乙烯生物合成关键酶基因研究进展

ACC合成酶(ACS)和ACC氧化酶(ACO)是催化乙烯生物合成的两个关键酶，这两个酶的基因在调控果品和花卉完熟及衰老方面具有重要意义，本文综述了ACS和ACO基因最新研究进展。     

HXKs基因在香蕉果实后熟过程中功能初探

香蕉是世界主要水果之一,对乙烯非常敏感,属于呼吸跃变型果实。目前,香蕉果实后熟机理还未完全探明,后熟过程中己糖激酶(HXK)与乙烯的关系还不清楚。通过BLAST比对从香蕉基因组数据库中发现HXK基因家族的11个成员,经克隆获得这些基因的序列,并研究HXKs基因在香蕉果实自然后熟过程中的转录表达谱,重点研究乙烯利、甘露糖、NAG和1-MCP对HXKs基因表达、HXK酶活和内源乙烯生物合成的影响。结果表明:在香蕉果实后熟过程中HXKs基因呈现差异性表达,乙烯利上调大多数HXKs基因的表达和HXK酶活,1-MCP的作用正好相反,这表明外源乙烯正作用于HXK。另一方面,甘露糖加快内源乙烯生物合成,NAG却推迟内源乙烯生物合成,这说明HXK正作用于内源乙烯生物合成。所以,在香蕉果实后熟过程中乙烯和HXK之间存在相互促进的关系。这将为进一步阐明香蕉果实后熟机制提供理论依据,也将为香蕉果实保鲜新技术的挖掘提供新思路。     

香蕉MuMADS1与泛素激活酶(MuUBA)在采后果实中的相互作用

以香蕉MuMADS1为诱饵载体,采后2 d的香蕉果实的cDNA文库为猎物,采用酵母双杂交的方法得到了泛素激活酶E1的基因片段,命名为MuUBA。采用双分子荧光互补技术进一步验证MuMADS1与MuUBA在植物体内的相互作用。荧光实时定量PCR结果表明,MuMADS1和MuUBA在香蕉中子房发育第4个阶段的表达量最高,但是在茎中的表达量很低,表明其在不同的组织和发育的果实中的表达具有协同性。MuMADS1和MuUBA的表达都受外源乙烯和1-MCP的高度调控,推测MuMADS1和MuUBA在香蕉果实的采后成熟过程中具有很重要的作用。     

Determination of Antioxidant Constituents in Cactus Pear Fruits

An analytical study was carried out on the presence of antioxidant constituents and the in vitro antioxidant capacity in the extracts of three species of Spanish red-skinned cactus pear fruits (Opuntia ficus-indica, Opuntia undulata and Opuntia stricta). The cactus pear fruit extracts were analyzed for determined constituents: ascorbic acid, flavonoids (quercetin, isorhamnetin, myricetin, kaempferol and luteolin), betalains, taurine, total carotenoids and total phenolics. The antioxidant capacity was assessed by means of two different methods: the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (Trolox equivalent antioxidant capacity) method and the 2,2-diphenyl-1-picrylhydrazyl radical method. Opuntia ficus-indica fruit extract had the strongest antioxidant capacity and taurine content. O. stricta fruits were the richest in ascorbic acid and total phenolics, whereas O. undulata fruits showed the highest carotenoid content. Quercetin and isorhamnetin were the main flavonoids detected. This study provides basic information on the presence of bioactive compounds and antioxidant capacity in extracts of cactus pear fruits, in order to consider these extracts as ingredient for the production of health-promoting food.     

Crises respiratoires et maturation chez deux fruits (la poire et la cerise) cueillis à divers stades et conservés ensuite à temperature constante,en présence ou non d'éthylène

Respiratory behaviour and response to ethylene of two fruits was compared during growth and maturation. The first one, the cherry (var. Bigarreau Napoleon) is ‘non climacteric’. The second one, the pear (var. Passe-Crassane) is ‘climacteric’ and needs a certain period of cold for its usual maturation. Young cherries exhibit a natural respiratory rise after 4 or 5 days of storage but young pears do not. Breaf ethylene treatments involve a rise of respiration, more intense with the pear. As for ‘adult’ fruits the respiratory response varies but the effect on ripening is not established and, in our conditions, does not seen to be really significative. The following question can be asked. What are the relations between respiration (with or without a climacteric rise), the different ripening phenomena and the action and role of ethylene? More generally, it would be very interesting to study, in a comparative way, the ripening of climacteric and non climacteric fruits, with a special look at the factors promoting, delaying or preventing the normal course of ripening.     

