Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect

Introduction of a constitutive antisense full-length chalcone synthase (CHS) cDNA gene in petunia can result in an inhibition of flower pigmentation. We have evaluated some of the factors which may be important for the effectiveness of an antisense CHS gene.Antisense CHS genes encoding half-length or quarter-length RNA complementary to the 3 half of CHS mRNA are able to affect flower pigmentation, while a gene encoding RNA complementary to the 5 half of CHS mRNA did not show phenotypic effects in transgenic petunia plants. We demonstrate that the RNA encoded by the latter gene has a much lower average steady-state level in leaf tissue than the RNAs encoded by the other antisense gene constructs. We have compared the CaMV 35S and endogenous CHS promoter strengths and intrinsic stabilities of sense and antisense CHS RNAs. From the data we conclude that the constitutive antisense CHS genes are not likely to provide an excess of antisense RNA compared to the CHS mRNA derived from the endogenous genes.Effective inhibition of flower pigmentation is also observed when the antisense CHS gene is under control of the homologous CHS promoter. The results indicate that the mechanism of antisense inhibition cannot solely operate via RNA duplex formation between sense and antisense RNA.     

Physical, chemical and microscopic characterization of a new starch from chayote (Sechium edule) tuber and its comparison with potato and maize starches

In this work, the chayote tuber starch (CHS) was isolated and its chemical composition and its physical and microscopic characteristics were determined, and compared with potato (PS) and maize (MS) starches. The starch content in chayote tubers (728 g kg⁻¹ dry weight) was similar to potato tubers (700 g kg⁻¹ dry weight), with a high level of purity (>98%), while its phosphorous content was higher (0.15%) than PS (0.08%) and MS (0.01%). Starch granules were oval, irregular, truncated and rounded with sizes between 7 and 50 μm with smooth surfaces. CHS dispersions (1% and 4%, w/w) showed higher viscosity (75 and 1715 mPa s), than PS (350% and 50% lower) and MS (715% and 600% lower). The gelatinization temperature (65 to 74 °C) was similar in CHS and PS. The pasting properties (RVA) of the starches suggest that CHS showed better characteristics than the commercial potato and maize starches. Therefore, CHS could be used as a thickening agent and a substitute to PS in food dispersions where a high viscosity is needed.     

甜荞查尔酮合酶基因Chs的克隆及序列分析

利用RT-PCR技术从甜荞中克隆得到查耳酮合酶(CHS)的cDNA开放阅读框(ORF)序列,命名为FeChs,NCBI登录号为GU172166.1.该序列长1 179 bp,编码392个氨基酸,与其它植物CHS基因的同源性为78%～92%,其推导的氨基酸序列含有CHS高度保守的活性位点及CHS的标签序列GFGPG.     

Five Nodulation Mutants of White Sweetclover (Melilotus alba Desr.) Exhibit Distinct Phenotypes Blocked at Root Hair Curling,Infection Thread Development,and Nodule Organogenesis

Flavonol aglycones are required for pollen germination in petunia (Petunia hybrida L.). Mutant plants lacking chalcone synthase (CHS), which catalyzes the first committed step in flavonoid synthesis, do not accumulate flavonols and are self-sterile. The mutant pollen can be induced to germinate by supplementing it with kaempferol, a flavonol aglycone, either at the time of pollination or by addition to an in vitro germination system. Biochemical complementation occurs naturally when the mutant, flavonol-deficient pollen is crossed to wild-type, flavonoid-producing stigmas. We found that successful pollination depends on stigma maturity, indicating that flavonol aglycone accumulation may be developmentally regulated. Quantitative immunoblotting, in vitro and in vivo pollen germination, and high-performance liquid chromatographic analyses of stigma and anther extracts were used to determine the relationship between CHS levels and flavonol aglycone accumulation in developing petunia flowers. Although substantial levels of CHS were measured, we detected no flavonol aglycones in wild-type stigma or anther extracts. Instead, the occurrence of a conjugated form (flavonol glycoside) suggests that a mechanism may operate to convert glycosides to the active aglycone form.     

Cloning of cDNA for UDP-glucose pyrophosphorylase and the expression of mRNA in rice endosperm

Rice endosperm UDP-glucose pyrophosphorylase (UGPase) cDNA clones were isolated by screening a lambda ZAP II library prepared from poly (A(+)) RNA of japonica rice (cv Sasanishiki) endosperm with a probe of potato UGPase cDNA. One cDNA clone, possessing about 1,700 nucleotides, contained the complete open reading frame of rice UGPase. At the nucleotide-sequence level, the UGPase cDNA of rice endosperm had high homology with the UGPase cDNA of barley endosperm (84%) and potato tuber (71%). The calculated molecular weight (50 kDa) agrees with the value determined by SDS-PAGE (51 kDa). At the amino-acid sequence level, rice UGPase has high homology with the UGPase of barley (92%) and potato (85%). The enzyme contained conserved sequence elements which are thought to be involved in substrate binding and catalytic activity. A Southern-blot analysis indicated that the gene existed as a single copy. Expression of the enzyme in rice endosperm examined by Northern-blot analysis was high at 10-15 days after heading.     

