Expression Pattern Of GPAT Gene And Its Promoter Identification In Xinjiang Azolla

Plant growth is affected by some environmental stresses in the natural environment,including cold,low temperature,high salt and drought.Plants adapt to the effects of environmental stress on some complex molecular mechanisms such as signal transduction,perceptual stress,and activation of specific gene expression.Under low temperature stress,the transcription initiation site of the cold response-related gene is regulated as a cold-inducible promoter of the molecular switch,thereby enhancing the plant's resilience to cold stimulation.There are few studies on the function of promoter structures.In this study,the cold-resistant plant A.nanus was used as a test material to clone the promoter sequence of glycerol triphosphate acyltransferase gene(GPAT)by using chromosome walking technology,and its function was studied.The main experimental results are as follows:1.The expression levels of GPAT gene at different time and under low temperature and exogenous ABA treatment were detected by real-time fluorescence quantitative(q PCR).The expression of GPAT gene in root and stem tissues of A.sinensis was basically the same.It showed a downward trend before 48 h,then increased,and the expression level in the leaves was an upward trend before 24 h,and it was a downward trend after 24 hours.The expression changes of the three tissues were higher than the control.It can be seen that low temperature induces an increase in the expression level of the GPAT gene.Under the treatment of ABA,the expression of GPAT gene in roots,stems and leaves showed a significant increase and then decreased.The expression of the GPAT gene in roots and stems reached the highest value at 48 h,which was 23 times of the control.At 29 times,the change in expression level reached the highest value at 24 h in leaf tissue,which was 42 times that of the control.In summary,ABA induced an increase in the expression of GPAT gene.2.The pipstream promoter of the GPAT gene was cloned,and a 1695 bp sequence fragment upstream of ATG was obtained.The sequence was compared with the original sequence by DNAMAN software,except for the initial 100 bp.In addition,there is no repeated sequence,which is compared with the database,and there is no sequence of homology.It is proved that the obtained promoter sequence was cloned for the first time and named GPATp1.3.Predictive analysis of cis-acting elements that may be present in the GPAT gene promoter GPATp1 sequence was performed using the plant promoter prediction database PLACE and Plant CARE.It was then found that TATA-box,CAAT-box,two conserved and other upstream cis-acting elements,including the ABA response element ABRE,the cold response element MYB/MYC and the light response element G-box,and some elements that enhance transcription levels And so on in the promoter region.4.The upstream fragment of the GPAT gene promoter ATG was cloned by chromosome walking technique and named as GPATp1.The vectors lacking the promoter region fragments were constructed and named as GPATp1-1,GPATp1-2,GPATp1-3,and GPATp1-4,respectively.For the four different lengths of the deletion fragment GUS fusion vector,the Agrobacterium-mediated transient transformation of tobacco leaves was used.After infection,the tobacco plants were treated with low temperature and ABA.The deletion fragments of the four promoters did not have a promoter at low temperature.Activity,under ABA treatment,the longer the length of the deletion fragment,the stronger the promoter activity.