Expression of structural genes related to anthocyanin biosynthesis of Vitis amurensis

This research was designed to assess the changes in anthocyanin content in grape skins of Vitis amurensis and to explore m RNA transcriptions of 11 structural genes（PAL,CHS3, CHI1, F3H2, F30 H, F3050 H, DFR, LDOX, UFGT,OMT and GST） related to anthocyanin biosynthesis during grape berry development, by the use of HPLC-MS/MS and real-time Q-PCR analysis. Accumulation of anthocyanins began at veraison, continued throughout the later berry development and reached a peak at maturity. Veraison is the time when the berries turn from green to purple. Expression of PAL, CHI1, and LDOX were up-regulated from 2 to4 weeks after flowering（WAF）, down-regulated from6 WAF to veraison, whereas DFR was up-regulated at8 WAF, and then up-regulated from veraison to maturity.CHS3, F3050 H, UFGT, GST, and OMT were down-regulated from 2 WAF to veraison, and then up-regulated from veraison to maturity. The transcriptional expressions of the11 structural genes also showed positive correlations with the anthocyanin content from veraison to maturity. Positive correlations were also observed between OMT transcriptional level and the content of methoxyl-anthocyanins, and between F3050 H transcriptional level and the content of delphinidin anthocyanins. F3H2 and F30 H expression was up-regulated at 2 WAF. F3H2 expression was down-regulated from 4 WAF to veraison and then up-regulated again from veraison to maturity. F30 H expression was down-regulated at 4 WAF and then up-regulated again from 6 WAF to maturity. F30 H transcriptional level was correlated positively with the cyanidin anthocyanin concentration from veraison to maturity. These results indicate that the onset of anthocyanin synthesis during berry development coincides with a coordinated increase in the expression of a number of genes in the anthocyanin biosynthetic pathway.

Cloning and Expression Analysis of <i>RrMYB</i>113 Gene Related to Anthocyanin Biosynthesis in <i>Rosa rugose</i>

Anthocyanin is one of water-soluble natural pigments widely existing in flowers, fruits, stems, leaves and seeds of plants, and it is the major factor conferring pink or red to the petals of Rosa rugose. MYB TFs play an important role in the anthocyanin synthesis in plants. This work aimed to clone the MYB gene related to anthocyanin synthesis in the petals of Rosa rugose, and explore the relationship between them to lay a good foundation for gene engineering improvement of R. rugose. Based on the transcriptional data, a full-length cDNA sequence of MYB Gene, RrMYB113 (GenBank accession Nos MG720012), was cloned at the first time from the petals of Rosa rugose “Zi zhi” with RT-PCR and RACE methods. The full-length cDNA is 885 bp with an open reading frame of 654 bp, encoding 216 amino acids. The derived RrMYB113 protein has a molecular weight of 25,297.64 Da, a calculated pI of 9.61, a R2R3-MYB domain and bHLH binding domain, and it also has the signature motifs ((A/S/G)NDV and KPRPR(T/S)), thus belonging to Sg6 R2R3-MYB subfamily. In the secondary structure of RrMYB113 protein, there is 37.04% α-helix, 39.81% random coil, 14.81% extended peptide chain, and 8.33% β-corner. There is no transmembrane domain and no signal peptide cleavage site, seventeen Ser phosphorylation sites, fifteen Thr phosphorylation sites, four Tyr phosphorylation sites, and no O-glycosylation sites. The expression of RrMYB113 increased with the color deepening in petals, and it expressed at a higher level in petals than in other tissues of R. rugose “Zi zhi”. These results are meaningful to reveal that RrMYB113 might be an important regulator in anthocyanin biosynthesis and coloration in the petals of R. rugose.

