Molecular Cloning And Functional Study Of Genes Related To Seed Dormancy And Germination In Mingxian 169 Wheat

Mingxian 169 is a stripe rust inducing material widely used in wheat breeding in Huanghuai wheat region of China,which has strong dormancy,can avoid the occurrence of pre-harvest germination caused by high temperature and rainy weather during seed development and maturity,and reduce the loss of agricultural production.It is an ideal material for studying crop pre-harvest germination resistance.However,the long dormancy time of seeds will affect the rapid and uniform germination,growth and development after sowing,and delay agricultural production.The fallen seeds will also easily lead to biological mixing in autumn breeding fields.Therefore,it is of great practical significance to excavate the seed germination related genes and improve the dormancy of Mingxian 169in actual breeding and agricultural production.In this study,by screening the transcriptome data of the germinated and dormant seeds of Mingxian 169,we cloned jasmonic acid signal pathway genes:TaTIFY3B,TaTIFY10A,TaJAZ1,which were highly expressed in the germinated seeds,and analyzed their bioinformatics characteristics,subcellular localization and expression pattern,over-expressed in rice and Arabidopsis,combined with Arabidopsis T-DNA insertion mutant to verify the function of the gene,in order to provide candidate genes for wheat genetic breeding.The main results are as follows:（1）The CDS sequences of TaTIFY3B,TaTIFY10A and TAJAZ1 were obtained by cloning.Bioinformatics analysis showed that the CDS sequence of TaTIFY3B gene was606bp long,and the protein molecular weight was about 20k Da,consisting of 201 amino acids.It was an unstable hydrophobic protein.The CDS sequence of TaTIFY10A gene is696bp long,and the protein molecular weight is about 24k Da.It consists of 231 amino acids and is a stable hydrophobic protein.The CDS sequence of TaJAZ1 gene is 1230bp long,and the molecular weight of the protein is about 43k Da,encoding 409 amino acids.It is an unstable hydrophobin.All three proteins did not contain transmembrane regions,and all contained the core motif of the TIFY family,TIF[F/Y]XG,and the Jas core motif unique to the JAZ subfamily,CCT-2,which were highly conservative in wheat crops.The promoter regions of the three genes all contained multiple hormone-responsive elements such as methyl jasmonate and abscisic acid and multiple non-biological stress-responsive elements.（2）Subcellular localization analysis showed that the fusion proteins with TaTIFY3B,TaTIFY10A and TAJAZ1 connected to the fluorescent vector could detect fluorescence in cell membrane and nucleus.（3）The analysis of expression patterns showed that TaTIFY3B,TaTIFY10A and TAJAZ1 were all expressed in the whole growth stage of wheat,but their expression levels were different,and their expression levels in the germination stage of wheat seeds were significantly higher than those in the seed development to maturity stage.The experiment further verified that when the seed development entered the mature dormancy stage（40d after anthesis）,the expression levels of TaTIFY3B,TaTIFY10A and TAJAZ1 genes were significantly decreased,which was speculated to be the negative regulatory factor for seed dormancy.At the early germination stage of wheat seeds,treatment with water,50μmol·L^-1abscisic acid（ABA）and 50μmol·L^-1 methyl jasmonate（Me JA）could induce the up-regulated expression of TaTIFY3B,TaTIFY10A and TAJAZ1.（4）Construct an overexpression vector,overexpressing TaTIFY3B,TaTIFY10A and TAJAZ1 in rice Nipponbare,and obtaining 10 T₁ generation positive plants respectively,and selecting three strain with high expression respectively for phenotypic analysis,and that results are as follows:after treatment with water and 20μmol·L^-1 ABA,The germination rate,root length andα-amylase activity and soluble sugar content in seeds of rice overexpressed with TaTIFY3B,TaTIFY10A and TAJAZ1 were significantly higher than those of the receptor Nipponbare,and the induction of ABA signaling pathway gene ABI5was significantly lower than that of the receptor Nipponbare,and the three genes were less sensitive to ABA.However,under the action of Me JA,the germination rates of overexpressed rice and Nipponbare were not significantly different,but they promoted the leaf growth of rice.Theα-amylase activity and soluble sugar content in overexpressed rice seeds were significantly higher than those in Nipponbare.The above results preliminarily indicated that the selected three jasmonic acid signaling pathway genes could promote seed germination.（5）Three genes of TaTIFY3B,TaTIFY10A and TAJAZ1 were overexpressed in Arabidopsis to obtain five positive plants,and two high-expression lines were randomly selected respectively and phenotypic analysis was performed combined with T-DNA insertion mutants of corresponding genes.The results showed that:Under the action of different concentrations of ABA,the seed germination rate and root length of TaTIFY3B overexpressed Arabidopsis are significantly higher than those of wild-type（WT）Arabidopsis and tify3b mutant,and there is no significant difference between the two.After treatment with 0.75μmol·L^-1 ABA,the induction amount of ABA signaling pathway gene ABI5 presents the trend of over-expression>WT>the mutant,and the difference is significant.The overexpression of TaTIFY10A and TaJAZ1 in Arabidopsis and the corresponding Arabidopsis mutants（tify10a and jaz3）had similar phenotypes,and the three genes were less sensitive to ABA.Under the action of Me JA,the seed germination rates of Arabidopsis overexpressing TaTIFY3B,TaTIFY10A and TAJAZ1 were higher than those of WT and the corresponding mutants,but the differences between the latter two were not significant.The root lengths of Arabidopsis overexpressed by TaTIFY3B and TaJAZ1 were significantly longer than those of WT and the mutant,while that of Arabidopsis overexpressed by TaTIFY10A was shorter than that of WT and the mutant.Under normal growth conditions,the flowering time of Arabidopsis overexpressed by TaTIFY3B,TaTIFY10A and TAJAZ1 was earlier,while that of the mutant was delayed.The above results preliminarily showed that TaTIFY3B,TaTIFY10A and TAJAZ1 played important roles in promoting seed germination and plant growth.In summary,the selected jasmonic acid signaling pathway genes TaTIFY3B,TaTIFY10A and TAJAZ1 played an important role in promoting seed germination and plant growth,and were conservative in both dicotyledonous and monocotyledonous functions in this study,which provided a reference for improving crops such as Mingxian 169 that had strong dormancy and delayed agricultural production.