菠萝蜜果实PG基因的克隆与表达分析

为明确PG基因在菠萝蜜果实后熟软化过程中的作用,本研究以‘海大2号’菠萝蜜果实为材料,采用0.5 mg/L 1-MCP和1000 mg/L ETH处理,研究了室温（20℃）条件下果实成熟过程中硬度和果胶的动态变化,克隆获得4个PG基因,并对其进行了生物信息学和表达分析。结果表明：随着菠萝蜜果实的成熟,果肉硬度快速下降,可溶性果胶和离子型果胶不断增加,共价态果胶有所下降;ETH处理促进了果实WSP和ISP含量的上升,加速了软化进程,1-MCP处理抑制了贮藏前期果肉硬度的下降,推迟了软化进程,但明显提高了3种果胶的含量。AhePG1~AhePG4基因的开放阅读框（ORF）长度1221~1434 bp,编码406~477个氨基酸。AhePG1蛋白含有4个保守结构域（Ⅰ~Ⅳ）,AhePG2和AhePG3只含结构域Ⅰ和Ⅱ,AhePG4缺失结构域Ⅲ;AhePG基因分别与桃（AF095577.1）、菜豆（XM_007162208.1）、葎草（MN971583.1）、菜豆（XM_007151391.1）PG基因编码的氨基酸序列的亲缘关系较近,相似度分别达75.63%、73.11%、79.71%、70.75%。qRT-PCR分析结果显示：AhePG1基因在果实成熟前期表达量低,在后期高表达,而AhePG2/3/4基因的表达量在果实成熟过程中总体较低。ETH处理抑制了AhePG1基因的表达量,而1-MCP处理延缓了4个AhePG基因表达量的增加,但增加了成熟后期AhePG2、AhePG3、AhePG4基因的表达。相关性分析发现,果肉硬度与水溶性果胶含量和AhePG1基因表达呈显著和极显著负相关,而水溶性果胶含量又与AhePG1基因表达呈显著正相关。本研究说明,菠萝蜜果实的软化与果胶降解有关,AhePG1可能是菠萝蜜果实果胶降解和果实软化的关键PG基因之一,控制着果实成熟后期的软化;1-MCP处理能延缓菠萝蜜果实的成熟,但并不影响果实后期成熟时的软化,AhePG1、AhePG2、AhePG3、AhePG4基因可能对其软化均有作用。     

2个14-3-3蛋白基因在香蕉果实成熟过程中的表达分析

通过简并引物和降落PCR方法结合RACE(rapid amplification of c DNA end)技术在香蕉果实c DNA文库中获得2个14-3-3基因,分别命名为Ma14-3-3d和Ma14-3-3h,把Ma14-3-3d和Ma14-3-3h与Ma14-3-3a、Ma14-3-3c、Ma14-3-3e、Ma14-3-3i进行多重序列比对和同源性比较。结果表明:Ma14-3-3d和Ma14-3-3h与Ma14-3-3a、Ma14-3-3c、Ma14-3-3e、Ma14-3-3i具有较高的同源性,同时具有一定的差异性。遗传进化分析结果表明,Ma14-3-3d与Ma14-3-3a、Ma14-3-3c、Ma14-3-3e、Ma14-3-3i同属于non-ε类14-3-3基因,Ma14-3-3h属于ε类14-3-3基因。RT-PCR分析结果表明,Ma14-3-3d和Ma14-3-3h在香蕉不同器官中差异表达,Ma14-3-3d在香蕉的根、茎、叶、花和果中均有表达,且在茎、叶和花中的表达量高于根和果;Ma14-3-3h在根、花和果中的表达量较高,而在茎和叶中的表达量较低。q RT-PCR分析结果表明,Ma14-3-3d基因的表达明显受乙烯的诱导,而Ma14-3-3h在正常成熟、乙烯处理与1-MCP处理的果实中表达量均很低,与果实成熟的相关性不明显。推测Ma14-3-3d可能与香蕉果实成熟密切相关,可能参与乙烯调控果实成熟过程中的生物合成与信号转导。     

