Analysis Of AnDHN From Ammopiptanthus Nanus In Arabidopsis Improves Tolerance To Abiotic Stress

Dehydrin(DHN)is the second family of proteins rich in late embryonic development(LEA-II)and main class of dehydration stress-responsive genes.Abiotic stresses such as drought,high salt,and low temperature can cause dehydration stress in plants,induce large amounts of dehydranin expression,thus protect proteins,nucleic acids,and cell membranes.However,the functions of different types of DHN proteins in plant growth and resistance to abiotic stress are quite different.Ammopiptanthus nanus,as a kind of desert vegetation with strong stress resistance,exploring its molecular mechanism of stress resistance will help us to improve crop stress resistance,so as to improve crop yield and quality.In the previous research of the research group,we cloned and obtained a dehydratin gene from Ammopiptanthus nanus(An DHN).In order to determine the function of An DHN gene in the resistance of Ammopiptanthus nanus to abiotic stress,we constructed an An DHN overexpression vector and transferred it into Arabidopsis.The T3 generation homozygous of An DHN transgenic Arabidopsis as test materials and various abiotic stress resistance analyses were performed to observe the resistance of transgenic Arabidopsis thaliana to explore the function of An DHN genes in abiotic stress.The main results are as follows:1.In the experiment,the recombinant plasmid p CAMBIA1302-An DHN-GFP was constructed by in-fusion method,and agrobacterium containing the recombinant plasmid vector was injected into tobacco leaves by transient expression method.Meanwhile,using PAD62 Marker as a control,the subcellular localization analysis of An DHN protein was performed using a confocal microscope.The results show that PAD62 Marker is only distributed on the cell membrane,and it is not distributed on other organelles.However,the distribution of An DHN protein from Ammopiptanthus nanus is very similar to that of PAD62 Marker,and it is also mainly distributed on the cell membrane,which suggested that An DHN protein may play an important role in protecting plant cell membranes.2.The constructed recombinant p B7YWGII-An DHN was overexpressed into Arabidopsis plants by agrobacterium tumefaciens inflorescence staining,and selection of T0 generation An DHN transgenic Arabidopsis plants by spraying with herbicides.In order to quickly identify positive transgenic plants,among T1,T2 and T3 generation plants,in addition to spraying with herbicides,we also screened positive plants by using the bar gene test strip detection and PCR amplification detection method using genomic DNA as a template.Finally,An DHN transgenic Arabidopsis T3 generation homozygous plants were successfully obtained,which will provide important experimental materials for the next study of the function of An DHN in the resistance of plants to abiotic stress.3.Under drought,high salt and low temperature stress treatments,the T3 generation homozygous lines D1,D2 and D3 seeds of An DHN transgenic Arabidopsis as test materials were used to analyze the germination ability.The results showed that under normal conditions,the growth of seedlingswas not significantly different among the transgenic and wild-type Arabidopsis;however,under salt,drought,and low temperature stresses,the An DHN transgenic seedlings showed the more tolerance.In addition,under drought,high salt and low temperature stress,An DHN overexpression can improve the antioxidant activity of transgenic plants by increasing different antioxidant substances or enzymes.For example,Under drought stress,An DHN overexpression can reduce MDA content,while increasing RWC,PRO,GSH content,CAT and POD enzyme activity to increase the antioxidant activity of transgenic plants,thereby improving the drought resistance of transgenic plants.Furthermore,In addition,the results of DAB and NBT tests also indicated that An DHN overexpression enhanced the scavenging of ROS in the transgenic plants.Thus,these results indicated that An DHN overexpression significantly improvedthe antioxidant activity of the transgenic plants toprotect plants under drought,high salt,and low temperature stresses.