京白梨果实采后PG、糖苷酶和LOX活性变化及其基因表达特性

 以京白梨为试材,测定贮藏期间果实多聚半乳糖醛酸酶（PG）、糖苷酶（β-Gal、α-L-Af）及脂氧合酶（LOX）活性及其基因表达变化及对1-MCP和低温的响应。结果表明,果实软化呈慢（3 d内）、快（3 ~ 9 d）、慢（9 d后）的变化趋势;PG、β-Gal、α-L-Af和LOX活性及其基因表达量均随果实软化呈不同程度增加,且β-Gal和LOX的活性均在果实采收后3 d内上升快,其基因的表达量亦迅速增加,由此认为β-Gal和LOX可能在果实软化早期起着更为重要的作用。经1-MCP处理和冷藏后,β-Gal、α-L-Af、LOX和PG的活性及基因表达显著受抑,延缓了果实软化进程,且1-MCP对β-Gal和LOX的抑制作用最强烈,尤其在处理前期。在果实冷藏过程中PG和β-Gal活性基本维持原有水平,其基因表达在贮藏前期受到抑制,LOX活性和基因表达均维持在极低水平,说明LOX对低温较其它酶敏感。因此认为,PG、β-Gal、α-L-Af和LOX均不同程度地参与了京白梨果实的后熟软化,在早期软化阶段β-Gal和LOX可能起着更为重要的作用,1-MCP对β-Gal和LOX活性和基因表达具有更显著的调控作用,而低温可能主要是通过抑制LOX活性和基因表达来抑制果实软化。     

白菜BcMF2基因在大肠杆菌中的高效表达

通过PCR方法扩增BcMF2cDNA完整的编码区序列,构建pETBcMF2重组质粒,转化大肠杆菌BL21(DE3),IPTG诱导融合蛋白高效表达;SDSPAGE电泳检测发现在70kD处有一条蛋白质特异条带,与预期的目的产物蛋白带大小一致,同时发现,其阳性克隆在30℃和IPTG终浓度为0.4～1.0mmol·L-1时诱导6h,均能获得较高的表达量。     

The manipulation and modification of tomato fruit ripening by expression of antisense RNA in transgenic plants

Summary The common cultivated tomato (Lycopersicon esculentum Mill.) provides a major focus for improvement of crop quality through genetic engineering. Identification of ripening-related cDNAs has enabled the modification of specific aspects of ripening by manipulating gene expression in transgenic plants. By utilizing antisense RNA to modify expression of ripening genes, we have inhibited the production of the cell wall-metabolising enzymes polygalacturonase and pectinesterase and created transgenic plants that contain, effectively, single, targeted mutations affecting these genes. Furthermore, this approach has been used with previously unidentified cDNA clones to enable both functional identification and manipulation of genes involved in ethylene production (ACC oxidase) and carotenoid biosynthesis (phytoene synthase). The use of antisense RNA targeted to specific genes to alter ripening phenotypes and improve commercial utility of fruit by affecting shelf-life, processing characteristics and nutritional content is discussed.We have used the extreme ripening-impaired mutant, ripening inhibitor (rin) to identify additional genes implicated in the ripening process. This approach has resulted in the cloning of several novel ripening-related mRNAs which are now being studied by antisense experiments. This may enable identification and manipulation of additional genes involved in processes such as softening, flavour and aroma generation and susceptibility to pathogens.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - PE pectinesterase - PG polygalacturonase - SAM S-adenosyl methionine - SARs scaffold attachment regions     

Control of ripening in transgenic tomatoes

Major progress has been made over the last few years in the identification and regulation of tomato ripening genes. At least 25 genes showing elevated expression during ripening have been cloned and several, including polygalacturonase, which modifies fruit textures, have been shown to be ripening-specific. In addition, genes have been cloned for ACC synthase and ACC oxidase, which control the synthesis of ethylene, which plays a critical role in ripening. Inhibition of expression of polygalacturonase, pectinesterase, ACC synthase, ACC oxidase and phytoene synthase has been achieved in transgenic plants, using antisense technology. The expression of several genes has also been inhibited by sense gene suppression. New traits caused by these transgenes are stably inherited. Antisense tomatoes with reduced polygalacturonase have improved textural qualities which are being exploited commercially for the fresh and processed markets. Overexpression of phytoene synthase has been shown to restore carotenoid production in the yellow flesh mutant and can be used to enhance colour in other cultivars. Antisense fruit in which ACC synthase or ACC oxidase are inhibited show slower ripening and reduced over-ripening. ACC oxidase antisense genes have also been shown to delay leaf senescence. It is to be expected that further genes determining other quality traits will be identified and manipulated soon.     

