1、1Title:Cell-wall invertases from rice are differentially expressed in caryopsis during the grain filling stageAuthors:Yong-Qin WANG1, 2, Xiao-Li WEI1, Hong-Lin XU1, Cheng-Lin CHAI1, 2, Kun MENG1, 2, Hong-Li ZHAI1, 2, Ai-Jun SUN1, 2, Yong-Gang PENG1, 2, Bin WU1, 2, Gui-Fang XIAO1, Zhen ZHU1*Received:
2、 6 Feb. 2007 Accepted: 6 Mar. 2007Handling editor: Tai WangAddresses:1State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China2Graduate School of the Chinese Academy of Sciences, Beijing 100101, ChinaCorresponding aut
3、hor:Zhen ZHUMailing address: 601 Group, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, ChinaE-mail: Tel: (+8610) 648734902Fax: (+8610) 648528903Cell-wall invertases from rice are differentially expressed in caryopsis during the grain filling
4、 stageYong-Qin WANG1, 2, Xiao-Li WEI1, Hong-Lin XU1, Cheng-Lin CHAI1, 2, Kun MENG1, 2, Hong-Li ZHAI1, 2, Ai-Jun SUN1, 2, Yong-Gang PENG1, 2, Bin WU1, 2, Gui-Fang XIAO1, Zhen ZHU11State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Bei
5、jing 100101, China2Graduate School of the Chinese Academy of Sciences, Beijing 100101, ChinaAbstractCell-wall invertase plays an important role in sucrose partitioning between source and sink organs in higher plants. To investigate the role of cell-wall invertases for seed development in rice (Oryza
6、 sativa L.), cDNAs of three putative cell-wall invertase genes OsCIN1, OsCIN2 and OsCIN3 were isolated. Semi-quantitative RT-PCR analysis revealed different expression patterns of the three genes in various rice tissues/organs. In developing caryopses, they exhibited similar temporal expression patt
7、erns, expressed highly at the early and middle grain filling stages and gradually declined to low levels afterward. However, the spatial expression patterns of them were very different, with OsCIN1 primarily expressed in caryopsis coat, OsCIN2 in embryo and endosperm, and OsCIN3 in embryo. Further R
8、NA in situ hybridization analysis revealed that strong signal of OsCIN2 mRNA was detected in the vascular 4parenchyma surrounding the xylem of the chalazal vein and the aleurone layer, whereas OsCIN3 transcript was strongly detected in the vascular parenchyma surrounding the phloem of the chalazal v
9、ein, cross-cells, the aleurone layer and the nucellar tissue. These data indicates that the three cell-wall invertase genes play complementary/synergetic roles in assimilate unloading during the grain filling stage. In addition, the cell type-specific expression patterns of OsCIN3 in source leaf bla
10、des and anthers were also investigated, and its corresponding physiological roles were discussed. Key words: Cell-wall invertase; Gene expression; Oryza sativa L.; RNA in situ hybridization; seed developmentFinancial grants:This work was financially supported by grants from National Program on Key B
11、asic Research Projects (2004CB720406), Innovation Foundation of Chinese Academy of Science (KSCX2-SW-306 and KSCX1-SW-03), Program for Strategic Scientific Alliances between China and the Netherlands (KNAW-PSA, 04-PSA-BD-04 for P.B.F.O.; KNAW-CEP, 04CDP022 for Y.X.).5题目: 水稻细胞壁转化酶基因在灌浆期的水稻颖果中具有不同表达模式
12、作者: 王永勤 1, 2，魏晓丽 1，徐鸿林 1，柴成林 1, 2，孟昆 1, 2，翟红利 1, 2，孙爱君 1, 2，彭永刚 1, 2，吴斌 1, 2，肖桂芳 1，朱祯 1*单位:1 中国科学院遗传与发育生物学研究所，植物基因组学国家重点实验室，北京，1001012 中国科学院研究生院，北京，100101摘要: 在高等植物中，细胞壁转化酶对蔗糖在源-库器官的分配起着重要作用。为研究细胞壁转化酶对水稻种子发育的作用，克隆了 3 个水稻细胞壁转化酶基因（OsCIN1，OsCIN2 和 OsCIN3）的 cDNAs。半定量 RT-PCR 分析表明，3个基因在多个水稻组织/器官中具有不同的表达模式。
13、在发育的颖果中，它们具有相似的时间表达模式，在籽粒灌浆的早期和中期表达量高，之后表达量逐渐降至低水平；但空间表达模式不同，OsCIN1 主要在颖果皮中、OsCIN2 在胚和胚乳中、OsCIN3 在胚中表达。进一步 RNA 原位杂交分析表明，在围绕合点维管束木质部的薄壁细胞和糊粉层中检测到 OsCIN2 的强烈表达，而在围绕合点维管束韧皮部的薄壁细胞、横细胞、糊粉层和珠心组织中检测到OsCIN3 的强烈表达。上述结果表明，这 3 个水稻细胞壁转化酶基因在水稻籽粒灌浆期间对同化物的卸载起着互补或协同的作用。此外，还检测了 OsCIN3在源叶片和花药中的细胞特异表达模式，并对它可能起的生理作用进行了
