Dehiscence-related expression of an Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus

Pod dehiscence in Arabidopsis thaliana is accompanied by an increase in the expression of a polygalacturonase (PG). The gene encoding this mRNA has been characterized and shown to have extensive homology to a similar PG gene from Brassica napus . The A. thaliana PG promoter was fused to β -glucuronidase (GUS) and the expression of this reporter gene analysed in transgenic B. napus plants. The GUS activity was detected throughout the dehiscence zone of pods from 35 d after anthesis and expression was restricted to those cells that undergo cell separation. Expression was also detectable at the junction between the seed and the funicular tissue and in mature anthers of flowers. Transgenic plants containing the PG promoter fused to barnase were sterile as a consequence of the anthers failing to undergo dehiscence. Fertilization of PG-barnase plants resulted in the development of pods that exhibited a reduced capacity to shatter. The role of PG during cell separation processes in plants is discussed.     

人工合成甘蓝型油菜特长角变异系的选育

用中国西藏东部采集的1个白菜型油菜地方品种与云南甘蓝类蔬菜白花芥蓝远缘杂交,人工合成了甘蓝型油菜新材料,从杂种后代中通过系统选育,获得了1个特长角变异系,其主花序中部角果平均长度20cm左右,果身长16～18cm,果喙长3cm左右.从中获得的极端最长角果达31.5cm,果身长26.1cm,果喙长5.4cm.这是迄今芸苔属植物中很少见到的长角果油菜材料.该材料的平均角果长度大约为普通甘蓝型油菜的3倍左右,遗传已基本稳定,定名为川农特长角.本文报导其选育经过和主要特征特性,并对其育种和研究利用价值作了简要分析.     

人工合成甘蓝型油菜特长角性状的遗传分析

以人工合成甘蓝型油菜特长角品系H218普通甘蓝型油菜中油821和MSP334为材料, 配制成两个杂交组合的各6个世代群体, 即P1、P2、F1、F2、B1、B2, 研究了全果长、果身长、果喙长3个角果性状的遗传特点。结果表明, 两个组合的全果长、果身长、果喙长在F2群体中近乎呈现连续正态分布, 显示为多基因控制的数量性状。两个组合的全果长广义遗传力为65.89%~70.77%、狭义遗传力为44.01%~46.78%, 基因数目均为5对; 果身长广义遗传力为60.14% ~ 63.38%、狭义遗传力为46.89%~47.38%, 基因数目均为6对; 果喙长广义遗传力为26.36% ~ 46.44%, 狭义遗传力为18.08%~37.87%, 基因数目均为2对。基因效应初步分析表明, 两个组合的3个性状基因效应均符合加性-显性二基因互作模型, 存在显著的上位性效应。两个组合的全果长、果身长、果喙长加性效应、显性效应都很重要, 全果长、果身长的加性效应大于显性效应, 果喙长显性效应大于加性效应。     

白菜和白菜型油菜角果相关性状遗传分析

以种荚差异显著的白菜自交不亲和系（P1）和白菜型油菜自交系（P2）为亲本及杂交获得的4个基本世代（P1、P2、F1、F2）为材料,应用植物数量性状主基因+多基因混合遗传模型对其种荚相关性状进行遗传分析。结果表明,芸薹种作物的种荚长度(SPL)性状及喙长（SBL）性状均受加性-显性-上位性多基因控制（C-0模型）,多基因遗传率分别为83.16%和68.67%;种荚宽度(SPW)性状受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制（E-0模型）,其主基因遗传率为20.40%,多基因遗传率为78.34%。种荚相关各性状均以多基因遗传为主,种荚宽度性状受环境因素影响较小,为1.26%;种荚长度、喙长受环境因素影响较大,分别达16.89%和25.36%。针对芸薹种作物种荚性状的改良要以多基因为主,并注意环境条件影响。以种荚差异显著的白菜自交不亲和系（P1）和白菜型油菜自交系（P2）为亲本及杂交获得的4个基本世代（P1、P2、F1、F2）为材料,应用植物数量性状主基因+多基因混合遗传模型对其种荚相关性状进行遗传分析。结果表明,芸薹种作物的种荚长度(SPL)性状及喙长（SBL）性状均受加性-显性-上位性多基因控制（C-0模型）,多基因遗传率分别为83.16%和68.67%;种荚宽度(SPW)性状受2对加性-显性-上位性主基因+加性-显性-上位性多基因控制（E-0模型）,其主基因遗传率为20.40%,多基因遗传率为78.34%。种荚相关各性状均以多基因遗传为主,种荚宽度性状受环境因素影响较小,为1.26%;种荚长度、喙长受环境因素影响较大,分别达16.89%和25.36%。针对芸薹种作物种荚性状的改良要以多基因为主,并注意环境条件影响。     

