Study On The Cloning Of The Rice Leaf Shape Gene NL7 And Its Molecular Regulation Mechanism

Leaf morphology is an important part of plant architecture in rice.It is also an important consideration trait for breeders in achieving super high-yielding breeding and molecular breeding of ideal plant architecture.Isolation and cloning the gene related to leaf morphology through forward genetics,which is not only contribute to analyzing the genetic regulatory network of rice leaf morphology development,but helpful to improve rice plant architecture and molecular design breeding of ideal plant type.In this study,narrow leaf 7（nl7）,a genetically stability mutant,was identified from a mutated indica cultivar SH527 by EMS.Morphological observation showed that there was no significant difference in the top of three leaf length,while width was decreased significantly;compared with wild type,the effective tiller number increased significantly,the yield per plant decreased significantly;the internode length and width of stem decreased in varying degrees,except for the grain width were no significant difference,the other traits of nl7（including number of grains per spike,seed setting rate,thousand grain weight,length and thickness of grains）were decreased significantly or very significantly.Leaves histological and glumes electron microscopic scanning showed that there was no significant difference in bulliform cells,mesophyll cells and the size of vascular bundles in nl7 leaves,but the number of large and small vascular bundles decreased significantly.There was no significant difference in the length and width of glume cells,presumably the change of grain morphology caused by the number of cells.Physiological experiments showed that there was no significant difference between chlorophyll content and SPAD value at tillering stage;stomatal conductance and transpiration rate of nl7 decreased significantly,but intercellular CO₂ concentration was significantly higher than that of wild type.Genetic analysis showed that the narrow leaf trait of nl7 was controlled by a pair of recessive nuclear gene.NL7 were cloned by map-based cloning technique,which was positioned at an interval about 36.405 Kb,between markers C7-8 and C7-9,with five open reading frames.According to the sequencing results,there was a single base substitution（T/G）which occurred in the 127 bp downstream of the LOC_Os07g09450,predicted that the gene encoded a heat shock protein（DnaJ）,and it’s a new leaf shape gene that hasn’t been cloned.Therefore,we preliminarily speculate that LOC_Os07g09450 is a candidate gene of NL7.Real-time PCR results showed that NL7 was constitutively expressed in all tissues and organs of SH527,with the highest expression level in young leaves.Using GFP as a label,it was determined that NL7 protein was expressed in cytoplasm.Molecular evolutionary analysis of NL7protein showed that the sequence of NL7 protein was highly conserved among different species.Knockout LOC_Os07g09450 showed that loss-of-function lead to a decrease in leaf width.The content of endogenous hormones determination showed that the strigolactones and trans-zeatin-riboside of nl7decreased very significantly and significantly.By analyzing the transcription levels of some genes at tillering stage showed that NL7 might be related to cellulose synthase,strigolactones,gibberellin and auxin biosynthesis.The screening results of yeast libraries showed that seven genes might interact with NL7.By yeast two hybridization showed that NL7 protein interacted with LOC_Os01g03820 and LOC_Os01g31690.