Physiological And Molecular Mechanisms Of TaPRK In Winter Wheat In Response To Low Temperature Stress

In some areas like Heilongjiang province of China,the poor quality and low yield of wheat comply with the extremely cold environment in winter.Dongnongdongmai 1(Dn1)is the first strong coldresistant winter wheat(Triticum aestivum L.)variety,which is safe in winter in Heilongjiang Province.The species has a reviving rate as high as 85%.Jimai22(J22)cultivar is planted over a wide area in northern China,but cannot survive the cold winter temperatures in Heilongjiang.The species has an 2% winter survival rate.Therefore,it is of great scientific and practical significance to carry out research on the cold-resistance mechanism of winter wheat in agricultural production in the northern high-cold regions.As an important signaling regulator,ABA,SA,and JA plays a key role in plant regulation of abiotic stress.For exploring the change of photosynthetic performance under cold stress(5?,0?,-10? and-25?),the enzyme activity and expression levels of 4 key genes of carbon assimilation(Ta Rbc L,Ta RCA,Ta Rbc S and Ta PRK)in Dn1 and J22 were studied after treated by ABA,SA and JA.Therefore,this experiment will explore the effects of 4 key enzyme genes of carbon assimilation under low temperature stress to determine its relative expression level at low temperature by quantitative method,and bioinformatics prediction analysis and expression pattern analysis of Ta PRK,construction of Ta PRK plant expression vector and its genetic transformation.The study also can complement key enzyme genes of carbon assimilation regulation of winter wheat cold-resistant mechanism.The results are as follows:(1)The Rubisco activity(Ribulose-1,5-bisphosphate carboxylase/oxygenase)in two winter wheat leaves increased first and then decreased with the fall of temperature,and peaked at 0 ?.The activity of Rubisco in Dn1 was significantly higher than that in J22.The RT-PCR results showed that the expression levels of Ta Rbc L(ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit)and Ta PRK(phosphoribulokinase)were shown a change of descending first and then ascending as the temperature dropped,and reached a peak at-10?;The expression levels of Ta Rbc S(ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit)and TaRCA(Rubisco activating enzyme)declined at lower temperature.But exo-ABA significantly enhanced the Rubisco activity of winter wheat as well as the m RNA expression of Ta Rbc L,Ta RCA,Ta Rbc S and Ta PRK under low temperature stress,which Ta PRK increased most significantly.(2)Ta PRK bioinformatics analysis: Ta PRK full-length c DNA with an open reading frame of 1215 bp encoding 404 amino acids,and expression product is a stable hydrophobic protein,localized in the chloroplast,with chloroplast transit peptide.the homologous amino acid protein multi-sequence comparison indicates that the Ta PRK protein contains typical amino acid sequence ATP domains;phylogenetic analysis shows that: Ta PRK is closely related to PRK clusters of monocotyledons,in which wheat and Aegilops tauschii are gathered.(3)Ta PRK expression pattern: During the wintering period,the relative expression levels of Ta PRK in tiller node of Dn1 significantly higher than that in J22.The relative expression levels of Ta PRK in leaves of Dn1 were significantly higher than that in at-10 and-25?,while it lower than that in J22.With the decrease of temperature in field,the relative expression of Ta PRK in tiller node of Dn1 and J22 under SA and Me JA treatment showed a trend of first increasing and then decreasing,peaked at-10?,and were significantly higher than the control group atdifferent temperature.The relative expression of Ta PRK in tiller node of Dn1 and J22 under SA treatment showed that in the reactions decreased-increased-decreased,and were significantly higher than the control group at 10?,which was no significant difference in the other three temperatures;the relative expression of Ta PRK in leaves of Dn1 and J22 under Me JA treatment significantly higher than the control group at 0? and-25?,reached the peak at 0?.(4)The Ta PRK gene of Dn1 was cloned and its length was 1215 bp.It was linked to the plant expression vector and the overexpression vector p CMBIA2300 u of Ta PRK was successfully constructed and 5 positive over-transformed Ta PRK plants the seedlings that had grown to about 4 weeks were treated at low temperature.Physiological indicators showed that the chlorophyll content in the transgenic oxTa PRK-1 and ox-Ta PRK-2 strain was higher than In the WT and prk plants at 0? and-10?,the MDA content and relative conductivity of the transgenic ox-Ta PRK-1 and ox-Ta PRK-2 strain were lower than those of the WT and prk plants at -10 ?.