Expression of ACC Oxidase Gene from Sugarcane Induced by Hormones and Environmental Force

In the present study, a full-length cDNA encoding 1-aminocyclopropane-1-carboxylic acid oxidase gene has been cloned from sugarcane （named GZ-ACO）. Two primers were designed for coding the ORF in the full-length cDNA of GZ-ACO gene from sugarcane. PCR amplification was performed with sugarcane DNA template, and a fragment of 1 104 bp （GZ34） was obtained. GZ34 was labeled with [α-32P] dCTP as the probe and used for hybridization after cloning and sequencing. Southern blotting analysis indicated that there were at least three other sequences, which weakly hybridized with the GZ34. Northern analysis showed that GZ34 was strongly induced by treatment with IAA, BA, ethephon, LiC1 and cold stress, respectively. As a contrast, the mRNA for ACO gene was at lower levels for both the light-grown and dark-grown plants without additional treatment. There were two transcripts in the dark-grown plants and three transcripts in the treatments with IAA, BA and cold stress, but there was only one transcript in ethephon treatment. It showed that GZ-ACO might be a gene connected with ethylene formation and take part in response to the induction of plant hormone and environmental stress.

Isolation and Characterization of Cucumber ACC Oxidase Gene and Its Upstream

Ethylene has been implicated as a sex-determining hormone in cucumber. Its exogenous application increases femaleness,and gynoecious genotypes were reported to produce more ethylene. 1-aminocyclopropane-1-carboxylate (ACC) oxidase(ACO) is the key enzyme in ethylene biosynthesis. In this study, a 1200 base pair (bp) candidate fragment was amplifiedfrom the cucumber genome with degenerated primers derived from the ACO amino acid consensus sequence amongdifferent plant species. The coding region and its upstream (1 155 bp) were obtained by vector-mediated inverse PCR. Thenovel gene was analyzed by bioinformatics tools. Four exons and three introns were identified in the coding sequence.The spliced length of mRNA was 933 nucleotides (nts) and it encoded 311 amino acids. Phylogenic analysis result of thenew gene (CsACO4, GenBank accession number AY450356) was in accordance with the evolution relationship of geneticsamong various plant species. Northern blotting showed that the gene expressed among female flowers of gynoecious andmonoecious genotypes, it could not express in other organs. This implied that the gene might be correlated with the femalebehavior positively. Further work is on the way to demonstrate the complexity of the relationship between the endogenousethylene and the sex determination.

Propylene Effects on Ethylene Biosynthesis in Relation to Gene Expression of ACS,ACO in Persimmon Fruit

Influences of propylene treatment on fruit softening and ethylene biosynthesis of persimmon genotype, 'Hiratanenashi' were investigated. The treatment with propylene could accelerate the softening and enhance ethylene biosynthesis, with higher sensitivity of immature fruit to the propylene was consistent with mRNA increase derived from transcription of ACS and ACO genes. Furthermore, ethylene synthesis increase in immature fruits was controlled and regulated mainly by DK-ACS1, DK-ACS2, DK-ACO1 and DK-ACO2, but regulated only by DK-ACS1 and DK-ACO1 in mature fruits.

Ethylene Production and 1-Aminocyclopropane-l-Carboxylate （ACC） Synthase Gene Expression in Tomato （ L ycopsicon esculentum Mill.） Leaves Under Enhanced UV-B Radiation

Tomato （Lycopslcon esculentum Mill.） plants grown in a greenhouse were irradiated with two different levels of UV-B, namely 8.82 （T1） and 12.6 kJ/m^2 per day （T2）. Ethylene production, 1-aminocyclopropane-1-carboxylate （ACC） content, 1-（malonylamino） cyclopvopane-1-carboxylic acid （MACC） content, gene expression of ACC aynthase （EC 4.4.1.14）, and ACC oxidase activity in tomato leaves were determined. The results Indicated that ACC content, the activity of ACC synthase and ACC oxidase, and ethylene production Increased continuously under low doses of UV-B radiation, whereas at high doses of radiation these parameters Increased during the first 12 d and then started to decrease. The MACC content increased continuously over 18 d under both doses of UV-B irradiation. The changes in ACC content, ACC synthaae activity, ACC oxidase activity, the transcriptional level of the ACC synthase gene, and ethylene production were consistent with each other, suggesting that ACC synthase was the key enzyme in ethylene biosynthesis and that ethylene production in tomato leaf tissues under UV-B radiation could be regulated by the expression of the ACC synthase gene. The results also indicate that the change in ethylene metabolism may be an adaptive mechanism to enhanced UV-B radiation.

