Identification Of Drought-resistant Lines And Analysis Of Its Molecular Mechanism Of Transgenic Sorghum SbSNAC1 Gene In Maize

Maize(Zea mays L.)is one of the most important crops,which plays an important role in agricultural and industrial production in China.Drought is one of a main stresses that lead to the damage of maize production.Therefore,improving the drought resistance of maize is of great significance to maize production.With the wide application and development of transgenic technology,transgenic drought-resistant crops can be obtained quickly and contribute to the crop production.On the other hand,the safety assessment of transgenic crops has also been paid more and more attention.In our previous study,a SbSNAC1 gene was identified in a drought-resistant sorghum variety "XGL-1" in Xinjiang.In the study of gene expression pattern,it was found that SbSNACl gene could respond to abiotic stress such as drought stress,high salinity,low temperature,and plant hormone ABA.Overexpression of the gene in Arabidopsis can significantly improve the survival rate of Arabidopsis under drought treatment compared with the control group.In this study,the elite drought-resistant transgenic maize lines with SbSNAC1 gene were identified and characterized.Furthermore,the molecular characteristics of the transgenic lines were identified by using Western,Southern,and third-generation sequencing techniques.The molecular mechanism of drought resistance of SbSNACl gene in maize was further analyzed by RNA-seq ChIP-seq,Y1H and other techniques.The main results are as follows:1.Two elite transgenic lines with drought-resistance,SbSNAC1-382 and SbSNAC1-466,were screened out from several transgenic lines by drought-resistance analysis.Under the drought treatment,the survival rate and relative water content of the leaves of the two transgenic lines were significantly higher than that of the control lines,the water loss rate and stomatal conductance were significantly lower than that of the control lines,and the ABA and H2O2 Content in the leaves of the transgenic lines were significantly increased compared with the control lines.The stomatal closure rate of the transgenic lines was significantly higher than that of the control lines accord to the results of electron microscope analysis.It might be the changes of these physiological characters which improved the drought resistance of transgenic maize.2.Southern blot showed that SbSNACl gene could be inherited stably in SbSNAC1-382 and SbSNAC1-466 transgenic lines,which has 2 and 1 copy respectively.And it was found that SbSNAC1 gene could be expressed stably at the transcription and translation levels in the two transgenic lines by using RT-PCR and Western blot techniques.Furthermore,with genome walking and Third-generation sequencing techniuqes,it was found that the integration of exogenous insertion sequence of SbSNAC1 transgenic maize lines SbSNACl-382 and SbSNAC1-466 were located at 177155650 to 177155696 on chromosome 5 and 205240398 to 205240482 on chromosome 1,respectively.3.Using ChIP-seq technology,we found that the core binding sequence of SbSNAC1 protein was ACGT in transgenic maize.According to the results of Chip-seq,11 downstream genes related to drought-resistance which were regulated by SbSNAC1protien in transgenic maize lines,and 10 genes were assured by Y1H analysis.