁迫下,该基因的表达量显著降低。研究认为,NtPHT2;1基因主要是负责烟株正常生长发育条件下磷酸盐的转运与利用。     

菘蓝CYP83B1基因的克隆与表达分析

对菘蓝（Isatis indigotica Fort.）CYP83B1基因进行了克隆与表达模式分析。结果显示,IiCYP83B1基因全长为1652 bp,包含2个外显子和1个内含子;cDNA全长为1500 bp,编码499个氨基酸。IiCYP83B1编码的蛋白没有跨膜结构域和信号肽,主要定位于内质网膜,属于亲水性蛋白,二级结构主要由无规则卷曲螺旋和α-螺旋组成,与萝卜（Raphanus sativus Linn.）、欧洲油菜（Brassica napus L.）、甘蓝（Brassica oleracea L.）和芜菁（Brassica rapa L.）等植物的CYP83B1蛋白具有较高的同源性。qRT-PCR分析结果表明,IiCYP83B1基因在菘蓝的根、茎、叶、花和果中均有表达,且以叶中的表达量最高;在幼苗期、生长期和花期稳定表达且均显著高于萌芽期;茉莉酸甲酯（methyl jasmonate,MeJA）和葡萄糖（glucose,Glu）能够显著促进该基因的表达,而低温（4℃）和水杨酸（salicylic acid,SA）处理对其表达具有一定的抑制效应。本研究结果可为进一步探讨IiCYP83B1基因的功能提供参考。     

陆地棉纤维优势表达基因GhRACK1的克隆与序列分析

纤维品质改良是我国棉花育种的主要目标之一,纤维特异或优势表达基因的挖掘是利用基因工程手段改良纤维品质的关键。根据苏棉12纤维中优势表达的GhRACK1 EST序列设计引物,通过RACE技术克隆了GhRACK1基因的全长cDNA。推导的氨基酸序列含有4个串联的WD基序,属于WD40重复家族,与已知的RACK1蛋白同源性达70％以上,PDB模拟的蛋白三维结构也与已知的RACK1蛋白结构相似。荧光定量PCR分析表明GhRACK1在纤维中的表达量比叶片中高20倍以上。研究结果为棉花纤维品质改良基因工程提供了新的基因资源。     

Transparent Testa Glabra 1 (TTG1) and TTG1-like genes in Matthiola incana R. Br. and related Brassicaceae and mutation in the WD-40 motif

TTG1 (Transparent Testa Glabra 1), a WD-40 repeat protein, is involved in regulation of flavonoid/anthocyanin biosynthesis, seed coat (mucilage) development/pigmentation and trichome formation in leaves. Here, we characterized the TTG1 gene of Matthiola incana wild type ( e locus), showing 85.3% similarity to TTG1 of A. thaliana on the nucleotide level and 96.2% on the protein level. A white-flowered and glabrous mutant, line 17, of M. incana exhibits one nucleotide change, leading to an amino acid substitution directly in the WD motif (W158R). Correspondingly, the DFR (dihydroflavonol 4-reductase) gene, in which the expression is known to be dependent on TTG1, is not expressed in Matthiola mutant lines 17 (and 19). Comparison of the GC content of the Matthiola TTG1 (54.1%) and Arabidopsis TTG1 (46.1%) genes revealed a strong difference, mostly obtained by neutral substitutions (C to T transitions). To examine whether this is an ecologically influenced trend, a fragment of TTG1 was characterized from another Matthiola species ( M. tricuspidata ) and from Malcolmia flexuosa subsp. naxensis from the eastern Mediterranean, near a beach with sandy and salty soils. Both Matthiola species have a higher GC content in the TTG1 gene than Arabidopsis and the closer-related Malcolmia , indicating that the GC content is rather an evolutionary than an ecological signal. A similar WD-40 repeat protein gene (containing no intron in the 3' untranslated region) with high similarity to the Arabidopsis TTG1 -like ( AtAN11 ) gene was found in Matthiola .     

The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.     

Mutations in the pale aleurone color1 regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1 gene in Arabidopsis thaliana

The pale aleurone color1 (pac1) locus, required for anthocyanin pigment in the aleurone and scutellum of the Zea mays (maize) seed, was cloned using Mutator transposon tagging. pac1 encodes a WD40 repeat protein closely related to anthocyanin regulatory proteins ANTHOCYANIN11 (AN11) (Petunia hybrida [petunia]) and TRANSPARENT TESTA GLABRA1 (TTG1) (Arabidopsis thaliana). Introduction of a 35S-Pac1 transgene into A. thaliana complemented multiple ttg1 mutant phenotypes, including ones nonexistent in Z. mays. Hybridization of Z. mays genomic BAC clones with the pac1 sequence identified an additional related gene, mp1. PAC1 and MP1 deduced protein sequences were used as queries to build a phylogenetic tree of homologous WD40 repeat proteins, revealing an ancestral gene duplication leading to two clades in plants, the PAC1 clade and the MP1 clade. Subsequent duplications within each clade have led to additional WD40 repeat proteins in particular species, with all mutants defective in anthocyanin expression contained in the PAC1 clade. Substantial differences in pac1, an11, and ttg1 mutant phenotypes suggest the evolutionary divergence of regulatory mechanisms for several traits that cannot be ascribed solely to divergence of the dicot and monocot protein sequences.     

Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis

We use Arabidopsis thaliana as a model to investigate coordination of cell proliferation and cell elongation in the three components that develop side by side in the seed. Two of these, the embryo and its nurturing annex, the endosperm, are placed under zygotic control and develop within the seed integument placed under maternal control. We show that integument cell proliferation and endosperm growth are largely independent from each other. By contrast, prevention of cell elongation in the integument by the mutation transparent testa glabra2 (ttg2) restricts endosperm and seed growth. Conversely, endosperm growth controlled by the HAIKU (IKU) genetic pathway modulates integument cell elongation. Combinations of TTG2 defective seed integument with reduction of endosperm size by iku mutations identify integument cell elongation and endosperm growth as the primary regulators of seed size. Our results strongly suggest that a cross talk between maternal and zygotic controls represents the primary regulator of the coordinated control of seed size in Arabidopsis.