Cloning of ACC Oxidase （ACO） Gene from Dendrobium officinale

Many studies suggest that ethylene plays an important role in regulating metabolite synthesis. Dendrobium plants are traditional Chinese medicine and nowadays its medicinal components are known to be secondary metabolites. In present study, a homolog of ACC oxidase （ACO） gene was isolated from flowers of Dendrobium officinale Kimura et Migo by PCR-method. The obtained cDNA of DoACO is 970 bp long and contains an open reading frame （ORP） encoding a protein with 314 amino acid residues. The DoACO shows high identity to its homologues from other plant species, that has 94.8% closest amino acid sequence of related protein with the ACO from Dendrobium hybrid cultivar. The putative ORP of the obtained sequence could encode a proper protein in respect of molecular weight under T -Lac promoter in E. coli.

Exogenous ethylene influences flower opening of cut roses (Rosa hybrida) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1- aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh- ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-in- duced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhib-ited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.

Cytokinins regulate spatially specific ethylene production to control root growth in Arabidopsis

Two principal growth regulators,cytokinins and ethylene,are known to interact in the regulation of plant growth.However,information about the underlying molecular mechanism and positional specificity of cytokinin/ethylene crosstalk in the control of root growth is scarce.We have identified the spatial specificity of cytokinin-regulated root elongation and root apical meristem(RAM)size,both of which we demonstrate to be dependent on ethylene biosynthesis.Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE(IPT)in proximal and peripheral tissues leads to both root and RAM shortening.By contrast,IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to that of mock-treated controls.We show that cytokinins regulate two steps specific to ethylene biosynthesis:production of the ethylene precursor 1-aminocyclopropane-1-carboxylate(ACC)by ACC SYNTHASEs(ACSs)and its conversion to ethylene by ACC OXIDASEs(ACOs).We describe cytokinin-and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes.Using direct ethylene measurements,we identify ACO2,ACO3,and ACO4 as being responsible for ethylene biosynthesis and ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis.Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2(ARR2),a member of the multistep phosphorelay cascade,and the C-terminal portion of ETHYLENE INSENSITIVE 2(EIN2-C),a key regulator of canonical ethylene signaling,is involved in the cytokinin-induced,ethylene-mediated control of ACO4.We propose tight cooperation between cytokinin and ethylene signaling in the spatially specific regulation of ethylene biosynthesis as a key aspect of the hormonal control of root growth.

Functional mechanism study of the allelochemical myrigalone A identifies a group of ultrapotent inhibitors of ethylene biosynthesis in plants

Allelochemicals represent a class of natural products released by plants as root,leaf,and fruit exudates that interfere with the growth and survival of neighboring plants.Understanding how allelochemicals function to regulate plant responses may provide valuable new approaches to better control plant function.One such allelochemical,Myrigalone A(MyA)produced by Myrica gale,inhibits seed germination and seedling growth through an unknown mechanism.Here,we investigate MyA using the tractable modelDictyostelium discoideum and reveal that its activity depends on the conserved homolog of the plant ethylenesynthesis protein 1-aminocyclopropane-1-carboxylic acid oxidase(ACO).Furthermore,in silico modeling predicts the direct binding of MyA to ACO within the catalytic pocket.In D.discoideum,ablation of ACO mimics the MyA-dependent developmental delay,which is partially restored by exogenous ethylene,and MyA reduces ethylene production.In Arabidopsis thaliana,MyA treatment delays seed germination,and this effect is rescued by exogenous ethylene.It also mimics the effect of established ACO inhibitors on root and hypocotyl extension,blocks ethylenedependent root hair production,and reduces ethylene production.Finally,in silico binding analyses identify a rangeof highlypotentethylene inhibitorsthatblock ethylene-dependent responseand reduce ethyleneproduction in Arabidopsis.Thus,we demonstrate a molecular mechanism by which the allelochemical MyA reduces ethylene biosynthesis and identify a range of ultrapotent inhibitors of ethylene-regulated responses.

花椰菜ACC氧化酶基因的克隆及其RNAi对内源基因表达的抑制作用

根据亲缘关系较近的几种作物ACC氧化酶氨基酸序列,设计一对简并引物,从花椰菜(Brassicaoleraceavar.botrytis)基因组中获得长1202bp的候选片段。序列分析表明该基因含有3个外显子(Exon)、2个内含子(Intron),编码区序列长756bp,编码252个氨基酸。同源性分析发现其与青花菜(Brassicaoleraceavar.Italica)上已发表mRNA序列同源率为99%(GenBank序列号:X81629),推断该基因为花椰菜ACC氧化酶基因,命名为BoACO(GenBank登录号:AY676466)。在此基础上,用BP克隆的方法构建BoACO的RNA干涉(RNAi)载体pHBACO,对花椰菜进行遗传转化,获得卡那霉素抗性转化植株5棵,分子检测证实外源片段成功导入其中3棵花椰菜基因组中。Northern杂交分析显示:转基因植株内源ACO基因转录的mRNA被降解。ACC氧化酶活性分析进一步表明,外源基因的导入大大地降低了ACC氧化酶活性。

