| Rice (Oryza sativa L.) is one of the most widely-grown staple crops in the world,and is also the most important model plant species for molecular biology and functionalgenomics. Since world population is continuously growing and per capita cultivatedland area is consequently declining, rice breeding for high yield and quality as long termobjectives is receiving great attention by plant breeders. Leaves are the major organs ofphotosynthesis in higher plants, leaf characters are closely correlated with formation ofgrain yield, and leaf rolling in rice has direct effects on plant architecture, and canopyphotosynthetic characteristics. Recent studies indicate that moderate leaf rolling mayeffectively improve canopy structures and significantly raising light energy utilizationrate. Therefore, leaf rolling has been considered as one of the most importantmorphological characters in ideal plant architecture breeding for super high yield in rice.Mining and identification of potential rolled-leaf mutants, and molecular geneticanalysis of leaf-rolling phenotype will not only provide new germplasm resources forrice architecture breeding, but lead to greater understanding of the molecularmechanisms underlying the formation of leaf rolling.A rolled-leaf rice mutant that phenotypically showed longitudinal and inwardrolling in leaves was obtained by Ac/Ds transposon mutagenesis methods. Thephenotypes of this mutant were preliminarily examined in this research, and the Dsinsertion was determined by PCR-based molecular methods. The nucleotide sequenceflanking of the Ds element was amplified and isolated by TAIL-PCR from the genomeof the mutant, and site of the Ds element on rice chromosome was characterized by thebioinformatics tool. Finally, the functions of the Ds-mutated gene were predicted. Themain results were summarized as follows:1. The phenotypic identification based on morphological traits and leaf anatomicalcharacteristics of the rolled-leaf mutant. One hundred of plants were randomly chosenfrom population of mutants and wild-type rice lines to determine the plant height and tiller number, their differences in these morphological parameters were compared andstatistically analyszed by using SPSS software. The1000-grain weight and grainplumpness were measured and compared between wild-type and rolled-leaf mutant.Anatomical structures of the wild-type and rolled-leaf mutant leaves were carefullyexamined using optical microscope and paraffin section technology, and theiranatomical differences in shape, size, and number of bulliform cells were compared.The results showed that there were significant differences both in plant height andtiller number between mutant and wild-type. The plant height in rolled-leaf mutant (76±3.93cm) was significantly lower than that in wild-type rice (88±4.38cm).Comparison of average tiller number showed the mutant exhibited more tillers (13±2.76) than wild-type rice (10±2.40). Results also indicated that the1000-grain weightswere25.1g,22.4g repcetively for the wild-type and the mutant, while the values ofgrain plumpness were95%and89%, repcetively in wild-type and the mutant. Both ofthese two parameters were lower in the mutant than those in the wild-type. Theanatomical examination showed that bulliform cells arranged normally to form a typicalfan-shaped structure in the wild-type leaf with larger parenchyma cells armed by severalsmaller ones, whereas in the mutant leaf, there were less and abnormally-shapedbulliform cells that showed highly-ragged cell margin and chimerically connectedthough parenchyma cells arranged a pattern similar to that in wild-type plant. Ourresults imply that abnormal structures in bulliform cells may be directly related torolling-leaf phenotype in the mutant.2. Amplification of Ds-flanking sequence and structural and functional predictionof the Ds-tagged gene. The Ds-flanking sequence was isolated from the mutant by usingthermal asymmetric interlaced PCR (TAIL-PCR) technique, and sequenced. Thesequenced Ds-flanking sequence was used as a query to carry out online homologoussequence search against nucleotide sequence database by NCBI/BLAST, and the Dsinsertion site, and the chromosomal localization of Ds-tagged gene were analyzed, thestructure and function of Ds-tagged gene were predicted by bioinformatics methods.The results indicated that the Ds-flanking sequence showed99%identity at thenucleotide level to the sequence of clone OSJNBa0003P07of rice chromosome10. The structure of the Ds-tagged gene was predicted by using software of FGENESH,GeneMark.hmm and Bioinformatics WEB server, respectively, all of which gave highsimilarities in the size and location of exons within the gene. Moreover, the productencoded by the gene was predicted by NCBI Entrez server and Pfam. It was revealedthat the deduced product was a member of rice hAT family, which was a pseudo protein.Taking all these morphological, anatomical and molecular data together, we candraw a conclusion that the Ds insertion affects the expression and regulation of the hATfamily protein that involved in the development and differentiation of bulliform cells,and eventually confers the rolled-leaf phenotype in rice. |
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