高粱不同类型胚性和非胚性愈伤组织的生理生化差异

植物体细胞胚胎发生过程中伴随着复杂的生理生化变化,为进一步揭示胚性愈伤组织的再生潜力,该研究以高粱Sb19未成熟胚诱导产生的两种胚性愈伤组织和一种非胚性愈伤组织为材料,通过测定各愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量,采用方差分析法对高粱体细胞胚胎发生过程中不同类型愈伤组织的生理生化指标进行了差异比较研究。结果表明:(1)高粱两种胚性愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量均显著高于非胚性愈伤组织,表明胚性愈伤组织中的代谢活性高于非胚性愈伤组织,能够为体细胞胚胎发生提供更多的物质能量基础。(2)两种类型胚性愈伤组织之间生理生化差异显著,其中,Ⅱ型胚性愈伤组织中可溶性蛋白和游离脯氨酸含量均显著高于Ⅰ型胚性愈伤组织,相反,Ⅱ型胚性愈伤组织中可溶性糖含量显著低于Ⅰ型胚性愈伤组织,这种生理生化差异在一定程度上影响了后期的分化。该研究结果为愈伤组织的胚胎发生能力与生化代谢的关系提供理论依据。     

GFP基因转化香樟胚性愈伤组织的研究

以香樟胚性愈伤组织作为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因(GFP)的遗传转化研究。经农杆菌侵染后的胚性愈伤组织通过共培养、选择培养后获得抗性愈伤组织和体胚,对抗性愈伤组织及体胚的诱导过程进行了GFP荧光检测。结果表明,GFP基因能在抗性愈伤组织和体胚中强烈表达,证明GFP基因能够在香樟遗传转化中得到应用。对抗性愈伤组织的PCR检测初步证实外源GFP基因已整合到香樟胚性愈伤组织的基因组中。     

尾巨桉胚状体诱导及愈伤组织差异研究

以尾巨桉优良无性系无菌苗茎段为外植体,通过对多种不同浓度生长调节剂组合的优化,进行胚状体诱导研究;并对胚性与非胚性愈伤组织进行形态解剖学观察、相关生理指标检测以及相关基因荧光定量PCR分析,以揭示尾巨桉胚性愈伤组织非胚性化发生的机理,为建立尾巨桉体细胞胚胎再生体系提供参考。结果表明:(1)胚性愈伤组织在MS+0.1mg/L NAA+0.01mg/L TDZ培养基中诱导得到胚状体,外植体经过0.5mol/L蔗糖处理12h有助于胚性愈伤组织产生胚状体,胚状体最高发生率为16.7%。(2)尾巨桉胚性与非胚性愈伤组织石蜡切片观察发现,两者的细胞形态特征存在明显的差异,胚性愈伤组织细胞体积小,排列紧密,表现出典型的胚性细胞特征,而非胚性细胞比较大,排列疏松,细胞呈不规则形状。(3)生理生化指标检测结果表明,非胚性愈伤组织中蛋白质含量、SOD、PPO及CAT活性均显著低于胚性愈伤组织,非胚性愈伤组织中木质素、可溶性糖含量以及PAL和POD活性要高于胚性愈伤组织,二者的反肉桂酸4-单加氧酶基因、淀粉磷酸化酶基因、谷胱甘肽硫转移酶基因、葡萄糖-1-磷酸腺苷酸转移酶基因、葡萄糖六磷酸异构酶基因、分支酸合酶基因以及苯丙氨酸解氨酶基因表达差异也达到显著水平。     

