Map-based Cloning Of A Leaf Tip Necrosis Mutant And Gene LTN2 Functional Analysis In Rice(Oryza Sativa L.)

Programmed cell death exists in every stages of plant growth and development. It is not only controlled by genes, but also affected by environment.Plant leaf is the most important organ of photosynthesis, and leaf senescence will shorten the filling time of rice grain the filling period, resulting in reduction of yeild. As a model plant,using rice mutant to research leaf wither caused by programmed cell death has great theoretical significance to reasearch rice leaf senescence mechanism and improve rice yields.A leaf tip necrosis and dwarf mutant t207 was discovered from the rice mutant library which our laboratory constructed, and its genetic background is japonica Nipponbare. Leaf tip necrosis phenotype appears in eight-leaf stage and it has more tillers than the wild type.It has dwarf phenomenon and its chlorophyll content is lower than the wild type. Genetic analysis showed that t207 mutant phenotype was controlled by a recessive gene. We made the combination of t207/Dular to establish mapping population and used map-based cloning method to locate the gene on a small range of rice chromosome 11.By sequencing the candidate genes, we found a 42 bp deletion of a gene exon and named the gene LTN2. The complementation experiment proved that the dwarf and leaf tip necrosis phenotype were indeed controlled by LTN2. Construct LTN2-GFP fusion protein vector and transform rice protoplasts. The subcellular localization result showed that LTN2 was a membrane located protein. Use intracellular domain of LTN2 as the bait protein to screen the library, and gain the LTN2 interacting protein OsPIN4. OsPIN4 is a auxin efflux carrier protein, and plays an important role in auxin polar transport. And then the analysis of some auxin signal gene expression showed that the expression of OsPIN4, LTN2 interaction protein, decreased in the mutant, while the expressions of other auxin efflux carrier proteins, like OsPIN1 b, OsPIN2, OsPIN5 a and OsPIN5 c, increased. So the dwarf and leaf tip necrosis phenotype may be due to the expression changes of OsPIN4 and its family members.Auxin polar transport changes lead to PCD acceleration of leaf tip, making leaf tip necrosis and wilt symptoms. Whether the mutation of LTN2 really influence the content and distribution of auxin needs further research. This study explains the genetic regularity and phenotype characteristics. The mutated gene is isolated, and the gene function is initial clarified. This research lays the foundation for further study of the programmed cell death mechanism and rice leaf senescence.