Vector Construction And Expression Analysis Of Cold-Related MiR164 And Its Interacting LncRNA And Target MRNA In Winter Wheat

Low temperature stress is one of the important obstacles that affect the normal growth of crops.The yield of crops is directly affected by the resistance to cold stress.Dongnong Dongmai 1(Dn1)is a strong cold-resistant variety cultivated by Northeast Agricultural University.It is of great theoretical and practical significance to carry out research on cold resistance mechanism.MicroRNA(miRNA)is closely related to the cold resistance of winter wheat;lncRNA can be used as ceRNA(competing endogenous RNAs),competitive binding miRNA,and regulate the expression of target genes together with miRNA and regulate the cold resistance of wheat.Dn1 has important resources miRNA and lncRNA.about cold-resistant.In this study,miRNA libraries and lncRNA libraries differentially expressed at different temperatures(5°C、-10°C、-25°C)constructed in the laboratory were used.Cold-related miR164 was screened through bioinformatics software.Further predict its target genes and lncRNA that interacts with miR164,The expression of the three genes was detected by RT-PCR.Construction of an expression vector of miR164 and its interaction with lncRNA laid the foundation for the breeding of wheat cold resistance molecules by means of biotechnology.The main findings are as follows:(1)In the lncRNA-miRNA database of Dn1 interaction,lncRNA-1156,lncRNA-1061,and lncRNA-0104 were screened for interaction with miR164.Bioinformatics software predicts that the target gene for miR164 is TaNAC6 A.Homology analysis showed that the homology was higher with the NAC gene in barley.(2)QRT-PCR quantitative analysis of TaNAC6 A,miR164 and lncRNA-1156 genes in Dn1 tiller nodes at different temperatures.The results showed that the relative expression of lncRNA-1156 showed an overall trend of increasing with decreasing temperature,miR164 showed a decreasing trend,and TaNAC6 A showed a rising trend.The negative correlation between miR164 and lncRNA-1156 was determined,and the negative correlation between miR164 and TaNAC6 A.The results preliminarily coincided with miR164 negatively regulated TaNAC6 A,lncRNA-1156 and TaNAC6 A competitive miR164 binding.The relative expression of TaNAC6 A in fast-tolerant cultivars showed a trend of rapid increase under conditions of decreasing temperature.It is proved that it is related to cold resistance and may improve the cold resistance of plants.(3)Analysis of conserved sequences in the miR164 mature region revealed that miR164 is highly conserved,and its secondary structure predicts that it has a typical stem loop structure.Cloning,ligation and transformation of miR164 based on miRNA precursor sequence and obtaining expression vector of pCAMBIA330035sU-pre-tae-miR164.expression of pre-tae-miR164 Arabidopsis T1 generation transgenic plants was obtained.(4)The secondary structure of lncRNA-1156 was predicted to be complex.The pCAMBIA330-035sU-lncRNA-1156 expression vector was constructed.The lncRNA-1156 overexpression vector and the pre-tae-miR164 overexpression vector obtained in this experiment have important theoretical and practical significance for the further adoption of biological technology to carry out cold resistance molecular breeding.