Cloning and Expression of Anthocyanin Biosynthesis Related Gene RrMYB6 in <i>Rosa rugosa</i>

R2R3-MYB transcription factor plays an important role in plant anthocyanin synthesis. Based on the transcriptional database of Rosa rugosa, one MYB transcription factor related to floral color, RrMYB6, was cloned. By using bioinformatics analysis method, cloning MYB gene and analyzing its function in anthocyanin biosynthesis regulation, we hope to lay a solid foundation for new color variety breeding of R. rugosa. Using the R. rugosa “Zi zhi” as the material, we obtained the total length of cDNA of RrMYB6 by RT-PCR and RACE. By analyzing its bioinformatics, we found that the formula of the protein was C1491H2368N452O470S17, molecular weight was 34690.97 Da, the theoretical pI was 8.74. In addition, it belonged to unstable protein with an unstable index at 50.59, and it was also a hydrophilic protein with the total average hydrophobic index at -0.847. In the secondary structure of RrMYB6 protein, the Alpha helix accounted for 32.35%, random coil was 47.39%, extended strand was 11.11%, and beta turn was 9.15%. The sequence analysis showed that RrMYB6 had a typical R2R3-MYB domain and bHLH binding domain, and it also had an N1, C1, C2 inhibitory motif, belonging to the Sg4 subfamily MYB protein. What’s more, evolutionary analysis indicated that the RrMYB6 protein was closely related with the MYB protein in Rosacea family, while it was far from those in other families. The expression analysis showed that RrMYB6 protein decreased with the color of petals deeping, and its expression was the lowest in the petals while the highest in stamens. According to the above results, it was speculated that RrMYB6 was involved in regulating the anthocyanin synthesis of R. rugosa, which belonged to negative regulatory mechanism.

Cloning and Expression Analysis of <i>RrGT2</i>Gene Related to Anthocyanin Biosynthesis in <i>Rosa rugosa</i>

At present, the research about flower color of Rosa rugosa is a very inno-vative and practical study. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1422bp, encoding 473 amino acids, designated as RrGT2, were isolated from flowers of R. rugosa ‘Zizhi’ and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT2 gene is C2334H3628N602O711S18, the relative molecular mass is 52,075.17 Da, and the theoretical isoelectric point is pI = 4.76. The result of the RrGT2 protein 3D model construction showed that it had the highest homology with the UDP-glycosyltransferase 74F2 protein model in the database (39.53%). Sequence alignments with the NCBI database showed that the RrGT2 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT2 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT2 enzyme activity. RrGT2 transcripts were detected in five flowering stages and seven tissues of R. rugosa ‘Zizhi’, R. rugosa ‘Fenzizhi’ and R. rugosa ‘Baizizhi’, and their expression patterns corresponded with the accumulation of antho-cyanins. Therefore, we speculated that glycosylation of RrGT2 plays a crucial role in anthocyanin biosynthesis in R. rugosa.

Effects of Cerium on Accumulation of Anthocyanins and Expression of Anthocyanin Biosynthetic Genes in Potato Cell Tissue Cultures

The effects of Ce （Ⅳ） on callus growth, anthocyanin content, and expression of anthocyanin biosynthetic genes in callus suspension cultures of Solanum tuberosum cv. Chieftain were studied by the measurement of fresh weight, spectrophotometric assays, and semiquantitative RT-PCR. The results indicate that 0.1 mmol·L^- 1 Ce （ Ⅳ ） can promote callus growth, increase the accumulation of anthocyanins, and enhance the expression of five anthocyanin biosynthetic genes （ CHS, F3H, F3＇5＇H, DFR, and 3 GT） most efficiently. At high concentrations of 1 mmol·L^- 1, Ce （Ⅳ） partially inhibits callus growth and at 2 mmol· L^-1 eventually lends to cell death. The results show that Ce（ Ⅳ ） can induce the expression of anthocyanin biosynthetic genes to produce and accumulate anthocyanins and increase the yield of anthocyanins.

