Improvement Of Drought Resistance And Low Nitrogen Stress Tolerance By SiWLIM2b Gene In Rice

Foxtail millet(Setaria italica L.)originated in China and has the characteristics of drought resistance and barrenness resistance.Foxtail millet genome is small.With the release of foxtail millet genome,foxtail millet has become an ideal material for studying stress resistance of monocotyledon crops.However,functional gene research in Foxtail millet has just started,and the unique stress-resistant regulation mechanism of foxtail millet is still poorly understood.Research on functional genome of stress resistance in foxtail millet will provide new ideas for molecular breeding of stress resistance in monocotyledon crops.In this study,we performed a transcriptome analysis of foxtail millet under drought and low-nitrogen conditions,and identified a gene SiWLIM2 b,which was down-regulated under drought and up-regulated under low-nitrogen stress.SiWLIM2 b belongs to LIM(Mec-3,Lin-11,Isl-1)gene family.Here we took advantage of the foxtail millet genome sequence to identify all the members of the LIM gene family.We analyzed LIM gene of foxtail millet expression under abiotic stress and hormone treatments using qRT-PCR.In order to further analyze the functions of SiWLIM2 b in drought resistance and low-nitrogen conditions,Phenotypic analysis was performed using homozygous T3 seeds of transgenic rice lines expressing SiWLIM2 b.The main test results obtained are as follows:1.By searching the foxtail millet genome,we identified a total of 10 putative LIM genes.A phylogenetic tree of the LIM domain proteins was constructed,and the result indicated that the LIM gene family differentiated after the divergence of monocotyledons and dicotyledons.In this study,chromosomal distribution analysis of the LIM gene family of foxtail millet indicated that the formation of LIM genes family of foxtail millet occurred after the chromosome duplication events.QRT-PCR analysis showed that the LIM gene of foxtail millet was induced by various stresses and exogenous hormones.2.The CDs region of SiWLIM2 b gene in foxtail millet consists of 600 bp bases,encoding 199 amino acids,with a relative molecular weight of 22.02 kD and a predicted isoelectric point of 8.98.The SiWLIM2b-GFP fusion protein was mainly distributed in both the cytoplasm and nucleus.The SiWLIM2 b gene had the highest expression levels in roots.3.The pot experiment and hydroponics experiment results showed that WT plants withered and died earlier than the transgenic rice plants.Under drought treatment,the MDA content of the three transgenic rice lines was significantly lower than that of WT,Chlorophyll and proline content of the three transgenic rice lines was significantly higher than that of WT.These results indicate that over-expression of the foxtail millet SiWLIM2 b gene enhances drought tolerance in transgenic rice.The qRT-PCR data showed that these phenylpropane synthesis-related genes were more highly expressed in the transgenic rice lines than in WT under both normal growth conditions and under drought treatment.The results of dual-luciferase report assay system experiment showed that the SiWLIM2 b gene of foxtail millet could directly bind the promoter regions of CAD,PAL,CCR2 and 4CL2 genes related to phenylpropane pathway.The flavonoid content of the SiWLIM2 b transgenic rice lines was significantly higher than that of recipient rice.These results indicate that over-expression of the foxtail millet SiWLIM2 b gene enhances drought tolerance in transgenic rice by influencing the expression of genes related to phenylpropane secondary metabolic pathway and enhancing the content of flavonoids.4.Under nitrogen deficiency treatment,the hydroponics experiment results showed that WT plants growth worse than the transgenic rice plants.The content of glutamine synthetase,the key enzyme of ammonia assimilation pathway in transgenic rice lines,was significantly higher than that in recipient rice.These results indicate that SiWLIM2 b gene plays a role in resisting low nitrogen stress by enhancing ammonia assimilation in transgenic rice.The SiWLIM2 b gene can potentially be used for molecular breeding of crops with increased resistance to abiotic stress.