14、讨论。6关键词：细胞壁转化酶；基因表达；水稻；RNA 原位杂交；种子发育基金项目：国家重大基础研究项目(2004CB720406)；中国科学院创新基金(KSCX2-SW-306 and KSCX1-SW-03)；中荷科学战略联盟项目(KNAW-PSA, 04-PSA-BD-04 for P.B.F.O.; KNAW-CEP, 04CDP022 for Y.X.)。* 通讯作者。E-mail: zz 。7In most plant species, assimilated carbon is transported as sucrose. Utilization of sucrose as a
15、source of carbon and energy depends on its cleavage by either invertase or sucrose synthase, which is crucial for development, growth and carbon partitioning (Sturm and Tang 1999). Invertase (-fructofuranosidase; EC.3.2.1.26) is a hydrolase, catalyzing the irreversible cleavage of sucrose into gluco
16、se and fructose (Copenald 1990). Based on their subcellular locations, plant invertase can be classified into cytosolic invertase, vacuolar invertase and cell-wall (extracellular) invertase (Sturm 1999). Cell-wall invertase is a glycosylated form with an acid pH optimum and a basic isoelectric point
17、, which allows it ionically bound to the cell wall via positive charges at low pH (Kim et al. 2000).Cell-wall invertase is considered to be one of the key enzymes involved in establishing sink strengths in various sink tissues (Sturm and Tang 1999). The importance of cell-wall invertase to regulate
18、seed development is underlined by mutant analysis in maize. In the maize (Zea may L.) mutant miniature-1, the lack of expression of an endosperm specific cell-wall invertase Incw2 in pedicel and endosperm causes interruption of photoassimilate transport into developing kernel, and as a consequence,
19、the seeds are only one fifth of the normal weight (Miller and Chourey 1992; Cheng et al. 1996). In fava bean (Vicia faba L.), a cell-wall invertase gene VfCWINV1 is observed to be expressed at the pre-storage phase of seed development in the thin-walled parenchyma of seed coat, a region known to be
20、the site of photoassimilate unloading, and a model of invertase-mediated unloading process has been proposed (Weber et al. 1995). The essential function of cell-wall 8invertase for supplying carbohydrates to sink organs has also been demonstrated by transgenic approaches. Apoplastic expression of a
21、yeast (Saccharomyces cerevisiae L.) invertase in tuber of potato (Solanum tuberosum L.) increases tuber size (Sonnewald et al. 1997), and ectopic expression of a plant cell-wall invertase in roots of Arabidopsis (Arabidopsis thaliana) leads to early flowering and an increase in whole plant biomass (
22、von Schweinichen and Bttner 2005). In carrot (Daucus carota L.), antisense expression studies show that cell-wall invertase plays a crucial role in sucrose partitioning to developing tap roots (Tang et al. 1999). Suppressed expression of an anther-specific cell-wall invertase Nin88 in transgenic tob
23、acco (Nicotiana tabacum L.) results in a block during pollen development (Goetz et al. 2001). All these results support the essential function of cell-wall invertase in carbon partitioning between source and sink organs.New progresses have been made in studies on the role of cell-wall invertase in d
24、evelopment of cereal seeds besides the work in maize described above. In barley (Hordeum vulgare L.), temporal and spatial coordinated expression of cell-wall invertases and hexose transporters in caryopses during the early developing stage suggests a role in carbohydrate supply (Weschke et al. 2003
25、). In rice, four of the nine members of the rice cell-wall invertase gene family have been observed to be expressed in immature seeds (Hirose et al. 2002; Cho et al. 2005; Ishimaru et al. 2005; Ji et al. 2005). RNA in situ hybridization analysis of OsCIN1 suggested its critical role in carbohydrate
