Genetic Transformation Of ZmPOT Gene In Maize By TALEN And Overexpression Vector

Maize test weight not only is an important yield index, also is the important target character of crop breeding programs. It can reflect the corn maturity, integrity, uniformity, grain structure compactness and use value, it is an important indicators of corn commodities and processing quality. It had became an important factor of quality grading in international trade. For a long time, researchers focused on how to improve the nutritional value and processing quality about the quality of corn.But they ignored the impact of commodity quality, especially kernel volume weight of corn quality. The study for the genetic basis of complex traits of maize test weight is not enough in-depth, it is very slow to improve volume weight of corn in our country, put another words, it seriously affected the competitiveness of the international market. In this study, maize test weight is used as the research object, firstly, to find the main effect gene of ZmPOT on chromosome 7 in maize through QTL mapping. Then through prediction, the expression product of this gene belongs to POT family protein which is the key cell membrane proteins and could transport amino acid into cells.In this study, the gene of ZmPOT was cloned, and the plant overexpression vector pCUB-GT1-ZmPOT-bar with endosperm-specific prpmoter GT1 was constructed. Also, to construct the plant expression vector of TALEN. The overexpression vector and TALEN expression vector was then introduced into maize immature embryo tissue and embryonic callus by Agrobacterum-mediated transformation.Expecting study the gene under the expression and knockout mutant’s contribution to the maize test weight, which further research ZmPOTgene’s function. This study has obtained the following results:1.Total RNA of maize B73 kernel was conducted to reverse transcription for getting cDNA, we cloned the full-length of ZmPOT gene with 2084bp. It shared about 100% similarity with the gene of B73 in Gramene database by sequencing.2. We successfully constructed the overexpression vector with endosperm-specific prpmoter GT1 and named pCUB-GTl-ZmPOT-bar using in-fusion technology. Through PCR detection, restriction analysis and sequence analysis, the results showed that the expression vector was constructed successfully.3. Through the software analysis, in order to ensure the specificity, we designed a target site of TALEN in the second exon based on the CDS sequence of ZmPOT By the method of "Golden-gate", we succeeded in assembling TALEN plasmid module. Then TALEN plasmid module respectively was built into the left and right arm frame carrier.4. Respectively to cut the left and right arm frame carrier and pCAMBIA1301 plant expression vector using enzymes, then TALEN left and right arm module is connected to the pCAMBIA1301 plant expression vector. Restriction analysis and module sequence analysis showed that the expression vector were constructed successfully.5.The overexpression vector pCUB-GT1-ZmPOT-bar and TALEN expression vector introduced into maize immature embryo tissues and embryonic calluses by Aggrobacterum-mediated transformation. Transgenic resistance plants were obtained after screening by herbicide Basta and Hygromycin. A total of 9 and 146 resistant plants of To generation were obtained from the overexpression vector pCUB-GT1-ZmPOT-bar and TALEN expression vector respectively.6. To resistance plants transformed with pCUB-GT1-ZmPOT-bar and TALEN expression vectorwere certified to be positive by PCR detection.However there was no positive plants obtained from immature embryo tissues.15 positive plants of To generation were obtained from the resistant calluses transformed with TALEN expression vector.7. Fifteen of To positive plants with TALEN expression vector were seeded to Yunnan. 330 of the T1 plants were subjected to PCR for identification of positive transgenic plants. As a result,43 positive plants of T1 generation were obtained. Then using enzymes to identify the 43 positive plants, we did not find any mutations in target gene.8. Testing positive FokI expression content in plant leaf by qRT-PCR technology. Test results showed that some transgenic material FokI gene has high expression.9. To determine the basic phenotypic of T1 generation transgenic grain, we suggested there were significant changes in kernel length and kernel width.