Research On Resistance Of G Protein α Subunit RGA1 To Salt Stress In Rice

Heterotrimeric GTP-binding protein,abbreviated as G protein,is a type of signal transduction protein,which plays an important role in the signaling process of animals and plants from extracellular to intracellular conduction.For example,the mutant G_αsubunit gene in rice can cause dwarfing rice plants,erecting ears and decreasing grain size.Recently,it has also been found to play a certain role in drought tolerance.Its alleles are widely used in japonica rice production.With the gradual reduction of cultivated land area and the increasingly serious problem of soil salinization,it is of great significance to cultivate new varieties of salt-tolerant rice.The key to the cultivation of salt-tolerant varieties is the identification of salt-tolerant genetic resources.In order to clone salt-tolerant rice genes,we screened the mutant library contain the descendant derired form the treating of Xida 1B with EMS（Ethal Malthane Sulfonate）,and a semi-dwarfed plant,smaller grain,and enhanced salt tolerance mutant was identified,named s525.The study was conducted in terms of cytology,genetics and molecular biology.The main findings are as follows:1.Phenotyping of s525When planting in the field,from the seedling stage,the plant height of the mutant was significantly lower than that of the wild type and continued until maturity,and the plant height of the mutant was only about 61.8%of the wild type at the mature stage.The reason for the dwarfing is that the length of the downside and downside of the mutant is extremely shortened,and the length of the downfall and downfall is significantly shorter.In addition,the spike length was shortened,but there was no significant change in the number of effective panicles and seed setting rate.Compared with the wild type,the length and width of the mutant seeds were significantly reduced,and the thickness did not change significantly,showing the characteristics of small round kernels,and the 1000-grain weight was also significantly lighter than that of the WT.2.Photosynthetic and flag leaf width of s525The length of flag leaf at the filling stage of the mutant was significantly shorter than that of the wild type,but the leaves became extremely significantly wider.In addition,the main reason for the widening of the leaves is the increase of large vascular bundles in the mutant leaves.In the frozen section of the flag leaf,it was found that the number of mesophyll cells in the mutant leaves was significantly higher than that in the wild type.The photosynthetic parameters measured subsequently showed that the net photosynthetic rate,transpiration rate,and stomatal conductance of the mutant leaves increased by 8.21%,28.99%,and 48.88%,respectively,compared with the wild type,and they all reached extremely significant differences.3.Gene mappingGenetic analysis using the F₁ and F₂ populations that were hybridized with the mutant s525 and Suihui 10 revealed that the s525 mutation was regulated by a pair of recessive nuclear genes.Using 609 F₂ recessive individuals,the s525 gene was finally located within the 179.81 kb physical distance between the fifth chromosome,S113-38and RM18413.There are 18 genes in this mapping interval,including the RGA1 gene（LOC_Os05g26890）.4.Candidate gene screening and transcriptional analysisAfter sequencing,it was found that the first base of the eleventh intron,G of the RGA1 gene was replaced by A in the mutant,and RGA1 was initially determine to the candidate gene.At the same time,it was also found that the mutation site affected the normal shearing of RGA1 mRNA,that is,the mutant did not produce normal D1 CDS,but produced two kinds of abnormal CDS.In the first type of CDS,exon 11 and exon12 were both cut off at the same time,and the total length of CDS was 892 bp.The second type of CDS directly retains the eleventh intron,and contain exhibited 1109 bp in length.5.s525 possesses high salt toleranceAfter immersed in 50 mM,100 mM,150 mM and 200 mM NaCl solutions,the germination rate of the WT seeds was lower than that of the mutant seeds.Even at 200mM,only the germination rate of the mutants was half.Among isolate leaves under salt stress,it was found that when the wild-type leaves turned yellow,the mutant leaves remained green.And in the salt stress experiment of living seedlings,it was also found that the mutants have lower sensitivity to salt stress.Under the treatment with 200 mM NaCl for 3 day,the whole plant of WT dehydrated and wilted,but that of s525 were upright and maintained vigor.6.QPCR analysis of salt-tolerance related genesThe RGA1 expression was up-regulated in the wild type treated with salt stress but down-regulated in the mutant,indicating that RGA1 is involved in the signal transduction of salt stress.The expression levels of other three salt-tolerance related gens,i.e.OsSIPP2C1,OsJAZ13 and Dehydrin were up-regulated in the mutants treated with salt stress,and the up-regulation level was much higher than that of the WT.Indicating that the salt-tolerance of the mutants was stronger than that of the WT.