| Wheat is a glycophyte and the widely cultivated wheat varieties usually shows low tolerance to salt and alkali stress,and often results in a significant decrease in yield under adverse environmental conditions such as salt and alkali.Salt-alkali stress often occurs hand by hand,but the damage caused by alkali stress to plants is more serious than salt stress.QTL mapping of wheat alkali-tolerant traits and identification of alkalitolerant functional genes is an effective way to ensure stable wheat production under adverse conditions such as salt and alkali.Shanrong 4(SR4)is a new saline-alkali tolerant introgression line selected from the asymmetric somatic hybrids of wheat/tall wheatgrass.Field and saline-alkali pond planting experiments have confirmed that SR4 has strong alkali tolerance ability,which is an ideal material for studying wheat alkali tolerance gene.In this paper,the function of alkali stress responsive gene TaCCD1 from SR4 was studied,and the QTL mapping for alkali tolerance was carried out using the recombinant inbred lines of SR4 and Chinese Spring.1.Functional study of the alkali stress responsive gene TaCCD1 from wheat introgression line SR4The TaCCD1 was identified from SR4 as an alkali stress responsive gene through transcriptome analysis.We analyzed the expression level of the TaCCD1 homologous genes in the A,B and D genomes of wheat and found that the expression level of the gene on chromosome 7B was the highest.Through protein sequence analysis,we found that TaCCD1 has a conserved EF-hand domain,which may function as a calcium ion binding protein.In order to verify the calcium ion-binding activity of TaCCD1,we expressed TaCCD1-GST fusion protein in E.coli and obtained the TaCCD1 protein through affinity purification,and then the calcium ion-binding activity of TaCCD1 protein was verified by different methods.First,the in vitro calcium ion binding migration experiment confirmed that TaCCD 1 has the ability to bind calcium ions;second,the excitation light intensity measurement of the Fura2 calcium ion affinity probe under in vitro incubation verified that TaCCD1 can bind calcium ions in vitro;finally,affinity purification of TaCCD1 by calcium ion affinity magnetic beads confirmed the calcium ion binding ability of TaCCD1.Through transient expression in wheat protoplasts,TaCCD1 was found to be located in the nucleus and cytoplasm.In order to study the function of TaCCD1,we constructed TaCCD1 Arabidopsis heterologous overexpression transgenic lines(OE).The study found that under the conditions of high pH and alkaline salt treatment,the root length of the OE lines is longer than that of the wild type,and the leaves are also larger than that of the wild type.Under H2O2 treatment,the number of lateral roots of the OE lines was significantly higher than that of the wild type;under ABA treatment,the root length of the OE lines was significantly increased compared to the wild type,indicating that TaCCD1 can enhance the plant’s ability to resistant alkali,oxidative stress and ABA.Through qPCR analysis,we found that calcium ion can significantly increase the expression of TaCCD1 in SR4.In order to further analyze the relationship between TaCCD1 and calcium ions,we used calcium ion fluorescent probes to detect the content and distribution of calcium ions in live cells of wild-type and OE lines.Under normal conditions,there is no significant difference in fluorescence intensity between wild-type and overexpression lines,but under alkaline conditions,the overexpression lines have stronger fluorescence intensity than wild-type,indicating that TaCCD1 promotes intracellular release of calcium ion.Further analysis found that alkali stress treatment will promote the calcium ion fluorescence signal gradually enriched from the cytoplasm to the cell membrane,and this phenomenon is more significant in the TaCCD1 overexpression line.In order to study the molecular mechanism of TaCCD1 to improve the resistance of plants to alkali stress,we used yeast two-hybrid to screen TaCCD1 interacting proteins.About 700 positive clones were preliminarily screened out and about 300 possible interacting proteins were selected by sequencing the positive yeast plasmids.By predicting the function of the possible interacting proteins,we selected some candidate proteins and cloned their full-length coding sequences for further interaction verification.Full-length sequence yeast two-hybrid analysis showed that TaCCD1 interacted with TaCTR1,TaPID and TaPP2C in yeast.Further bimolecular fluorescence complementary experiment(BiFC)verified that TaCCD1 interacts with TaPID and TaPP2C,and the interaction occurs mainly on the cell membrane.Studies in Arabidopsis have confirmed that the phosphorylation of PIN2 will lead to the change of its polar localization,and PIN2 is the phosphorylation substrate of PID protein.We treated the PIN2-GFP Arabidopsis lines with alkali stress and found that PIN2 appeared to change their polar localization,which indicates that PIN2 will be phosphorylated under the treatment of alkali stress,thus affecting the polar transport of auxin.Whether the interaction between TaCCD1 and TaPID affects the phosphorylation of PIN2 to further affects the polar transport of auxin and thus regulates the plant’s alkali tolerance is worthy of further study.In addition,TaCCD1 interacts with the negative regulator of ethylene signal CTR1.Exogenous application of ethylene precursor ACC can inhibit the alkali tolerance of TaCCD1 transgenic lines,suggesting that TaCCD1 may affect the ethylene signaling pathway through interaction with CTR1 to enhance the alkali tolerance of plants.2.SR4 alkali-resistant QTL positioningSR4 is a saline-alkali resistant wheat introgression line selected by asymmetric somatic hybridization between common wheat Jinan 177 and tall wheatgrass.We used SR4 and Chinese Spring to establish a recombinant inbred line population(RIL),and used a 55K SNP chip to perform SNP genotyping on 138 RIL lines.In the linkage groups,the average distance between two markers is 0.1-0.3 cM.By using the yellow leaf rate at the seedling stage as the alkali tolerance index for QTL mapping,three major QTLs for alkali tolerance were initially identified,and the synergistic sites were derived from SR4.These three QTL loci are located on 5A,1D,and 3D chromosomes respectively.The major alkali-tolerance gene on the 5A chromosome is located in a 9 cM intervals,explaining about 19%of the genetic variation.The major alkali-tolerance gene on the 1D chromosome is located in a 17.8 cM intervals,explaining about 10%of the genetic variation.The major alkali-tolerance gene on the 3D chromosome is located in a 6.5 cM intervals,explaining about 15%of the genetic variation.By using the ratio of fresh weight of SR4 and Chinese Spring as the alkali tolerance index,three major QTLs were initially identified,which were located on chromosomes 1A and 2B.The major gene on chromosome 1A is located in a 11 cM intervals,explaining about 18%of genetic variation,and the major gene of locus 2B-1 is located in a 9 cM intervals,explaining about 18%of genetic variation,while the major gene at the 2B-2 locus is located in a 14 cM intervals,explaining about 15%of the genetic variation.The next step is to establish secondary mapping populations by backcrossing RIL lines with Chinese Spring,and fine-mapping different alkali-resistant QTLs. |
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