Functional Analysis Of Wheat Salt-Alkali Stress Responsive Serine Threonine Protein Kinase Gene TaSTPKB

Abiotic stress has a great impact on plant growth and crop yield.Saline-alkali stress and oxidative stress are common abiotic stresses.Wheat is a common crop in China,but the wheat varieties are sensitive to salt and alkali stress,resulting in a significant reduction in production under saline-alkali environment.Characterizing genes related to abiotic stress resistance and exploring their mechanism has become an effective way for crop breeding to achieve stable and increased yields.Asymmetric somatic hybridization between common wheat and tall wheatgrass generated a variety of introgression wheat lines with different traits.Among them,Shanrong 4 showed obvious resistance to salt-alkali stress,which provided us a special material for studying the regulation mechanism of wheat in response to salt-alkali stress.Previously,the saline-alkali tolerant candidate gene TaSTPKB was selected by analyzing the Shanrong 4 transcriptome,but its function and mechanism of action is still unclear.In this thesis,we explored the saline-alkali tolerant function and mechanism of TaSTPKB.The results of the study are as follows:1.Bioinformatic analysis for TaSTPKBThe open reading frame of TaSTPKB is 1206 bp,encoding 402 amino acids,containing STKC-IRAK domain,ATP binding site and peptide substrate binding site.Alignment with genome sequences of wheat variety Chinese Spring revealed that TaSTPKB may be located on the 6B chromosome of wheat.The homologous multiple sequence alignment showed that this gene is closer to the STPK gene of Aegilops,Trichosanthes,and Barley,suggesting that TaSTPKB may have similar functions to STKB of Aegilops,Trichosanthes,and Barley.Analysis of the promoter sequence showed that the promoter of TaSTPKB gene has light response,ABA response and low temperature response elements,indicating that this gene may be involved in the response of wheat to stress.Subcellular localization analysis indicated that TaSTPKB protein is located in the cytoplasm and nucleus.2.TaSTPKB makes plants sensitive to abiotic stressIn order to study the function of TaSTPKB gene,we constructed wheat overexpression lines（OE）and wheat RNAi lines.We found that the aboveground part of the wheat OE lines of TaSTPKB was shorter than that of the control line Yangmai 20（YM20）under alkali stress.Under salt,oxidative and ABA stress,the growth of the wheat OE lines was also weaker than that of YM20,as the aboveground part of OE lines was shorter and the fresh weight was lighter than YM20.TaSTPKB RNAi lines showed resistance to saline-alkali,oxidative and ABA stress compared with control line YM20,as the aboveground part of RNAi lines was longer and the fresh weight was heavier than that of YM20.3.Mechanism analysis of TaSTPKBThe H2O2 content in the wheat OE lines was higher than that of YM20;DAB and NBT staining also showed that TaSTPKB-OE was darker than YM20,and TaSTPKBRNAi was lighter than YM20;We detected the expression level of ROS synthesis and scavenging related genes through qRT-PCR.The results indicated that the expressions of ROS scavenging related genes was down-regulated in OE plants,and the expression of ROS synthesis related genes increased in OE lines under alkaline stress treatment.The activities of ROS scavenging enzymes SOD and POD were all decreased in OE wheat lines.Based on the above results,we speculate that the overexpression of TaSTPKB may result to the accumulation of too much ROS,which damages the normal life activities of plant cells and makes the plants sensitive to stress.To exploring the possible mechanism of TaSTPKB under stress,we identified TaSTPKB interacting proteins through screening the yeast cDNA library.Through yeast two-hybrid assay and split-luciferase complementation assay（BILC）,we confirmed that TaSTPKB interacted with TaHMP,and TAHMP interacted with TaCSD.TaHMP is a copper transporter,and TaCSD is a member of the SOD family,and the activity of TaCSD requires the participation of copper ions.Therefore,we hypothesized that TaSTPKB may phosphorylate TaHMP toinhibit its activity,so that TaCSD could not receive the necessary copper ions for its work,and the decreased activity led to the accumulation of ROS,which made plants sensitive to stress.