To explore flower development molecular mechanism of the Double lily,the AGL6 homologous gene was cloned from Double lily cv. Belonica. The open reading frame of LiAGL6 gene was 744 bp,encoded a protein of 247 amino acids. The LiAGL6 protein molecular weight was 28.3 ku,the grand average of hydropathicity was-0.748 and the theoretical pI was 8.23. The protein structure analysis showed that LiAGL6 protein had a typical MADS-box domain,a K-box region and two AGL6 motifs. Phylogenetic tree analysis showed that LiAGL6 belonged to monocotyledon group,AGL6 branch of the AP1/AGL9 subfamily,and had the highest similarity with Hyacinthus orientalis which was also belonged to monocotyledons,the similarity reached 78%. All the results indicated LiAGL6 was the homologous gene of AGL6,so named it LiAGL6. Cellular localization assay revealed LiAGL6 expressed in the nucleus of onion epidemical cells,which was the basic characteristics of the transcription factors. The qRT-PCR re-sult showed the highest expression of LiAGL6 was in flowers while no expression occurred in leaves and stems. LiA-GL6 was expressed most in the seventh whorl petals followed by the sixth and third whorl petals,however,there was almost no expression in the second whorl petals. The research showed the LiAGL6 gene might play a regulatory role on formation of Double lily flowers.

[目的]克隆百合LiSEP3基因并确定其在百合不同地上器官及不同花瓣中的表达差异,探讨SEP3基因在重瓣花中的表达模式.[方法]利用RACE方法从重瓣百合品种‘比罗尼卡’中克隆得到LiSEP3基因核苷酸序列,利用SMART软件对其蛋白结构进行分析,以MEGA 5.0软件进行系统进化树分析,用基因枪轰击法进行亚细胞定位分析,采用荧光定量PCR进行不同地上器官及不同花瓣中LiSEP3基因表达差异分析.[结果]LiSEP3基因属于MADS-box家族E类基因,其开放阅读框为729 bp,共编码242个氨基酸;LiSEP3基因编码的蛋白具有典型的MADS结构域、K-box区及2个SEP基序;LiSEP3与同为单子叶植物的六出花的SEP3亲缘关系最近;LiSEP3蛋白定位于细胞核中;LiSEP3基因在花中的相对表达量最高,在茎、叶中几无表达;在1～7轮花瓣中均能检测到LiSEP3表达,且位于最内侧的第7轮花瓣中相对表达量最高.[结论]LiSEP3基因属于MADS-box家族E类基因,主要在花中表达,其中在最内侧花瓣中表达量最高,其表达模式与双子叶植物花中SEP3基因表达模式存在不同.

通过RT-PCR与RACE技术从唐菖蒲(Gladiolus hybridus)品种‘Rose Supreme’球茎中克隆茉莉酸生物合成途径中的关键酶——12-氧-植物二烯酸还原酶(12-oxo-phytodienoic acid reductase,OPR)基因的1 489 bp全长cDNA序列,quantitative RT-PCR结果表明,GhOPR3在唐菖蒲叶、花、根、匍匐茎、新球茎和籽球中都表达,其中在籽球和匍匐茎中相对表达量较高;0.1～0.5 mmol.L-1的茉莉酸甲酯(Methyl jasmonate,MJ)处理后,提高了GhOPR3在球茎中的表达量和内源MJ含量;采用农杆菌介导侵染拟南芥花粉,进行GhOPR3基因的过表达分析,过表达拟南芥株系较野生型提高了耐盐性和抗旱性;提高了机械损伤后相关基因的表达水平和内源MJ含量.

Virus-induced gene silencing (VIGS) system could be performed successfully in Gladiolus hybridus with vacuum infiltration of cormels and young plants.

Jasmonic acid (JA) plays a regulatory role in plant development such as tuber and corm formation and their responses to environmental stresses. The action of JA in regulating plant growth and stress responses often requires the elevation of endogenous levels by de novo synthesis. Allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase 3 (OPR3) are the key enzymes involved in jasmonic acid biosynthesis in plants. Here, we isolated the AOS, AOC and OPR3 genes (GhAOS, GhAOC and GhOPR3) in full length from in vitro developing corms of Gladiolus hybridus. The subcellular localization analysis indicated that both GhAOS and GhAOC were chloroplast localization proteins whereas GhOPR3 was localized within the peroxisome. Real-time quantitative PCR showed that GhAOS,GhAOC and GhOPR3 genes were expressed constitutively in all organs with different levels having a relatively higher level in corms and cormels. Additionally, we observed that MJ treatments contributed to increasing the amount of three genes, mRNA level and endogenous MJ content thus promoting corm formation and enlargement. These results revealed that GhAOS, GhAOC and GhOPR3 were genes of significant importance and their expression contributed to JA biosynthesis thus playing an important role in corm formation and enlargement. (c) 2013 Elsevier B.V. All rights reserved.