Cloning and Expression of One Anthocyanin-Related R2R3-MYB Gene in <i>Rosa rugosa</i>

Based on the transcriptome of Rosa rugosa, one anthocyanin-promoting R2R3-MYB gene, RrMYB10.1 (Accession Nos:MH717244), was cloned from the petals of Rosa rugosa ‘Zizhi’. Sequence analysis results showed that RrMYB10.1 had a full length opening reading frame of 747bp, encoding 249 amino acids. Sequence analysis revealed that RrMYB10.1 contained the conserved R2R3-MYB domain, two atypical anthocyanin-promoting motifs and a conserved amino acid signature for the interaction with bHLH protein. The results of phylogenic tree revealed that RrMYB10.1 showed high homology with other anthocyanin-promoting proteins in Rosacea, and sharing the highest identity (98.39%) with RhMYB10. RT-PCR results showed that RrMYB10.1 was mainly expressed in petals among various tissues and expressed significantly higher in petals in bud stage than in opening period. To sum up, these results showed that RrMYN10.1 may play a key role in regulating anthocyanin concentration, thus providing a certain foundation on regulating flower color formation in Rosa rugosa.

An acyltransferase gene that putatively functions in anthocyanin modification was horizontally transferred from Fabaceae into the genus Cuscuta

Horizontal gene transfer(HGT) refers to the flow of genetic materials to non-offspring,and occasionally HGT in plants can improve the adaptation of organisms in new niches due to expanded metabolic capability.Anthocyanins are an important group of water-soluble red,purple,or blue secondary metabolites,whose diversity results from modification after the main skeleton biosynthesis.Cuscuta is a stem holoparasitic genus,whose members form direct connection with hosts to withdraw water,nutrients,and macromolecules.Such intimate association is thought to increase the frequency of HGT.By transcriptome screening for foreign genes in Cuscuta australis,we discovered that one gene encoding a putative anthocyanin acyltransferase gene of the BAHD family,which is likely to be involved in anthocyanin modification,was acquired by C.australis from Fabaceae through HGT.The anthocyanin acyltransferase-like(AT-like) gene was confirmed to be present in the genome assembly of C.australis and the transcriptomes of Cuscuta pentagona.The higher transcriptional level in old stems is consistent with its putative function in secondary metabolism by stabilizing anthocyanin at neutral pH and thus HGT of this AT-like gene may have improved biotic and abiotic resistance of Cuscuta.

Cloning and Expression Analysis of <i>RrGT1</i>Gene Related to Anthocyanin Biosynthesis in <i>Rosa rugosa</i>

Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1161 bp, encoding 386 amino acids, designated as RrGT1, was isolated from flowers of R. rugosa ‘Zizhi’ and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT1 gene is C1879H2964N494O556S14, the relative molecular mass is 41,820.02 Da, and the theoretical isoelectric point is pI = 5.03. The result of the RrGT1 protein 3D model construction showed that it had the highest homology with the UDP-glucose: anthocyanidin 3-O-glucosyltransferase protein model in the database (47.01%). Sequence alignments with the NCBI database showed that the RrGT1 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT1 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT1 enzyme activity. RrGT1 transcripts were detected in five flowering stages and seven tissues of R. rugosa ‘Zizhi’, R. rugosa ‘Fenzizhi’ and R. rugosa ‘Baizizhi’, and their expression patterns corresponded with the accumulation of anthocyanins. Therefore, we speculated that glycosylation of RrGT1 plays a crucial role in anthocyanin biosynthesis in R. rugosa.

Overexpression of the <i>Rosa rugosa RrGT1</i>Gene Induces Anthocyanin Accumulation in Tobacco

Rosa rugosa has always been an important plant in landscape application, and the improvements and innovations about its flower color are particularly important. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1161 bp, encoding 386 amino acids, designated as RrGT1, were isolated from flowers of R. rugosa “Zizhi” and then functionally characterized. Sequence alignments with the NCBI database show that the RrGT1 protein is a member of the GTB superfamily and has typical conserved amino acid residues called PSPG that are crucial for RrGT1 enzyme activity. RrGT1 transcripts were detected in five flowering stages and seven tissues of R. rugosa “Zizhi” and their expression patterns corresponded with the accumulation of anthocyanins. Additionally, the in vivo function of RrGT1 was investigated via its overexpression in tobacco. Transgenic tobacco plants expressing RrGT1 induced anthocyanin accumulation in flowers, indicating that RrGT1 could encode a functional glycosyltransferase (GT) protein for anthocyanin biosynthesis and could function in other species. Therefore, we speculated that glycosylation of RrGT1 played a crucial role in anthocyanin biosynthesis in R. rugosa.

