Cloning And Expression Analysis Of DREB2 Gene From Paspalum Notatum

Drought and saline-alkali stress have serious effects on the growth and development of plants.Under extremely harsh environmental conditions,they can directly cause plant death.In the process of long-term evolution of higher plants,various complex defense mechanisms have been formed to resist stress,DREB transcription factor is a kind of peculiar to the plant,with DRE cis element of specific binding of transcription factors.This type of transcription factor regulates the expression of a series of stress-resistance genes in abiotic stresses.Therefore,genetic manipulation of DREB transcription factors are widely and successfully used in anti-abiotic stress breeding in virous plants which can obtain a better comprehensive effect.As a popular grass species,Paspalum notatum has strong resistance ability to and can be used as a good material for studying the stress resistant mechanism in plants.In order to excavate stress-resistance genes and explore the stress resistance mechanism of Paspalum notatum.In this study,based on cloning of the DREB2 gene of Paspalum notatum,the PEG-6000(20%)and Na Cl(300 mmol·L-1)solutions were used to simulate the stress and the tissue expression and physiological index changes of DREB2 gene were analyzed.The results are as follows:(1)A total of 42,844,132 sequence reads were obtained from transcriptome sequencing,which including sequence information of 6,426,619,800 bp(6.42 GB).Reads were sequence assembled to acquire 99,235 unigenes with an average length of641.96 bp.(2)53,886 unigene of Paspalum notatum can find similar sequences in the NR database.Among the closely related species that match similar sequences,the highest levels of Setaria italica,Sorghum bicolor,and Zea mays.The classification notes of GO,COG and KEGG database all indicate that a large number of unigenes are relatedto the information transmission mechanism,transportation and metabolism.(3)Using RNA-Seq combined with RT-PCR technology,the DREB2 gene was obtained from Paspalum notatum and named Pn DREB2(Gen Bank accession number:MH150946).Amino acid sequence analysis showed that the ORF of Pn DREB2 was774 bp,encoding 257 amino acids with a relative molecular mass of 28.09 k D and a theoretical isoelectric point of 5.25.This plant protein has a conserved domain AP2 domain typical of the DREB family of genes.(4)The results of evolution and cluster analysis showed that the genetic relationship between the Pn DREB2 gene and Sorghum bicolor,Saccharum spontaneum,Zea mays,Setaria italica and Hemarthria compressa closer,amino acid sequence homology were 91.10%,89.50%,88.20%,87.60% and 87.60%.(5)q RT-PCR analysis showed that under drought and salt stress,the expression of Pn DREB2 gene was the highest in the stem of Paspalum notatum,followed by young ear and leaf.Pn DREB2 m RNA expression was strongly induced by drought(PEG-6000,20%)and high salt(Na Cl,300 mmol·L-1),it showed a trend of increasing first and then decreasing.(6)With the extend of PEG stress time,the soluble protein content,soluble sugar content,POD activity,SOD activity and chlorophyll a content all showed a trend of increasing first and then decreasing,while CAT activity showed a trend of decreasing firstly and then increasing.Chlorophyll b and total chlorophyll content increased first and then decreased,followed by a small increase;the proline and malondialdehyde increased with increasing stress time.(7)With the extend of Na Cl stress,the content of soluble protein showed a decreasing trend.The contents of proline and malondialdehyde increased with the extension of Na Cl stress time,the other indicators showed a trend of increasing first and then decreasing.(8)Under Na Cl and PEG stress,the expression of DREB2 gene showed a significant positive correlation with antioxidant enzymes,and negative correlation with total chlorophyll,chlorophyll a,and chlorophyll b.Differential expression of DREB2 gene and soluble protein,soluble sugar,proline,malondialdehyde differencebetween two stress treatments.Under salt stress,the expression of DREB2 gene was not significantly positively correlated with soluble sugar and soluble protein.There was a negative correlation between amino acid and malondialdehyde.Under drought stress,DREB2 gene expression was positively correlated with soluble sugar and proline,not significantly correlated with malondialdehyde,and negatively correlated with soluble protein.