26、supply for developing caryopsis during the pre-storage phase (Hirose et al. 2002). However, cell type-specific expressions of other members in 9developing caryopses have not been examined. Therefore, more detailed analysis of expressions of other cell-wall invertases will help to elucidate their rol
27、es in rice caryopsis development. In this study, cell type-specific expressions of OsCIN2 and OsCIN3 in developing caryopses, anthers and/or source leaf blades were investigated.ResultscDNA cloning and sequence analysisA 560-bp fragment was amplified through RT-PCR by using degenerate primers design
28、ed from the conserved -fructosidase motif and cysteine catalytic site of known cell-wall invertases (Sturm 1999). Sequence of this cDNA fragment showed 80% and 83% identities with the corresponding region of the maize cell-wall invertase genes Incw1 and Incw2 (GenBank accession number (ACC): AF05012
29、9 and AF165180), respectively. After the rice genome sequence was published (Yu et al. 2002), this partial cDNA sequence was used to search the rice genome sequence, and three high homologous sequences were identified, showing 98.5%, 72.7% and 65.3% identity to this partial cDNA sequence and located
30、 at contiguous DNA sequences #13373, #35123 and #7732 (http:/ respectively. Full-length cDNAs of the three putative cell-wall invertases were isolated by RT-PCR using gene-specific primers and RNA from two-week-old seedlings. cDNAs of the three genes contain ORFs 1734, 1797 and 1761 bp in length, re
31、spectively. They show a high degree of homology to each other and to other homologues from maize, barley and wheat at both the nucleotide level and the amino acid level, with highest homology to the maize Incw1, Incw2 and Incw3 (GenBank ACC: AF050129, AF165180 and 10AF043346), respectively. And the
32、three putative rice cell-wall invertase genes were correspondingly named as OsCIN1, OsCIN2 and OsCIN3 (GenBank ACC: AY342319, AY340072 and AY342320), respectively. Analysis of the deduced proteins of OsCIN1, OsCIN2 and OsCIN3 revealed many typical features of cell-wall invertases, including the cons
33、erved -fructosidase motif (NDPNG/A), the catalytic cysteine (MWECP/V) site, a basic isoelectric point, a conserved N-glycosylation site and a putative signal peptide probably required for extracellular localization of the protein (Figure 1). There are another two potential N-glycosylation sites (NXS
34、/T) located at the same positions of the three genes, and two specifically located in OsCIN3 (Figure 1). Comparing the cDNA sequences and genomic DNA sequences (NCBI database) revealed that the three genes are all organized into six introns and seven exons, and contain a 9-bp exon 2, encoding three
35、amino acids (DPN) of the conserved -fructosidase motif (NDPNG/A) (Sturm 1999).Expression of OsCIN1, OsCIN2 and OsCIN3 in different rice tissues/organsTissue-specific expressions of OsCIN1, OsCIN2 and OsCIN3 were investigated by semi-quantitative RT-PCR. As shown in Figure 2, the three genes show dif
36、ferent expression patterns in various tissues/organs. OsCIN1 is expressed in all sink and source tissues/organs tested at very different levels, strongly expressed in sink leaf blades, shoots and roots of seedlings, and weakly expressed in source leaf blades, leaf sheaths, uppermost internodes and anthers. OsCIN2 is specifically expressed in sink tissues/organs, expressed highly in sink leaf blades, uppermost internodes and anthers, and lowly in shoots and roots of seedlings, but not expressed in source leaf blades and