土壤含水量对玫瑰茄花青素含量及DFR基因表达水平的影响

【目的】分析土壤含水量对玫瑰茄萼片花青素含量以及二氢黄酮醇⁃4⁃还原酶(DFR)基因表达水平的影响,为玫瑰茄栽培时的水分管理以及进一步明确DFR基因在花青素合成中的功能提供参考。【方法】以玫瑰茄品种‘广东早熟’为试验材料,分别设置了33%(极度干旱)、55%(轻度干旱)、80%(渍涝)3个土壤含水量处理,分别测定各处理下开花后10(前期)、20(中期)、30 d(后期)玫瑰茄萼片中花青素的含量以及5个DFR基因的表达量,并对花青素含量与DFR基因表达量的相关性进行了分析。【结果】(1)不同土壤含水量下,随着开花时间的推移,玫瑰茄萼片花青素含量均呈下降趋势,且开花前期与开花后期的差异显著;含水量为80%时,花青素含量下降幅度最大;开花前、中期,土壤含水量为33%的花青素含量明显低于土壤含水量为55%、80%的含量;开花后期,3个土壤含水量处理的花青素含量差异不显著。(2)不同土壤含水量下,DFR基因的表达量随着开花时间的推移呈现不同的变化趋势。土壤含水量为33%时,5个DFR基因的表达量高于其他两个含水量处理,且在开花后30 d显著上调,达到最高值,其中,DFR4表达量最高,DFR5表达量最低;土壤含水量为55%、80%时,5个DFR基因的表达量均较低。(3)土壤含水量为55%时,DFR4、DFR5表达量与花青素含量显著相关,表明其为调控花青素合成的关键基因。【结论】严重的干旱和渍涝都不利于玫瑰茄萼片花青素的合成;干旱胁迫促进了DFR基因的表达,而渍涝情况下DFR基因的表达受到了抑制;DFR4、DFR5基因为调控玫瑰茄花青素合成的关键基因。

遮阳对马瑟兰葡萄花色苷组分及合成相关基因表达的影响

【目的】宁夏贺兰山东麓金山产区栽培的酿酒葡萄成熟期花色苷积累过快,降解严重,探讨不同转色期遮阳对葡萄果实花色苷组分及合成相关基因表达的影响,为提升葡萄色泽稳定性、改善品质提供理论依据。【方法】以马瑟兰酿酒葡萄为试材,在转色初期-采收期(T1)和完全转色期-采收期(T2)采用遮阳网对葡萄树体顶端进行遮阳处理,以正常生长不遮阳的树体为空白对照。采用HPLC方法分析花色苷组分与含量,利用RNA-seq技术和qRT-PCR技术研究花色苷合成相关基因表达水平。【结果】遮阳可显著降低马瑟兰葡萄果实成熟期可溶性固形物含量,缓解滴定酸含量下降趋势,各处理检测到的16种花色苷种类均一致;完全转色期-采收期遮阳处理下提高了总花色苷含量,明显提高了Mv类花色苷含量,同时也提高了酰化花色苷的修饰比例;使得PAL、CHS3、F3’H、F3’5’H、UFGT、GST4和MYB90等花色苷合成相关基因的表达量上调,CHI基因表达水平与花翠素类(Dp)和花青素类(Cy)含量呈显著正相关,MYB5b基因表达水平与Dp、Cy、甲基花青素类(Pn)及甲基花翠素类(Pt)含量显著正相关。qRT-PCR分析发现,筛选的15个基因中CHI、F3H、F3’5’H、F3’H、DFR、LDOX、UFGT和OMT在各处理中的表达水平的变化趋势和转录组测序不一致,其他基因均一致。【结论】完全转色期-采收期遮阳,促进了花色苷的合成,提高了相关基因的转录水平,提升了酰化花色苷比例,增加了花色苷颜色的稳定性,进而提升了葡萄及葡萄酒的色泽稳定性。