柑桔果实浮皮发生机理及控制途径研究Ⅱ．特早熟温州蜜果实浮皮与衰老的关系

为探明柑桔果实浮皮与衰老的关系，以特早熟温州蜜柑品种胁山为试材，采前对其幼果和枝叶喷施赤霉素、亚精胺、醋酸钙，测定果皮及果肉的多聚半乳糖醛酸酶（PG）、超氧化物歧化酶（SOD）和丙二醛（MDA）等生理生化指标。结果表明：胁山品种随着果实浮皮的出现，PG活性提高，果皮的细胞膜脂质过氧化作用产物MDA逐渐积累，而果皮和肉的SOID活性则急剧下降。胁山果实浮皮的根本原因是膜脂过氧化作用引 果皮早衰。采前喷施赤霉素、亚精胺、醋酸钙对MDA含量，PG，SOD活性均有一定调节作用，其中以赤霉素效果最佳。     

苹果全基因组多聚半乳糖醛酸酶基因家族的鉴定及进化分析

从苹果全基因组中鉴定出85个多聚半乳糖醛酸酶(PG)基因,通过聚类分析将其分成了7个组(Group A~Group G),其分子量在176~1 125 aa之间,等电点在4.68~9.58之间。鉴定出来的Md PG基因除在14号染色体没有分布外,其余都有分布。系统分析了Md PG蛋白的保守基序和Md PG基因结构,发现苹果PG蛋白除包含有广泛存在于各种PG蛋白的4个保守基序以外,还含有其他相对特异的保守基序,Md PG75具有最多的外显子数,达到18个。对85个苹果PG蛋白之间的功能联系网络进行了系统研究,分析预测了相关基因的功能和多个基因间功能的联系,并作了初步验证。     

番茄果实软化过程中钙处理对多聚半乳糖醛酸酶、脂氧合酶、伸展蛋白的影响

为探明番茄果实的一些生理特性,研究了4% CaCl2 溶液真空渗透处理的番茄果实,在软化过程中多聚半乳糖醛酸酶(PG)、脂氧合酶(LOX)、伸展蛋白(EXP)的变化。结果表明:在处理初期Ca2+ 对LOX活性及LeLOXB基因的表达有显著的抑制作用;在处理的后期,Ca2+ 对PG活性及PG、LeEXP1 基因表达有明显的抑制作用。并就Ca2+对PG、LOX、EXP及果实软化机理的影响进行了讨论。     

柿果实采后软化中PG酶活性及其基因DkPG1的表达

为了进一步探索多聚半乳糖醛酸酶（PG）在柿（Diospyros kaki L.）采后软化中的分子调控机制,应用实时荧光定量PCR技术和DNS比色法检测常温下丙烯和1–甲基环丙烯（1-MCP）处理‘富平尖柿’果实中PG基因DkPG1的表达量和PG酶活性的变化。结果显示,随着柿果实成熟软化,DkPG1基因转录水平呈先上升后下降的趋势,其中丙烯处理4 d出现表达高峰,比对照提前12 d且峰值极显著高于对照,而1-MCP处理的果实中的表达高峰出现在处理后28 d,比对照推迟12 d峰值极显著低于对照。丙烯处理果实PG酶活性迅速增加,活性峰与DkPG1表达高峰出现在同一天,1-MCP处理的果实PG酶活性明显降低,活性峰出现在DkPG1表达高峰前4 d。此外,对不同处理下乙烯释放量和果胶组分变化分析的结果显示DkPG1基因的表达受乙烯调控,进而影响果胶组分代谢。     

EDTA和金属离子对多聚半乳糖醛酸酶酶活力的影响及其动力学研究

实验以EDTA和不同金属离子为效应物,果胶为反应底物,研究不同浓度的EDTA,Na+,K+,Ca2+,Cu2+,Zn2+,Mn2+,Fe2+,Fe3+,Ag+和Ba2+对多聚半乳糖醛酸酶(PG)催化活性的影响。结果表明,一定浓度的Cu2+,Zn2+,Mn2+,Fe2+和Fe3+对PG有激活作用,对PG产生最大激活作用的浓度分别为0.3mmol/L,0.4mmol/L,0.3mmol/L,0.3mmol/L和0.6mmol/L,EDTA,Ca2+,Ag+和Ba2+对PG有抑制作用,Na+和K+对PG活性没有影响。动力学研究表明,0.3mmol/LNa+和K+处理后,PG的Km和Vmax基本没有发生变化,2mmol/LEDTA和0.3mmol/LCa2+处理后,PG的Km和Vmax均下降,0.3mmol/LCu2+,Zn2+,Mn2+,Fe2+,Fe3+处理后,PG的Km和Vmax都上升,0.3mmol/LAg+和Ba2+处理后,PG的Km上升,Vmax下降。     

绿色木霉菌对丝核菌多聚半乳糖醛酸酶活性的影响

就丝核菌(Rhizoctonia)胞外多聚半乳糖醛酸酶(Polygalacturonase,PG)及绿色木霉菌(Trichoderma viride)对PG活性的影响进行了研究.结果表明,双核禾谷丝核菌(Rhizoctonia cerealis)AG-D融合群WK-47、WK-141、WK-160菌株,多核立枯丝核菌(R.solani)AG1-IA融合群YW-2菌株的胞外PG有较高的活性,不同核型的丝核菌胞外PG的活性差异显著;绿色木霉菌TCS-1菌株不同时间的发酵液对PG的活性具有明显的抑制作用.