Screening Of Key Metabolic Pathways For Late Spring Cold Tolerance Of Wheat Panicles And Functional Identification Of TaJAZ6 Gene

Wheat(Triticum aestivum L.)is one of the three major crops in the world and the staple food of 60 % of the world ’s population.Its high and stable yield is an important guarantee for the national economy and people ’s livelihood.In the context of climate warming,the late spring cold weather is one of the important weathers that endanger the Huang-Huai wheat region.The late spring cold weather leads to frostbite of young ears and poor floret development,resulting in a significant decrease in yield.Frequent late spring cold disasters urgently need to carry out research on the related mechanisms of wheat resistance to late spring cold.Studying the cold tolerance mechanism of wheat and excavating cold response genes are not only of great significance to reveal the cold tolerance mechanism of wheat,but also provide candidate genes for the study of wheat resistance to late spring cold,so as to provide important theoretical basis for the genetic improvement of stress resistance and the cultivation of high-quality wheat varieties resistant to late spring cold。In this experiment,ZM366 and AK58 were used as experimental materials,and a variety of physiological indexes were measured after low temperature stress treatment at the pistil and stamen primordium differentiation stage,aiming to comprehensively and systematically reveal the physiological mechanism of wheat panicle defense against low temperature damage;The metabolic pathways of wheat were explored by transcriptome sequencing and the key JAZ genes were screened out.The functions of JAZ genes were identified by bioinformatics analysis,subcellular localization and fluorescence quantitative PCR,which was helpful to reveal the molecular regulation network of wheat in response to low temperature stress as a whole and provide new gene resources for cold tolerance breeding.The findings are as follows :1、Analysis of seed setting rate and physiological characteristics of two wheat varieties under low temperature stressAfter 72 h of 0℃ low temperature treatment,low temperature had a slight effect on the spike shape of AK58,but had a significant effect on the spike shape of ZM366.The seed setting rate of AK58 and ZM366 decreased by 12% and 89%,respectively;The soluble sugar content of AK58 and ZM366 decreased by 39% and 43% respectively;Under the same treatment,the soluble sugar content of AK58 was higher than that of ZM366;Soluble protein content increased by 1.3 times and 1.1 times,respectively.The MDA content of young spikes of AK58 decreased significantly,while that of ZM366 was opposite;Compared with the control,low temperature treatment increased the activity of POD and SOD in AK58 but decreased the activity in ZM366.The CAT activity of ZM366 increased,but the increase was less than that of AK58.According to the above results,AK58 is more resistant to spring cold than ZM366.2、Screening of key metabolic pathways for wheat resistance to late spring cold based on transcriptomicsThe young panicles of AK58 and ZM366 treated at 0 ℃ for 72 h were used as materials.The transcriptome sequencing of young panicles of AK58 and ZM366 treated at low temperature and control group was performed using Illumina platform.Compared with the normal temperature control,2468 up-regulated genes and 3688 down-regulated genes were identified in AK58.A total of 4286 up-regulated and 5145 down-regulated DEGs were found in ZM366.In the two genotypes,down-regulated DEGs were more than up-regulated DEGs.Among the genes responding to low temperature,there were 3642 DEGs in the two materials,and 2514 and 5789 DEGs were specifically expressed in AK58 and ZM366,respectively.KEGG enrichment analysis of differentially expressed genes showed that Wheat young ears can respond to and adapt to spring freeze stress by changing Starch and sucrose metabolism,Photosynthesis-antenna proteins pathway,Circadian rhythm-plant,Plant-pathogen interaction,Amino sugar and nucleotide sugar metabolism,Fatty acid elongation,Ascorbate and aldarate metabolism,Pentose and glucuronate interconversions,Phenylalanine metabolism,RNA degradation,SNARE interactions in vesicular transport,Plant hormone signal transduction,Phenylpropanoid biosynthesis pathways.Further comparison of the metabolic pathways of two wheat varieties in response to spring freeze stress revealed that the young spike of wheat can resist the spring freeze stress mainly by changing the starch and sucrose metabolism,the antenna protein pathway of photosynthesis,the circadian rhythm plants,the plant hormone signal transduction,and the proline metabolism pathway.These metabolic pathways and signal transduction pathways play an important role in spring freeze stress response.3、Effects of MeJA on physiological characteristics of wheat under low temperature conditionsThere were significant differences in the response of AK58 and ZM366 to low temperature stress.The changes of soluble sugar,soluble protein,malondialdehyde(MDA),superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)activities showed that the normal metabolism of the two genotypes of wheat was inhibited under low temperature stress in spring,and the frost resistance of the two genotypes was different.ZM366 was sprayed with methyl jasmonate(MeJA).Under low temperature stress,300μmol / L and350μmol / L exogenous methyl jasmonate could effectively increase soluble protein content,peroxidase(POD)and catalase(CAT)activity,and reduce malondialdehyde(MDA)content.4、Bioinformatics analysis of TaJAZ6 proteinIn this study,the full-length sequence of TaJAZ6 gene was cloned from Zhengmai 366.The open reading frame encodes 179 amino acid residues.The full-length CDS sequence of the gene is 537 bp,the isoelectric point is 5.07 PL,and the relative molecular mass is44535.24 MW.The instability coefficient of negatively charged residues is 44.51,the total average hydrophobicity is-2.522 < 0,and the unstable hydrophilic protein with signal peptide.The results of protein interaction showed that TaJAZ6 may be directly or indirectly involved in the regulation of jasmonic acid signaling pathway.The protein was mainly composed of α-helix,extended strand and random coil,which had 66.67 % similarity with the template 5t0 q.1.B.A total of 12 types of cis-acting elements were identified,including light response,low temperature response,low temperature stress induction,participation in defense and stress response,anaerobic induction,MeJA response,flavonoid response,abscisic acid response,etc.Evolutionary analysis showed that it had high homology with wild emmer wheat.5、Subcellular localization of TaJAZ6The subcellular localization of TaJAZ6 protein was carried out by Agrobacterium-mediated transient transformation onion technology.The results showed that the protein was in the nucleus,indicating that it mainly performed its function in the nucleus,which was the same as the subcellular localization prediction results.6、The expression of TaJAZ6 gene under different stressesIn response to MeJA and low temperature stress,TaJAZ6 gene was expressed in roots,stems,leaves and ears,with the highest expression level in roots.Low temperature treatment significantly increased the expression of TaJAZ6 in roots,stems,leaves and ears.Spraying300μmol/L MeJA significantly increased the seed setting rate of wheat under low temperature stress.Meanwhile,300μmol/L MeJA decreased the expression of TaJAZ6 gene in roots,stems,leaves and young ears under low temperature stress.It is speculated that TaJAZ6 gene may participate in jasmonic acid signal transduction and negatively regulate the metabolism of resistance to cold spring inversion in wheat.