柚多聚半乳糖醛酸酶基因家族的鉴定和分析

草莓、桃、苹果等果实在生长后期和贮藏过程中逐渐软化,而柑橘类特别是柚在成熟后期和贮藏过程中出现粒化,严重影响其经济价值.本研究分析和鉴定了柚基因组中多聚半乳糖醛酸酶基因(CgPG)家族成员以及PG酶活的变化,旨在揭示柚果实成熟过程中CgPG表达与汁胞粒化形成的关系.本研究对柚基因组库中CgPG基因家族成员的数量、基因定...     

香蕉乙烯受体基因cDNA的克隆及其表达分析

提取香蕉果实总RNA，根据有关文献报道的乙烯受体基因序列设计引物，通过RT-PCR方法扩增其开放读框(open reading frame，ORF)，结果同时扩增出2个长短不同的特异cDNA序列。测序结果表明：其中较长的cDNA序列与该报道的香蕉乙烯受体cDNA序列的对应区段(ORF)长度一致，同源性为99．1％；另一较短cDNA序列为新序列，其与该报道序列的同源性达97．2％，但缺少该报道序列中的19和1036 bp的区段。RT-PCR扩增结果显示，报道的cDNA序列在香蕉果实的几个成熟阶段均有表达，而在根和叶片等组织中检测不到其表达，说明其表达具有果实组织特异性。Southern杂交结果显示，该报道基因序列在香蕉基因组中为单拷贝存在。因此，推测新克隆的缺失cDNA序列可能来自同一基因的转录后剪辑，其可能为1个新的香蕉乙烯受体基因。     

不同催熟条件对香蕉后熟均匀性的影响研究

粉、蔗糖、果糖和葡萄糖含量作为参考指标,研究采收后贮藏时间、催熟时间和催熟温度对2种香蕉果实后熟速度的影响。结果表明,采收后仅贮藏5 h的香蕉,在22 ℃、12 h乙烯催熟处理的条件下,尾蕉的成熟指数、表皮颜色、可溶性总糖和葡萄糖的含量和头蕉相比有显著差异,尾蕉成熟速度显著低于头蕉;22 ℃下贮藏5 d后再进行乙烯催熟处理,头蕉和尾蕉的成熟速度基本一致;适当提高催熟温度或延长催熟时间至24 h,也能提高头蕉和尾蕉的后熟一致性     

Composition of pulp,skin and seeds of prickly pears fruit (Opuntia ficus indica sp.)

The proximate composition of pulp, skin and seeds of prickly pear cactus (Opuntia ficus indica) was investigated and is reported on a dry weight basis. The most abundant component of the pulp and skin was ethanol-soluble carbohydrates. Pulp contained glucose (35%) and fructose (29%) while the skin contained essentially glucose (21%). Protein content was 5.1% (pulp), 8.3% (skin) and 11.8% (seeds). Starch was found in each of the three parts of the fruit. Pulp fibers were rich in pectin (14.4%), skin and seeds were rich in cellulose (29.1 and 45.1%, respectively). Skin was remarkable for its content of calcium (2.09%) and potassium (3.4%). Prickly pear is a neglected nutritional source which should be more widely used because of its potential nutrient contribution.     

番木瓜NAC转录因子的克隆与表达分析

为探究番木瓜NAC转录因子的序列特征及功能,以‘大庆7号’番木瓜果肉为试验材料,采用RTPCR克隆出2个不同的NAC类基因,命名为CpNAC1(Gene Bank KT364871)和CpNAC2(Gene Bank KT372241),其开放阅读框(ORF)长度分别为609 bp和805 bp,分别编码202个和268个氨基酸,其N端含有NAM保守结构域。采用实时荧光定量PCR研究其在乙烯利及清水对照处理后果实不同成熟时期中的表达情况,结果发现,CpNAC1和CpNAC2基因随着处理后时间的增加,表达量呈先下降后缓慢上升的趋势,且均与果实成熟呈负相关。但CpNAC1表达趋势与果实成熟过程中乙烯的表达量相反,受乙烯抑制降低表达量,从而参与了番木瓜果实的成熟衰老进程,而CpNAC2基因在乙烯处理后番木瓜果实中表达量与对照处理相比没有显著变化,说明CpNAC2基因不是通过乙烯信号传导途径来调控果实成熟。     

