Study On Molecular Mechanism Of Drought Resistance Of GGB In Arabidopsis Thaliana

Drought is a kind of adversity factor affecting the normal growth and development of plants,which can cause serious yield reduction or even death of crops.Therefore,improving the drought resistance of plants is of great significance to promote agricultural development.Plant ROP(Rho-related GTPases from plant)protein,also known as RAC,is a kind of G protein involved in signal transduction widely in higher plants.It is also called plant signal molecular switch which plays a key role in physiological and biochemical processes such as plant growth and development,stomatal regulation and so on.For example,ROP2 and ROP6 are involved in stomatal regulation.Type ? geranyl transferase that consists of alpha subunit PLP and beta subunit GGB and play a significant role in ABA-mediated drought control is involved in protein isoprenylation modification in Arabidopsis thaliana.However the mechanism by which GGB gene regulate drought resistance is not clear.Studies have shown that some ROPs can be isoprenylated,but it is not clear whether ROP2 and ROP6 or more other ROPs proteins are the target proteins of GGB isoprenylated modification and whether they can interact to participate in drought control.Therefore,the interaction between GGB and ROPs proteins in Arabidopsis thaliana was investigated and the molecular mechanism of GGB regulating stomatal closure was preliminarily explored,which laid a theoretical foundation for crop drought resistance breeding.The main results are as follows:(1)The ROP2,ROP3,ROP5 and ROP6 genes of Arabidopsis thaliana were cloned and inserted to pGBKT 7 vector.The pGADT7 yeast two-hybrid vector with GGB gene and pBridge yeast three-hybrid vector with PLP and ROPs were constructed.The results of yeast two-hybrid showed that GGB could not bind to ROP2,ROP3,ROP5 and ROP6 in yeast.The results of three-hybrid also showed that GGB could not bind to ROP2,ROP3,ROP5 and ROP6 in the presence of PLP subunits.Finally,the bimolecular fluorescence complementation expression vector of GGB and ROPs gene was constructed.The results showed that there was interaction between ROP2,ROP3,ROP5,ROP6 and GGB in tobacco.(2)A 35S-GFP-ROP6-P1300 vector was constructed.The tobacco leave was co-transformed with mixed Agrobacterium tumefaciens containing m55 marker gene and ROP6.The final results showed that ROP6 was located on the cell membrane.35S-GFP-ROP6-P1300 was transformed into ggb mutants and wildtype of Arabidopsis thaliana.The results showed that the subcellular localization of ROP6 in mutants and wildtype did not change significantly.(3)Homozygous rop6 T-DNA insertion mutant was successfully identified by the method of "double primers" and RT-PCR and then rought resistance were investigated.The results showed that water loss rate of leave and survival rate after rehydration were no significant difference between rop6 mutant and wild type.It was speculated that other ROPs genes may be involved in drought resistance and ROPs was is redundant in the resistance to drought.(4)In order to further identify other ROPs genes involved in drought resistance,CRISPR/Cas9 gene editing technology was used to design two target loci according to each gene of ROP3,ROP5,ROP3/ROP5,ROP6 CDS and genome sequence.AtU6::SgRNA-Cas9-P1300 gene editing vector with AtROPs target sequence was const ructed,and 70 transgenic Arabidopsis plants were obtained by flower dipping method.Target sequence mutation were testing,which provides a material basis for identification of more drought-resistant ROPs genes.