Conserved structure and function of the Arabidopsis flowering time gene CONSTANS in Brassica napus

The Arabidopsis thaliana CONSTANS (CO) gene which promotes flowering in long days was recently isolated by chromosome walking. The mapping of QTLs controlling flowering time in Brassica species has identified genomic regions that contain homologues of the CO gene. Four genes homologous to the Arabidopsis CO gene were isolated from a pair of homoeologous loci in each of two doubled-haploid Brassica napus lines displaying different flowering times, N-o-1 and N-o-9. The four genes, BnCOa1, BnCOa9, BnCOb1 and BnCOb9, are located on linkage groups N10 and N19, and are highly similar to each other and to the Arabidopsis CO gene. Two regions of the proteins are particularly well conserved, a N-terminal region with two putative zinc fingers and a C-terminal region which may contain a nuclear localization signal. All four genes appear to be expressed in B. napus. The BnCOa1 allele was shown to complement the co-2 mutation in Arabidopsis in a dosage-dependent manner causing earlier flowering than in wild type under both long- and short-day conditions.     

Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers

To gain new insights into the mechanism underlying cytoplasmic male sterility (CMS), we compared the nuclear gene expression profiles of flowers of a Brassica napus CMS line with that of the fertile B. napus maintainer line using Arabidopsis thaliana flower-specific cDNA microarrays. The CMS line used has a B. napus nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of both B. napus and A. thaliana DNA. Gene expression profiling revealed that a large number of genes differed in expression between the two lines. For example, nuclear genes coding for proteins that are involved in protein import into organelles, genes expressed in stamens and pollen, as well as genes implicated in either cell-wall remodeling or architecture, were repressed in the CMS line compared with B. napus. These results show that the mt genome of the CMS line strongly influences nuclear gene expression, and thus reveal the importance of retrograde signalling between the mitochondria and the nucleus. Furthermore, flowers of the CMS line are characterized by a replacement of stamens with carpelloid organs, and thus partially resemble the APETALA3 (AP3) and PISTILLATA (PI) mutants. In accordance with this phenotype, AP3 expression was downregulated in the stamens, shortly before these organs developed carpelloid characteristics, even though it was initiated correctly. Repression of PI succeeded that of AP3 and might be a consequence of a loss of AP3 activity. These results suggest that AP3 expression in stamens depends on proper mt function and a correct nuclear-mt interaction, and that mt alterations cause the male sterility phenotype of the CMS line.     

拟南芥FRUITFULL(FUL)基因的表达调控模式

FRUITFULL(FUL)基因是一类MADS box基因,在控制开花时间、花分生组织分化、茎生叶形态以及心皮和果实的发育中发挥重要作用。为了阐明FUL的表达调控模式,克隆了拟南芥Arabidopsis thaliana FUL启动子区(-2148bp~+96bp)及其第一内含子,并构建一系列启动子分段缺失表达载体及含FUL第一内含子的融合载体。并进一步构建了各顺式作用元件融合拟南芥TUBULIN和ACTIN启动子的表达载体。转基因拟南芥分析结果表明,FUL启动子的上游存在2个抑制其表达的顺式作用元件,其中一个很可能与转录因子AP1的结合有关;2个存在于上游调控区的CArG-box对FUL基因表达起到重要的调控作用;FUL基因第一内含子参与拟南芥心皮和雄蕊的发育调控,而且有增强基因表达的作用。     

Two Homologs of the FLOWERING LOCUS C Gene from Leaf Mustard (Brassica juncea)

Using the direct amplification of genomic DNA from two cultivars of leaf mustard (Brassica juncea), we obtained two homologs of the MADS-box gene FLOWERING LOCUS C(FLC), which regulates flowering time in arabidopsis. Nucleotide and deduced amino acid sequences of two cloned FLC fragments (from exon 2 to exon 7) were compared to the previously characterized FLC genes in arabidopsis and FLC homologs in other Brassicaceae species. The homolog AY266265 is an ortholog of the FLC3 gene from Brassica rapa (95% identity), whereas the function of the homolog AY268931 has not been established conclusively. The FLC gene and its homologs were used to compare the variability in the primary structures of exons and introns.     

