Detection Of Maize Transgenic Plants And Optimization Of Methods

With the development of the biology technology,genetically modified(GM)technology had been utilized to alter the genetic character of various crops over the past decades,including maize、wheat and other major food crops,which has made great contributions to improve the production of grain.But the safety problem of GM food is paid much closer attention in recent years,so it is urgent to take measures to manage GM food security.However,the foundation is to build comprehensive and scientific GM detection methods,which can provide basic technology service for management of GM food security.We take advantage of maize as materials to research the application、comparasion and assessment of GM detection methods in this paper,just offering some simple methods and stratagems for GM maize detection.The followings are major results:1、We treated the GM T3 generation plants with TsVP and bar gene by spraying herbicide glyphosate when they grew at 3rd-leaf stage.And then we detected some plants with better glyphosate resistance among them by PCR method.The positive GM lines of PCR results are 31 among 110 TsVP gene lines,so the positive rate of TsVP gene lines is 28.1%;The positive GM lines of PCR results are 61 among 190 bar gene lines.And then we took advantage of lateral flow strips to detect 6 positive samples of PCR detection,the results showed that both control line and test line appeared on the strips which demonstrated that all of the 6 samples expressed the PAT protein encoded by bar gene,this results matched well with PCR detection.However,other 4 wild type samples just revealed control line without test line,therefore they were negative.2、We extracted the agarose gel with positive PCR electrophoresis band of TsVP gene to sequence in case of false positive results.Comparing the sequence of sequencing with primal sequence,we found that the match rate reached 99%,which fully suggested that the results of PCR detection were reliable.3、We detected the relative expression level of Zmdal in Zmdal overexpressing plants and ZmbZIP4 in ZmbZIP4 overexpressing plants via real-time RT-PCR.The results showed that expression level of Zmdal in GM plants was significantly higher than that in wild type;Because the ZmbZIP4 was promoted by a stress response promoter,the expression level of ZmbZIP4 in GM plants was different with wild type to a certain extent under normal condition;but after stressing by 150mM NaCl for 12h,the expression level of ZmbZIP4 in GM plants was significantly different with wild type.These results suggested that the insertion of single copy affirmed by Southern blot hybridization expressed normally in plants,which proved they were GM plants for further.4、Southern blot was used to confirm the GM lines with single copy of target gene for further.According to the different T-DNA insertion region sequences,we designed appropriate and special Southern blot hybridization probes.Those DNA probes were labeled with Dig-11-dUTP alkali-labile using random oligonucleotides as primers,then the efficiency of labeled probes was tested by dot hybridization;The whole genome DNA was extracted from fresh leaves using CTAB method,we examined the purity、concentration and integrity of DNA via ultraviolet spectrophotometer and agarose gel electrophoresis;And then the DNA fragments with different size which resulted from that genome DNA was digested by restriction enzyme were separated by agarose gel electrophoresis,then they were transferred to Nylon membrane with positive ion.Next,the Nylon membrane was dealt by a series of procedures including fixing、hybridizing、blocking、immunizing and color-developing.We detected 6 different types of GM materials by Southern blot,the results confirmed that the these genes were integrated into the genome DNA,and existed in the form of only one copy.5、Utilizing the ZmbZIP4 standard protein to establish ELISA quantitative standard curve,we analyzed the level of ZmbZIP4 protein in overexpressing plants and wild type.The ELISA results showed that the protein concentration in GM plants is lto 2 times as much as it is in wild type under normal condition;however,the level of ZmbZIP4 protein in overexpressing plants increased obviously after treatment of salt stress,and the protein concentration in GM plants reached 2.7、4.8、5.8 times compared with wild type,the statistical analysis results reached a significant difference.In general,We made use of the antibody of ZmbZIP4 protein to quantify the concentration of the ZmbZIP4 protein in GM plants and wild type under normal condition and salt stress by ELISA method,then made comparison between them.