Physiological And Molecular Mechanisms In Enhanced Salt And Drought Tolerance Of Pgtip1-Transgenic Soybeans

Tonoplast intrinsic proteins(TIPs)are important functional proteins that are located on the tonoplasts of plants and mediate the transport of water and other small molecules across the membrane.In the process of research and discovery,they can be divided into five subfamilies:TIP1,TIP2,TIP3,TIP4 and TIP5,which are mainly involved in the physiological processes of plant growth and development,such as water absorption and root nutrient absorption,tissue expansion,transpiration and Photosynthesis etc.Secondly,TIPs perform important functions in response to a variety of abiotic stresses such as drought,salt and oxidative stress.Previous studies have shown that,the Panax ginseng TIP gene PgTIP1 was previously demonstrated to have high water channel activity by its heterologous expression in Xenopus laevis oocytes and in yeast;Transgenic PgTIP1 Arabidopsis plants grew better under normal environmental conditions and their tolerance to salt or drought stress was significantly enhanced.However,its role in the adaptation of crops,such as soybeans,to salt or drought stress has not been studied and reported.In this study,the cultivated soybean Lee68 was used as the experimental material,while screening transgenic soybean(T2 positive strains)of Nannong 8831 original species and 4076 original strains,mediate by Agrobacterium rhizogenes K599,using the marker-free plant expression vector pCAMBIA0390 to study the transformation method and efficiency of transgenic PgTIP1 gene in soybean,at the same time,preliminary verification of the tolerance of PgTIP1 function to salt stress of soybean materials was carried out.In the process,the existing hair root experiment technique was optimized from the preparation,infestation and culture conditions.Secondly,the PgTIP1-transgenic soybean plants mediated by the pollen tube pathway,represented by the lines N and J11,were analyzed at the physiological and molecular levels for enhanced salt tolerance.The results showed that PgTIP1-trasgenic soybean lines were found to possess stronger root vigor,reduced root and leaf cell membrane damage(REL),increased SOD,POD,CAT,and APX activities,steadily increased leaf Tr,RWC,and Pn values,and smaller declines in chlorophyll and carotenoid content when exposed to salt stress compared to wild type.Moreover,the distribution patterns of Na+,K+,and Cl-in the roots,stems,and leaves of salt stressed transgenic plants were readjusted,in that the absorbed Na+and Cl-were mainly restricted to the roots to reduce their transport to the shoots,and the transport of root-absorbed K+ to the shoots was simultaneously promoted.PgTIP1 transformation into soybean plants enhanced the expression of some stress-related genes(GmPOD,GmAPX1,GmSOS1,and GmCLC1)in the roots and leaves under salt treatment.Finally,PgTIP1 gene was further transformed into the hybrid strain 4076(F5),which was selected for salt tolerance generation by generation from the cross assembly G.max N23674×G.soja BB52 accession.The PgTIP1-transgenic soybean plants mediated by the pollen tube pathway,represented by the lines L19 and L29,were simultaneously analyzed at the physiological and molecular levels for enhanced salt and drought tolerance.The results showed that,the salt-stressed PgTIP1-transgenic L19 and L29 have acquired better leaf stomatal movement and water-gas exchange capacity,obvious drops of absorption and accumulation of Na+and Cl-in shoots,and enhanced antioxidase activities of SOD,POD,CAT,and APX in the roots or leaves in contrast to WT.When suffered from drought stress,the strengthened leaf water-retention capacity and the alleviated cell membrane damages(REL and MDA)of roots and leaves were displayed for PgTIP1-transgenic soybean plants.Thus,the PgT1P1-transgenic soybean lines could exhibit salt and drought tolerance together,which are not only related to the primary functions of PgTIP1 gene involved in water status regulation,but also to its secondary roles in up-regulated expression of stress-related genes(GmNHX1,GmSOS1,GmCLC1,GmCAT1,GmAPX1,GmNCED1 and GmP5CS1).In addition,we also conducted field surveys and routine agronomic traits surveys of transgenic PgTIP1 soybean strains.We discover in addition to 100-seed weight(three materials were consistent),the plant height,number of branches,number of stem nodes,number of plants per plant,total grain number per plant,and total grain weight per plant were significantly increased compared with the original species(WT);Compared with the original lines(WT),strains L19 and L29 had only significant increases in plant height and 100-seed weight,and no changes in other indicators.In terms of agronomic traits,the necessity of simultaneously researching cultivated soybeans and wild soybeans(or hybrids of wild soybeans)for the selection of new stress-resistant varieties and their significance for production practices can be demonstrated.In summary,heterologous expression of PgTIP1 gene in soybean materials can improve the salt or drought tolerance of transgenic soybeans by improving water relationship,cell membrane damage,photosynthesis,ion homeostasis and ROS scavenging ability.It is a good candidate gene for molecular genetic improvement of salt-tolerant and drought-tolerant crops in the future.