The Resistance Test, Genetic Analysis And Backcross Of Transgenic Glyphosate-resistant Maize

Maize ?Zea mays L.?, as the third largest crop food in the world, which ranks only next to rice and wheat, is very important fodder, industrial raw material, and biological energy source. The weeds in the farmlands compete with maize plants for space, water, sunshine, and fertilizer. Furthermore, maize can be infected with some disease caused by some insects on the weeds. The damage to the maize caused by weeds has led to a drastic reduction of the yield, which has seriously affected maize industry. The herbicide resistance maize plants which achieved through transformation will not be damaged when extirpate weeds by using herbicide. However, there are limited kinds of maize plants which are suitable for transformation system. And it is very difficult to put the transformed maize plants into practical use, as the yield of the transformed plants usually is quite low, and their comprehensive traits are not excellent. Therefore, achieving maize plants which not only possess traits expressed by exogenous gene but excellent comprehensive traits is of great significance.In this study, the male parents are the T₃ generation transformed glyphosate resistance maize plants of inbred line 18-599R, and the female parents are sweet corn, Mo17, R08, R18, RP125 and RP128 which with excellent agronomic traits and availabilities for practical use are female parents. Cross and backcross are proceeded with the expectation of obtaining transgenic maize plants which can be applied into practical use with excellent comprehensive traits and stable inheritance. Spraying glyphosate to test the resistance of F1, BC₁, BC₂ generation plants at three leaf stage and make statistics of survival plants and dead plants of BC₁ and BC₂ generation plants after spraying glyphosate to analyze the segregation ratio and explore the inheritance law. Furthermore, molecular detecting methods are used to track the exogenous target gene by PCR, RT-PCR and strip test, to test the backcross generations from DNA level, transcription level and protein level. The main results are as follows:1. The glyphosate resistance test in the farmlands and statistics:The segregation BC₁ and BC₂ plants of 6 female parents are obvious after spraying glyphosate, the result of the analyzing shows that the segregation ratio is almost 1:1, which conforms to one dominant allele gene inheritance law. While the ratios in the SH008 and BC₁ and RP125 BC₂ are not 1:1.2. The results of PCR detection:The PCR amplifications of some BC₁ and BC₂ generation maize plants show that the target gene is detected in all the samples. Which indicate that the target gene is transferred to the next generation through backcrossing in DNA level.3. The results of RT-PCR and sequencing:PCR amplify the DNA of the sweet corn, RP125 and RP128 of BC₁ and BC₂ generations which are chosen randomly, sequencing the detected gene and analyze the alignment with the sequence of2mG2-epsps gene published. The sequencing result shows that the alignment is 100% in both SH008 and RP125 BC₁ and BC₂ and 99% in RP128 BC₁ and BC₂. Extract RNA from the plants which target gene sequencing alignment is correct to proceed RT-PCR. The result of RT-PCR shows that target gene is detected in all the samples, which indicates that the exogenous gene is normally transcripted in the backcross offsprings.4. The result of protein test with the strip:Firstly PCR amplify part of the 6 female parents of BC₁ and BC₂ plants, the result shows that the target gene is detected in all the samples. And then the protein is tested by using the strip, the result shows that the protein is identified in SH008 BC₂, Mo17 BC,, Mo17 BC₂, R08 BC₁, R08 BC₂, R18 BC₁,R18 BC₂, RP125 BC₁ and RP125 BC₂. Whereas target protein is not detected in sweet corn BC₁, RP128BC₁ and RP128BC₂, which possibly caused by the less content of protein in these samples and the strips are not available for detecting. In the following experiment, it is of great necessity to detect the protein by using other molecular methods.