The Preliminary Function Analysis Of OsPCR1 Gene In Cd Transport

PCRs?plant cadmium resistance?are a kind of protrin,influences grain weight and Zn accumulation in grains.They widely distributed in fungi,algae,higher plants and animals.OsPCR family has been six members and some of members were clarified in the ionic transport activities.OsPCR1 is a member of the OsPCR family of proteins,Although previous reports have revealed that OsPCR1 is involved in the transport of zinc in rice and regulates the grain size of rice,the related studies on cadmium ions are rarely mentioned.Cadmium?Cd?is one of the most harmful heavy metal ions to humans and plants.To study the transport cadmium mechanism of OsPCR1 in rice cadmium,it is possible to reduce the cadmium content of rice grain,thus greatly reducing the risk of this heavy metal accumulation in the human body.In this paper,the ion transport function of OsPCR1 in rice was studied using the rice mutant pcr1 obtained by CRISPR/Cas9 gene editing technology and the overexpression of OsPCR1 in Arabidopsis to obtain heterologous overexpression lines.The results are as follows:Firstly,the knockout mutant of OsPCR1 makes it more insensitive to cadmium stress.By adding a certain concentration of CdSO₄ to the culture medium,we found that knockout mutants of OsPCR1 are more insensitive to cadmium than the wild type through plate experiments.We measured its fresh weight and plant height,indicating that under the stress of cadmium,the mutants are less toxic.Secondly,quantitative and semi-quantitative analysis of tissue expression patterns of OsPCR1.OsPCR1 was expressed in both above-ground and underground parts of rice,and the amount of expression in the underground part was much higher than that in the aerial part.The expression of the underground part of OsPCR1increased with the induction of cadmium,while the expression level of the aboveground part did not change much,which proved that OsPCR1may be involved in the transport of cadmium ions in rice.Thirdly,a 35S::PCR1-GFP subcellular localization vector was constructed to transform Arabidopsis mesophyll protoplasts and found that OsPCR1 was localized on the cell membrane.Fourthly,OsPCR1 was expressed in yeast in vitro and it was found that the yeast strain transferred to OsPCR1 was less sensitive to cadmium.Further determination of cadmium ions in yeast showed that the cadmium content of the yeast strain transferred to OsPCR1 was much lower than that of the control group,and the results of the liquid growth curve experiment were also consistent,indicating that OsPCR1is an outward-transporting protein.Cadmium ions are transported extracellularly.Fifthly,the heterologous overexpression of OsPCR1 in Arabidopsis revealed that the heterologous overexpressing strain was less toxic than the control under cadmium stress.Its biomass,whether fresh or rooted,is better than the control after being stressed.Further determination of Arabidopsis ions showed that the cadmium content of the heterologous overexpression line was lower than that of the control group.Sixthly,the knockout mutant seedlings were treated with different cadmium ions and found that the growth status of the pcr1 mutant was better than that of the wild type control.Then the Cd ion content was measured.The results showed that the accumulation of cadmium in the underground part of the mutant was higher than that in the wild type,but the aboveground part showed the opposite result.The zinc ion is lower in the underground part of the mutant than in the wild type,but the ground part shows the opposite result.Seventhly,mutant pcrl and wild type were planted in soil containing cadmium,and the cadmium content in different parts was measured.It was found that in the stems,leaves,husks,and grains,the cadmium ion content of the knockout mutants was lower than that of the wild type Nipponbare,but the opposite occurred in the roots.