拟南芥角果发育和开裂的调控机制及在油菜育种中的应用

十字花科芸苔属甘蓝型油菜(Brassica napus)在角果发育成熟过程中,因角果开裂种子散落引起产量损失最低达20%左右,最高可达50%左右。模式植物拟南芥在角果形态和开裂机制方面与甘蓝型油菜具有相似性,角果开裂主要与果瓣、胎座框和果瓣边缘层的细胞发育有关。通过对拟南芥角果发育期间基因对果瓣、胎座框和果瓣边缘层的识别和发育进行综述,明确了拟南芥角果发育和开裂的基因调控机制,提出了从模式植物到大田作物甘蓝型油菜抗裂角育种的新策略。     

A Brassica napus skp1-like gene promoter drives GUS expression in Arabidopsis thaliana male and female gametophytes

We isolated a gene, BnSKP1γ1, expressed in rapeseed (Brassica napus) microspores, which encodes a protein closely related to the Saccharomyces cerevisiae Skp1p protein previously shown to play a role in cell cycle regulation. Twelve SKP1-related genes have already been identified in the Arabidopsis thaliana genome. Using a PCR-based strategy, we isolated three other genes. To date, most data available concerning the function of the SKP1-related genes in plants are indirect. Studies on transgenic A. thaliana plants showthat a 1100-bp BnSKP1γ1 promoter fragment can direct GUS expression in female gametophytes soon after the first haploid mitosis and in male gametophytes from the tetrade stage. No GUS expression can be detected in sporophytic tissues. RT-PCR experiments suggest that this gene is expressed in a similar way in rapeseed. This is the first reported case of a gene exhibiting such an expression pattern in angiosperms. Received: 5 October 1999 / Revision accepted: 28 March 2000     

Molecular cloning and characterization of a KCS gene from Cardamine graeca and its heterologous expression in Brassica oilseeds to engineer high nervonic acid oils for potential medical and industrial use

Nervonic acid 24:1 Δ15 ( cis -tetracos-15-enoic acid) is a very long-chain monounsaturated fatty acid and exists in nature as an elongation product of oleic acid. There is an increasing interest in production of high nervonic acid oils for pharmaceutical, nutraceutical and industrial applications. Using a polymerase chain reaction approach, we have isolated a gene from Cardamine graeca L., which encodes a 3-ketoacyl-CoA synthase (KCS), the first component of the elongation complex involved in synthesis of nervonic acid. Expression of the Cardamine KCS in yeast resulted in biosynthesis of nervonic acid, which is not normally present in yeast cells. We transformed Arabidopsis and Brassica carinata with the Cardamine KCS under the control of the seed-specific promoter, napin. The T₃ generations of transgenic Arabidopsis and B. carinata plants expressing the Cardamine KCS showed that seed-specific expression resulted in relatively large comparative increases in nervonic acid proportions in Arabidopsis seed oil, and 15-fold increase in nervonic acid proportions in B. carinata seed oil. The highest nervonic acid level in transgenic B. carinata lines reached 44%, with only 6% of residual erucic acid. In contrast, similar transgenic expression of the Cardamine KCS in high erucic B. napus resulted in 30% nervonic acid but with 20% residual erucic acid. Experiments using the Lunaria KCS gene gave results similar to the latter. In both cases, the erucic acid content is too high for human or animal consumption. Thus, the Cardamine KCS: B. carinata high nervonic/highly reduced erucic transgenic seed oils will be the most suitable for testing in pharmaceutical/nutraceutical applications to improve human and animal health.     

甘蓝型油菜长角果变异体的遗传研究

用在中国发现的甘蓝型油菜长角果变异体的优良后代“鄂油长角”及短角果品种“中油821”为亲本,研究了角果长及与角果长密切相关的两个性状――千粒重和每果粒数的遗传。结果表明,角果长、千粒重、每果粒数3个性状受核基因控制,不存在任何形式的母性遗传。供试双亲间角果长性状存在2对基因的差异。长角果对短角果为显性。数量遗传分析表明,角果长、千粒重的遗传符合加性显性二基因互作模型,存在显著的上位性效应。角果长性状在两个基因座上呈联合分布,显性效应均大于加性效应,且存在超显性。千粒重性状以加性效应为主。 Abstract：Eryou long-pod,which was derived from the unique long-pod mutant discovered in China,was crossed with the commercial broad rapeseed cultivar Zhongyou 821.From the crossing six basic generations were generated for studying the genetical behaviors of pod length and two related characters i.e.1000?seed weight and seed number per pod.The results showed that the three characters were governed by nuclear genes.The difference in pod length between two parents was related to two loci.Generation mean analyses revealed that the 6?parameter mode was to interpret the inheritance of the characters studied and the epistasis was significant.Pod length mainly showed association of its genes and highly significant dominant effect,and even superdominant effect too.Meanwhile,additive effect was predominant in 1000-seed weight.     

