| Crops will encounter many stresses during normal growth.As a large country for wheat cultivation,low temperature stress has a great impact on wheat crops.Dongnong Dongmai No.1（Dn1）is the first winter wheat variety in China that can be safely winter-resistant and cold-res istant（30°C low temperature）in the high and cold regions of Heilongjiang Province.It is a valuable material for cold tolerance research.Micro RNA（mi RNA）is closely related to the cold resistance of winter wheat.mi RNA regulates its target gene to initiate cold-resistance response;while lnc RNA interacts with mi RNA,it affects the expression of mi RNA on its target gene,and then makes cold resistance of winter wheat.Make a difference.Screening analysis was carried out in the strong cold-resistant winter wheat variety Dn1,and mi RNA and lnc RNA related to its cold resistance were found.In this study,6PGL with significant differential expression under mi RNA regulation was discovered by KEGG pathway.According to the mi RNA and lnc RNA databases established in different low temperature environments in the laboratory,the mi RNAs with significant difference in expression of Ta6 PGL can be found from the mi RNA database,which is tae-mi RNA5049-3p,and then found lnc RNA interacting with tae-mi RNA5049-3p from lnc RNA database,using RT-PCR and transgenic technology to detect the expression of the three.The plant expression vector of tae-lnc RNA-0310 was constructed,and Arabidopsis thaliana was infested by dip flowering method.The phenotype of T2 plants was observed and the phenotype of plants was analyzed.Semi-quantitative PCR analysis of transgen ic Arabidopsis T2 generation under cold stress The expression characteristics of tae-lnc RNA-0310-tae-mi RNA5049-3p-Ta6 PGL lays a fou-ndation for cold-resistant molecular bree ding of wheat.The main research results are as follows:（1）6PGL with significant expression under mi RNA regulation was found by KEGG path-way.Two lnc RNAs interacting with tae-mi RNA5049-3p which regulates Ta6 PGL expression were screened in Dn1 mi RNA and lnc RNA database: tae-lnc RNA-0310,tae-lnc RNA-0150,and the hom-ology of the target gene Ta6 PGL,showed that it had higher homology with the Bd6 PGL gene in Brachypodium serrata in other species.（2）The expression levels of tae-lnc RNA-0310 and tae-lnc RNA-0150 genes at different tem-peratures in Dn1 bifurcation were analyzed by q RT-PCR.The results showed that the expressionlevel of tae-lnc RNA-0310 was firstly decreased with temperature.The over all trend of increasing,then decreasing,and increasing,the relative expression of tae-lnc RNA-0150 showed a decreasing trend with the gradual decline of temperature.The negative correlation between tae-mi RNA5049-3p and tae-lnc RNA-0310 and the positive correlation between tae-lnc RNA-0310 and Ta6 PGL were preliminarily determined.The results were initially consistent with the competi tion between tae-lnc RNA-0310 and Ta6 PGL and tae-mi RNA5049-3p binding relationship.It is proved to be rela-ted to cold resistance and can improve the cold resistance of plants.（3）The secondary structure prediction of tae-lnc RNA-0310 and tae-lnc RNA-0150 showed that it has a typical neck ring structure,and the neck ring structure of tae-lnc RNA-0150 is more complicated.According to the full length of tae-lnc RNA-0310 and tae-lnc RNA-0150,the tae-lnc RNA-0310 was ligated and transformed,and p CAMBIA330035 s U-lnc RNA-0310 expression vector was constructed and infiltrated by dip flower method.Wild type Arabidopsis thaliana was cultured to obtain T2 generation tae-lnc RNA-0310 Arabidopsis thaliana transgenic plants.（4）The leaf phenotypes of wild-type and transgenic plants were observed,and the transgenic tae-lnc RNA-0310 Arabidopsis thaliana T2 plants were treated with low temperature stress at 4 °C for 72 h.The expression of tae-lnc RNA-0310 and its target gene Ta6 PGL was observed.The results showed that there was a positive correlation between the two;indicating that the cold resist-ance of the tae-lnc RNA-0310 transgenic plants was improved.RT-PCR and over-expressing plants confirmed that tae-lnc RNA-0310 can increase the expres sion of Ta6 PGL by competitively binding Tae-mi RNA5049-3p with Ta6 PGL under low tempera ture stress,thus mediating PPP pathway to improve plant cold resistance Sex.This study prelimi narily revealed that nc RNA regulates the expression of 6PGL in winter wheat under cold stress,and has important theoretical significance and good application prospects for molecular breeding of new cold-resistant winter wheat varieties in the northern cold regions. |