Genetic stability of long-term micropropagated Opuntia ficus-indica (L.) Mill. plantlets as assessed by molecular tools: Perspectives for in vitro conservation

Randomly amplified polymorphic DNA markers (RAPD) were employed to assess the level of genetic stability of long term micropropagated prickly pear (Opuntia ficus-indica) plantlets.Thirteen micropropagated plantlets were chosen from a clonal collection of shoots that originated from a single mother shoot. This clonal collection had been maintained under in vitro culture conditions for at least 5 years, as achieved for the time by axillary branch multiplication in Opuntia ficus-indica.Twenty arbitrary primers were used to compare RAPD patterns between in vitro raised material and the mother plant. Only 11 primers were found to yield distinct and reproducible amplification products resulting in a total of 87 amplified products, out of which 82 bands were monomorphic across all the plantlets and 5 showed polymorphisms.Cluster analysis performed on the basis of similarity indices indicated that all micropropagated plantlets and their mother plant grouped together in one major cluster with a 91% level of similarity.Low level of genetic variation has been detected, as polymorphic bands accounted for just 2.79% of the total genetic variation. This very low level of genetic variation, despite more than 5 years of in vitro culture, demonstrates the genetic stability of Opuntia ficus-indica and indicates that the axillary branch multiplication method is highly reliable for the multiplication of genetically true-to-type plant material.The high degree of clonal fidelity detected here, recommend the use of axillary-branching micropropagation technique for the safe in vitro conservation of prickly pear interesting genetic resources.     

1-MCP处理对‘油木奈’果实采后生理和品质的影响

研究1.2 μL/L的1-甲基环丙烯（1-methylcyclopropene,1-MCP）处理对在（25±1）℃下贮藏的‘油木奈’（Prunus salicina Lindl. cv. Younai）果实采后生理和品质的影响。结果表明,与对照果实相比,1-MCP处理可有效降低‘油木奈’果实呼吸强度、呼吸峰值和乙烯释放量,延缓果实细胞膜相对渗透率升高和果实表面色调角h°值的下降,保持较高的果实硬度、可滴定酸和果皮叶绿素含量,延迟果实外观颜色转变,减少果实失重和腐烂。经1-MCP处理的果实在（25±1）℃下贮藏15 d时的好果率为80％,而对照果实只有58％。因此认为,1-MCP处理可以延缓采后‘油木奈’果实后熟衰老和保持果实品质,延长果实保鲜期。     

Biochemical and Nutritional Characterization of Three Prickly Pear Species with Different Ripening Behavior

Biochemical and nutritional changes were studied during the ripening process of three Opuntia morphospecies with different ripening behavior: Naranjona (O. ficus-indica), Blanca Cristalina (Opuntia sp.), and Esmeralda (Opuntia sp.) of early, early-intermediate, and intermediate-late ripening, respectively. In loss of fresh weight, Naranjona showed the highest values, while in Blanca Cristalina and Esmeralda, a discrete weight loss was found. No significant differences were found among morphospecies in soluble solids, total titratable acidity and pH during the postharvest days. Blanca Cristalina and Esmeralda showed an increase in the content of carotenoids, while these diminished in Naranjona. The cell wall enzymes evaluated showed particular behaviors during the ripening of each morphospecies suggesting a fine biochemical control and not a clear relationship between fruit softening and enzyme activity. This study provides basic information on prickly pear ripening, in order to understand this process for its control and for improving shelf life.     

