Screening Of Heat-resistant Wheat Germplasm Resources And Study On Heat-tolerance Of Heat-related Gene TaHsfA2-1

Wheat?Triticum aestivum L.?is an important food crop in China.It is vulnerable to subject to high temperature stress in the later growth stage,which accelerates the grain filling rate and causes early plant death,resulting in unsaturated grains,reduced yield and quality.In this study,10different winter wheat varieties widely planted in Hebei province were used.Field experiments and laboratory tests were combined to investigate the heat tolerance of wheat at different growth stages in the field.The number of grains per spike,1000-grain weight,heat-lethal time and plumpness were measured,then the heat-tolerant wheat variety was screened by analyzing and comparing the thermosensitivity index.Based above,heat shock transcription factor gene TaHsfA2-1 was isolated from heat-tolerant variety Cang 6005.Based on the analysis of gene biological characterization,the thermotolerances of gene TaHsfA2-1 were assayed through transformation into yeast and Arabidopsis,respectively.The results will provide heat-tolerance wheat varieties and strong responsive gene for heat-tolerant breeding and genetic improvement of wheat.The results are as follows:1.The time of heat death among different wheat varieties were significant difference during the same growth period under the normal conditions.There was a significant difference between Cang 6005 and the other 9 varieties while subjected to heat treatment during grain filling period,the time of heat death of Cang 6005 was longest,the value is 40.8 min.Cang 6005 has relative stronger heat-resistance in late growth stage.2.Among the 10 wheat varieties planted in the field?normal growth conditions?and in the automation greenhouse?heat stress conditions?,there were significant differences of grain number per spike between Cang 6005 and other varieties under the same conditions.The number of grains per spike of Cang 6005 was the highest both under heat treatment and normal field conditions with the value of 39.333 and 40.667 grains,respectively.No significant difference of the grains per spike in automatic greenhouse and field could be observed in Ji 5265,the number of grains per panicle was 33 and 33.667 grains,respectively.The number of grains per panicle of other varieties under heat treatment was 3⁶ grains less than that in field.Heat stress took no significant effect on grain number per panicle of Cang 6005 and Ji 5265.3.While subjiected to heat treatment in automatic greenhouse,the grain plumpness of all varieties were lower than that planted under the normal conditions in field.The change rate of grain plumpness of Cang 6005 was the smallest,which was 0.69%.Heat stress took little effect on grain plumpness of Cang 6005.4.The 1000-grain weight of variety Jinhe 9123 was the largest among the 10 varieties planted in the field?normal growth conditions?and in the automatic greenhouse?heat stress conditions?,which were 45.177 g and 49.613 g,respectively,with significant difference.The 1000-grain weight of Cang 6005 were 39.033 g in the automation greenhouse and 40.312 g in the field,the difference between them is 4.436 g.The 1000-grain weight of other varieties in the automation greenhouse was 4.43⁷.84 g,which was less than that in the field.Heat stress took no significant effect on1000-grain weight of Cang 6005.5.Among the 10 Wheat varieties planted in the field?normal growth conditions?and in the automatic greenhouse?heat stress conditions?,variety Jinhe 9123 had the highest yield,which was8337.0 Kg·hm^-22 in the field and 7833.0 Kg·hm^-22 in the automation greenhouse.The yield of Cang6005 was 7850.0 Kg·hm^-22 in the field and 7579.0 Kg·hm^-22 in the automation greenhouse,no significant difference exists.The yield difference of other varieties ranged from 410 to 1288.5Kg·hm^-2.Heat stress took no significant effect on the yield of Cang 6005.6.Cang 6005,a heat-tolerant cultivar,was screened through the thermosensitivity index.Its1000-grain weight-thermosensitivity index was 0.26,yield-thermosensitivity index was 0.39 and total thermosensitivity index was 0.65.7.TaHsfA2-1 was cloned from Cang 6005 leaves.Its coding sequence was 1041 bp in length,encoding 346 amino acid residues,containing DBD,OD and NLS domains.The results of qRT-PCR showed that the expression of TaHsfA2-1 gene in mature leaves was the highest,the value was 10 times of that of control?young roots?.Heat shock at 37?significantly upregulated the gene expression both leaves and roots.The expression of TaHsfA2-1 gene in leaves reached a peak at 90 minutes after heat shock,with a value of 44 times of that in control?0 min?.The expression of TaHsfA2-1 gene in roots reached peak at 60 minutes after heat shock,with the 12times expression level of that in control?0 min?.The expression of TaHsfA2-1 could be significantly up-regulated by 10 mmol·L^-11 SA,0.8 mmol·L^-11 H₂O₂ and 20%PEG,respectively,and the expression reached its peak at 120 min,the expression level were 5,8 and 4 times of the control?0 min?,respectively.8.Subcellular localization by transient expression in tobacco epidermic cells revealed that TaHsfA2-1 was located in the nucleus.TaHsfA2-1 was transformed into yeast and Arabidopsis by Agrobacterium-mediated method,respectively.It was found that the growth of transgenic yeast and Arabidopsis seedlings under heat stress were stronger than that of the control?empty vector?.After the basal and acquired heat treatments for 2 h,the expression of multiples Hsp genes of transgenic Arabidopsis seedlings could be up-regulated to different degrees,suggesting the thermotolerance mechanism of Cang 6005.