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Karnal bunt of wheat is an important quarantine disease that interrupts India’s wheat trade in the international market. The whole transcriptome of germinating and dormant teliospores of Tilletia indica was performed using the RNA Seq approach to identify germination-related genes. Approximately 63 million reads were generated using the RNA sequencing by the Illumina NextSeq500 platform. The high-quality reads were deposited in NCBI SRA database (accession: PRJNA522347). The unigenes from the pooled teliospores were 16,575 having unigenes length of 28,998,753 bases. The high-quality reads of germinating teliospores mapped on to 21,505 predicted CDSs. 9,680 CDSs were common between dormant and germinating teliospores of T. indica. 11,825 CDSs were found to be in germinating teliospores while only 91 were unique in dormant spores of pathogen. The pathway analysis showed the highest number of pathways was found in germinating spores than dormant spores. The highest numbers of CDSs were found to be associated with translation (431 in number), transport and catabolism (340), signal transduction (326), and carbohydrate metabolism (283). The differential expression analysis (DESeq) of germinating and dormant teliospores showed that 686 CDS were up-regulated and 114 CDS were down-regulated in the germinating teliospores. Significant germination-related genes in the spores were validated using qPCR analysis. Ten genes viz. Ti3931, Ti6828, Ti7098, Ti7462, Ti7522, Ti 9289, Ti 8670, Ti 7959, Ti 7809,and Ti10095 were highly up-regulated in germinated teliospores which may have role in germination of spores.Further, these differentially expressed genes provide insights into the molecular events. This first study of transcriptome will be helpful to devise better management strategies to manage Karnal bunt disease.
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Objective: To clone CDS sequence of Dioscorea opposita SUS gene and analyze the protein structure,in order to provide a theoretical basis for the regulation mechanism of SUS gene and the synthesis mechanism of D.opposita polysaccharides.Method: Total RNA in D.opposita was extracted and reverse-transcribed into first strand of cDNA.Specific primers were designed according to an annotated SUS gene sequence obtained from the laboratory D.opposita genome database,and the coding region of the SUS gene was obtained by gene cloning technique and the protein sequence characteristics were analyzed by protein prediction analysis software.Result: A 2 448 bp gene sequence was cloned with a complete open reading frame (ORF).The gene was named DoSUS1.The formula of protein encoded by DoSUS1 gene in D.opposita was C4209H6534N1115O1205S23,and the molecular weight was 9 788.32,the total number of amino acids was 815,the theory isoelectric point (PI) was 6.10,the extinction coefficient was 110 505,the aliphatic index (AI) was 94.15,the instability index was 32.18,and the grand average of hydropathicity (GRAVY) was-0.225.It was a stable and soluble acidic protein.There were phosphorylation sites in the DoSUS1 amino acid sequence,with no transmembrane region and signal peptide.The secondary structure and the tertiary structure showed that DoSUS1 was an α class protein.Functional domain predictions showed that DoSUS1 had sucrose synthesis domain and glycosyl transfer domain.The homology alignment showed that the amino acid sequence of DoSUS1 was more than 80% similar to the amino acid sequence of the aligned monocots.The phylogenetic tree showed that DoSUS1 was closely related to SUS of monocotyledon evolution.Conclusion: The coding sequence of SUS gene was cloned from D.opposita for the first time,and its protein structure was analyzed to lay a foundation for further studying the roles of SUS in the growth and polysaccharide synthesis of D.opposita.
