Functional Analysis Of Spartina Alterniflora SabZIP19 And SaTRA2 Genes In Rice Salt Tolerance

Soil salinization slow the growth and development of crops and reduces crop yields,which seriously threatens the national food security of our country.As our major crops are monocotyledonous plants,it is of great significance for national food safety to dig deep into high-salt stress response genes from monocotyledonous halophytes and engineering salt tolerant crops.Spartina alterniflora is an important invasive monocotyledonous halophyte in the coastal salt marshes of China.It has excellent wide adaptability and high salt tolerance.Our previous study showed that Spartina and rice transcriptome have high similarity.Therefore,cloning the high-salt tolerance gene from Spartina using molecular biology and applying it to the improvement of rice salt tolerance is an effective method for breeding salt-tolerant rice,which has certain practical significance in agricultural production.This study based on the high salt stress related second and third-generation transcriptome data from our previous studies in Spartina,and used bioinformatics methods to mine high salt stress response genes.These genes were then further functional characterizated in both model plant Arabidopsis and crop-rice,with a view to providing certain genetic resources for improving salt tolerance in rice.The relevant content and results of this study briefly described as follows:(1)Integrated the second-generation RNA-seq data of Spartina from our previous study,and identified 66 high salt stress positive response genes from the list of differential expressed transcripts under different salinity conditions(control,350 m M,500 m M,800 m M NaCl)with the criteria: FDR < 0.05 and | Fold change |> =2.Among them,six genes(Cluster12777,Cluster3405,Cluster17656,Cluster15847,Cluster23863,Cluster28253)were used for further study.(2)The gene expression of six high salt tolerant candidate genes verified by RT-PCR using Spartina alterniflora seedlings under different salinity conditions(350 m M,500 m M,800 m M NaCl).The results showed that under high salt treatment,the expression of the above genes increased significantly,indicating that these genes are potential high salt stress response genes.(3)The sequence of SabZIP19 transcription factor encoding gene(Cluster12777)extracted from our previous third-generation full-length transcriptome of Spartina alterniflora.The gene then cloned and transformed into model plant Arabidopsis to characterize its function on salt tolerance.Western blot analyses showed that SabZIP19 successfully transformed into Arabidopsis and expressed correctly.The high salt-tolerant study with the T3 generation of SabZIP19 over-expression lines showed that SabZIP19 grew better and had longer root length than the control under high salt stress,which preliminary verification that SabZIP19 can improve the salt tolerance of plants.In addition,the study also found that SabZIP19 overexpressing plants flowered earlier than the control group,indicating that SabZIP19 can participate in the regulation of plant growth cycle.(4)The Spartina salt-tolerant candidate gene list were then screening again to select genes that have not been studied for salt tolerance in plants,and preliminary results showed that the SaTRA2(Cluster3405)is a newly discovered high salt stress response gene.Both SaTRA2 and SabZIP19 then over-expressed into Kitaake rice.The T1 generation of SabZIP19 and SaTRA2 over-expression transgenic lines used for functional characterization of their roles in salt tolerance.The results showed that under 200 m M salt stress,the SabZIP19 and SaTRA2 overexpression lines had longer root length than the control group.Moreover,SabZIP19 overexpression lines had higher Plant height.Furthermore,SabZIP19 and SaTRA2 overexpression lines had higher chlorophyll content than the control group.In addition,SabZIP19 overexpression lines had significantly increased dry weight compared to the control group.The above results indicated that SabZIP19 and SaTRA2 could enhance the salt tolerance of rice to a certain extent.(5)In order to explore the potential molecular mechanism of SaTRA2 involved in plant salt tolerance,this study carried out a quantitative expression study on the salt stress genes SOD1,SOS1,NHX1,and P5CS1 in SaTRA2 overexpressing plants,and found that these key salt stress responsive genes were increased in a certain degress compared to the control under high salt stress.The results indicated that SaTRA2 can alleviate the high salt toxicity by enhancing the expression of the key salt stress responsive genes.