Cloning Of Genes Related To Phosphorus Efficiency And Genetic Transformation Of Maize

Phosphate is one of the most crucial elements for plants to grow and develop. It is not only the structural components of the cell, but also plays an important role in cell metabolism and signal transduction. The lack of phosphorus has been one of main factors that limit plant growth and productivity because of the relative inmobility of phosphorus and the low level of phosphate in soil. Maize is one of the most widely distributed grain crop and China has the second largest annual production in the world. Maize is also a very valuable feed for livestock and an essential raw material for producing industrial alcohol and liquor. A set of mechanisms has evolved in maize to deal with the low phosphorus stress in the soil, including some appropriate adjustments of root structure, phosphate absorption, the phosphorus turnover utilization, and so on. Understanding the relevant mechanisms and cloning genes involved in low phosphorus tolerance is of great importance to improve the grain yield.There are a large number of documents that describe DKEB1b gene participating in some stress-resistant process including low temperature resistance, drought tolerance, etc through a variety of pathways. So presumably, it may also has a certain effect on the tolerance to low phosphorus stress. Phosphate transporters play an important role in uptaking and transporting the phosphate. They help plants to transport phosphorus to important positions and maintain growth and metabolism, especially in the low phosphorus conditions.In this work, I cloned three genes, DREB1b, PHT1-1and PHT1-6from maize by PCR technique. All the genes might be involved in low phosphate tolerance. Firstly maize elite inbred line Qi319, which is sensitive to low phosphorus stress, was c ultured hydroponicly to3-leaf stage, and then transferred to low phosphorus conditions for eight days, and roots were sampled and RNA and genomic DNA was extracted as template for PCR reaction. DREBlb, PHT1-1and PHT1-6were then recombined into plant expression vectors, respectively. The recombinant plasmids were transformed into maize inbred Qi319and H99through agrobacteria-mediated genetic transformation. The transformed seedlings were transplanted to pots after co-c ulture with agrobacteria and then selected by spraying glufosinate at the stage of three-leaf. Only those transformed co uld survive. At last, for DREB1b, I obtained30glufosinate-resistant plants for H99and95for Qi319. Genomic DNA was extracted from2-3cm2of new-sprouting leaves sampled from the glufosinate-resistant plants and PCR was performed. The PCR-positive plants were transplanted to the field and T1were harvested. Genome DNA extracted from T1endosperm was used as template for PCR to identify the positive cases of seeds and the positive seeds were to plant in field to harvest T2for further research. For PHT1-1and PHT1-6genes, the genetic transformation of maize was ongoing, and transformed seedlings would be transplanted and selected soon.