外源ABA及其抑制剂钨酸钠对木薯块根类胡萝卜素相关基因和蛋白的影响

旨在研究外源ABA及其抑制剂钨酸钠(Na_2WO_4)对木薯块根类胡萝卜素相关基因和蛋白的影响。以木薯品种华南9号(Manihot esculenta Crantz SC9)为材料,HPLC测定块根β-胡萝卜素;Western blot分析相关蛋白表达量;QPCR分析相关基因表达量。结果显示,外源ABA及钨酸钠能提高块根β-胡萝卜素的含量;其中外源ABA与钨酸钠都提高了类胡萝卜素合成途径基因PSY2与LCYB表达,并抑制降解相关的ZEP与NCED3基因的表达,从而能够在转录水平的变化上解释β-胡萝卜素含量的变化,且LCYB与ZEP在转录与翻译水平具有一致性;与质体相关PAP1基因和FBN1a在处理下上调表达在一定程度上增加了质体小球的数量;外源ABA及钨酸钠使活性氧(ROS)相关的超氧化物歧化酶(Fe-SOD和Cu/Zn-SOD)表达量升高保护了块根中类胡萝卜素的稳定。这三者共同作用,提高了块根β-胡萝卜素的含量。     

木薯淀粉合成相关酶基因的克隆及表达分析

根据核苷酸同源性设计简并引物,克隆了一段大小为665 bp的木薯腺苷二磷酸葡萄糖焦磷酸化酶小亚基基因(sAGP)片段,并根据已知序列设计特异引物,克隆分离得到GBSS基因和SBE基因的cDNA片段.半定量RT-PCR分析结果表明,在木薯不同生长时期,3种淀粉合成相关酶基因的时空表达规律不同,GBSS基因表达有时期特异性和组织特异性,只在块根形成期和块根成熟期表达,在组织部位茎中表达最多,并且在这3种基因中表达水平相对较低.而sAGP基因在这3种基因中表达水平最高,在木薯决根成熟期竟达超量表达水平,并且在华南124和辐选0l中有品种差异,差异明显,在辐选01中的表达要优于华南124的表达.SBE基因表达较平稳,在各个时期表达水平都处于中间水平,在4个时期中,块根成熟期时的表达水平相对较高,块根形成期和苗期其次,收获期最低,也具有品种间差异,在辐选01中的表达要优于华南124的表达.     

西葫芦CCD基因家族鉴定及其在果实发育中的表达北大核心CSCD

类胡萝卜素裂解双加氧酶(carotenoid cleavage dioxygenase,CCD)是类胡萝卜素氧化裂解途径中的关键酶,在植物生长发育、香气形成及胁迫响应等过程中均发挥着重要作用。该研究运用生物信息学方法从西葫芦全基因组中鉴定出13条具有完整RPE65保守结构域的CCD基因,为进一步解析CCD基因家族在西葫芦中的功能奠定基础。结果表明:(1)聚类分析显示,13个西葫芦CCD基因编码的蛋白可分为CCD1、CCD4、CCD7、CCD8、NCED、CCD1-like共6个亚组,且CCD8和CCD7亚组与其他家族成员的遗传距离较远。(2)顺式作用元件预测分析发现,CCD基因启动子中含有光信号、激素、环境胁迫和生长发育响应元件。(3)转录组数据分析显示,CCD家族基因具有组织表达特异性,其中3个CCD基因在组织中不表达,CpCCD1基因在叶和果实中显著高表达。(4)在果实发育过程中,8个CCD基因呈现上调表达,2个CCD基因呈现下调表达,其中CpCCD1、CpCCD4a、CpCCD4b、CpCCD8a这4个CCD基因在果实膨大生长期或成熟期出现显著高表达,推测它们可能在西葫芦果实发育过程中具有重要的调控作用。     