碳氮源对花榈木胚性愈伤组织诱导、发育及有机物积累的影响

为探讨花榈木体胚发生过程中不同碳氮源处理对胚性愈伤组织诱导、发育和有机物积累的影响,并筛选出有利于花榈木体胚发生的碳氮源,优化体胚发生体系,该研究以成熟胚为外植体,通过单因素试验分析3种碳源、4种蔗糖浓度和6种氮源处理下胚性愈伤组织诱导、发育和有机物积累的差异。结果表明:(1)蔗糖中胚性愈伤组织诱导率显著高于葡萄糖和麦芽糖,但其体胚诱导率、体胚分化率、胚性愈伤组织可溶性糖、淀粉和可溶性蛋白含量差异不显著。(2)随着蔗糖浓度的升高,胚性愈伤组织、体细胞胚(体胚)诱导率、体胚分化率、胚性愈伤组织重量和可溶性蛋白含量呈先升高后降低的趋势,均以添加30 g·L^-1蔗糖最高,而胚性愈伤组织可溶性糖和淀粉含量呈增加的趋势。(3)在6种氮源处理中,胚性愈伤组织诱导率以添加500 mg·L^-1谷氨酰胺的处理最高,体胚诱导率则以添加谷氨酰胺和水解酪蛋白的处理较高,但不同氮源处理间体胚分化率无差异; 添加有机氮源的处理其胚性愈伤组织可溶性蛋白含量显著高于无氮源处理。总之,不同的碳氮源通过影响花榈木胚性愈伤组织的诱导、发育和有机物的积累,从而影响其体胚诱导率,但对体胚分化率影响不显著。初步认为30 g·L^-1蔗糖和500 mg·L^-1谷氨酰胺作为碳氮源可促进花榈木体胚发生诱导。     

龙眼胚性愈伤组织DRM1基因的克隆及其定位与表达分析

该研究以龙眼胚性愈伤组织的转录组数据为基础,对龙眼胚性愈伤组织DlDRM1基因进行克隆和生物学信息分析,并检测其在体胚发生过程中不同发育阶段、不同浓度的外源激素(2,4-D、IAA、KT)处理下及不同组织部位的表达,以揭示DlDRM1基因在龙眼体胚发生过程中的功能。结果表明:(1)从龙眼转录组unigene序列筛选获得龙眼结构域重排甲基化酶1基因(命名为DlDRM1)全长序列,并利用RT-PCR法从‘红核子’龙眼胚性愈伤组织中克隆获得DlDRM1基因的cDNA全长序列(GenBank登录号为KY990493);DlDRM1基因cDNA全长2 574bp,包括494bp的5′UTR,184bp的3′UTR,可编码包含631个氨基酸的蛋白质。(2)生物信息学分析显示,DlDRM1是一个不稳定的亲水蛋白,不含信号肽,不存在跨膜结构域,其分子式为C_(3104)H_(4839)N_(851)O_(984)S_(28);序列比对和系统进化分析表明,龙眼DlDRM1与脐橙DRM相似度最高(76.85%),二者亲缘关系也最为接近。(3)实时荧光定量PCR分析发现,DlDRM1在龙眼各组织器官中均有表达,且在果肉中表达量最高,其次是花蕾,在叶中的表达量最低;DlDRM1基因在非胚性愈伤组织中表达量最高,而且在非胚性愈伤向胚性愈伤转变过程中DlDRM1基因的表达量呈逐步下降趋势,说明DlDRM1基因与体胚胚性呈负相关关系,在龙眼体胚发生过程中可能发挥着重要的作用;一定浓度的IAA和2,4-D能够促进DlDRM1基因的表达,而KT则抑制DlDRM1的表达。(4)亚细胞定位结果表明,DlDRM1定位于细胞核和细胞膜上。     

植物胚性愈伤诱导的机理及提高途径

对植物胚性愈伤诱导的研究结果进行综合分析,寻找植物胚性愈伤诱导参与的基因、诱导的机理、甲基化与提高植物胚性愈伤诱导途径之间的联系,探索提高植物胚性愈伤诱导几率的可能途径,为转化困难的植物的基因工程操作奠定基础。LBD (laterial organ boundaries domain)家族基因、伤口诱导脱分化蛋白WIND1 (wound induced dedifferentiation 1)、AP2/ERF转录因子BABYBOOM等参与了植物胚性愈伤诱导的过程;DNA甲基化在这个过程中也发挥了重要作用,植物胚性细胞通过PCG和PKL通路两种表观遗传途径促使胚性细胞保持胚性。这些研究结果表明,通过筛选距离合子胚最近的体细胞作为外植体,利用分子标记进行筛选具有胚性的细胞或组织作为外植体进行培养,在培养基中添加去甲基化的试剂等方法可能能够提高植物胚性愈伤诱导的比例,转化困难的农作物的基因工程育种有望得到显著改进。     

