Study On The Regulatory Network Of Rice To Enhance Cadmium Resistance By Overexpression Of Lsi1

Heavy metal pollution in soil is one of the major environmental hazards in the world,while Cadmium(Cd)pollution ranks first in all heavy metal pollution.Cadmium has a great impact on the growth and development of rice,which can damage the chlorophyll structure of rice leaves,reduce the chlorophyll content,resulting in slow growth,decrease plant height,inhibited root growth and development of rice.Previous studies have shown that silicon treatment not only significantly reduces the amount of cadmium but also reduces the ratio of cadmium accumulated in the aboveground and below ground and in leaves and stem.Silicon can alleviate the toxicity of cadmium on rice by inhibiting the uptake of cadmium by rice and upward transport of cadmium,increasing the deposition of cadmium in cell walls and the proportion of exchangeable cadmium in free space.Previously,it was found that after inhibiting the expression of Lsi1 gene in rice,resistance to cadmium stress in rice was decreased.After enhancing the expression of Lsi1 gene in rice,the ability of rice to resist cadmium stress was enhanced,suggesting that Lsi1 can regulate rice cadmium resistance.In order to reveal that Lsi1 regulates the rice cadmium-resisting protein action network,the Lsi1 expression system for stabilizing FLAG and GFP-tagged proteins was cloned by Gateway cloning technology to genetically transform Lemont and Dular rice,respectively.PCR amplification of genomic DNA and Western blotting of proteins were performed.It was confirmed that the Lsi1 fusion gene was successfully integrated into the rice genome and expressed,and positive transgenic rice plants were obtained by screening.The transgenic plants were treated with 1 mM cadmium for seven days.The results showed that the leaves of FLAG-Lsi1 Dular rice and Lemont rice transgenic plants exhibited milder symptoms of cadmium poisoning than wild-type plants,and had strong phenotypic tolerance to cadmium toxicity.Lsi1 gene can increase the resistance of rice to cadmium and reduce the toxicity of cadmium on rice.In order to further study the regulation of Lsi1 protein,the co-protein of NIP protein encoded by Lsi1 gene was obtained by immunoprecipitation.The results showed that 583 NIP-interacting proteins were identified in Cd-transgenic rice plants,and 272 NIP-interacting proteins were identified in the cadmium-treated transgenic rice.Thus,after cadmium treatment,the number of proteins interacting with NIP in transgenic rice leaves was significantly reduced,suggesting that cadmium stress inhibited the expression of interacting proteins.A comparative analysis of the interacting proteins of the Cd-treated and non-Cd-treated samples revealed that 183 interacting proteins were identical.In the samples without Cd treatment,400 proteins were not detected in the Cd-treated samples.These proteins include ferredoxin-NADP reductase,serine hydroxymethyltransferase,fructose diphosphate aldolase,soluble starch synthase,glucose-6-phosphate isomerase,pyruvate dehydrogenase,acetyl-CoA synthetase,glutamate dehydrogenase,xylanase inhibitory protein,GTP-binding protein and aquaporin PIP2-7,etc.It suggesting that the cadmium-stressed interactions between the original protein interactions of rice were inhibited or destroyed by cadmium stress.At the same time,89 interaction proteins were found only in Cd-treated samples,these proteins include ferredoxin-NADP reductase,serine hydroxymethyltransferase,fructose diphosphate aldolase,soluble starch synthase,glucose6-phosphate isomerase,pyruvate dehydrogenase,acetyl-CoA synthetase,glutamate dehydrogenase,xylanase inhibitory protein,GTP-binding protein and aquaporin PIP2-7,etc.It indicating that the interaction process of these proteins occurred after Cd stress,suggesting that cadmium stress stimulated the interaction of these proteins with Lsi1 protein,presumably under cadmium stress these proteins are likely to interact with the Lsi1 protein to enhance rice resistance to cadmium stress.The function of 272 proteins interacting with NIP proteins in cadmium-treated transgenic rice was classified.The results showed that these proteins are mainly involved in photosynthesis,carbohydrate metabolism,lipid metabolism,nitrogen metabolism,amino acid metabolism,secondary metabolism,and hormones.Metabolism,stress response,redox regulation and signal transduction processes,among which 5 proteins are consistent with the interaction protein of the NIP protein provided in the STRING database.In addition,this study also identified an aquaporin PIP2-7.The non-invasive micro assay technique was used to determine the cadmium ion flux in Lsi1 gene over-expression,interfering materials,and wild-type material.The results showed that the cadmium uptake of rice plants with both Si and Cd at the same time was overexpressed in rice and wild-type plants.In contrast,simultaneous addition of silicon and cadmium and addition of cadmium alone did not significantly affect the uptake of cadmium in rice by the Lsi1 gene,but it was significantly higher than that of the Lsi1 gene overexpressing rice and wild type,suggesting that silicon still exists in the underground part of rice.The antagonism of cadmium reduces the absorption of cadmium by rice.Through this study,it can be seen that rice expression of Lsi1 not only regulates silicon uptake and antagonizes cadmium accumulation,but Lsi1 gene can also regulate the ability of rice to resist cadmium stress through the interaction of protein and protein in leaf expression,suggesting that Lsi1 may have a cause multiple positive effects.