Biotic and abiotic stress-responsive genes are stimulated to resist drought stress in purple wheat

Triticum aestivum L. cv. Guizi 1(GZ1) is a drought-tolerant local purple wheat cultivar. It is not clear how purple wheat resists drought stress, but it could be related to anthocyanin biosynthesis. In this study, transcriptome data from droughttreated samples and controls were compared. Drought slightly reduced the anthocyanin, protein and starch contents of GZ1 grains and significantly reduced the grain weight. Under drought stress, 16 682 transcripts were reduced, 27 766 differentially expressed genes(DEGs) were identified, and 379 DEGs, including DREBs, were related to defense response. The defense-response genes included response to water deprivation, reactive oxygen, bacteria, fungi, etc. Most of the structural and regulatory genes in anthocyanin biosynthesis were downregulated, with only Ta DFR, Ta OMT, Ta5,3GT, and Ta MYB-4 B1 being upregulated. Ta CHS, Ta F3H, TaCHI, Ta4CL, and TaF3’H are involved in responses to UV, hormones, and stimulus. Ta CHS-2D1, Ta DFR-2D2, Ta DFR-7D, TaOMT-5A, Ta5,3 GT-1B1, Ta5,3GT-3A, and Ta5,3GT-7B1 connect anthocyanin biosynthesis with other pathways, and their interacting proteins are involved in primary metabolism, genetic regulation, growth and development, and defense responses. There is further speculation about the defense-responsive network in purple wheat. The results indicated that biotic and abiotic stress-responsive genes were stimulated to resist drought stress in purple wheat GZ1, and anthocyanin biosynthesis also participated in the drought defense response through several structural genes.

Effects of Abscisic Acid Treatment on Berry Coloration and Expression of Flavonoid Biosynthesis Genes in Grape

In order to enhance berry coloration of bright-red grape cultivars, the effects of abscisic acid (ABA) treatment on the quantity and composition of anthocyanins as well as the expression of genes related to flavonoid biosynthesis in the berry were examined. Exogenous ABA treatment increased anthocyanin content, especially petunidin- and malvidin-type anthocyanins. Quantitative real-time PCR analysis revealed that ABA treatment around véraison resulted in the upregulation of genes encoding enzymes responsible for both general flavonoid and anthocyanin biosynthesis. On the other hand, the gene expressions of enzymes involved in proanthocyanidin synthesis were drastically decreased at véraison and remained extremely low even with ABA treatment. Thus, increases in the total amount and composition ratios of petunidin- and malvidin-type anthocyanins were mainly caused by ABA-induced upregulation of uridine diphosphate glucose flavonoid glucosyl transferase, glutathione S-transferase 4, O-methyl transferase and flavonoid 3’, 5’ hydroxylase expression, resulting in the deep coloration of berry of skin.

Establishment of Virus-Induced Gene Silencing (VIGS) System in Perennial <em>Rosa</em>Plants under Field Conditions

Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing mechanism. Compared with the traditional transgenic technology, VIGS is a transient expression system, which can achieve good results in a short time. At present, it is widely used to study the function of plant genes, but most of them are model plants, and the experiments are carried out always in the indoor environment with controlled light and temperature conditions. In this study, we creatively provided a method to establish VIGS system using perennial Rosa plants as experimental materials under field conditions. The recombinant virus vector was constructed with RrGT1 gene as reporter gene and modified TRV-GFP virus as vector, and the perennial R. rugosa “Zizhi” and R. davurica were used as experimental verification materials. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems such as the confirmation of the inoculation period, the preparation of the infective fluid, the inoculation technology of the virus vector and the light and temperature conditions of plant materials cultured after inoculation were solved one by one. When the RrGT1 gene was silenced, the Rosa plants showed a pale petal color phenotype. By detection, it was found that the expression of endogenous RrGT1 gene was significantly down-regulated, and the content of all anthocyanins also decreased significantly. Therefore, we believed that the attempt to establish VIGS system in perennial Rosa plants under field conditions was very successful.