中华猕猴桃品种‘Hort16A’果肉颜色形成的分子机制

对中华猕猴桃(Actinidia chinensis Planch.)品种‘Hort16A’果实发育过程(花后2~140 d及采后7~16 d)中果肉颜色的变化进行了观察,并对果实采后果肉中叶绿素和类胡萝卜素含量以及叶绿素和类胡萝卜素生物合成和降解相关基因的表达进行了研究。结果表明:花后2~140 d,中华猕猴桃品种‘Hort16A’的果肉为绿色;采后7~16 d,果肉颜色由绿变黄,果肉中叶绿素含量先升高后显著降低,类胡萝卜素含量无显著变化;类胡萝卜素/叶绿素比在采后16 d显著升高。采后12 d果肉中叶绿素生物合成相关基因中CAO1、Glu TR1、LHCB1、LHCB2、CBR1和CLH1基因的相对表达量较采后7 d显著升高,之后显著降低;采后12~16 d果肉中叶绿素降解相关基因中PAO1、PAO2、PPH1、PPH_2、PPH3、SGR1和SGR2基因的相对表达量总体上显著高于采后7 d,说明果肉中叶绿素含量降低是叶绿素的生物合成减少及其降解增加共同作用的结果。采后12~16 d果肉中类胡萝卜素生物合成相关基因中Crt ISO1、ZISO1、LCYB2、CYP1和CHY1基因的相对表达量较采后7 d显著升高,类胡萝卜素降解相关基因中NCED1、NCED2、ZEP1和CCD2基因的相对表达量也显著升高,说明类胡萝卜素生物合成和降解达到平衡,导致类胡萝卜素含量无显著变化。相关性分析结果显示:中华猕猴桃品种‘Hort16A’果实采后果肉中叶绿素含量与PAO2基因的相对表达量在0.05水平上显著正相关,但与SGR1基因的相对表达量显著负相关;类胡萝卜素含量与LCYB2和CYP1基因的相对表达量在0.05水平上显著正相关;个别叶绿素和类胡萝卜素生物合成和降解相关基因相对表达量间的相关性在0.05或0.01水平上显著。研究结果显示:中华猕猴桃品种‘Hort16A’果实采后果肉中叶绿素的生物合成减少及其降解增加是导致叶绿素含量降低和果肉变黄的主要原因。     

木薯蔗糖合成、转运与块根淀粉积累关系研究

该研究以淀粉含量不同的两个木薯品种(辐选01和华南124)为材料,通过测定各品种不同生育期叶、茎和根的蔗糖含量及块根淀粉含量,分析了蔗糖合成、转运和块根淀粉积累过程的相关性.结果表明:与华南124相比,在整个生育期内辐选01叶、茎的蔗糖含量均较高,块根蔗糖含量在块根膨大初期以前高于华南124,块根膨大初期以后则相反.在木薯的整个生育期,与辐选01相比,华南124的淀粉合成量和淀粉合成速率均较低.叶和茎蔗糖含量的变化规律与淀粉合成速率的变化规律相反,即块根淀粉积累明显加快时叶和茎的蔗糖含量略呈下降趋势,而块根淀粉合成减慢时叶、茎的蔗糖含量又开始上升.随着生育期的延后,块根蔗糖含量越来越低.在块根形成初期,蔗糖含量最高的组织部位为块根,其次为茎秆,最低的是叶片;而在块根成熟期时则相反,即蔗糖含量最高的部位是叶片,其次为茎杆,块根的蔗糖含量最小.相关性分析结果表明,木薯叶片蔗糖含量与块根淀粉含量呈显著的正相关;茎秆蔗糖含量与块根淀粉积累量呈不显著的正相关;块根蔗糖含量与淀粉积累量呈显著的负相关.由此可见,木薯叶、茎和根蔗糖与块根淀粉积累过程密切相关,其中叶片合成蔗糖的能力与块根利用蔗糖的能力在淀粉的积累过程中发挥关键作用.该研究结果为木薯的生产选育与高效栽培提供了理论依据.     