【Objective】The objective of this study is to analyze the expression pattern of GhAOS and the effect of this gene on JAs biosynthesis in Gladiolus hybridus,and study the response mechanism of the GhAOS gene to damage stress in plant.【Method】The technology of gene gun bombardment was used to analyze the sub-cellular localization of GhAOS.The real time RT-PCR was used to analyze the expression pattern of GhAOS.Agrobacterium-mediated genetic transformation system of Arabidopsis was also used to analyze the overexpression of GhAOS.【Result】The result of sub-cellular localization showed that the GhAOS-GFP fussion protein was targeted into the chloroplast thylakoid membrance.Real time RT-PCR analysis showed that the relatively high expression level of GhAOS was observed in stolons and cormels,and the expression level in corms steadily increased under MJ treatment with a raising concentration gradients from 0.1 mmol·L-1 to 0.5 mmol·L-1.Meanwhile,the endogenous MJ content was also correlated with the expression level of GhAOS.In addition,the salt tolerance and drought-resistance were increased in the overexpression Arabidopsis plants,and the related resistance genes and the endogenous MJ content were increased after mechanical damage.【Conclusion】GhAOS gene promoted JAs biosynthesis in Gladiolus hybridus,and increased the salt tolerance and drought-resistance in Arabidopsis.AOS gene expression may play a major readjustment function on the signal of JA in Gladiolus hybridus,and it may be closely related with the activation of some defense genes.After mechanical damage,the expression level of related resistance genes and the content of endogenous MJ were increased.

To clone the full-length cDNA of GhAOC and analyze the expression patterns of GhAOC and the effect on the biosynthesis of jasmonic acid.The full length cDNA of GhAOC was cloned in Gladiolus hybridus'Rose Supreme'corms by RT-PCR and RACE.The technology of gene gun bombardment was used to analyze the sub-cellular localization of GhAOC.The real time RT-PCR was used to analyze the expression pattern of GhAOC.A full-length cDNA named GhAOC encoding a key enzyme of the biosynthesis of jasmonic acid was cloned in Gladiolus.The open reading frame encompassed 735 bp encoding a polypeptide of 244 amino acids with calculated protein molecular mass of 26.52 ku.The sub-cellular localization analysis indicated that GhAOC was a chloroplast protein.Real time RT-PCR analysis showed that GhAOC gene was expressed in leaf,flower,root,stolon,corm and cormel,and the relatively high expression level of GhAOC was observed in corm and cormel.Meanwhile,the expression level and the endogenous MJ content in corms steadily increased under MJ treatment with a raising concentration gradients from 0.1 mmol/L to 0.5 mmol/L.Based on the results of our pilot study,the expression of GhAOC promoted the the biosynthesis of jasmonic acid in Gladiolus hybridus.

A full length cDNA named LlHSF1,belonging to Heat shock transcription factor(HSF),was cloned from Lilium longiforum‘White Heaven’by RT-PCR and RACE(rapid amplification of cDNA ends). The gene consisted of 1 068 bp and open reading frame encompassed 780 bp encoding a polypeptide of 260 amino acids with calculated protein molecular mass of 30.123 kD. Cluster analysis showed that this gene was highly homologous to known HSF from other organisms. RT-PCR analysis showed that expression of LlHSF1 could be detected in leaf,root and bulb at 22 ℃,and it was expressed highly in the leaf.Theresults with SYBR GREEN real-time quantitative PCR showed that the gene expression level increased in leaves during 1–12 hours at 42 ℃.

Abscisic acid (ABA) is one of the essential hormones involved in many developmental and physiological processes during plant life cycle, such as seed development, bud dormancy, fruit maturation and responses to abiotic stresses. To better understand the physiological role of ABA during Gladiolus hybridus cultivar 'Rose supreme' cormels dormancy release, the key genes involved in ABA biosynthesis (GhNCED) and catabolism (GhCYP707A1 and GhCYP707A2) were isolated and characterized. The deduced amino acid sequence of GhNCED shared high similarity to Crocus sativus NCED, which belongs to the same Iridaceae family as G. hybridus. Furthermore, changes in cormels sprouting. ABA content and GhNCED. GhCYP707A1 and GhCYP707A2 expression patterns during postharvest storage at 4 degrees C and 22 degrees C were investigated. The results showed that 4 degrees C storage condition was more effective to break cormels dormancy than 22 degrees C storage. ABA content gradually decreased to a level significantly in cormels stored at 4 degrees C. The expression of GhNCED in Gladiolus cormels stored at 4 degrees C gradually decreased and the transcription level was lower than cormels stored at 22 degrees C. Similarly, GhCYP707A2 expression level was found significantly higher in cormels kept in 4 degrees C storage temperature as compared to that stored at 22 degrees C. Moreover GhCYP707A1 was almost constantly expressed during storage at both temperature conditions. These findings indicated that ABA content may be co-regulated by GhNCED and GhCYP707A2 gene expression during postharvest storage and dormancy release of Gladiolus cormels. (C) 2012 Elsevier B.V. All rights reserved.

