Gene Editing Of Jasmonic Acid Signal Pathway Gene OsbHLH010 To Cultivate Low-Cadmium Rice Material

In China,the rapid development of industrialization and urbanization has consequently led to the problem of soil cadmium exceeding the standard which is becoming increasingly serious.Cadmium is a accumulative toxic heavy metal.Cadmium in soil can flow to the table through the food chain and ultimately endanger human health.Rice is one of the most important grain crops in China,and it is also a crop that is easy to accumulate cadmium in grain.Therefore,the selection and cultivation of low cadmium rice by biotechnology is not only conducive to improving the quality and safety of crops,but also conducive to human health.Jasmonic acid(JA)is an endogenous plant hormone that plays an important role in plant response to biotic and abiotic stresses.At present,the research on jasmonic acid and cadmium in rice mainly focuses on the changes of jasmonic acid content and some antioxidant enzyme activities under cadmium stress.The effects of jasmonic acid on cadmium absorption,especially on cadmium accumulation in grain,are rarely reported.here,we investigated the effect of jasmonic acid on cadmium absorption in rice and found that knockdown of jasmonic acid signaling pathway gene OsbHLH010 has potential application value in reducing cadmium content in grains.The main findings are as follows:Two-week-old rice seedlings were transferred to a nutrient solution with low cadmium(0.05 μM CdCl2)and treated with different concentrations of methyl jasmonate(MeJA)for 10 days to analyze the elemental content in the shoot and root.The results showed that after MeJA treatment,the cadmium content in rice shoot increased significantly.In addition to the significant increase in cadmium content after MeJA treatment,the manganese content also increased significantly after MeJA treatment.However,the treatment of OsNramp5 mutants with different concentrations of MeJA for 10 days did not increase the content of manganese and cadmium in the aerial parts of the mutant OsNramp5.After that,we detected the expression of OsNramp5 in rice roots at different time periods(12,24,48 h)under different concentrations of MeJA.Fluorescence quantitative PCR results showed that the expression of OsNramp5 was induced by MeJA,and the higher the concentration of MeJA,the more obvious was the induction effect.These results indicate that jasmonic acid promotes the accumulation of manganese and cadmium in rice by promoting the expression of OsNramp5.OsbHLH010 is a homologous protein of the core transcription factor OsMYC2 of jasmonic acid signal.The promoter of OsbHLH010 was fused with the reporter gene GUS,and pro010:GUS transgenic material was developed.GUS staining results showed that OsbHLH010 was expressed in roots and leaves,and its expression in the middle column of the root was stronger than its expression in other parts of the root.Transfecting tobacco leaf with the green fluorescent label 35S-sGFP-OsbHLH010 vector through Agrobacterium,we found that OsbHLH010 is located in the nucleus.We constructed and screened the OsbHLH010 mutants without transgenic components by CRISPR-Cas9 gene editing technology.Compared with wild type,the contents of manganese and cadmium in roots and stems of mutants decreased significantly under low cadmium treatment.The results PCR real-time fluorescence quantification showed that the expression of OsNramp5 in mutants decreased significantly.these results suggest that OsbHLH010 mutations cause decreased OsNramp5 expression and reduced manganese and cadmium uptake.In order to analyze the application prospects of this mutant,a soil pot experiment was conducted.OsbHLH010 mutant was grown in a cadmium-contaminated soil,after the rice maturation,the agronomic trait statistics and element contents were determined.The results showed that there was no significant change in plant height,1000-grain weight,tiller number,seed setting rate,yield per plant.Compared with the control group,the content of manganese in straw decreased by 25%,cadmium content decreased by 40%-50%,while the content of iron,copper,zinc and other elements did not change significantly.the important thing is that the cadmium content in the mutant grains decreased by 40%-50%,but the manganese,iron,copper,zinc and other elements did not change significantly.Above results show that the mutant osbhlh010 can reduce the cadmium content of nearly half of rice without reducing the yield of rice and the content of essential trace elements in grain,which indicates that the mutant has a good application prospect.To sum up,the research in this paper will lay a solid foundation for the breeding of low cadmium rice.