Cloning and Expression Analysis of <i>RrRUP</i>2 Gene Related to Photomorphogenesis Biosynthesis in <i>Rosa rugosa</i>

Plants have evolved and perfected a series of light receptors to feel the light at different bands and regulate the expression, modification and interaction of related genes in plants through signal transduction. So far, many photoreceptors have been identified in plants, UVR8 has recently been identified as a receptor for UV-B light. This paper cloned a WD40 gene related to UVR8 protein subunit, named RrRUP2, based on the Rosa rugose transcriptome data, using Rosa rugose “Zi zhi” as experimental materials. The full length of cDNA of the gene was obtained by RT-PCR and RACE methods. The total length of this gene is 1173 bp, and it encodes 390 amino acids. After bioinformatics analysis, the molecular formula C3415H5659N1173O1434S313 was predicted;the relative molecular weight was 96129.27 Da;the theoretical isoelectric point PI value was 5.00;and its instability index was 47.06. The total average hydrophobic index was 0.750. In the secondary structure of RrRUP2 protein, there are 10 α-helix, 45 β-helix, 181 Random coil, and 154 Extended strand. Gene Bank Blast results showed that the amino acid sequence encoded by RrRUP2 was more than 90% homologous with the RUP2 protein of Rosa chinensis, Fragaria, Malus, Pyrus, Prunus, Juglans, Arabidopsis and Tobacco, so it can be inferred that the RrRUP2 gene is a WD repeat-containing protein. Regarding to fluorescence quantitative expression analysis of RrRUP2, we find its experssion pattern is corresponded with the accumulation of anthocyanins.

不同叶片颜色的木薯MeANR基因克隆及表达分析

【目的】对木薯花青素还原酶基因(MeANR)进行克隆及表达分析,为解析木薯叶片花青素生物合成的分子机制、叶色改良及新品种培育提供理论依据。【方法】以3种叶片颜色的木薯华南9号(SC9,绿叶)、花叶木薯(黄绿叶)和紫叶木薯(紫叶)为材料,测定其花青素含量和原花青素含量,并对MeANR基因进行克隆及生物信息学分析,采用实时荧光定量PCR检测MeANR基因在不同组织及激素处理下的表达模式,通过转基因酵母胁迫试验验证MeANR基因在不同激素处理下的表达情况。【结果】SC9、花叶木薯和紫叶木薯叶片中的花青素含量和原花青素含量均存在显著差异(P<0.05),其中紫叶木薯叶片的花青素含量最高,达444.59 ng/g,分别为SC9和花叶木薯的24.48和28.32倍。从3种叶片颜色的木薯叶片中分别克隆到1个MeANR基因,其编码区(CDS)序列为1041 bp,序列完全一致,但与Phytozome数据库中公布的ANR基因序列(登录号:Manes.16G016400)存在7个碱基差异。MeANR蛋白由346个氨基酸组成,属于稳定的非分泌型亲水性蛋白,二级结构主要由无规则卷曲(42.77%)和α-螺旋(37.28%)构成,延伸链和β-转角占比较小,亚细胞定位于叶绿体。MeANR基因在芽中的相对表达量最高,其次是叶片和花,在茎中的相对表达量最低。MeANR基因在不同激素处理下呈不同的表达模式,水杨酸(SA)处理后随着处理时间的延长,MeANR基因的相对表达量呈先下降后上升再下降的变化趋势,处理后24 h MeANR基因的相对表达量比处理0 h显著下降68%,而在脱落酸(ABA)和茉莉酸甲酯(MeJA)处理后MeANR基因的相对表达量总体上呈上升趋势,但ABA处理后24 h达峰值,MeJA处理后9h达峰值。在SA处理下转MeANR基因酵母菌的存活率低于对照(转pDR196空载体的酵母菌),而在ABA和MeJA处理下转MeANR基因酵母菌的生长情况优于对照。【结论】木薯叶片颜色深度与花青素含量存在正相关性。MeANR是木薯叶片花青素积累的负调控基因,具有明显的组织表达特异性,其表达受SA抑制,受ABA和MeJA诱导。

