Functional Analysis Of Thaumatin-like Protein Involved In Fiber Secondary Cell Wall Synthesis Through Genetic Transformation Of Cotton And Tobacco

Reverse genetics is a powerful approach to verify the functions of novel genes. In this approach, the functional study of a gene starts with the gene sequence and the function of a gene can be altered to analyse its effect on the development and behaviour of the organism. Gene of interest can be transformed into host genome through Recombinant-DNA technology to elucidate its role under different set of conditions. In this way, genes of interest can be transferred into the same and different species and even other kingdom.We have isolated two thaumatin-like protein(GbTLPl and GbTLP2) cDNAs from sea-island cotton(Gossypium barbadense L.) previously (Tu et al.,2007), which showed 97% protein sequence identity to each other (GenBank DQ912960, DQ912961). In this study, we have we have explored the role of a Thaumatin-like protein gene by its over-expression and RNA interference. Under the control of CaMV 35S promoter, we over-expressed a full-length GbTLPl cDNA, in both cotton and tobacco, which is 966 bp in length with an open reading frame (ORF) of 735 bp encoding a polypeptide of 244 amino acids. For RNA interference of GbTLP gene in transgenic cotton, mismatching regions of both GbTLP1 and GbTLP2 were used to induce post-transcriptional gene silencing (PTGS).Following is the summary of results so far, obtained through the functional analysis of thaumatin-like protein gene(GbTLP) in transgenic tobacco and cotton.1. Relative quantification of GbTLP1 revealed its higher level expression by Real-time PCR in both Gossypium hirsutum and Gossypium barbadence (donor species). The expression level of GbTLPl increased significantly in the fiber of both cotton species at 20 and 25 days post anthesis while there was no or negligible expression of GbTLPl at other developmental stages. Expression studies of this gene at different fiber developmental stages in cotton plant revealed a possible role of GbTLPl in secondary cell wall development and thickening.2. The GbTLPl expression profile in cotton fiber suggested a possible role in cell wall thickening which is a "protective shield" to resist fungal invasion. Transgenic tobacco plants were verified by Southern blotting. The constitutive and higher GbTLP1 expression plants were revealed by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis. In this experiment, transgenic plants without GbTLPl expression were also used which behaved similar to non transgenic control plants. Transgenic tobacco plants constitutively over-expressing GbTLPl gene, showed remarkable resistance against Verticillium dahliae, which can be useful for the further control of this fungus.3. These plants also presented an enhanced resistance against other biotic stress agents like Fusarium oxysporum and abiotic stresses like salinity and drought which shows positive involvement of this gene in plant defense mechanism. GbTLPl over-expressing plants showed drought tolerance by avoiding lipid peroxidation which resulted into less malondialdehyde (MDA) accumulation. These transgenic plants maintained lower Na+and higher K+contents which seems to be a possible reason for their salinity tolerance.4. To make this study more comprehensive, we over-expressed as well as down regulated (RNA interference) this gene in cotton. Transgenic cotton plants are grown and self-crossed in the field for two consecutive years to fix traits and do experiments on T2 generation. At the moment, T2 generation of transgenic cotton plants (with over-expression and also RNA interference of GbTLP) is in the field. The fiber quality studies in T1 generation of transgenic cotton (over-expressed and RNA interference) show that the GbTLP gene has apparently decreased fiber length and increased the micronaire value which reflects greater fineness and maturity of the fiber. These studies need further investigation.5. The over-expression of GbTLP1 did not influence plant growth of both cotton and tobacco which makes this gene a safe choice for genetic engineering.The purpose of incorporating this gene into tobacco genome was to observe the behavior and influence of this gene under different biotic and abiotic stress conditions. We used tobacco because it can grow quickly and its leaves are broader which can be used to see the growth pattern of fungus. Thaumatin-like protein is a member of Pathogenesis-related proteins family which motivated us to see the role of this gene in creating resistance against Verticillium dahliae and Fusarium oxysporum f.sp. vasinfectum. By the over-expression and RNA interference of GbTLP in transgenic cotton, we are expecting some conclusive differences which can help us to deduce its role because this gene is present in cotton genome. In this way, the pleiotropic effect of this gene in disease resistance and fibre development can open new research areas to sort out the involvement of other PR proteins in cell wall development.