康乃馨ACC氧化酶反义基因遗传转化康乃馨的研究

在建立康乃馨从愈伤组织到完整植株的高频再生体系的基础上,用花特异表达启动子驱动的康乃馨ACC氧化酶反义基因通过农杆菌介导法对康乃馨进行遗传转化,获得了3株转基因植株,经多重PCR及PCR-Southem杂交检测,证实康乃馨ACC氧化酶的反义基因已整合进康乃馨的基因组中。

反义ACC氧化酶基因的导入对番茄乙烯生成的抑制作用

将反向克隆的ＡＣＣ氧化酶基因通过Ｔｉ质粒导入到番茄基因组中，转基因番茄植株叶片和果实中ＡＣＣ氧化酶活性和乙烯产生速率均受到显著抑制，显示出反义基因能抑制靶基因（ＡＣＣ氧化酶）的表达。子代抗性标记基因的分离结果表明呈单基因遗传。

ACC氧化酶cDNA克隆及其对美洲黑杨体内乙烯产生的反义抑制

利用ＲＴ－ＰＣＲ技术克隆了番茄ＡＣＣ氧化酶基因ＬＥＥＴＨＹＢＲ编码区０．９ｋｂ的ｃＤＮＡ片段，经酶切图谱分析和序列分析鉴定后，反向插入到植物表达载体ｐＢｉｎ４３８中，构建了表达ＡＣＣ氧化酶反义ＲＮＡ的二元载体。用农杆菌侵染美洲黑杨叶片，在含卡那霉素的ＭＳ培养基上选择转化子和植株再生，通过ＰＣＲ检测筛选到１６株转基因杨树植株，Ｓｏｕｔｈｅｒｎｂｌｏｔ分析初步确证了外源基因是以单拷贝插入到杨树基因组中；对杨树幼苗乙烯释放量的测定结果表明转基因杨树的乙烯释放量为对照植株的２８％。

甘蔗ACC氧化酶基因片段的克隆与序列分析

1 -氨基环丙烷 - 1-羧酸 (ACC)氧化酶是植物乙烯合成的一个关键酶 ,乙烯作为一种内源激素 ,对植物生长、老熟过程有多方面的调节作用。根据报道的各种植物ACC氧化酶氨基酸序列上前后两个保守区设计两个简并引物 ,以甘蔗总DNA为模板 ,通过PCR扩增到一个 940bp的基因片段。将片段序列在NCBI的BLAST软件上进行同源性搜寻 ,显示的 63个序列全部是ACC氧化酶基因 ,因而认为克隆到的片段就是甘蔗ACC氧化酶基因的一个成员。经对不同植物来源的ACC氧化酶基因家族进行比较分析 ,去除一个 10 3bp的“内含子”后 ,推导的氨基酸序列为 2 79个残基 ,占推测全长氨基酸残基总数的 86%左右。经同源性分析 ,序列与毛竹和水稻ACC氧化酶的同源率达到 86%。系统进化分析表明 ,该序列最先与水稻、其次和香蕉的ACC氧化酶聚类 ,然后再与双子叶植物的ACC氧化酶聚类 ,符合按形态特征分类的血缘关系。基因的获得对下一步了解乙烯的合成表达与甘蔗生长。

桃果实ACC氧化酶基因的克隆及序列分析

乙烯是调控果实成熟衰老的内源激素,有效地调控乙烯的生物合成便是控制果实成熟的关键,而ACC氧化酶是植物乙烯生物合成途径中的限速酶之一。本研究以成熟的白粉桃为材料提取果实总RNA,克隆ACC氧化酶基因,旨在利用反义技术抑制乙烯合成,延迟桃果实成熟,提高硬度。将克隆片段与pMD18-T载体连接并转化到E.coliDH5α,经PCR、酶切和测序鉴定。分析表明该基因全长为1246bp,其中编码区长960bp,共编码319个氨基酸,与已登陆的ACC氧化酶基因(AF319166)核苷酸序列和氨基酸序列的同源性分别为99%和98%,表明成功的克隆了ACC氧化酶基因。经生物信息学分析,推测该蛋白的分子式为C1626H2542N420O485S12,相对分子质量为36.12kDa,等电点为5.27;该蛋白为亲水性非分泌型蛋白,无信号肽存在;其蛋白质二级结构以α-螺旋、β-折叠和无规卷曲为主,是一个紧凑型球状结构域。