羽衣甘蓝SEPALLATA-like基因的系统发育与表达分析

E类MADS-box基因SEPALLATA (SEP)-like在被子植物生殖生长特别是花器官发育方面具有重要作用。为分析羽衣甘蓝E功能MADS-box基因SEP-like基因的序列特征及其在花发育过程中的时空表达模式,以羽衣甘蓝品系‘14 line’为试材,利用cDNA末端快速扩增(Rapid amplification of cDNA ends,RACE)技术克隆了SEP直系同源基因BroaSEP1/2/3 (GenBank登录号:KC967957、KC967958、KC967960)。序列和系统进化树分析表明,这3个基因分别与野甘蓝(Brassica oleracea var. oleracea)、芜菁Brassica rapa、萝卜Raphanus sativus、甘蓝型油菜Brassica napus的SEP1、SEP2、SEP3基因具有很高的同源性。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每一基因都有其亚家族特异的C-末端功能域SEPⅠ和SEPⅡ基序。BroaSEP1、BroaSEP2、BroaSEP3基因的开放阅读框长...     

Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana and Brassica napus contain complex mixtures of aliphatic monomers derived from non-extractable lipid polyesters. Most of the monomers are deposited in the seed coat, and their compositions suggest the presence of both cutin and suberin layers. The location of these polyesters within the seed coat, and their contributions to permeability of the seed coat and other functional properties are unknown. Polyester deposition was followed over Brassica seed development and distinct temporal patterns of monomer accumulation were observed. Octadecadiene-1,18-dioate, the major leaf cutin monomer, was transiently deposited. In contrast, the saturated dicarboxylates maintained a constant level during seed desiccation, whereas the fatty alcohols and saturated omega-hydroxy fatty acids continually increased. Dissection and analysis of Brassica seed coats showed that suberization is not specific to the chalaza. Analysis of the Arabidopsis ap2-7 mutant suggested that suberin monomers are preferentially associated with the outer integument. Several Arabidopsis knockout mutant lines for genes involved in polyester biosynthesis (att1, fatB and gpat5) were examined for seed monomer load and composition. The variance in polyester monomers of these mutants is correlated with dye penetration assays. Furthermore, stable transgenic plants expressing promoter::YFP fusions showed ATT1 promoter activity in the inner integument, whereas GPAT5 promoter is active in the outer integument. Together, the Arabidopsis data indicated that there is a suberized layer associated with the outer integument and a cutin-like polyester layer associated with the inner seed coat.     

A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression

During Arabidopsis flower development a set of homeotic genes plays a central role in specifying the distinct floral organs of the four whorls, sepals in the outermost whorl, and petals, stamens, and carpels in the sequentially inner whorls. The current model for the identity of the floral organs includes the SEPALLATA genes that act in combination with the A, B and C genes for the specification of sepals, petals, stamens and carpels. According to this new model, the floral organ identity proteins would form different complexes of proteins for the activation of the downstream genes. We show that the presence of SEPALLATA proteins is needed to activate the AG downstream gene SHATTERPROOF2, and that SEPALLATA4 alone does not provide with enough SEPALLATA activity for the complex to be functional. Our results suggest that CAULIFLOWER may be part of the protein complex responsible for petal development and that it is fully required in the absence of APETALA1 in 35S::SEP3 plants. In addition, genetic and molecular experiments using plants constitutively expressing SEPALLATA3 revealed a new role of SEPALLATA3 in activating other B and C function genes. We molecularly prove that the ectopic expression of SEPALLATA3 is sufficient to ectopically activate APETALA3 and AGAMOUS. Remarkably, plants that constitutively express both SEPALLATA3 and LEAFY developed ectopic petals, carpels and ovules outside of the floral context.     

Redirection of tryptophan metabolism in tobacco by ectopic expression of an Arabidopsis indolic glucosinolate biosynthetic gene

Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [²H₅] and [¹³C₁₀¹⁵N₂]IAOx and [¹³C₆], [²H₅] and [2′,2′-²H₂]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2′,2′-²H₂]IAN overestimated the amount of IAN by 2-fold compared to when [²H₅]IAN was used as internal standard, presumably due to protium-deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [¹³C₁]IAN underestimated the amount of IAN when the ratio of [¹³C₁]IAN standard to endogenous IAN was greater than five to one, whereas either [²H₅]IAN or [¹³C₆]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection ∼ 100 pg/g fr. wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1-9 μg/g fr. wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/g fr. wt, and IAA levels were ∼9-14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (∼200 and 400 ng/g fr. wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis.