棕色棉类黄酮3'-羟化酶基因(F3'H)的克隆及色素合成途径中相关基因表达特性研究

为探讨花色苷途径在彩棉色素形成中的作用及彩棉色素形成规律,本研究根据葡萄(Vitis vinifera)的类黄酮3'-羟化酶(flavonoid3'-hydroxylase,F3'H)基因全长cDNA序列blast所得棉花(Gossypium hirsutum)的EST序列(GenBank登录号:DT545210,CO071403和BG447485)设计引物,以开花后16d的新彩棉5号(XC-5)纤维为材料,采用RACE(rapid amplification of cDNA ends)和RT-PCR方法分离得到了2个类黄酮3'-羟化酶基因的全长cDNA序列,长度为1761和1892bp,均含有一个97~1629bp、长度为1533bp的开放阅读框,编码510个氨基酸,将这两个序列命名为GhF3'H-1和GhF3'H-2,分别提交GenBank,登录号为HM598123和HM598124,此2个序列编码区完全相同,仅在3'非翻译区(UTR)存在片段长短的差异。半定量RT-PCR检测花色苷合成途径中查尔酮合成酶(chalcone synathase,CHS)基因、F3'H、类黄酮3',5'-羟化酶(flavon...     

茶树黄烷酮3-羟化酶基因(F3H)的克隆及功能分析

黄烷酮3-羟化酶(flavanone 3-hydroxylase,F3H)是类黄酮代谢途径中的关键酶。本研究采用逆转录PCR技术,获取了茶树(Camellia sinensis)F3H的开放阅读框(open reading frame,ORF)包含1 107个碱基,编码含有368个氨基酸的蛋白质,推测分子量为41.46 kD,等电点为5.61;实时荧光定量PCR表明,CsF3H在叶片中表达量较高,且受光照影响;将此基因重组到表达载体PRSF上,导入大肠杆菌(Escherichia coli)BL21中进行原核表达,优化原核表达的条件,结果表明,最佳诱导温度28℃、异丙基-β-d-硫代半乳糖苷(IPTG)浓度1.0 mmol/L、诱导时间5 h;利用高效液相色谱法(HPLC)对重组蛋白进行了体外酶活的检测,结果表明,重组目的蛋白具有F3H酶活性,可将反应底物柚皮素(N)和圣草酚(E)分别转化为二氢山奈素(DHK)和二氢槲皮素(DHQ);当E作为底物时,酶活性明显高于柚皮素。本研究结果为茶树F3H酶动力学研究和其器官组织特异性研究提供了基础资料。     

农杆菌介导的蓝色基因转化中国水仙

从矮牵牛(Petunia hybrida)花瓣中分离得到编码蓝色基因F3'5'H(类黄酮3',5'羟基化酶)的Hf2基因(1527 bp),从牵牛(Ipomoea nil)花瓣中分离得到dfr基因(二氢黄酮醇4-还原酶,1212 bp),将正向Hf2片段和反向dfr片段连接到pBRLys双元载体质粒上,得到具有Ubi(ubiquitin)组成型启动子的植物表达载体pU-bi-Hf2-dfr.利用冻融法将双价重组质粒pUbi-Hf2一afr导入农杆菌(Agrobacterium tumefaciens)菌株EHA105中,菌落PCR鉴定农杆菌转化子.以中国水仙(Narcissus tazetta var.chinensis)愈伤组织为材料,进行遗传转化.CTAB法提取130株中国水仙遗传转化再生苗的基因组DNA,进行PCR和PCR-Southem blot检测,获得3株阳性转基因植株.     

马铃薯愈伤组织中色素含量与4个花色苷合成相关基因的表达差异

本研究从马铃薯（Solanum tuberosum cv．Chieftain）茎的愈伤组织中分离得到绿色、白色和红色3种愈伤组织,并利用鲜重法和分光光度法分别测量愈伤组织的生长量、叶绿素和花色苷的含量;通过半定量RT—PCR法分析4个花色苷生物合成相关基因的表达差异。结果表明：绿色愈伤组织生长最快,叶绿素含量最高,红色愈伤组织生长最慢,叶绿素含量最低;绿色和白色愈伤组织中几乎不含花色苷,红色愈伤组织中花色苷含量最高,达2．1OD513/mgFw;对CHS、F3H、DFR和F3’5’H四个花色苷生物合成相关基因表达分析发现,绿色和白色愈伤组织不合成花色苷可能与DFR基因不表达有关。本实验结果为进一步阐明花色昔生物合成机理和花色苷色素的生产应用提供一定的理论依据。     

