Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2^nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.     

缙云山川鄂连蕊茶在不同群落类型中的RAPD分析

采用随机扩增多态性DNA(RAPD)对常绿阔叶林灌木层广布种川鄂连蕊茶(Camellia rosthorniana)在缙云山3个群落类型(毛竹林、针阔混交林和常绿阔叶林)中的遗传结构和DNA多样性进行了研究。10个引物共扩增出138个产物,其中115个是多态性的,多态位点比率为83．33％。Shannon指数估算的3个种群的遗传多样性为常绿阔叶林0．3345,毛竹林0．3148,针阔混交林0．3085,遗传分化为8．12％;Nei指数计算的3个种群的基因多样性为常绿阔叶林0．2095,毛竹林0．1981,针阔混交林0．1934,遗传分化为7．51％。种群间遗传距离在0．0177～0．0393之间。川鄂连蕊茶在3个群落中的种群大小和生长状况不同,各种群遗传多样性的高低可能是对所处群落生境的适应。     

山茶属山茶组植物的分类,分化和分布

山茶组Ｓｅｃｔ．Ｃａｍｅｌｉａ植物迄今已合格发表的名称有７２种,１亚种和７变种,其中Ｓｅｃｔ．Ｐａｒａｃａｍｅｌｉａ中的威宁短柱茶Ｃ．ｗｅｉｎｉｎｇｅｎｓｉｓ和Ｓｅｃｔ．Ｃｏｒａｌｉｎａ中的连山离蕊茶Ｃ．ｌｉｅｎｓｈａｎｅｎｓｉｓ应归属本组。经研究订正,确认该组共１２种和６变种,其余名称均作为相应种、变种和变型的同物异名,文中讨论了物种的形态变异与分化,分布与替代,自然杂交等问题。     

茶树CsMYB123转录因子的克隆及表达特性研究

该实验以茶树品种‘紫娟’为试验材料,利用RT-PCR方法,从茶树cDNA中克隆得到一个R2R3-MYB型基因(CsMYB123)。生物信息学分析显示,CsMYB123基因的开放阅读框为915bp,编码304个氨基酸,蛋白分子量约34.07kD,理论等电点为8.69,含有2个保守的MYB结构域,编码1个R2R3-MYB蛋白;CsMYB123蛋白与拟南芥(Arabidopsis thaliana)MYB转录因子家族第五亚组的AtMYB123亲缘关系最近;CsMYB123属于亲水性蛋白,无N端信号肽,可能定位于细胞核上。荧光定量PCR分析表明,CsMYB123基因在茶树各组织的表达量大小依次为:一芽一叶第二叶第三叶第四叶老茎嫩茎,且在一芽一叶中的表达量是嫩茎的15.68倍;但CsMYB123的表达受IAA、ABA、ETH和GA3的抑制。花青素含量检测显示,茶树‘紫娟’各组织中花青素的含量高低依次为:第二叶一芽一叶第三叶第四叶嫩茎老茎,且第二叶和一芽一叶的含量分别为老茎的15倍和11倍。研究发现,CsMYB123基因在茶树‘紫娟’的新稍中高水平表达,且其表达模式与不同组织中的花青素含量呈较好的正相关关系,推测CsMYB123基因与茶树花青素合成的调控相关。     

茶树原位转化方法的建立

解决茶树缺乏高效、稳定的遗传转化体系问题,建立了一种基于根癌农杆菌介导的茶树原位转化转基因方法,为茶树基因功能研究和种质创新奠定坚实的基础。以茶树品种‘铁观音’、‘白叶1号’和‘龙井43’的实生幼苗为受体材料,进行去顶芽和腋芽处理。利用根癌农杆菌菌液侵染实生苗伤口,通过抗性筛选获得再生芽,经分子生物学鉴定后,采用短穗扦插法获得茶树转基因植株。结果表明,再生芽中GUS(β-glucuronidase)和HYG(hygromycin)标记基因经多次PCR检测均为阳性,经测序验证PCR产物序列与标记基因序列一致。‘铁观音’、‘白叶1号’和‘龙井43’的转化率分别为8.14%、2.99%和2.53%。建立了不依赖茶树组织培养的原位转化转基因体系,具有操作简便、转化率高、成本低、试验周期短的特点。     

Pyrophosphate-dependent phosphofructokinase from pollen: properties and possible roles in sugar metabolism

To evaluate the role of pyrophosphate-dependent phosphofructokinase (PFP. EC 2.7.1.90) in the sugar metabolism of pollen. its occurrence and properties were studied in pollen grains of several plants including camellia ( Camellia japonica L.). In all pollen samples, PFP was strongly activated by fructose-2,6-bisphosphate (F2,6BP), and the activity of F2,6BP-activated PFP was higher than that of phosphofructokinase (PFK. EC 2.7.1.11). PFP partially purified from camellia pollen required Mg²⁺ for activity with an optimum at 1 m M . and was almost unaflected by a variety of metabolites at 1 m M . Its molecular mass was around 220 kDa, and apparent K_m values for F6P, PP_i. F1, 6BP and P_i were 294, 4, 20 and 580 u M , respectively. The levels of F2.6BP. PP_i and F6P in camellia pollen were sufficent to support the forward reaction by PFP, and PFP, was 20- to 40-fold more active than PFK during pollen growth. These results suggest that pollen PFP plays a role in glycolysis but not gluconeogenesis. and the possible relevance of this to pollen tube growth is discussed.     

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.     

农林废弃物混配基质的理化性质及其对油茶幼苗生长效应的综合评价

以腐熟的甘蔗渣( SB)、木薯皮( CP)、花生壳( PS)和火炭灰( BA)及园土( GS)为原料,按照不同体积比配制成9种混配基质,并以等体积比泥炭和园土混配基质为对照,对各基质的理化性质及基质中油茶( Camellia oleifera Abel)幼苗的生长状况进行比较;在对基质理化指标和油茶幼苗的壮苗指数进行线性回归分析和通径分析的基础上,采用主成分分析和综合评价法对各基质的育苗效果进行综合评价。结果表明：9种农林废弃物混配基质的容重、总孔隙度和毛管孔隙度分别为0.23~0.47 g·cm-3、60.90%~67.23%和46.53%~58.27%, pH 6.72~pH 7.14,各基质的速效氮含量、速效磷含量、速效钾含量、pH值、电导率和通气孔隙度均高于或显著高于对照,容重则显著低于对照。在不同基质中油茶幼苗的茎粗、单株叶片数、叶长、叶宽、叶绿素相对含量( SPAD)、单株茎叶干质量、单株根干质量和壮苗指数均存在一定差异,而株高和根冠比却无显著差异;其中S9混配基质也V( SB)：V( CP)：V( BA)=2：1：1页中幼苗的大部分生长指标较高,表现出明显的生长优势。线性回归和通径分析结果显示：除通气孔隙度外,基质的其他8个理化指标基本涵盖了影响幼苗壮苗指数的关键因素;其中,速效钾含量是对壮苗指数直接影响最大的负相关因子,而速效磷含量、电导率和总孔隙度对壮苗指数则有较大的正向直接作用,并且三者通过速效钾含量对壮苗指数产生较大的负向间接作用;此外,基质容重对壮苗指数也有一定的负向直接作用。综合评价结果显示：S9和S7混配基质也V( SB)：V( BA)：V( GS)=4：3：3页对油茶育苗效果的综合得分较高,分别为14.363和14.337,建议将S9混配基质作为油茶育苗的首选替代基质,S7混配基质作为备选基质。