Preliminary Analysis Of The Interaction Genes Of LRK2,the Leucine-rich Receptor-like Kinase Gene Of Rice,and The Functional Analysis Of A Yellow Green Leaf Gene YGL15 In Rice

Rice,one of the most important staple cereals,feeds nearly half population of the world.The tiller number is one of the important agronomic traits of rice.Tillering has a decisive effect on the yield composition of rice.Appropriate tillering ability is an important goal of breeding rice for the ideal plant architecture.In the previous study,it was found that the over-expression of LRK2 transgenic lines increased the effective tiller number and increased the resistance to drought stress.In addition,LRK2 was found to interact with eukaryotic translation elongation factor OsEF1A.In order to analyze the function of OsEF1A,the interaction gene of LRK2,OsEF1A was over-expressed in rice by transgenic technique.And the tissue specific expression analysis of OsEF1A was carried out.Those results will help us preliminarily understand the function of OsEF1A.In this study,to explore the biological function of OsEF1A in rice stress response,we investigated the adaptive response of OsEF1A over-expression lines under various stress conditions.In addition,the interaction between LRK2 and OsEF1A was further confirmed by yeast two-hybrid technique.Meanwhile verified other downstream proteins that interact with LRK2.This study provides new informations to understand the molecular mechanism of LRK2.The detailed research results are shown as follows:(1)Biostatistics were used for statistic agronomy traits of over-expression OsEF1A lines,including effective tiller number,plant height,thousand seed weight and so on.Statistical results showed that over-expression OsEF1A can significantly improve the tiller number,effectively reduce the plant height,thousand seed weight and seed setting rate.(2)20% PEG simulate drought,4 ? low temperature,0.15mol/L NaCl and 20?mol/L ABA were respectively used for abiotic stresses.We analyzed the expression levels of OsEF1A by qRT-PCR.The results showed that the expression levels of OsEF1A was induced by these environmental stresses,which is suggested that the induction of OsEF1A expression by abiotic stresses might reflect the general adaptive response of rice to the adverse circumstances.(3)The interaction between OsEF1A and LRK2 was further confirmed by yeast two-hybrid method.Other two downstream proteins that interact with LRK2 were verified at the same time.We also constructed vectors for transgenic into rice to study their expression patterns and functions.Leaves are the most important source of nutrients needed for the normal growth and development of plants.It is the main organ for photosynthesis,respiration and transpiration.Therefore,the study of plant leaf color is essential.In the previous study,we screened to a yellow-green leaf mutant ygl15.The YGL15 was cloned on chromosome 2 between RM279 and Pm6 by map-based cloning.In order to analyze the function of YGL15 gene,we analyzed the expression and localization of YGL15 by GUS and GFP transgenitic plants.At the same time,we analyzed the development of chloroplast in ygl15 and the expression levels of genes which relate to chloroplast development and photosynthesis in ygl15.The purpose of this experiment is to explore the function of YGL15 and provide valuable genetic information resources for the follow-up function analysis.The detailed research results are shown as follows:(1)The expression levels of YGL15 in different tissues and organs were detected by qRT-PCR and GUS staining.These results showed that YGL15 was predominantly expressed in green organs of rice,with the highest transcript levels in leaves,followed by root-shoot junctions,culms,roots and leaf collars,but with very low expression levels in young spikes.(2)We transfered YGL15 fused with GFP protein into rice and tobacco epidermal cells.Then confocal microscopy observation were used to analyze the YGL15 subcellular level expression.The results showed that YGL15 was located on the cell membrane and its localization showed a polarity.(3)We observed the phenotype of mutant ygl15 and investigated these main agronomic traits.Compared with wild-type GZ63S,ygl15 mutant exhibited a yellow-green leaf phenotype throughout the entire growth period.Its level of photosynthetic pigments was significantly decreased throughout the whole growth period.To investigate whether the ygl15 mutation affects chloroplast development,we compared the ultrastructure of chloroplasts in ygl15 and the wild-type GZ63S at the seedling stage using TEM.The results show that the chloroplast of ygl15 developed much loosed thylakoid lamellar structures.(4)We analyzed the expression levels of related genes of chloroplast development and photosynthesis in mutants ygl15 and wild-type GZ63S by qRT-PCR.The expression levels of nuclear-associated nuclear genes in photosynthesis,rbcS,RCA,Cab1R,Cab2R and V2,were significantly reduced in ygl15.Expression of the chlorophyll synthesis genes,including YGL1,HEMA1 and DVR,were also significantly reduced in ygl15.The expression of plastid genes which transcribed by NEP,RpoA,RpoB and Rpsl2,were increased.However,the expression levels of PEP-dependent genes(psaA,psbA,and rbcL)were significantly reduced in ygl15.