巴西橡胶树4－羟基－3－甲基－2-（E）－丁烯基－4－磷酸还原酶基因（Hb—HDR）的克隆及表达分析

4-羟基-3-甲基-2-（E）-丁烯基-4-磷酸还原酶（4-hydroxy-3-methyl-2-（E）-butenyl-4-diphosphate reductase,HDR）是异戊烯基焦磷酸合成途径之一甲基赤藓糖磷酸（methylerythritol phosphate,MEP）途径中的最后一个酶,催化4-羟基-3-甲基-（2E）-丁烯基-4-磷酸生成异戊烯基焦磷酸。根据植物HDR的同源序列设计引物,通过RT-PCR结合RACE的方法在橡胶树中获得了与其相应的HDR基因,命名为HbHDR。序列分析表明HbHDR长1627bp,编码462个氨基酸,属于LYTB家族,该氨基酸序列与烟草、长春花、胡黄连、拟南芥、银杏和火炬松的HDR同源性为79．1％、78．4％、76．5％、75．3％、72．2％和70．9％。半定量RT-PCR结果显示,乙烯诱导胶乳HbHDR的表达。     

植物二氢黄酮醇-4-还原酶基因的研究进展

二氢黄酮醇-4-还原酶(DFR)是花青素生物合成途径中的关键酶,在花色的修饰中起重要作用,目前,已从多种植物中分离得到。从DFR基因的结构和作用机制、底物特异性、时空表达模式及花色修饰等方面进行了概括和总结,为DFR基因的进一步研究和利用提供理论依据。     

水稻化感物质作用特性的研究

在对水稻化感作用生理生化特性研究的基础上，进行了水稻叶片浸提液的GC-MS分析和相关酶的活性测定。结果表明，水稻叶片中苯丙氨酸裂解酶（PAL）和肉桂酸-4-羟化酶（CA4H）活性的大小与酚的含量密切相关，同时水稻化感作用强度与其肉桂酸含量呈正相关关系。而与其水杨酸含量呈负相关关系，因此水稻化感作用是由许多化感物质共同作用的结果。     

植物表达载体pCAMBIA2301-del-ros的构建及其在草莓上的验证

为了研究多基因互作,减少多次转化的不确定性,本研究在花青素合成调节基因del(delila)和ros(rosea1)的上下游分别引入CaMV 35S启动子和nos终止子,以pBI121-del和pCAMBIA1301-ros做中间载体,并利用BamHⅠ和BglⅡ产生相同4碱基末端的片段,在连接酶的作用下将CaMV 35S启动子分别驱动的del和ros基因构建在同一个植物表达载体pCAMBIA2301上,利用所构建的载体pCAMBIA2301-delros通过根癌农杆菌(Agrobacterium tumefaciens)LBA4404介导的方法转化草莓(Fragaria ananassa Duch.),并进行PCR检测,获得转基因草莓。通过组织观察发现,转基因草莓的根和叶的颜色变成红紫色;实时RT-PCR分析表明,在转基因草莓中花青素合成的结构基因花色素合成酶(anthocyanidinsynthase,ANS)、查尔酮异构酶(chalcone-flavanone isomerase,CHI)、查尔酮合酶(chalcone synthase,CHS)、二羟黄酮醇-4-还原酶(dihydroflavonol-4-reductase,DFR)、黄烷酮-3-羟化酶(flavanone 3-hydroxylase,F3H)和类黄酮-3-O-葡糖基转移酶(UDP glucose-flavonoid 3-O-glcosyl-transferase,UFGT)在转基因株系中的表达均上调,这也进一步证实通过该方法构建的植物表达载体pCAMBIA2301-del-ros可以用来转化植物。本研究结果表明,植物表达载体pCAMBIA2301-del-ros成功构建,为多基因植物表达载体的构建提供了一种思路,也为在植物体内同时研究多个基因的互作提供借鉴。     

