Cloning And Functional Analysis Of GmNHL1 Gene In Soybean Under Drought And Salt Stress

Soybean is the main grain and oil crop in our country.In recent years,the climate in Northeast China is changeable,and abiotic stress such as drought,salt and alkali in the early growth and development of soybean has an important effect on its yield and quality.Studies have shown that the NDR1/HIN1-like(NHL)gene family plays an important role in various defense responses,growth and development,and resistance to abiotic stress in a variety of plants.In the early stage of this study,a yield-related gene GmNHL1 was screened through GWAS genome-wide association analysis.In this study,the CDS region of the target gene was cloned,and the plant expression vector was constructed and its gene function was analyzed.The main test results are as follows:1.The full length of the target gene GmNHL1 gene ORF was 624bp and encoded 207 amino acids from soybean material Z070 by RT-PCR.Bioinformatics predicted that the protein was a hydrophilic,non-secretory protein.The subcellular localization vector pCAMBIA1302-GmNHL1-GFP labeled with green fluorescence was constructed and transformed into the leaves of the tobacco to observe the fluorescence performance.The results showed that the protein expressed by the target gene was localized on the cell membrane.2.The expression of target genes under different stress was analyzed by fluorescence quantitative PCR.The results showed that GmNHL1 gene could be induced to express in different degrees under drought,low temperature,ABA and salt stress.Under drought stress and salt stress,the expression level continued to increase and significantly up-regulated.3.The target gene GmNHL1 was integrated into plant overexpression vector pCAMBIA3301-Bar and CRISPER/Cas9 vector pCBSG015.The construction of the vector was verified by enzyme digestion sequencing.The genetic transformation was performed by agrobacterium-mediated method and pollen tube channel method.The integration of the progeny of transgenic plants was determined by PCR,Bar strip and Southernblot hybridization.A total of 14 T2 transgenic transgenic plants were screened,and 7 T2 transgenic transgenic transgenic transgenic plants were screened.4.SouthernBlot hybridization showed that the target gene was successfully integrated in the genome of the tested plants,but the integration form and hybridization signal strength were different.5.Functional analysis and verification of genes:Stress stress tests were performed on different strains obtained by genetic transformation and positive by various identification methods.The test results showed that under drought and salt stress,the plants exposed to the expression vector pCAMBIA3301-GmNHLl showed strong drought and salt tolerance.At the same time,physiological and biochemical indexes were determined,and phenotype was analyzed under stress.The results showed that compared with the control,the contents of malondialdehyde and O2of overexpressed strains(OE)were significantly decreased under drought and salt stress,but the activities of antioxidant enzymes(SOD,POD),contents of proline and contents of chlorophyll in plants were significantly increased.On the contrary,the gene edited lines(KO)showed low drought resistance and salt tolerance in pCBSG015-GmNHL1.It was preliminatively verified that GmNHL1 is a functional gene that positively regulates plant response to stress,and increases plant resistance to drought and salt stress by reducing the accumulation of reactive oxygen species in soybean plants,so as to improve plant drought and salt tolerance.This experiment laid a theoretical foundation for breeding new varieties of resistant crops.