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Objective To compare the coding sequences (CDS) of Yersinia pestis D106004 strain from Yulong County in Yunnan Province and Z176003 strain from Qing-Tibet Plateau in order to find the differences between their genomes and the genetic characteristics. Methods The CDS of Yersinia pestis D106004 strain and Z176003 strain were searched and compared by BLAST. Twenty-two differential CDS were selected to design 22 pairs of primers. PCR amplification was carried out in 119 representative plague strains from different isolation sources (natural foci of Himalayan marmot plague in the Qinghai-Tibet Plateau, natural foci of Apodemus chevrieri and Eothenomys miletus plague in Yunnan), time span of about 50 years, and distribution in six ecological types including Tibet, Qinghai, Sichuan, Gansu and Yunnan, and PCR products were sequenced and verified. The strains were all from the State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Results In 119 representative plague strains of 6 ecological types, the cumulative sequence length of 22 differential CDS PCR amplification products was 2.13 × 106 bp. Among the 119 representative plague strains in the foci of Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain, 22 differential CDS had high homology, there was no difference in 78.2％ (2047/2618) sequences of differential CDS, and 21.8％ (571/2618) sequences had three types of gene mutations ( deletion , missense and frameshift mutations). The characteristics of the differences were stable in the 6 ecological plague strains of the foci, and they were divided into 6 geographical distributions. Conclusion Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain have high homology, close genetic relationship, and little difference in genome, but the genetic characteristics of different ecotype strains are stable.
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Objective: In order to study the function of geranylgeranyl pyrophosphate synthase (GGPPS) gene, the CDS nucleotide sequence of GGPS was cloned from Panax notoginseng, and its prokaryotic expression was performed. Methods: The primers were designed according to the reported GGPPS gene sequence in Genbank, and the coding sequence was obtained by RT-PCR. The prokaryotic expression vector was constructed and transformed into Escherichia coli BL21 for the expression under the induction of isopropyl β-D-1-thiogalactopyranoside (IPTG). Results: The CDS of GGPS gene had a full length of 1 032 bp coding for 343 amino acids. Results of SDS-PAGE showed that a 29 000—44 000 protein was achieved and the recombinant protein was mainly in the form of insoluble inclusion body. Conclusion: The CDS nucleotide sequence of GGPPS gene was successfully cloned, and the stable prokaryotic expression was established. This study will provide a foundation for the further functional researches of GGPPS gene in P. notoginseng.
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Background & objectives: Different developmental stages of Wuchereria bancrofti, the major causal organism of lymphatic filariasis (LF), are difficult to obtain. Beside this limitation, to obtain complete coding sequence (CDS) of a gene one has to isolate mRNA and perform subsequent cDNA synthesis which is laborious and not successful at times. In this study, an alternative strategy employing polymerase chain reaction (PCR) was optimized and validated, to generate CDS of Macrophage migration Inhibitory Factor-2 (wbMIF-2), a gene expressed in the transition stage between L3 to L4. Methods: The genomic DNA of W. bancrofti microfilariae was extracted and used to amplify the full length wbMIF-2 gene (4.275 kb). This amplified product was used as a template for amplifying the exons separately, using the overlapping primers, which were then assembled through another round of PCR. Results: A simple strategy was developed based on PCR, which is used routinely in molecular biology laboratories. The amplified CDS of 363 bp of wbMIF-2 generated using genomic DNA splicing technique was devoid of any intronic sequence. Interpretation & conclusions: The cDNA of wbMIF-2 gene was successfully amplified from genomic DNA of microfilarial stage of W. bancrofti thus circumventing the use of inaccessible L3-L4 transitional stage of this parasite. This strategy is useful for generating CDS of genes from parasites that have restricted availability.
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Objective: To provide the evidence for the identification and genetic diversity of Pinellia ternata by studying cpDNA non-coding sequences of P. ternata and its related species. Methods: Besides P. cordate and P. pedatisecta, 43 P. ternata samples were collected from the main habitats in China. psbK-psbI and atpF-atpH sequences in leaf genome DNA were cloned by PCR. Comparative analysis was carried out by bioinformatics software. Results: The sequence length of atpF-atpH in P. ternata was 337-342 bp and conservative. The numbers of variable sites and parsimony information sites were only 7 and 1, respectively. The genetic distance was 0-0.024.The length of psbK-psbI was 432-435 bp, with 37 variable sites, including 17 information sites, and the genetic distance was 0-0.069.Cluster analysis was not consistent with the phenotype or the geographical distributions. Conclusion: The discrimination of psbK-psbI is better than that of atpF-atpH, and there are more mutation sites in psbK-psbI sequence among species in P. ternata.