ABSTRACT
The sterile line is the basis of crop heterosis utilization. To broaden the sources of male sterility in tobacco, the Ntms1 (Nicotiana tabacum L. ms1) gene was cloned from the tobacco variety K326 by homologous cloning based on the Cams1 (Capsicum annuum L. ms1) gene sequence of male-sterility genes in pepper. The protein structure and physicochemical properties of the two genes were determined by bioinformatics analysis, and the function of the Ntms1 gene was verified by the CRISPR/Cas9 system. The results showed that the sequences of Ntms1 and Cams1 were 85.25% similar, and plant homeodomains were found in both genes; the physical and chemical properties were also very similar. It is speculated that the Ntms1 gene had the same function as the Cams1 gene in controlling male sterility. Compared to the wild-type plants, the filaments of the Ntms1 knockout mutant plants were shorter, and the stamen was shorter than the pistil. The anthers did not develop fully and had few viable pollen grains; the tapetum and the anther wall had developed abnormally, and the anther chamber was severely squeezed. The malondialdehyde content in the mutant plants was significantly higher than that in the wild-type plants, while self-fertility was significantly lower in the mutant plants. The results showed that the Ntms1 gene plays an important role in regulating fertility in tobacco.
ABSTRACT
BACKGROUND: As a unique biological phenomenon, heterosis has been concerned with the superior performance of the heterosis than either parents. Despite several F1 hybrids, containing supernal nicotine content, had been discovered and applied to heterosis utilization in Nicotiana tabacum L., nevertheless, the potential molecular mechanism revealing nicotine heterosis has not been illustrated clearly. RESULT: Phenotypically, the F1 hybrids (Vall6 × Basma) show prominent heterosis in nicotine content by 3 years of field experiments. Transcriptome analysis revealed that genes participating in nicotine anabolism (ADC, PMT, MPO, QPT, AO, QS, QPT, A622, BBLs) and nicotine transport (JAT2, MATE1 and 2, NUP1 and 2) showed an upregulated expression in the hybrid, a majority of which demonstrated an overdominant performance. RT-PCR confirmed that nicotine anabolism was induced in the hybrid. CONCLUSIONS: These findings strongly suggest that nicotine synthesis and transport efficiency improved in hybrid and overdominance at gene-expression level played a critical role in heterosis of nicotine metabolism.