百合查尔酮合成酶基因的克隆与分析

以西伯利亚百合为试材,通过半巢式PCR和RT-PCR技术分别克隆了查尔酮合成酶基因(CHS)的DNA和cDNA.生物信息学分析显示,CHS的DNA序列全长1 397 bp(登录号HM622754),包含2个外显子和1个内含子;cDNA序列编码区全长1 182 bp(登录号HQ161731),编码393个氨基酸,具有3个典型的CHS蛋白结构域:N-末端结构域(Lys3-Pro229)、C-末端结构域(Gln239-Pro389)和聚合酶Ⅲ结构域(Met1-Thr391);不同百合品种的CHS基因编码的氨基酸序列相似性高达98%,表明百合CHS基因在进化上呈现出十分保守的趋势;不同植物CHS基因序列的系统进化邻接树结果表明:百合与单子叶植物鸢尾及禾本科的水稻、大麦、玉米等亲缘关系更为接近.     

Molecular cloning, expression and mapping analysis of a novel cytosolic ascorbate peroxidase gene from tomato.

Ascorbate peroxidase (APX, EC 1.11.1.11) plays a major role in H(2)O(2)-scavenging in plants and can help to avoid reactive oxygen species (ROS) damage. A new cytosolic APX gene was cloned from tomato (designated LecAPX2) by RACE-PCR. The full-length cDNA of LecAPX2 contained a complete open reading frame (ORF) of 753 bp, which encoding 250 amino acid residues. Homology analysis of LecAPX2 showed a 94% identity with potato cAPX gene and 92% identity with another tomato cAPX gene (APX20), the deduced amino acid showed 88% homology with APX20 protein and 75-92% identity with cAPX from other plants such as potato, tobacco, broccoli, spinach, pea, rice, etc. LecAPX2 revealed the existence of a haem peroxidase and plant APX family signatures. Northern blot analysis showed that LecAPX2 was constitutively expressed in root, stem, leaf, flower and fruit of tomato, whereas the expression levels were different. LecAPX2 was mapped to 6-A using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.     

非洲菊查尔酮合酶基因的克隆、序列分析及在大肠杆菌中的表达

利用RT-PCR方法,从非洲菊(Gerbera hybrida)花瓣的CDNA中克隆到了查尔酮合酶(Chalcone Synthase,CHS)基因CHS,进行了序列分析。结果表明,克隆到的CHS基因全长为1197bps,编码一个由398个氨基酸残基组成的多肽,与Helariutta等发表的非洲菊查尔酮合酶CHSI基因的CDNA序列的CHS基因同源性高达99％。进一步将该基因克隆到表达载体pET32a上,经IPTG诱导表达,得到高效表达的融合蛋白。     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.     

Regulated expression of ADPglucose pyrophosphorylase and chalcone synthase during root development in sweet potato

The phase transition in sweet potato root during tuber differentiationis a complex developmental process that involves changes in gene expression andmorphogenesis. Among the three kinds of root in sweet potato (white fibrousroot, thick-pigmented root and lateral root), ADP-glucose pyrophosphorylase(AGPase) and chalcone synthases (CHS) are expressed only in thick-pigmentedroots after 3 weeks, and this also depends on the developmental stage. Sinceexposing roots to the light or culturing under hydroponic conditions inhibitstuber formation in sweet potato, the expression of AGPase and CHS was studiedunder light and dark conditions. AGPase and CHS expression in sweet potatorootswas suppressed very sensitively by light or water stress, similar to rootdevelopmental patterns. Based on an analysis of AGPase and CHS expression indifferent kinds of root tissues and in different developmental stages, thesegenes were shown to be closely associated with the differentiation ofthickeningpigmented roots.     

决明查尔酮合成酶基因的克隆及序列分析

以决明(Cassia tora)为实验材料,利用RT-PCR和RACE技术,从决明嫩叶中克隆出查尔酮合成酶(Chal-one synthase,CHS)基因,其cDNA全长为1 459 bp,编码一个由390个氨基酸残基组成的多肽.氨基酸序列分析表明,决明CHS基因的氨基酸序列中含有44.61%的中性疏水氨基酸,29.74%的中性亲水氨基酸,12.56%的酸性氨基酸和13.O8%的碱性氨基酸.决明CHS基因的氨基酸序列中具有CHS家族酶系的氨基酸保守残基,包括结合底物CoA的结合残基及催化聚酮合成的催化残基,表明其可能参与聚酮化合物的合成.决明与其它植物CHS的氨基酸序列的进化分析表明,其与同为豆科决明属的翼叶决明(Cassia alata)的同源性较近,并且CHS家族可以分为CHS亚家族与非CHS亚家族.将得到的序列提交GenBank,登录号为EU430077.