Functional Analysis Of NRAMP Family Candidate Genes In Maize And Virus Of Maize Rough Dwarf Disease Detection

Maize,as the most widely cultivated crop in China,is an important grain,feed and industrial raw material.It plays an essential role in promoting the national economic development and food security.However,at all stages of growth and development,maize is affected by biotic or abiotic stress factors like heavy metal pollution,disease and insect pests,which lead to the decrease of Yield and the decline of the quality.With the continuous development of modernization and industrialization in China,the environmental problems caused by heavy metals are becoming more and more serious,which poses a great threat to the food security.Cadmium(Cd)is the main heavy metal pollutant among the national soil pollution.Analysing its absorption,transport and grain enrichment in maize plants,identifing candidate genes involved in the process and improveing the tolerance and enrichment ability of cadmium in existing maize materials by means of molecular techniques can help to cultivate low-cadmium-accumulated maize materials.Maize rough dwarf disease,known as the"cancer of maize",is one of the most important diseases that seriously threatens maize production.It was reported that the rice black strip dwarf virus(RBSDV)is the culprit of maize dwarf disease in China,and RBSDV is spread by grey planthopper between crops.Therefore,it is very important to strengthen the preventive measures for the timely detection of RBSDV.As a powerful tool for genome editing,CRISPR/Cas system has made the genome editing technology simple and efficient.It has become a hot technology in the research field of human,animal and plant functional genes at the present stage.In this study,on the basis of preliminary verification of the function of Cd stress response candidate genes obtained from early screening,the CRISPR/Cas9 based on the knocking vector was constructed and genetic transformation was carried out.At the same time,RBSDV RNA was cut in CRISPR/Cas13a system to destroy RNA enzyme reporter molecules for RBSDV detection.The main results are as follows:1.Using bioinformatics analysis,the NRAMP family protein evolution tree was constructed,and 7 Zm NRAMP family genes were obtained.According to the protein prediction software CELLO,SUSUI,MEMSAT and so on,the Zm NRAMP family proteins were predicted to be membrane proteins,including 9～12 transmembrane helices.The expression of maize NRAMP family genes under Cd stress was analyzed by q RT-PCR assay.Results showed that all Zm NRAMP family genes were up-regulated under Cd stress,and the expression of Zm NRAMP family genes in roots was much higher than that of genes in stem and leaf,indicating that this gene family is involved in response to Cd stress and is more sensitive to the response to Cd stress in roots than in shoots.The up-regulated expression of ZmNRAMP2 and Zm NRAMP3 under Cd stress was significantly higher than that of other members of Zm NRAMP family.It was speculated that ZmNRAMP2 and Zm NRAMP3 were the two most sensitive genes of the Zm NRAMP family in response to Cd stress.The transient expression test of ZmNRAMP2 and Zm NRAMP3 showed that ZmNRAMP2 and Zm NRAMP3 were located on the plasma membrane and were consistent with the predicted sites.2.The total length of two genes of ZmNRAMP2 and Zm NRAMP3 were obtained,and the knockout sequences were screened according to the sequence characteristics,and the CRISPR/Cas9 knockout vectors of these two genes were successfully constructed.The vector was transformed into maize callus by Agrobacterium mediated transformation。In the 58 positive seedlings that survived,the sequencing analysis was conducted on two gene target sites of 20 materials,and the results showed that all of them had mutations.3.Construction of Cas13a protein expression vector:highly-purified Cas13a protein was obtained by building and purifying protein that expressed in prokaryotic.A shearing experiment was conducted to RNA fragment of the RBSDV,and results show that the combination of Cas13a and cr RNA can degrade the target RNA,which means that the CRISPR/Cas13a shear system has specific cutting activity.The target RNA concentration was amplified by RT-RPA and T7 transcripase transcriptional technology.Target RNA was identified and cut by CRISPR/Cas13a system.The incidental cutting effect of Cas13a was caused to destroy the RNA enzyme reporter,and the the green fluorescence was detected in reaction solution by the fluorescence detector.The detection of RBSDV through this method can detect RBSDV with a concentration of 1×10~1 copies/L,showing a very high sensitivity.