Functional Genomics

Abstract: CIPK ( calcineurin B-like calcium sensor interacting the protein kinase) is a group of typical serine / threonine protein kinase targeting calcium sensor calcineurin B-like protein-specific. In the previous study, we obtained the transcriptome of Gossypium.aridum associated with salt stress. It was founded that a CIPKrelated gene had high level of expression abundance. .Then, a full-length cDNA encoding a CIPKs homologue (GarCIPK) was isolated from G.aridum by in-silico cloning and RT-PCR techmology. The deduced GarCIPK protein had 449 amino acids with an estimated molecular mass of 51.12kD and an isoelectric point of 8.13. The putative kinase N-terminal catalytic domain of GarCIPK contained a serine / threonine protein kinase domain. The conserved 24-amino acid motif within C-terminal non-kinase regions was also found in GarCIPK. In order to comfirm its putative function, the GarCIPK gene which was driven by CaMV35S promoter and stress-specific promoter RD29A were transferred into tobacco separately by agrobacterium-mediated transgenic technology. The trangeneic plants of T1 generation were used to identify salt and drought tolerance. The results showed that transgenic plants had enhanced tolerance to salt and drought stress.

Abstract: Brassinosteroids (BRs) are a class of plant steroidal hormones that are involved in a wide variety of physiological and developmental programmes. The first step in BR biosynthesis is catalyzed by a steroid 5a-reductase (DET2), and DET2 gene controls the level of BR. In cotton, DET2 gene (GhDET2) has been cloned in G. hirsutum (AADD tetraploid species). The genus Gossypium includes diploid species in eight genomic groups and five tetraploid species. In the evolution process, many genes sequences changed. So the research selected some diploid species and tetraploid species for tested materials to study the change of DET2 gene squence in the cotton evolution process. Based on the full-length gene cDNA sequence of DET2 in Genebank, we designed PCR primers to amplify DET2 gene, and its sequences in different cotton species were analysed. The results showed that DET2 gene was not only in A, D genomic species, but also in B, C genomic species, and there were high similarity among tested species. This indicated that DET2 gene in cotton in the evolution process had minor change, this gene might be an indispensability gene during cotton growth and development. The analysis of DET2 gene expression level in different cotton species showed that DET2 gene expression level of D genomic species was significantly higher than that of the other cotton species in A、B、C genome. The results indicated that DET2 genes existed in all tested cotton species, but its importance in different species might be different. The research is propitious to understanding the change of DET2 gene during the cotton evolution, and provides more information of DET2 gene for improving cotton cultivars.

CLCV is one of the most severe causes of cotton damage for which no adequate remedy is available. The possible solutions to this problem may be the transfer of resistant genes in the background of agronomically and technologically good varieties. In response to viral infection, several plants may show quantitative and qualitative differences in gene expression. In the present work differential display reverse transcriptase PCR (DDRT) was used to compare overall differences in gene expressions between infected and healthy plants. Screening through 12 primer-pair combinations resulted in up-regulation of 26 cDNA transcripts and 7 transcripts have significant homology with protein sequences of known genes i.e. ribosomal protein, Photosystem protein unit and a predicted hypothetical protein. The transcripts identified are first time reported against CLCV infection and submitted to GenBank database as novel ESTs. The expression levels of 7 transcripts were evaluated by real-time RT-PCR. The GAPDH gene was used as the reference gene to normalize the expression levels. All transcripts showed different level of over-expression in healthy leaves as compared to CLCV diseased leaves. There is 1.6 fold more expression of predicted hypothetical protein, approx. 1 fold of ribosomal protein S14, 1 fold of photosystem reaction center subunit XI , 0.12 fold more expression of transcript A5B2, 0.62 fold more of uncharacterized membrane protein, 0.5 fold of transcript A5B1 as compared to the control. Transcript A6B2 showed homology with Vitis vinefera protein that was supposed to be involved against CLCV infection, was selected and full length gene, GhLCVR (Accession Number: HQ338125) has been amplified. The gene comprises a single open reading frame of 142 amino acids with molecular mass of 16.1 kDa Predicted amino acid sequence shares 80-33% identities with Glycine max, Populus trichocarpa, Vitis vinifera, Zea mays, Ricinus communis, Arabidopsis thaliana, Picea sitchensis, Physcomitrella patens and Staphylococcus aureus respectively. This gene and other identified transcripts could provide a valuable source for the future improvement of cotton varieties against CLCV infection.