Functional Analysis Of Wheat Auxin Response Pathway Gene TaIAA10 In Alkaline Stress

Wheat is a glycophyte with lower ability to tolerate salt-alkaline stress,and soil salinization often results a substantial reduction in wheat production.Although the main component of soil salinization is neutral salt NaCl,salinized soil is usually accompanied by a considerable amount of alkaline salt,mainly NaHCO3 and Na2CO3.This characteristic often causes more damage to plants.At present,there are more studies on the mechanism of plant salt stress tolerance,but less on the mechanism of plant alkaline stress resistance.Therefore,there is an urgent need to strengthen the research on plant alkaline stress tolerance.Shanrong NO.4（SR4）is a new salt-alkaline-tolerant wheat variety selected from the introgression lines derived from asymmetric somatic hybridization between common wheat and tall wheatgrass.Previous studies have shown that,compared with salt tolerant wheat variety SR3,SR4 has higher resistance to alkali stress,so it is an excellent material for studying wheat alkaline stress.TaIAA10 is a member of the early auxin responsive Aux/IAA protein family,which was found to response to alkaline stress through transcriptome analysis of alkaline stress treated SR4 seedlings.Previous studies have found that its ectopic overexpression in Arabidopsis improves the resistance of plants to alkaline stress.In this thesis,we further explored the function and mechanism of TaIAA10 in wheat alkaline stress tolerance.1.Sequence analysis of TaIAA10Previously,we identified a wheat Aux/IAA family gene TaIAA10 that is regulated by alkaline stress and showed different expression level between SR4 and JN177 based on the transcriptome data.Combined with the results of transcriptome sequencing,we obtained its full-length sequence through database search,and designed primers to amplify its full-length cDNA sequence.TaIAA10 is a classic Aux/IAA protein with a full-length gene coding sequence of 855 bp,which encoding a polypeptide of 284 amino acids and including all four conserved domains of the classic Aux/IAA protein.By comparing with the reference genome sequence of the wheat variety Chinese Spring,it is found that TaIAA10 is located on chromosome 6A,and there are also highly homologous genes located on chromosomes 6B and 6D.2.Subcellular localization of TaIAA10We constructed a transient expression vector pBI221-TaIAA10-GFP which recombined open reading frame of TaIAA10 and GFP to form a fusion protein and transformed the vector into wheat protoplasts.After culturing for about 24 hours under dark,we observed the distribution of green fluorescent protein using a laser confocal microscope.The results showed that the GFP signal was evenly distributed in the cells transformed with 35S::GFP vector,while the GFP signal was mainly distributed in the nucleus and cytoplasm of cells transformed with 35S::TaIAA10-GFP vector.3.TaIAA10 participates in wheat abiotic stress responseIn order to verify the function of TaIAA10,we constructed a wheat overexpression line（OE）of TaIAA10.The results of phenotypic analysis showed that after alkaline stress or ABA treatment for one week,the TaIAA10 OE lines had higher plant height and longer roots compared with the control line Yangmai 20（YM20）.The leaves of YM20 wilted more serious and contain lesser chlorophyll than OE lines,indicating that TaIAA10 overexpression improves the resistance of wheat to alkali stress and ABA.When treated with H2O2,wheat OE lines have shorter plant height and lighter fresh weight compared with YM20,although the root of OE lines were slightly longer than YM20,these results indicated that the overexpression of TaIAA10 may increase the sensitivity of wheat to H2O2.4.Screening of TaIAA10 interaction proteinsIn the classical process of auxin signal transduction,members of the Aux/IAA protein family usually act as negative regulators of auxin signal,and regulate auxin signal transduction by inhibiting the function of Auxin Response Factor（ARF）.Through yeast two-hybrid screening,we identified three ARF transcription factors that interacted with TaIAA10,and verified the interaction between them through split-luciferase complementation experiments.At the same time,we have verified that the three ARF transcription factors have transcriptional activation activity through the luciferase gene reporter system and the yeast system.5.TaIAA10 regulates TaABI5 expression through TaARF19Previous studies have found that overexpression of TaIAA10 in Arabidopsis can inhibit the expression of key transcription factors ABI3,ABI4 and ABI5 in the ABA signaling pathway,thereby enhancing tolerance to ABA,while its regulatory mechanism is unclear.We detected the expression levels of TaAB13 and TaABI5 in the TaIAA10 OE wheat lines and found that their expression levels in the OE lines were significantly reduced.In order to verify whether TalAA10 affects the expression of ABA signaling pathway genes through its interacting ARF transcription factors,we amplified the 3000 bp promoter sequence upstream of the TaAB15 coding region and found that its promoter region contains ARF transcription factor binding elements,which might be target of ARF transcription factors.The TaAB15 promoter was recombined into the luciferase reporter gene vector pGreenII0800 to construct a ProTaABI5::LUC luciferase reporter system,which was then expressed together with 35S::TaARF19 vector in tobacco leaves.The results show that TaARF19 can activate the expression of TaABI5,when TaIAA10 is expressed at the same time,the activation effect of TaARF19 on TaABI5 is suppressed.These results indicate that TaABI5 may be the target gene of TaARF19,and the interaction of TaIAA10 and TaARF19 inhibited the transcriptional activation of TaABI5 by TaARF19.