低纬高原紫色辣椒果实花色苷合成相关关键基因的挖掘

【目的】挖掘低纬高原生境中紫色辣椒果实花色苷合成相关的关键基因,为紫色辣椒果实花色苷合成分子机理研究和新种质创制奠定基础。【方法】以文山低纬高原主栽且抗性良好的紫色辣椒品种16HN01试材,用色价法和液相色谱串联质谱法分别测定16HN01各发育阶段果实[按长度并结合颜色将果实分为6个发育阶段(1~6),即长度≤1.5 cm、1.5~2.5 cm、2.5~3.5 cm、≥3.5 cm、青熟期与成熟期果实]的总花色苷含量(TAC)和主要花色苷单体含量(AMC),基于转录组测序和qRT-PCR与双因素相关性分析,挖掘果实花色苷合成相关的关键基因。【结果】16HN01果实TAC在发育阶段3最高,为4.626±0.202(A539-0.25A657)/g;主要花色苷单体为飞燕草素-3-O-(2'''-O-对香豆酰)芸香糖苷[Dp-3-O-(2'''-O-C)R]、飞燕草素-3-O-芸香糖苷(Dp-3-O-R)、飞燕草素-3-O-葡萄糖苷(Dp-3-O-G)和飞燕草素-3,5-O-二葡萄糖苷(Dp-3,5-O-DG),其含量分别在阶段3、阶段1、阶段1和阶段3最高;TAC、Dp-3-O-(2'''-O-p-C)R、Dp-3-O-R、Dp-3-O-G和Dp-3,5-O-DG含量与果实中UDPG类黄酮糖基转移酶基因(UFGT)转录水平之间的Pearson相关系数最高,分别为0.848(显著)、0.779、0.308、0.490和0.952(极显著),与WD40转录水平间的相关系数最高,分别为0.977(极显著)、0.702、0.163、0.357和0.674。【结论】低纬高原紫色辣椒果实花色苷合成相关关键结构基因主要包括UFGT、花色素合酶基因(ANS)65、ANS08和查尔酮合酶基因(CHS),转录因子基因WD40和bHLH分别极显著和显著正调控花色苷的合成,MYB和BBX负调控花色苷的合成。

