Rukai Chen

Isolation and identification of differentially expressed genes in sugarcane infected by Ustilago scitaminea.

Abstract The objective of this study was to survey the molecular mechanism of resistance to sugarcane smut (Ustilago scitaminea Syd.). Two sugarcane (Saccharum officinarum complex) varieties NCo376 with high resistance and F134 with susceptibility were infected by U. scitaminea and the genes associated with the smut resistance were detected with 12 anchored primers and 8 random primers via differential display reverse transcription PCR (DDRT-PCR). Seven differentially expressed fragments were obtained through cloning, sequencing, and semiquantitative RT-PCR validation. The results of Blast in GenBank showed that they shared high homology (35–99%) with cytochrome C oxidase (CCO) gene, ribosomal protein gene, NAD-dependent malic enzyme gene, aminotransferase gene, binding protein gene, RNA polymerase specific transcription initation factor and retrotransposon. The results of semiquantitative RT-PCR showed that CCO gene expression was regulated by U. scitaminea and salicylic acid, and was independent of H2O2. Besides, CCO gene was expressed in root, stalk, and leaf of sugarcane at relatively low levels. Thereby, the phytoalexin induced by CCO gene was inferred to inhibit the pathogen after infection.

Differential Protein Expression in Sugarcane during Sugarcane-Sporisorium scitamineum Interaction Revealed by 2-DE and MALDI-TOF-TOF/MS

To understand the molecular basis of a specific plant-pathogen interaction, it is important to identify plant proteins that respond to the pathogen attack. Two sugarcane varieties, NCo376 and Ya71-374, were used in this study. By applying 2-dimensional electrophoresis (2-DE), the protein expression profile of sugarcane after inoculating with Sporisorium scitamineum was analyzed. In total, 23 differentially expressed proteins were identified by MALDI-TOF-TOF/MS. Bioinformatics analysis revealed that the functions of these 20 differential proteins were associated with such functions as photosynthesis, signal transduction, and disease resistance, while the function of the remaining three proteins was not determined. From above, we can assume that the protein regulatory network during the interaction between sugarcane and S. scitamineum is complicated. This represents the first proteomic investigation focused on highlighting the alterations of the protein expression profile in sugarcane exposed to S. scitamineum, and it provides reference information on sugarcane response to S. scitamineum stress at the protein level.

Cloning and identification of promoter Prd29A and its application in sugarcane drought resistance

The 420 bp upstream regulatory region of rd29A gene from Arabidopsis thaliana genome was amplified by PCR. Sequence analysis showed that it shared 100% identity with the reported rd29A promoter and contained several cis-acting elements including dehydration responsive elements (DRE), ABA responsive elements (ABRE) and so on. The plant expression vector prd29A-S65T was constructed with this fragment linked up with green fluorescent protein (GFP) gene controlled by rd29A promoter, which was transferred to sugarcane callus by bombardment. The expressions of gfp in the transformed sugarcane callus and leaves treated with 6g/L PEG and 40% PEG, respectively, were detected by fluorescent microscope. The results showed that the expression vector had been transferred into sugarcane callus and GFP gene could be induced by PEG treatment. Through PCR analysis, 5 plants were identified to be the transgenic. At the same time, plant expression vector rd29a-dreb-hyg was constructed with this fragment linked up with DREB2B gene controlled by inducible promoter rd29A, which will lay the foundation for droughtresistant transgenic breeding in sugarcane. Molecular analysis showed that the rd29a-dreb-hyg has been successfully integrated into corresponding sugarcane variety.

Development of Loop-Mediated Isothermal Amplification for Detection of Leifsonia xyli subsp. xyli in Sugarcane

Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detecting Lxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification of Lxx and without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused by Lxx in sugarcane. This is the first report of LAMP-based assay for the detection of Lxx in sugarcane.

Molecular Variation of Sporisorium scitamineum in Mainland China Revealed by RAPD and SRAP Markers

Sugarcane smut caused by Sporisorium scitamineum occurs worldwide, causing serious losses in sugar yield and quality. To study the molecular variation of S. scitamineum, 23 S. scitamineum isolates collected from the six primary sugarcane production areas in mainland China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi provinces) were assessed by random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) markers. The results of RAPD, SRAP, and RAPD-SRAP combined analysis showed that, whereas the molecular variation of S. scitamineum was associated with geographic origin, there was no evidence of co-evolution between sugarcane and the pathogen. The results of RAPD, SRAP, or RAPD-SRAP combined analysis also did not provide any information about race differentiation of S. scitamineum. This suggests that the mixture of spores from sori collected from different areas should be used in artificial inoculations for resistance breeding and selection.

Isolation and Characterization of NBS-LRR Resistance Gene Analogs from Sugarcane

For the purpose of isolating resistance gene analogs (RGAs) from sugarcane (Saccharum officinarum Roxb.) with primers from the conservative sequences of nucleotide-binding site (NBS), 6 forward and 10 backward primers were designed according to the conserved motifs in the NBS regions of 3 typical NBS-LRR resistance genes RPS2, N, and L6. The homologous PCR was used to amplify NBS sequences from genomic DNA and cDNA of smut-resistant sugarcane variety NCo376. A total of 11 RGAs were obtained, of which 5 from the genomic DNA and 6 from the cDNA. Sequence analysis showed that these RGAs comprised of the conserved domains P-loop, Kinase-2a, Kinase-3a, and HD, which was conserved in NBS-LRR type of disease-resistance gene. The last amino acid in the alignment was residue W in LLVLDDV (W/D) motif, which is typical in non-TIR-NBS-LRR type of genes, indicating only non-TIR-NBS-LRR type resistance genes in the genome of sugarcane. The 11 RGAs, together with RPS2 and Xa1, were clustered into one group, and N and L6 were in another group. One RGA, termed PIC (EF059974), was validated through real-time PCR. The result showed that the expression of PIC gene was induced by Ustilago scitaminea and salicylic acid, but inhibited by hydrogen peroxide. The PIC gene had constitutive expressions in leaves, stalks, and roots of sugarcane, with the strongest expression in leaves.

SSR marker-based analysis of genetic relatedness among sugarcane cultivars (Saccharum spp. hybrids) from breeding programs in China and other countries

AbstractCapillary electrophoresis-based molecular genotyping was conducted on 35 sugarcane cultivars (Saccharum spp. hybrids) and 5 clones of related wild species with 20 polymorphic SSR DNA markers. A total of 251 alleles were identified with 248 alleles displaying varying degrees of polymorphism and the remaining three alleles being monomorphic. The total number of alleles by any SSR marker varied from as few as 7 to as many as 18, with an average of 12.5 alleles per marker. Diversity index (DI = 1 −

Productivity and Stability of Sugarcane Varieties in the 7th Round National Regional Trial of China

\sum\limits_{i = 1}^S {Pi^2 }