The expression level of GhLOX1 gene was analyzed in the corm of Gladiolus hybridus ‘Rose Supreme’by Real time RT-PCR,which were treated by different concentration of MJ(0.1,0.2, 0.5 mmol·L-1 )and SHAM(0.5,0.75,1.0 mmol·L-1)respectively. In addition,LOX activity, endogenesis MJ content,fresh weight and size of corms were also assayed. Analysis results showed that the expression level of GhLOX1 gene,LOX activity and endogenous MJ content,fresh weight and size of corms increased steadily with a MJ gradient from 0.1 mmol·L-1 to 0.5 mmol·L-1 . Conversely,these indexes steadily decreased with the increasing concentration of SHAM. At the same time,35S-GhLOX1 over expression vector was constructed,and GhLOX1 gene was transformed into the microtuber slices of potato cultivar‘Zhongshu 3’by Agrobacterium tumefaciens-mediated method. The results showed that the number,size and fresh weight of transgenic potato tubers had been increased to varying degrees.

Plant lipoxygenases (LOXs) is a functionally diverse class of dioxygenases involved in multiple physiological processes such as growth, senescence, and stress-related responses. Previously, based on the analysis of LOX1 activity and corm formation, it was depicted that LOX1 increased jasmonic acid (JA) synthesis and induced enlargement at top of stolon branches, forming cormels in Gladiolus hybridus. Here, LOX gene (GhLOX1) in full length was isolated from developing corms in G. hybridus. Sequence analysis showed that GhLOX1 was a novel LOX1 gene encoding 9-LOX. Transient expression analysis determined GhLOX1 to be a cytoplasm protein. Real time RT-PCR showed that the mRNA level of GhLOX1 gene correlated positively with LOX activity and was highest in cormels. GhLOX1 mRNA level. LOX activity and endogenous methyl jasmonate (MJ) content in corms increased steadily with a MJ gradient from 0.1 mmol/L to 0.5 mmol/L. Conversely, GhLOX1 mRNA level. LOX activity, and endogenous MJ content in corms steadily decreased with the increasing concentration of Salicylhydroxamic acid (SHAM). At the same time, we observed that MJ and SHAM treatments effected both LOX1 mRNA level and LOX activity, leading to promotion and inhibition of corm formation and enlargement, respectively. These data reveal that GhLOX1 is a novel LOX1 gene encoding a cytoplasm 9-LOX protein and its expression would contribute to LOX activity which plays an important role in corm formation and enlargement. In general, GhLOX1 is an ideal candidate for the promotion of corm formation and enlargement by gene manipulation in G. hybridus. (C) 2011 Elsevier B.V. All rights reserved.

抗坏血酸(ascorbic acid,AsA)作为植物细胞内重要的自由基清除剂,是通过参与一系列的氧化还原反应而发挥抗氧化剂的功能.单脱氢抗坏血酸还原酶(monodehydroascorbate reductase,MDHAR)在抗坏血酸-谷胱甘肽循环中,可以将氧化型抗坏血酸(MDHA)还原为还原型抗坏血酸(AsA),在维持植物体内抗坏血酸的正常代谢和植物体氧化还原平衡方面起着重要作用(Chen et al.,2003;2005).

An all-length cDNA of LlAPX gene was cloned from Lilium longiforum‘White Heaven’. The gene consisted of 1 074 bp encoding protein of 251 amino acids. Phylogenetic tree analysis showed that the APX had a highly conservative during evolution. We constructed a plant overexpression vector with LlAPX,and transgeniced to Arabidopsis thaliana. The APX activity in transgenic LlAPX Arabidopsis thaliana increased 4.7–7.8 fold,the AsA content increased 1.5–2.3 fold,and enhanced the salt tolerance in transgenic LlAPX Arabidopsis thaliana seeds.

Heat shock transcription factors (Hsfs) are the terminal components of the signal transduction chain mediating the activation of genes responsive to both heat stress and a large number of chemical stressors. This paper aims to clone Hsf from lily and characterize its function by analyses of mRNA expression, transactivation activity and thermotolerance of transgenic Arabidopsis. In this study, the gene encoding HsfA2 with 1,053 bp open reading frame (ORF) was cloned by rapid amplification of cDNA ends (RACE) technique from Lilium longiflorum 'White heaven'. Multiple alignment and phylogenetic analyses showed that the deduced protein was a novel member of the Hsf class A2. Expression analyses by RT-PCR indicated that LlHsfA2 expression was induced by heat shock and H(2)O(2) treatment, but not by NaCl. It was also found that the expression of LlHsfA2 correlated with thermotolerance in Lilium longiflorum 'White heaven' and Oriental hybrid 'Acapulco' under heat stress. Furthermore, yeast one-hybrid assay showed that LlHsfA2 had transactivation activity. In addition, overexpression of LlHsfA2 activated the downstream genes including Hsp101, Hsp70, Hsp25.3 and Apx2 and enhanced the thermotolerance of transgenic Arabidopsis plants. Taken together, our data suggest that LlHsfA2 is a novel and functional HsfA2, involved in heat signaling pathway in lily and useful for improvement of thermotolerance in transgenic plants.