茶树类黄酮3-ο-半乳糖基转移酶基因克隆及功能研究

糖基转移酶(UGT)是专门催化化合物糖基化反应的酶,能够有效提高类黄酮苷元化学稳定性并增加其水溶解度。为探究茶树中类黄酮糖基转移酶基因的功能,本研究以红叶2号紫芽茶品系紫色芽叶为材料,通过RACE技术,克隆了1个UGT基因。结果表明,UGT基因cDNA全长1 701 bp,包含一条大小为1 362 bp的CDS,编码454个氨基酸。该基因编码的蛋白与多个物种的类黄酮3-ο-半乳糖基转移酶(F3GalT)蛋白高度同源,且在其序列C端发现一段PSPG box特征序列,因此被命名为CsF3GalT。RT-qPCR结果表明,CsF3GalT基因在紫芽品种的表达水平均极显著高于绿芽品种,在同一紫芽品种紫叶中的表达水平也显著高于绿叶。对CsF3GalT基因构建原核表达载体,体外酶活试验表明CsF3GalT能够催化UDP-半乳糖与杨梅酮形成杨梅酮-3-ο-半乳糖,而不能催化UDP-葡萄糖和杨梅酮反应。CsF3GalT表达产物定位于细胞质、细胞膜和细胞核中。本研究为进一步阐明茶树UGTs的功能及其在花青素合成途径中的作用奠定了一定的理论基础。     

不同氮效率茄子基因型及其杂种F1的氮素吸收特性

为阐明杂种一代在氮素吸收方面的优势,研究了不同氮效率茄子基因型及其杂种 F1的氮素吸收特性。试验以3个典型氮效率的茄子基因型及其F1代为材料,研究其在正常供氮和低氮胁迫条件下的根系体积、根系干重、氮素吸收总量、根系活力、硝酸还原酶活性及谷氨酰胺合成酶活性。结果表明,与高氮低效-低氮低效基因型L相比,氮高效基因型H1、H2的单株根系体积、根系干重、根系活力以及氮素吸收总量均较大; 且具有较高的硝酸还原酶与谷氨酰胺合成酶活性。三个杂交组合F1-1（L×H1）、F1-2（L×H2）和F1-3（H1×H2）的单株根系体积、根系干重、根系活力、硝酸还原酶活性、谷氨酰胺合成酶活性以及氮素吸收总量的中亲优势（Hm）和超亲优势（Hp）多为正向优势; 其中,组合F1-3杂种优势最为明显。利用杂种在氮素吸收方面的优势,对于改善植株体内的氮代谢水平进而提高氮效率具有重要意义。     

红掌基因工程育种进展、问题与对策

红掌是极具观赏性和商业开发价值的盆栽植物和切花花卉,品种改良是推动其产业持续发展的根本。周期较长、生殖障碍、遗传基础不明晰等限制了红掌常规育种的发展,而基因工程具有高效、定向、打破种属隔离的优势,在红掌育种中有着广阔的应用前景。二十年来,红掌基因工程研究取得了巨大进展,本文回顾了该领域技术体系的发展历程,综述了转基因在红掌抗病害、虫害、低温等胁迫及花色、花期等多个性状改良中的应用研究现状,并对存在的问题和发展前景予以评析,为加快具有我国自主产权的红掌新品种选育提供参考。     

Silver nanoparticles preplant bulb soaking affects tulip production

Nanomaterials and particularly silver nanoparticles (AgNPs) have increased great interest in many fields of science. The element may affect plant growth and development but so far research studies have been scarce and their results disparate. Tulip is one of the most important ornamental plants and its production technology is constantly being improved. The aim of this study was to investigate the effects of AgNPs on growth and yield attributes of tulip cv. ‘Pink Impression’. Before planting, the tulip ‘Pink Impression’ bulbs were soaked in the following concentrations of AgNPs: 25, 50, 100 and 150?mg?L^-1. Control bulbs were soaked in deionized water. The plants were forced in a greenhouse for two growing seasons. The tulips treated with 100?mg?L^?1 AgNPs flowered earlier, had longer cut-flower stem, larger petals, greater stem diameter and cut-flower fresh weight. Moreover, at this concentration of AgNPs, they showed increased leaf greenness index (SPAD), stomatal conductance, root fresh weight, root length and produced daughter bulbs of the greatest weight. The study outcomes indicate that AgNPs may be used as plant growth promoters in horticulture.     

Two-phase flavonoid formation in developing strawberry (Fragaria x ananassa) fruit

Flavonoids are important secondary metabolites in strawberry as they fulfill a wide variety of physiological functions. In addition, they are beneficial for human health. Previous studies have shown for selected enzymes from the flavonoid pathway that flavonoid biosynthesis shows two peaks during fruit development. We provide optimized protocols for the determination of the activities of the key flavonoid enzymes: phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, flavonoid 3-O-glucosyltransferase, and flavonoid 7-O-glucosyltransferase. Using these protocols we were able to demonstrate two distinct activity peaks during fruit ripening at early and late developmental stages for all enzymes with the exception of flavonol synthase. The first activity peak corresponds to the formation of flavanols, while the second peak is clearly related to anthocyanin and flavonol accumulation. The results indicate that flavonoid 3-O-glucosyltransferase activity is not essential for redirection from flavanol to anthocyanin formation in strawberry.     