黄秋葵类胡萝卜素合成基因LCYB的克隆与分析

本研究应用RT-PCR和RACE技术克隆得到cDNA全长1797 bp的黄秋葵番茄红素β-环化酶基因LCYB,开放阅读框(ORF) 1509个碱基;预测编码503个AA,理论分子量(Mw)为56.288 kD,等电点(pI)为4.577;编码的蛋白与陆地棉(Gossypium raimondii)、黄麻(Corchorus olitorius)和可可(Theobroma cacao)同源蛋白的相似性均在88%以上,显示其高度的保守性,将基因命名为HyLCYB,GeneBank登录号为：KX257998。Motif Scan分析显示,蛋白氨基酸序列88 ~ 481位为HyLCYB保守结构域,并在88 ~ 113位含有1个FAD结合域。通过荧光定量PCR 分析表明,LCYB基因在黄秋葵根、茎、叶、花和果荚中都有表达;叶片生长中以成熟叶中表达最高,果实发育中以花后7天高表达。建立与优化了黄秋葵类胡萝卜素超高效液相检测体系,黄秋葵中主要含有β-胡萝卜素和叶黄素。β-胡萝卜素在成熟叶中含量最高, 果实以花后7天的果实含量最高,与LCYB基因的表达存在一定的相关性。     

八氢番茄红素合酶基因对人参的遗传转化

八氢番茄红素合酶(Phytoene synthase ,PSY)是类胡萝卜素生物合成的限速酶,通过建立PSY基因的人参转化体系,可促进相应类胡萝卜素的合成,从而提高人参的营养价值。本研究以人参愈伤组织为受体,以PSY 为目的基因,应用根癌农杆菌介导法进行遗传转化。以抗性筛选人参受体转染效率为指标,从菌液浓度、侵染胞龄、侵染时间、共培养时间四方面优化了转化体系。进行了PCR、PCR-Southern和RT-PCR分析鉴定及β-胡萝卜素含量测定,初步证明外源基因PSY 已整合到人参的基因组中并在转录水平上进行了表达,β-胡萝卜素含量平均提高了26倍。该研究为改善和提高人参中类胡萝卜素含量提供了一种新的途径。     

通过转基因提高β-胡萝卜素生物合成量

在黄花龙胆 (Getianalutea)花瓣中获得了植物类胡萝卜素生物合成途径中的 5个基因GGPS、PSY、ZDS、LycB、LycE ,它们分别位于类胡萝卜素合成途径中生成α 和 β 胡萝卜素的上游 .将其中的主要酶基因PSY、ZDS与 35S启动子和NOS终止子相连 ,通过根癌农杆菌 (Agrobacteriumtumefaciens)转入烟草 ,并通过RT PCR ,Northern分子杂交 ,Western分子杂交等证实这些基因在RNA、蛋白质水平能较好转录及表达 .高效液相层析法分析显示 ,这些基因的翻译产物具有酶的活性 .结果表明 ,PSY可使 β 胡萝卜素含量提高 1 0 8% .     

茶树种子发育过程的转录组分析

该研究以‘铁观音’茶树品种的种子为试验材料,采用转录组测序技术分析种子发育的3个时期(幼果期、膨大期、成熟期)的表达差异,探究茶树种子油脂代谢的分子机制。结果表明:(1)经转录组测序、组装后共获得30 940 581个clean reads,经数据合并拼接最终得到36 951条非冗余Unigene序列,其中28 476个Unigene可得到功能注释;在转录本中能够被注释到GO分类的Unigene有11 201条(30.3%),KEGG分析发现共有17 172个基因参与了127个代谢通路。(2)经KEGG通路筛选出14条与脂肪酸代谢相关的通路,且随着茶籽的发育,大部分脂肪酸调控途径相关基因呈下调趋势,其中上调基因数最多的有α-亚麻酸代谢途径和脂肪酸降解途径(有17个基因表达量上调),下调基因数最多的是甘油磷脂代谢途径(有58个基因表达量下调);在茶籽发育幼果期α-亚麻酸代谢途径中表达量上调的基因数超过表达量下调的基因数。(3)研究发现茶籽脂肪酸合成相关的基因涉及14个脂类调控途径,共409条差异基因;随着茶树种子发育到成熟期,上调的差异表达基因数量在减少,下调的差异表达基因数量增加,其中α-亚麻酸途径中的基因PLA2G16、DAD1、pldA、FabF、FabI表达量上调显著,随后表达量下调。(4)qRT-PCR检测结果表明,7个茶树FAD和1个ACP差异表达基因的水平与转录组测序结果基本一致;随着茶籽的发育,基因CsFAD7和Δ6-CsFAD从幼果期、果实膨大期至果实成熟期都为差异下调表达,CsFAD2、CsFAD6和Δ7-CsFAD为差异上调表达,CsFAD8、Δ8-CsFAD和CsACP在幼果期至果实膨大期差异上调表达,在果实膨大期至果实成熟期差异下调表达。     