芦苇胚性愈伤组织的形成及植株的再生

以芦苇种子为外植体,其愈伤组织的诱导率最高。叶鞘和叶片不发生脱分化。培养基中最合适的蔗糖浓度为4%。维生素 B 类、肌醇对愈伤组织的生长起促进作用。而酵母提取物对愈伤组织的诱导和生长具有明显的抑制作用。这种抑制效应,将随酵母提取物浓度的提高而增大。愈伤组织的继代培养,随培养基中2,4-D 浓度的提高,其平均鲜重明显降低。脱分化培养基中2,4-D 浓度对胚性愈伤组织的诱导形成具有一定的相关性。胚性愈伤组织经30代继代培养依然具有90%的分化频率,只是每块愈伤组织的分化苗数减少。反之,非胚性愈伤组织则完全丧失形态发生的能力。对两类愈伤组织进行扫描电镜的观察,发现其表面结构有很大差异。其过氧化物酶、酯酶同工酶谱以及可溶性蛋白的含量均有明显的差别。     

连香树胚性与非胚性愈伤组织生理生化差异及同工酶分析

本研究以连香树未成熟种子子叶胚诱导产生的胚性愈伤组织与非胚性愈伤组织为原材料,测定了两者的可溶性蛋白质、可溶性糖及游离脯氨酸的含量,并利用非变性聚丙烯酰胺凝胶电泳技术(polyacrylamide gelelectrophoresis, PAGE)对两者的过氧化物酶(peroxidase, POD)、超氧化物歧化酶(superoxide dismutase,SOD)、淀粉酶(amylase, AMY)同工酶进行了分析。比较发现胚性愈伤组织在可溶性蛋白质和游离脯氨酸的含量上均极显著高于非胚性愈伤组织。在可溶性糖含量上,胚性愈伤组织也显著地高于非胚性愈伤组织。胚性愈伤组织具有较高的代谢活性,为进一步胚性分化提供物质和能量基础。POD、SOD和AMY同工酶在连香树胚性愈伤组织与非胚性愈伤组织中酶带差异明显,表明这3种酶的活性与胚性愈伤组织的诱导密切相关,酶谱的差异可作为体细胞胚胎发生的参考标记。     

蔗糖转运蛋白基因VvSUC11和VvSUC12双价植物表达载体的构建及在甜菜中的遗传转化

将葡萄Vitis vinifera L.的蔗糖转运蛋白基因VvSUC11和VvSUC12与甘薯Ipomoea batatas L. Lam.的甘薯贮藏蛋白 (Sporamin) 基因的根部特异性启动子命名为SP1和SP2重组。以pCAMBIA2301为起始载体,构建了pCAMBIA2301- SP1-VvSUC11-SP2-VvSUC12用农杆菌介导法转化了甜菜Beta vulgaris L.品种KWS-9103,发现预培养4 d,侵染时农杆菌的浓度OD600值为0.5,附加0.005％表面活性剂Silwet L-77,延迟筛选4 d,转化效率最高,可达42％。对在卡那霉素中分化并生根的甜菜植株进行PCR和RT-PCR检测,证明目的基因已整合到甜菜中并表达,为进一步研究该基因在甜菜Beta vulgaris中的功能奠定了基础。     