红花花青素还原酶基因ANR的克隆及表达分析

花青素还原酶(ANR)是类黄酮代谢途径下游合成原花青素的关键酶,对花青素含量有一定的负调控作用。为探索ANR基因在红花(Carthamus tinctorius L.)中的序列特征及其与红花花色的关系,采用反转录PCR方法从红花中克隆ANR基因,对其进行生物信息学和系统进化分析,并检测了其在不同花色红花品种的不同组织部位及不同花期的表达量。结果表明,CtANR最长开放阅读框(ORF)为1 020 bp, 编码339个氨基酸,分子量为37 958.28,理论等电点为5.87,为不稳定的亲水性蛋白,属于NADB-Rossmann超家族,二级结构主要由α-螺旋和无规卷曲组成。CtANR和刺菜蓟ANR的同源性最高,为83.98%,与刺菜蓟ANR的亲缘关系最近,与梅花、欧洲甜樱桃ANR的亲缘关系较远,模体基序相差较大。定量分析表明,红色和白色红花品种中,CtANR基因都是在果球形成初期表达量最低,其次是根和茎中表达量较低,而在花中的表达量相对较高。在花发育的不同时期,红色红花品种中CtANR基因的表达呈现降低-升高-降低的趋势,而白色红花品种中CtANR基因的表达呈现降低-升高的趋势。在不同花色的红花品种中,CtANR基因的表达量在花发育的S1、S3和S5时期差异极显著(P<0.01),S4、苞片、根、叶中差异显著(P<0.05),其他时期与其他组织中差异不显著。本研究为解析红花类黄酮生物合成的分子机制及红花花色相关基因的研究提供了理论基础。     

Re-examination of chromogenic quantitative assays for determining flavonoid content

Flavonoids in plants have gained worldwide attention because of their benefits for human health. This study compared three analytical procedures commonly used for determining flavonoid content in plant samples in terms of chromogenic relationships and the reaction products of different flavonoid structures by means of using flavonoid standards with flavone, flavonol, flavanone, flavanol, and isoflavone and analytes such as phenolic acids commonly found in plant extracts. Procedure A produced a stable color reaction between 3-hydroxy-4-keto-flavonoids (flavonols) and 5-hydroxyflavones and was highly sensitive. Procedure B produced color reactions among most of the flavonoids, but the reaction products had different colors and faded over time. Procedure B also produced a color reaction with caffeic and chlorogenic acid. Procedure C was the most sensitive. It produced a color reaction and, like procedure A, could be used to quantify flavonols and 5-hydroxyflavones, but also showed color reaction toward caffeic and chlorogenic acid. On the basis of the results, the current three procedures are not satisfactory for determining all of the types of flavonoid. Two issues needed to be clarified before a promising determination of flavonoid content could be performed with chromogenic assays. The first is a survey of the literature to screen the possible predominant component of flavonoid in analytes. The other is guided by the predominant flavonoid; a promising calibration curve for flavonoid detection can be established on the basis of the selection of an appropriate method and a chemical standard with an equivalent dose response to the predominant flavonoid.     

Metabolite profiling of grape: Flavonols and anthocyanins

Flavonols are products of the flavonoid biosynthetic pathway, which also give rise to anthocyanins and condensed tannins in grapes. We investigated their presence in the berry skins of 91 grape varieties (Vitis vinifera L.), in order to produce a classification based on the flavonol profile. The presence of laricitrin 3-O-galactoside and syringetin 3-O-galactoside in red grapes is reported here for the first time. In red grapes, the main flavonol was quercetin (mean = 43.99%), followed by myricetin (36.81%), kaempferol (6.43%), laricitrin (5.65%), isorhamnetin (3.89%), and syringetin (3.22%). In white grapes, the main flavonol was quercetin (mean = 81.35%), followed by kaempferol (16.91%) and isorhamnetin (1.74%). The delphinidin-like flavonols myricetin, laricitrin, and syringetin were missing in all white varieties, indicating that the enzyme flavonoid 3',5'-hydroxylase is not expressed in white grape varieties. The pattern of expression of flavonols and anthocyanins in red grapes was compared, in order to gain information on the substrate specificity of enzymes involved in flavonoid biosynthesis.     