黄秋葵番茄红素β-环化酶LCYB基因的克隆与分析

应用RT-PCR和RACE技术克隆得到cDNA全长1797 bp的黄秋葵(Hibiscus esculentus L.)番茄红素β-环化酶基因LCYB,开放阅读框(ORF)包含1509个碱基;预测编码503个氨基酸,理论分子量(Mw)为56.288 kD,等电点(p I)为4.577;编码的蛋白与陆地棉(Gossypium hirsutum L.)、黄麻(Corchorus capsularis L.)和可可(Theobroma cacao L.)同源蛋白的相似性均在88%以上,显示其高度的保守性,将基因命名为HeLCYB,GenBank登录号为:KX257998。Motif Scan分析显示,蛋白氨基酸序列88~481位为HeLCYB保守结构域,并在88~113位含有1个FAD结合域。通过荧光定量PCR分析表明,HeLCYB基因在黄秋葵根、茎、叶、花和果荚中均有表达;叶片生长中以成熟叶中表达最高,果实发育中以花后7d高表达。建立与优化了黄秋葵类胡萝卜素超高效液相检测体系,结果显示黄秋葵中主要含有β-胡萝卜素和叶黄素。β-胡萝卜素在成熟叶中含量最高,果实以花后7 d含量最高,与HeLCYB基因的表达呈正相关。本研究揭示了HeLCYB基因表达和类胡萝卜素积累的特性,为开展黄秋葵类胡萝卜素分子调控机制研究奠定了基础。     

Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava is a widely grown staple in Sub-Saharan Africa and consumed as a cheap source of calories, but the crop is deficient in micronutrients including pro-vitamin A carotenoids. This challenge is currently being addressed through biofortification breeding that relies on phenotypic selection. Gene-based markers linked to pro-vitamin A content variation are expected to increase the rate of genetic gain for this critical trait. We sequenced four candidate carotenoid genes from 167 cassava accessions representing the diversity of elite breeder lines from IITA. Total carotenoid content was determined using spectrophotometer and total β-carotene was quantified by high-performance liquid chromatography. Storage root yellowness due to carotenoid pigmentation was assessed. We carried out candidate gene association analysis that accounts for population structure and kinship using genome-wide single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing. Significant SNPs were used to design competitive allele-specific PCR assays and validated on the larger population for potential use in marker-assisted selection breeding. Candidate gene sequencing of the genes β-carotene hydroxylase (crtRB), phytoene synthase (PSY2), lycopene epsilon cyclase (lcyE), and lycopene beta cyclase (lcyB) yielded a total of 37 SNPs. Total carotenoid content, total β-carotene, and color parameters were significantly associated with markers in the PSY2 gene. The SNPs from lcyE were significantly associated with color while those of lcyB and crtRB were not significantly associated with carotenoids or color parameters. These validated and breeder-friendly markers have potential to enhance the efficiency of selection for high β-carotene cassava, thus accelerating genetic gain.     

Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.     

Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutations in the Psy1 gene. The first mutation is a knockout allele (W180*) and the second mutation leads to an amino acid substitution (P192L). Plants carrying the Psy1 knockout allele show fruit with a yellow flesh colour similar to the r, r mutant, with no further change in colour during ripening. In the line with P192L substitution, fruit remain yellow until 3 days post-breaker and eventually turn red. Metabolite profiling verified the absence of carotenoids in the W180* line and thereby confirms that PSY1 is the only enzyme introducing substrate into the carotenoid pathway in ripening fruit. More subtle effects on carotenoid accumulation were observed in the P192L line with a delay in lycopene and β-carotene accumulation clearly linked to a very slow synthesis of phytoene. The observation of lutein degradation with ripening in both lines showed that lutein and its precursors are still synthesised in ripening fruit. Gene expression analysis of key genes involved in carotenoid biosynthesis revealed that expression levels of genes in the pathway are not feedback-regulated by low levels or absence of carotenoid compounds. Furthermore, protein secondary structure modelling indicated that the P192L mutation affects PSY1 activity through misfolding, leading to the low phytoene accumulation.