杉木未成熟胚胚性愈伤组织诱导影响因素探析

该研究从基因型、6-BA浓度、外植体接种方式和合子胚发育阶段等方面,分析杉木未成熟胚胚性愈伤组织诱导的影响因素。结果表明:基因型、6-BA浓度、外植体接种方式和合子胚发育阶段均对胚性愈伤组织诱导频率有不同程度影响。6种基因型中,有3种基因型诱导出胚性愈伤组织,其中基因型S18胚性愈伤组织诱导频率最高,为11.7%。6-BA浓度在1.0~1.5 mg·L~(-1)范围内时,基因型S18的胚性组织诱导频率较高。以在去皮种子的一端切开一个小口的接种方式为最优,将合子胚剥出的方式易造成合子胚褐化死亡,将未剥皮的种子切开一个小口后直接接入培养基的方式不利于愈伤组织生成。适合胚性愈伤组织诱导的合子胚发育阶段为受精至胚器官分化阶段,合子胚进入成熟阶段后不利于胚性愈伤组织诱导,合子胚易生长成完整植株。     

Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast

Large amounts of sugar are imported into grape berries from source leaves during ripening, and sucrose transporters play a key role during this process. In this study, a putative grape sucrose transporter gene VvSUC27, primarily expressed in sink tissue, was transformed into a yeast strain to characterize its function as a sucrose transporter. Sucrose was taken up by yeast transformed with VvSUC27 at an optimum pH of 4.0–5.0 and a K _m of 8.0–10.5 mM, indicating VvSUC27 is a LAHC (low-affinity/high-capacity) sucrose transporter. The ability of sucrose uptake in transformed yeast was activated by monosaccharides and inhibited by maltose and DEPC. Ya Li Zhang and Qing Yong Meng contributed equally to the paper.     

Functional Characterization of Two Ripening-related Sucrose Transporters from Grape Berries

The ripening of grape (Vitis vinifera L.) berries is associatedwith a large accumulation of glucose and fructose in the vacuolesof the fruit cells. These hexoses are derived from sucrose,which is released from the phloem and may be taken up by parenchymacells prior to hydrolysis. We have expressed two putative ripening-relatedsucrose transporters from grape berries, VvSUC11 (synonymouswith VvSUT1) and VvSUC12, in an invertase deficient yeast strainto characterize their transport activities. Sucrose was takenup by yeast transformed with either transporter at an optimumpH of <4.5 and with a Michaelis constant (K_m) of 0.9–1.4m M. The uptake of sucrose through VvSUC11 and VvSUC12 was inhibitedby protonophores and by vanadate. This is consistent with anactive uptake mechanism involving proton cotransport, typicalof sucrose/H⁺symporters. The transporters from grape berrieswere functionally similar to Scr1, a sucrose transporter fromRicinus cotyledons. It is likely that in grape berries VvSUC11and VvSUC12 facilitate the loading of sucrose from the apoplastinto the parenchyma cells. Copyright 2001 Annals of Botany Company Fruit, grape berries, plasma membrane, sugars, sucrose transporters, Vitis vinifera     

葡萄蔗糖转运蛋白在烟草中的反义表达及其对转基因烟草的影响

将葡萄蔗糖转运蛋白VvSUC27cDNA以反义方向插入到含有CaMV35s启动子的真核表达载体pBI121载体中,然后转化到烟草(Nicotianatobacumcv.Samsun)植株中.转反义VvSUC27cDNA的烟草植株在含有20g/L蔗糖的培养基上能够正常生长发育,但是通过切片观察,发现其根部发育较弱,且叶片叶绿体含量增加.可溶性糖测定发现,转基因烟草根部蔗糖含量只有野生型烟草的51%.14C蔗糖吸收实验发现转基因烟草转运外界蔗糖的能力大大降低.     

Comparative anatomy of embryogenic and non-embryogenic calli from<Emphasis Type="Italic">pimpinella brachycarpa</Emphasis>