百合ACC氧化酶基因全长cDNA的克隆及序列分析

以亚洲百合‘Polyanna’（Lilium spp．）花瓣为材料，根据已报道的百合ACC氧化酶基因片段设计1对末端扩增特异引物，采用RACE方法，获得百合ACC氧化酶基因的全长eDNA（GenBank登录号为EU296623）。该eDNA全长1152bp，具有一个954bp的开放阅读框，编码318个氨基酸。Blast搜索结果显示，百合ACC氧化酶基因核苷酸序列与其它植物已报道的ACC氧化酶基因具有71％～82％的相似性，氨基酸序列有70％～87％的相似性，聚类分析表明，与单子叶植物百合科郁金香首先聚类，其次与双子叶植物聚类，最后与单子叶禾本科和兰科植物聚类。

茶树ACC氧化酶基因全长cDNA的克隆与表达分析

ACC(1-氨基环丙烷-1-羧酸)氧化酶是植物乙烯合成过程中的关键酶之一,对乙烯的合成具有重要的调控作用。在茶树新梢cDNA文库测序所获得ESTs基础上,利用RT-PCR技术,克隆得到编码ACC氧化酶基因的全长序列,GenBank登录号为DQ904328。该基因长1232bp,编码320个氨基酸残基,预测分子量为36.2kD,等电点5.41。多序列联配表明茶树ACC氧化酶具有高度保守区域,基于邻接法的进化树显示与柿树ACC氧化酶的亲缘关系最近。对经高、低温后的不同品种进行RT-PCR分析,结果发现ACC氧化酶基因的表达量与品种的抗逆性有一定的相关性。

桃ACC氧化酶基因的克隆和植物表达载体的构建

以‘玉露’桃叶片基因组ＤＮＡ为模板，用ＡＣＣ氧化酶基因特异寡聚核苷酸为引物进行ＰＣＲ扩增，得到１．３ｋｂ左右的扩增产物，将其克隆到ｐＵＣ１９加Ｔ载体上，组建亚克隆并进行序列测定，结果表明，该基因全长有１２８８个碱基，由４个外显子和３个内含子组成。外显子由９５７碱基组成，编码３１９个氨基酸。该基因与桃、番茄、矮牵牛、康乃馨、苹果ＡＣＣ氧化酶ｃＤＮＡ氨基酸序列同源性分别为９９．３％，８３．０％，７６．８％，７４．０％，７５．０％。ＲＮＡ点杂交表明，该基因在未成熟果实检测不到杂交信号，随着成熟度增加，硬度下降，基因表达增强。将ＡＣＣ氧化酶基因分别正向和反向克隆到植物表达载体ｐＢＩ１２１中，并通过酶切分析，ＰＣＲ和点杂交鉴定重组质粒正确。将重组表达质粒导入根癌农杆菌中，经ＰＣＲ和Ｓｏｕｔｈｅｒｎ杂交鉴定证实质粒已被导入。

ACC氧化酶基因研究进展

乙烯作为植物的五大类激素之一,在植物的生长发育过程中发挥着重要作用.而乙烯在植物体内的生物合成主要是由ACC合成酶和ACC氧化酶这2个限速酶来控制的,通过调节它们的表达能有效地调节乙烯的合成量.本文就ACC氧化酶基因克隆及表达调控进行了综述,并对其应用前景进行了展望.

香石竹ACC氧化酶基因的克隆与植物表达载体构建

根据已发表的ACO核苷酸序列 ,设计一对引物 ,以香石竹品种‘American’基因组DNA为模板 ,扩增出香石竹ACC氧化酶基因 ,将其连接到pMD18加T质粒载体上 ,通过中间载体pBluescriptSK ,构建了ACO基因正向和反向插入的植物表达载体 ,并将其导入了农杆菌。酶切检测表明该片段已插入表达载体 ,序列测定结果显示所克隆基因片段含 3个外显子 ,2个内含子 ,其中

甜瓜ACC氧化酶反义基因植物表达载体的构建及转化烟草的研究

用限制性内切酶从目的基因供体质粒pBI-aACO1上切下大小约2.3kb的目的基因,将其定向连接在受体质粒pCAMBIA2301载体上,构建成含有GUS基因的甜瓜ACC氧化酶反义基因植物表达载体pCB-aACO1.采用直接转化法将pCB-aACO1导入根癌农杆菌菌株EHA105,并用新构建的工程菌对普通烟草进行了遗传转化研究.在Kanamycin选择压力下获得的烟草转化不定芽和完整植株,经过GUS基因组织化学法检测以及PCR方法鉴定,证实了该反义基因已导入烟草基因组中.此项研究为下一阶段用该反义基因转化甜瓜品种以改良甜瓜果实耐贮运性打下基础.