紫芽六堡茶花青素合成相关基因的转录组测序及表达分析

【目的】紫芽六堡茶(Camellia sinensis var. sinensis cv. Liupao)是六堡群体种茶树中芽叶呈紫色且富含花青素的特异品系,研究紫芽六堡茶花青素合成相关基因可为深入了解紫芽六堡茶花青素积累的分子机制奠定基础,为高花青素六堡茶树的分子育种提供遗传资源。【方法】利用盐酸乙醇分别提取紫芽和绿芽六堡茶芽中花青素,测定其含量;通过Illumina Hiseq 2 500高通量测序平台对紫芽和绿芽六堡茶进行转录组测序,分析花青素合成相关基因的表达水平,找出差异表达基因,进一步进行GO功能分析和KEGG富集分析;并通过荧光定量PCR对转录组测序结果进行验证。【结果】紫芽六堡茶花青素含量是绿芽的7倍;转录组测序共获得165 570条Unigene,平均长度1 450bp;对测序结果进行分析,筛选出与紫芽六堡茶花青素生物合成通路相关的基因243条,进一步筛选出43个差异显著表达基因,这些基因共编码14个关键酶,包括苯丙氨酸氨裂解酶(phenylalanine ammonialyase, PAL)、查尔酮合成酶(chalconesynthase,CHS)、查尔酮异构酶(chalconeisomerase,CHI)、肉桂酸4-羟化酶(cinnamateacid4-hydroxylase, C4H)、花色素苷还原酶(anthocyanidin reductase, ANR)、4-香豆酸CoA连接酶(4-coumarate-Co A ligase,4CL)、乙酰辅酶A羧化酶(acetyl-CoA carboxylase, ACC)、黄酮醇合成酶(flavonol synthase, FLS)、黄酮3′,5′-羟化酶(flavonoid-3′,5′-hydroxylase, F3'5'H)、黄酮3′-羟化酶(flavonoid 3'-hydroxylase, F3'H)、黄酮3-羟化酶(flavanone-3-hydroxylase, F3H)、二氢黄酮醇还原酶(dihydroflavonol 4-reductase, DFR)、花青素成合酶(anthocyanidin synthase,ANS)、无色花青素还原酶(leucoanthocyanidin reductase, LAR)。【结论】编码花青素生物合成的14个关键酶基因中有34个基因在紫芽六堡茶中上调表达,推测这些基因在紫芽花青素积累中起重要作用。

转录组和代谢组联合分析阐释木薯叶片花青素合成机制

木薯是世界上第六大粮食作物,其块根富含淀粉但缺乏蛋白质、花青素、胡萝卜素等营养物质。为了探索木薯花青素生物合成机制,本研究选取了两种不同颜色木薯种质资源叶片(FL与PL)为材料,进行转录组和花青素靶向代谢组及其联合分析。转录组分析结果显示在FL和PL中6864个差异表达基因,其中包含4112个上调表达和2752个下调表达。代谢组分析结果显示26种显著差异代谢物在PL中显著高于FL,其中21种属于花青素类。联合分析结果显示,其中7个差异表达的基因与花青素生物合成相关,且花青素含量与差异基因的表达呈正相关,尤其是MeANS1的表达差异最大。本研究结果为阐明木薯花青素的生物合成机制提供了候选基因,同时也为提高木薯花青素含量奠定科学基础。

马铃薯ARM基因家族的全基因组鉴定及表达分析

ARM蛋白重复序列(Armadillo repeats)广泛存在于高等植物中,它们参与多种细胞过程,如信号转导、核转运以及对多种生物/非生物胁迫的响应。本研究在马铃薯(Solanum tuberosum L.)全基因组水平下鉴定出了54个马铃薯ARM基因家族成员(StARMs),它们不均匀的分布在12条染色体上。根据其蛋白结构和系统发育特征,将54个StARMs分为3个亚家族。片段重复事件在马铃薯ARM基因家族的扩展中起主要作用。共线性分析发现,StARMs与番茄(Solanum lycopersicum)、拟南芥(Arabidopsis)、甘蓝(Brassica oleracea)、水稻(Oryza sativa)、玉米(Zea mays)分别有51对、17对、25对、6对和10对直系同源基因,这些基因均在纯化选择下进化。RNA-seq数据分析发现,4个StARM基因在匍匐茎中特异表达,2个StARM基因在根和心皮中特异表达,1个StARM基因在块茎中特异表,还有一些StARM基因参与了马铃薯对生物/非生物胁迫的响应。此外,本研究对3个不同颜色马铃薯块茎组织(薯皮和薯肉)进行了RNA-seq测序,分析了54个StARMs在不同颜色马铃薯块茎组织中的表达模式,并利用qPCR分析了StARMs在3个不同颜色块茎杂交子代薯肉中的相对表达量,筛选出了4个可能参与马铃薯块茎花色素苷生物合成的候选基因。本研究为进一步了解StARM基因家族的特征,深入分析StARM基因在马铃薯抵御生物/非生物胁迫和调控块茎花色素苷生物合成中的功能提供了理论依据。