Engineering flax plants to increase their antioxidant capacity and improve oil composition and stability

The composition of polyunsaturated fatty acids in the tissues is very important to human health and strongly depends on dietary intake. Since flax seeds are the richest source of polyunsaturated acids, their consumption might be beneficial for human health. Unfortunately, they are highly susceptible to auto-oxidation, which generates toxic derivatives. The main goal of this study was the generation of genetically modified flax plants with increased antioxidant potential and stable and healthy oil production. Since among phenylpropanoid compounds those belonging to the flavonoid route have the lowest antioxidant capacity, the approach was to inhibit this route of the pathway, which might result in accumulation of other compounds more effective in antioxidation. The suppression of the chalcone synthase gene resulted in hydrolyzable tannin accumulation and thus increased antioxidant status of seeds of the transgenic plant. This was due to the partial redirecting of substrates for flavonoid biosynthesis to the other routes of the phenylpropanoid pathway. Consequently, transgenic plants produced more (20-45%) polyunsaturated fatty acids than the control and mainly α-linolenic acid. Thus, increasing the antioxidant potential of flax plants has benefits in terms of the yield of suitable oil for human dietary consumption.     

慢照射诱发月季突变的研究

总照射量6kR—10kR、照射量率7.0R/h慢照射月季植株,经过反复修剪、转接,分离体细胞突变。目前已从中选出6个优良突变系。     

Phenolic compounds contribute to dark bran pigmentation in hard white wheat

Unacceptably dark bran color has prevented the white-kernelled variety Argent from meeting grain color marketing standards for hard white wheats (Triticum aestivum L.). The objective of this research was to identify phenolic compounds that negatively affect bran color in white wheat using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and vanillin-HCl and NaOH staining methods. In mature bran, FT-ICR-MS detected derivatives of the flavonol quercetin in varieties Argent and RL4137 (red-kernelled wheat) but not in W98616, a white wheat variety with acceptable grain color. Derivatives of the isoflavone formononetin were more abundant in W98616 relative to RL4137 and Argent. Vanillin-HCl staining indicated that RL4137 sequestered high levels of proanthocyanidin (PA) throughout its entire seed coat, whereas white wheats sequestered PAs as discrete speckles. Argent possessed abundant speckles over its entire seed coat, whereas speckles were almost undetectable in W98616. In mature kernels, flavonoids throughout the seed coat of RL4137 reacted with NaOH, but only the speckles appeared to react in white wheats. W98616 consistently had lighter grain than Argent before and after NaOH treatment. Free and bound phenolic differences in bran samples confirmed that the darker seed coat color of Argent, relative to W98616, was likely due to higher total phenolic acid content. Although isoflavones accumulated in Argent and RL4137, it appears that the majority of the flux through the flavonoid pathway ultimately accumulates quercetin derivatives and PAs. In W98616, PAs accumulate, but it appears that flavonoid biosynthesis ultimately accumulates isoflavones. Argent, compared to W98616, generally accumulated higher levels of total phenolics (flavonols, stilbenes, and PAs) within its darker pigmented bran.     

基于Super-GBS技术的高粱籽粒酿造相关性状QTL定位

为探究酒用高粱籽粒酿造性状的遗传基础,本研究利用超级基因分型技术（Supper-GBS）技术对BTx623×红缨子的205个家系的重组自交系（RIL）群体开展基因分型,构建了包含1 910个单核苷酸多态性标记（SNP）,标记平均间距为0.47 cM的连锁图谱。结合两年4个环境的表型数据,利用完备区间作图法（ICIM）对籽粒总淀粉含量、支链淀粉含量、单宁含量、硬度和颜色等5个酿造性状进行了数量性状位点（QTL）定位。结果表明,在高粱的1～9号染色体上检测到9、7、11、5和3个QTL分别与总淀粉含量、支链淀粉含量、单宁含量、籽粒硬度和籽粒颜色相关,一共涉及到35个不同的QTL,其中15个QTL与前人定位结果一致。在多个性状和环境中检测到3个重要遗传区段,分别位于1号染色体（66.30～71.55 Mb）、4号染色体（54.00～62.3 Mb）以及6号染色体（54.59～57.57 Mb）,其中包括已知的Tan1和Y1基因,以及6个与淀粉和类黄酮合成途径相关的候选基因,如编码β-淀粉酶、黄烷酮3-羟化酶和bHLH转录因子等。本研究结果为酿造性状相关基因的进一步克隆奠定了基础,也为利用标记...