| Wheat（Triticum aestivum）is one of the three major food crops in the world,and its yield and quality are crucial to ensuring global food demand.Under the background of population growth and environmental deterioration,low temperature stress has a great adverse impact on the growth and development of wheat and other crops,which are now facing the dilemma of reduced yield and quality.non-coding RNA（nc RNA）not only plays a key role in plant growth,development,differentiation,metabolism and other processes,but also has been confirmed by more and more studies that nc RNA plays an important role in plant stress response and regulation of plant stress resistance.nc RNA include Long non-coding RNA（lnc RNA）and small RNA（micro RNA,miRNA）.Micro RNA（miRNA）is a class of endogenous non-coding RNA with length of 21-25nt.The expression of target genes involved in plant development and stress tolerance is regulated by degrading m RNA or inhibiting translation,and miRNAs mainly negatively regulate target gene expression at the post-transcriptional level.Dongnongdongmai1（Dn1）is the first winter wheat variety in China with strong cold resistance（-30℃low temperature tolerance）that can safely overwinter in the alpine region of Heilongjiang Province（greening rate>85%）.It can improve the land utilization rate and achieve yield increase by changing the planting mode.It is of great theoretical and practical significance to reveal the molecular and physiological mechanism of strong cold resistance of Dn1 and to develop cold-resistance related gene resources.Four Dn1 cold-resistant response miRNAs libraries（5℃,0℃,-10℃and-25℃）were constructed in the previous stage.In this study,several miRNAs with significant expression differences were screened from the four libraries.Focusing on miR164 and its target m RNA（TaNAC6A）,bioinformatics analysis,q RT-PCR,5’RACE,tobacco transient co-transfection and dual luciferase were used to preliminically analyze the physiological and molecular mechanisms of miRNA-target m RNA（TaNAC6A）modulating Dn1 response to hypothermia stress.The main research results are as follows:（1）The expression patterns of tae-miR164 and its target gene TaNAC6A in Dn1 were detected by q RT-PCR under natural temperature drop in winter in field.The expression of tae-miR164 decreased with the decrease of temperature at 5℃,while the expression of TaNAC6A was the highest at-10℃and the lowest at 5℃.In leaves,the expression of tae-miR164 was the highest at 0℃and showed a downward trend with the decrease of temperature,while the expression of TaNAC6A was gradually increased with the decrease of temperature and significantly increased at-25℃.Both Tae-miR164 and TaNAC6A were expressed in response to low temperature stress.（2）Bioinformatics analysis showed that the full-length of pre-tae-miR164 was 156 bp and the mature sequence was 21 nt.The pre-tae-miR164 had stable stem-ring structure and high conservatism.The target gene of miR164 was predicted to be TaNAC6A by ps RNATarget software.The CDS of TaNAC6A was 918 bp long and encoded 306 amino acids.Here we add cis-actor prediction;TaNAC6A was predicted to be a stable hydrophilic protein with a typical conserved NAM domain and subcellular localization in the nucleus.（3）5’RACE accurately determined that TaNAC6A was cut at the 10th nucleotide cleavage site in the binding region of miRNA and target m RNA.The results of tobacco co-instantaneous expression and double luciferase experiment also confirmed that tae-miR164 inhibited the expression of TaNAC6A,which was consistent with the predicted results.（4）The results of promoter activity analysis showed that the expression of tae-miR164decreased and the expression of its target gene TaNAC6A increased under low temperature stress.As predicted.（5）The full-length of pre-tae-miR164 and TaNAC6A were cloned homologous,and the overexpressing and silencing vectors of pre-tae-miR164 and overexpressing TaNAC6A were constructed successfully.Arabidopsis thaliana was successfully transformed and screened to T3 generation,and 3 positive plants were obtained:OEtae-miR164,STTM-miR164 and OE-TaNAC6A.（6）After low temperature stress,the plant phenotypes of transgenic Arabidopsis Thaliana showed that the survival rate of OEtae-miR164 and nac6 mutant plants decreased,the leaf edges of OEtae-miR164 plants were serrated,and the root length was inhibited.The density of leaf surface trichoid of OEtae-miR164 Arabidopsis thaliana was decreased by optical microscopy.Scanning electron microscopy（SEM）showed that the leaf epidermal wax density of OEtae-miR164 Arabidopsis decreased,and the leaf stomatal opening decreased significantly after low temperature treatment,and the organelle structure was damaged obviously or even partially disorganized.（7）After low temperature stress treatment,physiological indexes of cold resistance of transgenic Arabidopsis thaliana were detected.The survival rate of OE-TaNAC6A and STTM-miR164 plants under low temperature stress was significantly higher than that of WT,and the contents of proline（Pro）,activities of superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）of both plants were significantly higher than those of WT.Conductivity,malondialdehyde（MDA）content and reactive oxygen species（ROS）content were significantly lower than those in WT.In contrast,in OEtae-miR164 and nac6 mutant plants,the changes of the above indexes showed an opposite trend,and the contents of Pro,SOD,POD and CAT decreased,while the relative conductivity,MDA content and ROS levels increased.（8）After low temperature stress treatment,the expression levels of stress-related genes At DREB1/2 and At ABI3/4/5 in OE-TaNAC6A transgenic Arabidopsis Thaliana were detected by q RT-PCR.The results showed that:The expression levels of At DREB1,At DREB2 and At ABI5 in OE-TaNAC6A transgenic Arabidopsis increased with decreasing temperature,and At ABI3 and At ABI4 showed a trend of increasing first and then decreasing.In conclusion,this study verified the regulation of cold resistance response of Dn1 by tae-miR164-TaNAC6A module through the application of allogenic expression strategies.By digging into the excellent cold-resistant miRNA resources of Dn1,the molecular mechanism of tae-miR164-TaNAC6A module in response to low temperature stress was preliminarily revealed,and the biological functions of tae-miR164 and TaNAC6A were allogeneously verified,providing theoretical basis for the improvement of cold-resistant crops. |