温度和乙烯处理对番荔枝软熟过程中多糖代谢的影响

以AP(African Pride)番荔枝冬季果为试材,研究不同温度、乙烯利处理下,番荔枝果实淀粉、可溶性糖、蔗糖等糖类以及果胶含量和细胞壁代谢相关酶(多聚半乳糖醛酸酶、纤维素酶、果胶甲酯酶)活性的变化,以期从多糖代谢角度探讨温度和乙烯对番荔枝冬季果软熟的影响。结果表明:低温(20℃)可延缓淀粉向可溶性糖的转化,高温(32℃)可促进淀粉向可溶性糖的转化,而28℃下番荔枝软熟最快。28、32℃下果实果皮变硬可能与果皮中原果胶的合成有一定关系。果实的软化与果肉中的原果胶降解有关,但和可溶性果胶含量上升无必然联系。温度和乙烯对酶活性均有不同程度影响,其中Eth-28℃处理对酶活性诱导最显著。Cx和PG活性的变化与果实软化最相关,而PME在果实软化中可能不起关键作用。      

菠萝PEPC基因家族生物信息学分析

磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase, PEPC）在C₄和景天酸代谢（CAM）光合途径吸收CO₂过程中发挥重要作用,并参与各种非光合作用过程,包括果实成熟、气孔开放、碳氮相互作用、种子形成和萌发以及调节植物对逆境胁迫的耐受性。菠萝为典型的景天酸代谢途径（CAM）植物,为了解磷酸烯醇式丙酮酸羧化酶（PEPC）在菠萝景天酸代谢途径（CAM）中的作用,本研究鉴定到3个菠萝PEPC基因,按照基因描述命名为AcPEPC1、AcPEPC3和AcPEPC4,进化树分析结果AcPEPC1和AcPEPC3为植物型PEPC（PTPCs）蛋白,序列同源性较高且基因结构、保守结构域及保守基序均一致。AcPEPC4为细菌型PEPC（BTPCs）蛋白,与AcPEPC1/AcPEPC3间的序列同源性低。组织表达分析表明AcPEPC1菠萝叶片表达量较高,而AcPEPC3和AcPEPC4在菠萝花和果实表达量较高。     

番茄甾体生物碱合成酶基因SlERT1b的功能验证及转录调控

绿色番茄中的甾体生物碱主要以α-番茄碱（α-tomatine）形式存在,使得果实带有苦味而不适宜食用。在果实成熟过程中,α-番茄碱会通过一系列酶促反应生成七叶皂苷A（esculeoside A）,使果实适宜食用,然而这一代谢途径尚未被完全解析。本课题组在先前研究中鉴定出一个参与这一代谢途径的糖基转移酶SlERT1b,本研究通过番茄的遗传转化实验研究SlERT1b的功能,并利用LC-MS检测其中的甾体生物碱,验证该基因可发挥糖基转移酶的功能。此外,施加外源乙烯和乙烯抑制剂1-MCP后,发现乙烯可调控SlERT1b基因,并影响甾体生物碱的合成途径。同时鉴定出可以调控SlERT1b基因的转录因子SlJERF1和SlAREB2。该研究结果为今后番茄遗传育种提供理论基础。     

番茄泛素活化酶基因家族进化及表达模式分析

泛素活化酶是蛋白泛素化所需的第一个酶,在泛素蛋白酶体途径中发挥重要作用。本文利用生物信息学方法从番茄基因组中鉴定出2个泛素活化酶(ubiquitin activating enzyme,UBA)基因,命名为Sl UBA1和Sl UBA2。序列分析表明Sl UBA1和Sl UBA2基因的CDS序列长分别为3 060和3 255 bp,分别编码1 019和1 084个氨基酸,编码蛋白分子量为114.15和120.46 ku,分别位于第6和9染色体。2个基因均含有5个内含子,编码蛋白均为酸性、疏水性蛋白;对蛋白二级结构分析发现2个UBA蛋白均以α-螺旋和无规则卷曲为主;亚细胞定位预测均定位于细胞核。序列比对和进化树分析表明番茄UBA基因与其他物种UBA基因相似性高,进化过程非常保守。番茄不同组织表达分析结果表明2个UBA基因在根系和花的表达量较高,果实成熟过程中的表达量相对较低。非生物胁迫试验结果表明:Sl UBA1基因在低温、干旱和盐胁迫下均上调表达;而Sl UBA2基因对非生物胁迫没有明显响应,这些结果表明Sl UBA1基因可能参与番茄对低温、干旱和盐胁迫的响应。