Anatomical differences between embryogenic and non-embryogenic calli ofPimpinella brachycarpa were investigated by light microscopy and electron microscopy. Initial callus tissue emerged from expiants after 14 d of culturing. The embryogenie calli (EC) were firm, rather opaque, and light yellow in color. The cells usually formed small, compact clusters. Nonembryogenic calli (NEC), however, were friable, semitransparent, and yellow or gray. These formed relatively larger and loosely held clusters. Scanning electron microscopy showed that EC were composed of individual compact and spherical cells that were rather regular in size and approximately 20 μm long. All were tightly held together and appeared to organize globular embryos. In contrast, the NEC comprised elongated and loosely held cells that were approximately 50 μm long. Tubular and u-shaped NEC cells protruded irregularly, and were of varying heights along the cell aggregates. Transmission electron microscopy of the EC revealed typical eukaryotic cytoplasmic components, including nuclei, mitochondria, and vacuoles in the cytoplasm enclosed by an electron-transparent cell wall. Based on the numerous ribosomes within the cytoplasm, these cells appeared to be well-organized and metabolically active. The NEC cells were much larger and more highly vacuolated than those of the EC. In ultrathin sections, the former seemed to be almost devoid of other cellular contents except for plastids and nuclei. Furthermore, EC and NEC showed different regeneration capacities in their somatic embryo formation. Most EC produced hyperhydric somatic embryos, followed by normal somatic embryos; whereas only a few shooted or rooted somatic embryos arose from the NEC.     

Role of ABA and Gibberellin A<Subscript>3</Subscript> on gene expression pattern of sugar transporters and invertases in <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. Malbec during berry ripening

Sugars are key constituents that affect quality of grape berries, and consequently the grape metabolic profile relevant to wine’s industry. However, enzymes and transporter genes expression involved in sugar transport at different phenological stages are scarcely studied. In addition, little is known about the role of the plant hormones ABA and Gibberellin (GA₃) as endogenous regulators, over the expression pattern of the sugars transporters genes in grapevine. The aim of this study was to analyze the expression pattern of the most relevant sugar transporters and invertases in leaves and berries of grapevine plants cv. Malbec during berry ripening stages and its shift after ABA and GA₃ sprays. In leaves, VvHT1 was the sugar transporter highly expressed, whereas VvHT6 was the most abundant in berries throughout berry ripening. Moreover, VvSUC12 and VvSUC27 were expressed at veraison greater in leaves than in berries, suggesting an active phloem loading at the onset of ripening. Applications of ABA and GA₃ enhanced the expression of VvSUC12 and VvSUC27 in pre-veraison leaves. Furthermore, hormones increased the expression of VvHT2, VvHT3 and VvHT6 in berries at different stages of ripening favoring sugar unloading from phloem. In conclusion, ABA and GA₃ are involved in the long-distance sugar transport from leaves to berries in Vitis vinifera L. cv. Malbec, and their exogenous application could be a suitable strategy to improve the process.     

枸杞体细胞胚发生中外源Ca2+的作用

脱分化的枸杞叶片外植体愈伤组织转入含有2,4-D的MS培养基上分化培养后有大量胚性细胞的分化和体细胞胚发生;加入一定量的外源Ca2 或45Ca2 ,明显地提高了胚性愈伤组织中体细胞胚发生的频率;加入Ca2 的鳌合剂EGTA则显著降低了体细胞胚发生频率;胚性愈伤组织中CaM的水平在多细胞原胚期和球形胚期显著升高,加入外源Ca2 后CaM含量几乎成倍增加;胚性愈伤组织中蛋白质组分与活性都远远多于或高于非胚性愈伤组织,加Ca2 后蛋白质组分种类也增加.     

NEC1, a novel gene, highly expressed in nectary tissue of Petunia hybrida

To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RT-PCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating that the protein is incorporated into the cell membrane or the cytoplast membrane. Immunolocalization revealed that NEC1 protein is present in the nectaries. Northern blot analyses showed that NEC1 is highly expressed in nectary tissue and weakly in the stamen. GUS expression driven by the NEC1 promoter revealed GUS activity in the outer nectary parenchyma cells, the upper part of the filament and the anther stomium. The same expression pattern was observed in Brassica napus. GUS expression was observed as blue spots on the surface of very young nectaries that do not secrete nectar and do accumulate starch. GUS expression was highest in open flowers in which active secretion of nectar and starch hydrolysis had taken place. Ectopic expression of NEC1 resulted in transgenic plants that displayed a phenotype with leaves having 3-4 times more phloem bundles in mid-veins than the wild-type Petunia. The possible role of NEC1 gene in sugar metabolism and nectar secretion is discussed.